Liquid phase peptide carrier synthesis of peptides and peptidomimetics

Abstract

The present invention provides compounds useful as carriers for liquid phase organic synthesis, such as liquid phase organic synthesis of peptides and peptidomimetics. In one aspect, the invention provides a compound having the structure of Formula (I): wherein R1, R2, R3, m and n are as described herein, as well as methods of making and using the compound.

Description

[0001] Field of the Disclosure

[0002] The present disclosure relates to liquid-phase peptide carrier synthesis of peptides and peptidomimetics, pharmaceutical compositions, and methods of treating GLP1R-related conditions.

[0003] Background of the Disclosure

[0004] The difference between liquid-phase peptide carrier (LPPS) synthesis and solid-phase peptide synthesis (SPPS) is that the latter is supported on a large solid polymer, while the former is supported on a well-defined molecule, and the difference between LPPS and CSPS is the phase separation as a separate precipitate in LPPS versus phase separation in CSPC. SPPS is the well-established and most common and popular method for making polypeptides (Jaradat D., “Thirteen Decades of Peptide Synthesis: Key Developments in Solid Phase Peptide Synthesis and Amide Bond Formation Utilized in Peptide Ligation,” Amino Acids 50(1): 39-68 (2018)). However, the heterogeneity of polypeptides requires a large excess of molar equivalents of amino acid building blocks to ensure completion of the peptide chain extension. On the other hand, CSPS requires fewer molar equivalents of building blocks, but separation and purification are tedious and time-consuming. There is a need to produce suitable chemicals related to LPPS to remove the need for purification stages and reduce the molar equivalents of building blocks, especially non-natural amino acids in the synthesis of peptidomimetics, which are problematic for scale-up and overall material consumption.

[0005] Initial methods for preparing a 10-mer peptidomimetic (M1) comprising five natural amino acid residues and five non-natural amino acid residues using the known LPPS tag 1) (Takahashi et al., “Novel Diphenylmethyl-Derived Amide Protecting Group for Efficient Liquid-Phase Peptide Synthesis: A JIPHASE,” Org Lett. 14(17): 4514-4517 (2012)) in Figure 1

[0006] ​Accordingly, there is a need in the art for new tag carriers that better allow for separation of the tag carrier peptide from the reaction mixture. In certain embodiments, the present disclosure meets this need and provides other advantages as well.

[0007] The foregoing discussion is presented solely to provide a better understanding of the nature of the problems faced by the art and should not be understood as an acknowledgement or a concession that any of the prior art is "prior art" with respect to the present application. References herein to references cited for the disclosure are not to be understood as an admission that such references are "prior art" with respect to the present application.

[0008] SUMMARY

[0009] Various non-limiting aspects and embodiments of the present disclosure are described below.

[0010] In one aspect, the present disclosure provides a compound having the structure of Formula (I):

[0011]

[0012] wherein:

[0013] m and n are independently an integer from 1 to 5, and (m+n) > 3;

[0014] R 1 is C 1-5 alkyl;

[0015] R 2 is, at each occurrence, independently optionally substituted C 10-30 alkyl; and

[0016] R 3 is, at each occurrence, independently optionally substituted C 10-30 alkyl,

[0017] or a salt thereof.

[0018] In some embodiments, R 1 is methyl.

[0019] In some embodiments, R 2 is, at each occurrence, independently optionally substituted C 18-26 alkyl, and R 3 is, at each occurrence, independently optionally substituted C 18-26 alkyl. In some embodiments, each R 2 is, independently, optionally substituted C 22 alkyl, and each R 3 is, independently, optionally substituted C 22 alkyl.

[0020] In some embodiments, the compound has the structure of Formula (Ia):

[0021]

[0022] or a salt thereof.

[0023] In another aspect, the present disclosure provides a process for preparing a compound of Formula (I), or a salt thereof:

[0024]

[0025] wherein:

[0026] m and n are independently an integer from 1 to 5, and (m+n) > 3;

[0027] R 1 is C 1-5 alkyl;

[0028] R 2 is, at each occurrence, independently optionally substituted C 10-30 alkyl; and

[0029] R 3 is, at each occurrence, independently optionally substituted C 10-30 alkyl. The process comprises:

[0030] (a) providing a compound of Formula (II) having the structure:

[0031]

[0032] wherein

[0033] PG is a suitable protecting group; and

[0034] (b) forming the compound of Formula (I) from the compound of Formula (II).

[0035] In some embodiments, PG is selected from the group consisting of allyloxycarbonyl (Alloc), benzyloxycarbonyl (Cbz), tert-butyloxycarbonyl (Boc), and 9-fluorenylmethoxycarbonyl (Fmoc).

[0036] In some embodiments, the compound of Formula (II) has the structure of Formula (IIa):

[0037]

[0038] In some embodiments, step (b) of forming the compound of formula (I) comprises reacting the compound of formula (II) with a protecting group-removing agent to produce the compound of formula (I). In some embodiments, the protecting group-removing agent is selected from the group consisting of Pd(PPh)3, PhSiH3, H2, piperidine, and trifluoroacetic acid (TFA).

[0039] In some embodiments, the process for preparing the compound of formula (I) further comprises the following steps:

[0040] Providing a compound of formula (III) having the structure:

[0041]

[0042] wherein

[0043] PG 1 is a suitable protecting group;

[0044] PG 2 is a suitable protecting group; and

[0045] forming the compound of formula (II) from the compound of formula (III) prior to step (a).

[0046] In some embodiments, PG 1 is independently selected from the group consisting of tert-butyldimethylsilyl (TBS), trimethylsilyl (TMS), triethylsilyl (TES), tert-butyldiphenylsilyl (TBDPS), and triisopropylsilyl (TIPS).

[0047] In some embodiments, PG 2 is independently selected from the group consisting of tert-butyldimethylsilyl (TBS), trimethylsilyl (TMS), triethylsilyl (TES), tert-butyldiphenylsilyl (TBDPS), and triisopropylsilyl (TIPS).

[0048] In some embodiments, each PG 1 and each PG 2 is the same. In some embodiments, PG 1 and PG 2 are different from each other.

[0049] In some embodiments, the compound of formula (III) has the structure of formula (Ilia):

[0050]

[0051] In some embodiments, the step of forming the compound of formula (II) prior to step (a) comprises:

[0052] reacting the compound of Formula (III) with a protecting group remover to obtain a deprotected intermediate;

[0053] reacting the intermediate with at least one compound of Formula (IV):

[0054] R-LG (IV),

[0055] wherein

[0056] R is an optionally substituted C 10-30 alkyl; and

[0057] LG is a suitable leaving group;

[0058] to produce a compound of Formula (II).

[0059] In some embodiments, LG is selected from the group consisting of halogen, OTf, OMs, and OTs.

[0060] In some embodiments, the protecting group remover is selected from the group consisting of tetrabutylammonium fluoride (TBAF), NH4F, SiF4, AcOH, HC1, LiAlH4, and K2CO3.

[0061] In some embodiments, the process for preparing a compound of Formula (I) further comprises the step of:

[0062] providing a compound of Formula (V) having the structure:

[0063] and

[0064] forming a compound of Formula (III) from the compound of Formula (V).

[0065] In some embodiments, the step of forming a compound of Formula (III) comprises:

[0066] reacting the compound of Formula (V) with a compound of Formula (Via) or Formula (VIb):

[0067] PG-X (Via) or PG2O (VIb),

[0068] wherein

[0069] X is CI or Br;

[0070] to produce a compound of Formula (III).

[0071] In some embodiments, the compound of Formula (Via) or Formula (VIb) is selected from the group consisting of AllocCI, Alloc20, Cbz20, CbzCI, FmocCI, and Boc20.

[0072] In some embodiments, the process for preparing the compound of Formula (I) further comprises:

[0073] providing a compound of Formula (VII) having the structure:

[0074]

[0075] wherein PG 3 is a suitable protecting group; and

[0076] forming the compound of Formula (V) from the compound of Formula (VII).

[0077] In some embodiments, PG 3 is selected from the group consisting of allyloxycarbonyl (Alloc), benzyloxycarbonyl (Cbz), tert-butyloxycarbonyl (Boc), and 9-fluorenylmethoxycarbonyl (Fmoc).

[0078] In some embodiments, the compound of Formula (VII) has the structure:

[0079]

[0080] In some embodiments, the step of forming the compound of Formula (V) comprises reacting the compound of Formula (VII) with a protecting group remover to produce the compound of Formula (V). In some embodiments, the protecting group remover is selected from the group consisting of Pd(PPh)3, PhSiH3, H2, piperidine, and trifluoroacetic acid (TFA).

[0081] In some embodiments, the process for preparing the compound of Formula (I) further comprises:

[0082] providing a compound of Formula (VIII) having the structure:

[0083] and

[0084] forming the compound of Formula (VII) from the compound of Formula (VIII).

[0085] In some embodiments, the process of forming the compound of Formula (VII) can comprise reacting the compound of Formula (VIII) with a compound of Formula (IX) having the structure:

[0086]

[0087] to produce a compound of Formula (VII).

[0088] In some embodiments, the compound of Formula (VIII) has the following structure:

[0089]

[0090] In some embodiments, the compound of Formula (IX) has the following structure:

[0091]

[0092] In some embodiments, the process for preparing a compound of Formula (I) further comprises:

[0093] providing a compound of Formula (X) having the following structure:

[0094]

[0095] wherein PG 4 is a suitable protecting group; and

[0096] forming a compound of Formula (VIII) from the compound of Formula (X).

[0097] In some embodiments, PG 4 is selected from the group consisting of allyl, allyloxycarbonyl (Alloc), benzyloxycarbonyl (Cbz), tert-butyloxycarbonyl (Boc), and 9-fluorenylmethoxycarbonyl (Fmoc).

[0098] In some embodiments, the compound of Formula (X) has the following structure:

[0099]

[0100] In some embodiments, the step of forming a compound of Formula (VIII) comprises reacting a compound of Formula (X) with a protecting group remover. In some embodiments, the protecting group remover is selected from the group consisting of Pd / K2CO3, 1,3-dimethylbarbituric acid / Pd(PPh3)4, Pd(PPh)3, PhSiH3, H2, piperidine, and trifluoroacetic acid (TFA).

[0101] In some embodiments, the process for preparing a compound of Formula (I) further comprises:

[0102] providing a compound of Formula (XI) having the following structure:

[0103] and

[0104] forming a compound of Formula (X) from a compound of Formula (XI).

[0105] In an embodiment, the compound of Formula (XI) has the following structure:

[0106]

[0107] In some embodiments, the process for forming a compound of Formula (X) comprises:

[0108] reacting a compound of Formula (XI) with a compound of Formula (XII) or a salt thereof:

[0109]

[0110] to produce a compound of Formula (X).

[0111] In some embodiments, the process for preparing a compound of Formula (I) further comprises:

[0112] providing a compound of Formula (XIII) having the following structure:

[0113] and

[0114] forming a compound of Formula (XI) from a compound of Formula (XIII).

[0115] In some embodiments, the compound of Formula (XIII) has the following structure:

[0116]

[0117] In some embodiments, the process for forming a compound of Formula (XI) comprises reacting a compound of Formula (XIII) with an oxidizing agent to produce a compound of Formula (XI). In some embodiments, the oxidizing agent can be selected from the group consisting of pyridinium chlorochromate (PCC), pyridinium dichromate (PDC), silver carbonate (Ag2CO3), tetra-n-propylammonium perruthenate (TPAP), and Dess-Martin periodinane (DMP).

[0118] In some embodiments, the process for preparing a compound of Formula (I) further comprises:

[0119] providing a compound of Formula (XIV) having the following structure:

[0120]

[0121] wherein

[0122] Y is -OC 1-6 alkyl; and

[0123] forming a compound of Formula (XIII) from a compound of Formula (XIV).

[0124] In an embodiment, the compound of Formula (XIV) has the following structure:

[0125]

[0126] In some embodiments, the process of forming a compound of Formula (XIII) comprises reacting a compound of Formula (XIV) with a reducing agent. In some embodiments, the reducing agent is selected from the group consisting of LiAlH4, DIBALH, and LiBH4.

[0127] In some embodiments, the process for preparing a compound of Formula (I) further comprises:

[0128] providing a compound of Formula (XV) having the following structure:

[0129] and

[0130] forming a compound of Formula (XIV) from a compound of Formula (XV).

[0131] In some embodiments, the compound of Formula (XV) has the following structure:

[0132]

[0133] In some embodiments, the process of forming a compound of Formula (XIV) comprises reacting a compound of Formula (XV) with a protecting group introducing agent to produce a compound of Formula (XIV). In some embodiments, the protecting group introducing agent is selected from the group consisting of TBSCl, TBSOTf, TMSCl, TMSOTf, TESCl, TESOTf, TBDPSCl, TBDPSOTf, TIPSCl, and TIPSOTf.

[0134] In yet another aspect, the present disclosure provides a product prepared according to any of the methods described in the present disclosure.

[0135] In some embodiments, the product is a compound of Formula (I):

[0136]

[0137] wherein:

[0138] m and n are independently an integer from 1 to 5, and (m+n) > 3;

[0139] R 1 is C 1-5 alkyl;

[0140] R 2 independently at each occurrence is optionally substituted C 10-30 alkyl; and

[0141] R 3 independently at each occurrence is optionally substituted C 10-30 alkyl,

[0142] or a salt thereof.

[0143] In one embodiment, the product is a compound of Formula (Ia):

[0144]

[0145] or a salt thereof.

[0146] In another aspect, the disclosure provides a method of using a compound of Formula (I) or a salt thereof for preparing a peptide or peptidomimetic:

[0147]

[0148] wherein:

[0149] m and n are independently integers from 1 to 5, and (m+n) > 3;

[0150] R 1 is C 1-5 alkyl;

[0151] R 2 independently at each occurrence is optionally substituted C 10-30 alkyl; and

[0152] R 3 independently at each occurrence is optionally substituted C 10-30 alkyl.

[0153] In some embodiments, the compound of Formula (I) is used as a carrier or tag for preparing a peptide or peptidomimetic.

[0154] In one embodiment, the peptidomimetic is a compound of the following formula:

[0155] or a salt thereof.

[0156] In one embodiment, the peptidomimetic is a compound of the following formula:

[0157] or a salt thereof.

[0158] In another aspect, the disclosure provides a compound prepared using a compound of Formula (I) or a salt thereof:

[0159]

[0160] wherein:

[0161] m and n are independently an integer from 1 to 5, and (m+n) > 3;

[0162] R 1 is C 1-5 alkyl;

[0163] R 2 is independently selected at each occurrence from optionally substituted C 10-30 alkyl; and

[0164] R 3 is independently selected at each occurrence from optionally substituted C 10-30 alkyl.

[0165] In one embodiment, the compound is of the formula:

[0166] or a salt thereof.

[0167] In one embodiment, the compound is of the formula:

[0168] or a salt thereof.

[0169] In yet another aspect, provided herein is a pharmaceutical composition comprising a peptidomimetic or compound of any of the embodiments described herein.

[0170] In a further aspect, provided herein is a pharmaceutical dosage form comprising a peptidomimetic or compound of any of the embodiments described herein.

[0171] In another aspect, provided herein is a method of selectively targeting GLP1R on the surface of a cell with a compound or peptidomimetic of any of the embodiments described herein. In some embodiments, the cell is a mammalian cell. In some embodiments, the cell is a human cell.

[0172] In another aspect, provided herein is a method of enhancing GLP1R activity in an individual in need thereof, the method comprising administering to the individual an effective amount of a peptidomimetic or compound of any of the embodiments described herein, a composition described herein, or a dosage form described herein.

[0173] In yet another aspect, provided herein is a method of reducing blood glucose levels in an individual in need thereof, the method comprising administering to the individual an effective amount of a peptidomimetic or compound of any of the embodiments described herein, a composition described herein, or a dosage form described herein.

[0174] In another aspect, provided herein is a method of reducing body weight in an individual in need thereof, comprising administering to the individual an effective amount of a peptidomimetic or compound of any embodiment described herein, a composition described herein, or a dosage form described herein.

[0175] In another aspect, provided herein is a method of treating a GLP1R-associated condition in an individual in need thereof, comprising administering to the individual an effective amount of a peptidomimetic or compound of any embodiment described herein, a composition described herein, or a dosage form described herein. In some embodiments, the GLP1R-associated condition is type II diabetes, obesity, liver disease, coronary artery disease, or kidney disease. In some embodiments, the GLP1R-associated condition is type II diabetes and / or obesity. In some embodiments, the peptidomimetic or compound of any embodiment described herein, the composition described herein, or the dosage form described herein is administered subcutaneously, intravenously, intradermally, intraperitoneally, or intramuscularly.

[0176] In yet another aspect, provided herein is a method of making a peptide or peptidomimetic, comprising the steps of:

[0177] (a) providing a compound of Formula (I):

[0178]

[0179] wherein:

[0180] m and n are independently an integer from 1 to 5, and (m+n) > 3;

[0181] R 1 is C 1-5 alkyl;

[0182] R 2 is, at each occurrence, independently selected from optionally substituted C 10-30 alkyl; and

[0183] R 3 is, at each occurrence, independently selected from optionally substituted C 10-30 alkyl,

[0184] or a salt thereof;

[0185] (b) providing a first amino acid (AA1); and

[0186] (c) coupling the first amino acid (AA1) to the compound of Formula (I) to form a peptide bond between the first amino acid (AA1) and the compound of Formula (I).

[0187] In some embodiments, the first amino acid (AA1) comprises a protected amino group.

[0188] In some embodiments, the first amino acid (AA1) is

[0189] In some embodiments, the method of making a peptide or peptidomimetic can comprise, prior to step (c), a step of activating the chemical group on the first amino acid (AA1) to make the first amino acid (AA1) for coupling with the compound of formula (I).

[0190] In some embodiments, the method of making a peptide or peptidomimetic can comprise the following steps:

[0191] (d) removing the protecting group from the first amino acid (AA1);

[0192] (e) providing a second amino acid (AA2); and

[0193] (f) coupling the second amino acid (AA2) to the first amino acid (I-AA1) to form a peptide bond (I-AA1-AA2) between the second amino acid (AA2) and the first amino acid.

[0194] In some embodiments, the second amino acid (AA2) is

[0195] In some embodiments, the method of making a peptide or peptidomimetic can comprise the following steps:

[0196] (g) removing the protecting group from the second amino acid (AA2);

[0197] (h) providing a third amino acid (AA3); and

[0198] (i) coupling the third amino acid (AA3) to the second amino acid to form a peptide bond (I-AA1-AA2-AA3) between the third amino acid (AA3) and the second amino acid.

[0199] In some embodiments, the third amino acid (AA3) is

[0200] In some embodiments, the method of making a peptide or peptidomimetic can comprise the following steps:

[0201] (j) removing the protecting group from the third amino acid (AA3);

[0202] (k) providing a fourth amino acid (AA4); and

[0203] (l) coupling the fourth amino acid (AA4) to the third amino acid to form a peptide bond (I-AA1-AA2-AA3-AA4) between the fourth amino acid (AA4) and the third amino acid.

[0204] In some embodiments, the fourth amino acid (AA4) is

[0205] In some embodiments, the method of making a peptide or peptidomimetic can comprise the steps of:

[0206] (m) removing the protecting group from the fourth amino acid (AA4);

[0207] (n) providing a fifth amino acid (AA5); and

[0208] (o) coupling the fifth amino acid (AA5) to the fourth amino acid to form a peptide bond between the fifth amino acid (AA5) and the fourth amino acid (I-AA1-AA2-AA3-AA4-AA5).

[0209] In some embodiments, the fifth amino acid (AA5) is

[0210] In some embodiments, the method of making a peptide or peptidomimetic can comprise the steps of:

[0211] (p) removing the protecting group from the fifth amino acid (AA5);

[0212] (q) providing a sixth amino acid (AA6); and

[0213] (r) coupling the sixth amino acid (AA6) to the fifth amino acid to form a peptide bond between the sixth amino acid (AA6) and the fifth amino acid (I-AA1-AA2-AA3-AA4-AA5-AA6).

[0214] In some embodiments, the sixth amino acid (AA6) is

[0215] In some embodiments, the method of making a peptide or peptidomimetic can comprise the steps of:

[0216] (s) removing the protecting group from the sixth amino acid (AA6);

[0217] (t) providing a seventh amino acid (AA7); and

[0218] (u) coupling the seventh amino acid (AA7) to the sixth amino acid to form a peptide bond between the seventh amino acid (AA7) and the sixth amino acid (I-AA1-AA2-AA3-AA4-AA5-AA6-AA7).

[0219] In some embodiments, the seventh amino acid (AA7) is

[0220] In some embodiments, the method of making a peptide or peptidomimetic can comprise the steps of:

[0221] (v) removing the protecting group from the seventh amino acid (AA7);

[0222] (w) providing an eighth amino acid (AA8); and

[0223] (x) coupling the eighth amino acid (AA8) to the seventh amino acid to form a peptide bond between the eighth amino acid (AA8) and the seventh amino acid (I-AA1-AA2-AA3-AA4-AA5-AA6-AA7-AA8).

[0224] In some embodiments, the eighth amino acid (AA8) is

[0225] In some embodiments, the method of making a peptide or peptidomimetic can comprise the following steps:

[0226] (y) removing the protecting group from the eighth amino acid (AA8);

[0227] (z) providing a ninth amino acid (AA9); and

[0228] (aa) coupling the ninth amino acid (AA9) to the eighth amino acid to form a peptide bond between the ninth amino acid (AA9) and the eighth amino acid (I-AA1-AA2-AA3-AA4-AA5-AA6-AA7-AA8-AA9).

[0229] In some embodiments, the ninth amino acid (AA9) is

[0230] In some embodiments, the method of making a peptide or peptidomimetic can comprise the following steps:

[0231] (bb) removing the protecting group from the ninth amino acid (AA9);

[0232] (cc) providing a tenth amino acid (AA10); and

[0233] (dd) coupling the tenth amino acid (AA10) to the ninth amino acid to form a peptide bond between the tenth amino acid (AA10) and the ninth amino acid (I-AA1-AA2-AA3-AA4-AA5-AA6-AA7-AA8-AA9-AA10).

[0234] In one embodiment, the tenth amino acid (AA10) is

[0235] In some embodiments, the method of making a peptide or peptidomimetic can comprise the following steps:

[0236] repeating the following steps: (i) providing an amino acid (AAi) having a protecting group; (ii) removing the protecting group from the amino acid (AAi); (iii) providing a second amino acid (AA2) having a protecting group; (iv) coupling the second amino acid (AA2) to the amino acid (AAi) to form a peptide bond between the second amino acid (AA2) and the amino acid (AAi); and (v) removing the protecting group from the second amino acid (AA2).n ) removing the protecting group;

[0237] (ii) providing a subsequent amino acid (AA n+1 ) ; and

[0238] (iii) coupling the subsequent amino acid (AA n+1 ) to the amino acid (AA n ) to form a peptide bond between the subsequent amino acid (AA n+1 ) and the amino acid (AA n ),

[0239] wherein the repeating can be performed from 1 to 100 times.

[0240] In some embodiments, the method of making a peptide or peptidomimetic can comprise the steps of:

[0241] (ee) removing the protecting group from the tenth amino acid (AA10);

[0242] (ff) providing a compound of Formula (B):

[0243]

[0244] wherein x is an integer from 1 to 15; and

[0245] (gg) coupling the compound of Formula (B) to one of the amino acids in the peptide or peptidomimetic to form a triazole ring.

[0246] In some embodiments, the compound of Formula (B) has the following formula:

[0247]

[0248] In some embodiments, the method of making a peptide or peptidomimetic according to any of the above embodiments further comprises:

[0249] (hh) cleaving the peptide bond between the first amino acid (AA1) and the compound of Formula (I) to obtain the peptide or peptidomimetic.

[0250] In some embodiments, each of the first amino acid (AA1), the second amino acid (AA2), the third amino acid (AA3), the fourth amino acid (AA4), the fifth amino acid (AA5), the sixth amino acid (AA6), the seventh amino acid (AA7), the eighth amino acid (AA8), the ninth amino acid (AA9), the tenth amino acid (AA10), and the subsequent amino acid (AA n+1 ) can be independently selected from the group consisting of:

[0251]

[0252] wherein PG5 are suitable protecting groups. In some embodiments, PG 5 is independently selected from the group consisting of triphenylmethyl (Trt), allyloxy carbonyl (Alloc), benzyloxy carbonyl (Cbz), tert-butyloxy carbonyl (Boc), and 9-fluorenylmethyloxy carbonyl (Fmoc) at each occurrence.

[0253] In some embodiments, each of the first amino acid (AA1), the second amino acid (AA2), the third amino acid (AA3), the fourth amino acid (AA4), the fifth amino acid (AA5), the sixth amino acid (AA6), the seventh amino acid (AA7), the eighth amino acid (AA8), the ninth amino acid (AA9), the tenth amino acid (AA10), and subsequent amino acids (AA n+1 ) can be independently selected from the group consisting of:

[0254] In one embodiment, the peptidomimetic has the formula:

[0255] or a salt thereof. In one embodiment, the peptidomimetic has the formula:

[0256] or a salt thereof.

[0257] In another aspect, provided herein is a compound having the structure of Formula (AI):

[0258]

[0259] wherein:

[0260] m and n are independently an integer from 0 to 5;

[0261] R 1a is C 1-5 alkyl;

[0262] R 2a is independently optionally substituted C 10-30 alkyl at each occurrence; and

[0263] R 3a is independently optionally substituted C 10-30 alkyl at each occurrence,

[0264] or a salt thereof.

[0265] In some embodiments, R 1a is methyl.

[0266] In some embodiments, R 2a is, at each occurrence, independently optionally substituted C 18-26 alkyl, and R 3a is, at each occurrence, independently optionally substituted C 18-26 alkyl. In some embodiments, each R 2a is, independently, optionally substituted C 22 alkyl, and each R 3a is, independently, optionally substituted C 22 alkyl.

[0267] In yet another aspect, provided herein is a compound having the structure of Formula (BI):

[0268]

[0269] wherein:

[0270] m is an integer from 1 to 5;

[0271] R 1b is C 1-5 alkyl; and

[0272] R 2b is, at each occurrence, independently optionally substituted C 10-30 alkyl;

[0273] or a salt thereof.

[0274] In some embodiments, R 1b is methyl.

[0275] In some embodiments, R 2b is, at each occurrence, independently optionally substituted C 18-26 alkyl. In some embodiments, each R 2b is, independently, optionally substituted C 22 alkyl.

[0276] In yet another aspect, provided herein is a compound having the structure of Formula (CI):

[0277]

[0278] wherein:

[0279] m and n are independently integers from 1 to 5, and (m+n) > 3;

[0280] R 2c is, at each occurrence, independently optionally substituted C 10-30 alkyl; and

[0281] R3c independently at each occurrence is optionally substituted C 10-30 alkyl,

[0282] or a salt thereof.

[0283] In some embodiments, R 2c independently at each occurrence is optionally substituted C 18-26 alkyl, and R 3c independently at each occurrence is optionally substituted C 18-26 alkyl. In some embodiments, each R 2c independently is optionally substituted C 22 alkyl, and each R 3c independently is optionally substituted C 22 alkyl.

[0284] In yet another aspect, provided herein is a method for preparing a compound of Formula (DI), or a salt thereof:

[0285]

[0286] wherein:

[0287] k is an integer from 1 to 15;

[0288] q is an integer from 0 to 10,

[0289] R 1d is C 1-6 alkyl;

[0290] PG 6 is a suitable protecting group; and

[0291] PG 7 is a suitable protecting group. The method comprises:

[0292] (a) providing a compound of Formula (DII) having the structure:

[0293] and

[0294] (b) forming the compound of Formula (DI) from the compound of Formula (DII).

[0295] In some embodiments, PG 6 is selected from the group consisting of allyloxycarbonyl (Alloc), benzyloxycarbonyl (Cbz), tert-butyloxycarbonyl (Boc), and 9-fluorenylmethoxycarbonyl (Fmoc).

[0296] In some embodiments, PG 7may be selected from the group consisting of allyloxycarbonyl (Alloc), benzyloxycarbonyl (Cbz), tert-butyloxycarbonyl (Boc), and 9-fluorenylmethoxycarbonyl (Fmoc).

[0297] In some embodiments, the compound of Formula (DI) has the structure of Formula (DIa) or Formula (DIb):

[0298]

[0299] In some embodiments, the compound of Formula (DII) has the structure of Formula (DIIa) or Formula (DIIb):

[0300]

[0301]

[0302] In some embodiments, step (b) to form the compound of Formula (DI) comprises:

[0303] reacting the compound of Formula (DII) with a compound of Formula (DIII):

[0304]

[0305] to produce the compound of Formula (II).

[0306] In one embodiment, the compound of Formula (DIII) has the structure of Formula (DIIIa):

[0307]

[0308] In some embodiments, the process for preparing the compound of Formula (DI) can comprise the step of providing a compound of Formula (DIV) having the structure:

[0309] and

[0310] forming the compound of Formula (DII) from the compound of Formula (DIV) prior to step (a).

[0311] In some embodiments, the compound of Formula (DIV) has the structure of Formula (DIVa) or Formula (DIVb):

[0312]

[0313]

[0314] In some embodiments, step (b) to form the compound of Formula (DII) comprises:

[0315] reacting a compound of Formula (DIV) with a compound of Formula (DVa) or Formula (DVb):

[0316] PG 6 - X (DVa) or PG 6 2O (DVb),

[0317] wherein

[0318] X is OSu, OTf, CI, or Br;

[0319] to produce a compound of Formula (DII).

[0320] In some embodiments, the compound of Formula (DVa) or Formula (DVb) is selected from the group consisting of AllocCI, Alloc20, Cbz20, CbzCI, FmocCI, FmocOSu, and Boc20.

[0321] In some embodiments, the process for preparing a compound of Formula (DI) can comprise the steps of:

[0322] providing a compound of Formula (DVI) having the structure:

[0323]

[0324] wherein

[0325] PG 8 is a suitable protecting group; and

[0326] forming a compound of Formula (DIV) from the compound of Formula (DVI).

[0327] In some embodiments, the step of forming a compound of Formula (DIV) comprises reacting the compound of Formula (DVI) with a protecting group remover to produce a compound of Formula (DIV). In some embodiments, the protecting group remover can be selected from the group consisting of Pd(PPh)3, PhSiH3, H2, HC1, piperidine, and trifluoroacetic acid (TFA).

[0328] In some embodiments, the compound of Formula (DVI) has the structure of Formula (DVIa) or Formula (DVIb):

[0329]

[0330] In some embodiments, the process for preparing a compound of Formula (DI) can comprise the steps of:

[0331] providing a compound of Formula (DVII) having the structure:

[0332] and

[0333] The compound of formula (DVI) is formed from the compound of formula (DVII).

[0334] In some embodiments, the step of forming the compound of formula (DVI) comprises reacting the compound of formula (DVII) with a base to produce the compound of formula (DVI). In some embodiments, the base is selected from the group consisting of LiOH, NaOH, and KOH.

[0335] In some embodiments, the compound of Formula (DVII) has the structure of Formula (DVIIa) or Formula (DVIIb):

[0336]

[0337]

[0338] In some embodiments, the process for preparing the compound of formula (DI) may include the following steps:

[0339] Provided are compounds of formula (DVIII) having the following structure:

[0340]

[0341] in

[0342] LG' is a suitable living group; and

[0343] The compound of formula (DVII) is formed from the compound of formula (DVIII).

[0344] In some embodiments, the step of forming a compound of formula (DVII) comprises reacting a compound of formula (DVIII) with an azido group introducing agent to produce a compound of formula (DVII). In some embodiments, the azido group introducing agent is selected from the group consisting of NaN3, TMSN3, (PhO)2P(O)N3, Zn(N3)2*2Py, and n-Bu4NN3.

[0345] In some embodiments, LG' is selected from the group consisting of halogen, OTf, OMs, and OTs.

[0346] In some embodiments, the compound of Formula (DVIII) has the structure of Formula (DVIIIa) or Formula (DVIIIb):

[0347]

[0348]

[0349] In some embodiments, the process for preparing a compound of Formula (DI) can comprise the following steps:

[0350] A compound of Formula (DIX) having the structure:

[0351]

[0352] wherein

[0353] LG" is a suitable leaving group; and

[0354] forming a compound of Formula (DVIII) from the compound of Formula (DIX).

[0355] In some embodiments, LG" is selected from the group consisting of halogen, OTf, OMs, and OTs.

[0356] In some embodiments, the step of forming a compound of Formula (DVIII) comprises:

[0357] reacting a compound of Formula (DIX) with a compound of Formula (DX):

[0358]

[0359] wherein

[0360] PG 8 is a suitable protecting group;

[0361] to produce a compound of Formula (DVII).

[0362] In some embodiments, PG 8 may be selected from the group consisting of allyloxycarbonyl (Alloc), benzyloxycarbonyl (Cbz), tert-butyloxycarbonyl (Boc), and 9-fluorenylmethoxycarbonyl (Fmoc). In some embodiments, PG 8 is tert-butyloxycarbonyl (Boc).

[0363] In some embodiments, the compound of Formula (DIX) has the structure of Formula (DIXa):

[0364]

[0365] In some embodiments, the compound of Formula (DX) has the structure of Formula (DXa):

[0366]

[0367] In some embodiments, the process for preparing a compound of Formula (DI) can comprise the following steps:

[0368] A compound of Formula (DXI) having the structure:

[0369]

[0370] wherein

[0371] R 2d is C 1-6 alkylene; and

[0372] forming a compound of Formula (DIX) from a compound of Formula (DXI).

[0373] In some embodiments, the step of forming a compound of Formula (DIX) comprises reacting a compound of Formula (DXI) with a reducing agent to produce a compound of Formula (DIX). In some embodiments, the reducing agent is selected from the group consisting of H2, NH4HCO2, NABH(OAc)3, and LiAlH4.

[0374] In some embodiments, the compound of Formula (DXI) has the structure of Formula (DXIa):

[0375]

[0376] In some embodiments, the process for preparing a compound of Formula (DI) can comprise the steps of:

[0377] providing a compound of Formula (DXII) having the structure:

[0378] and forming a compound of Formula (DXI) from a compound of Formula (DXII).

[0379] In some embodiments, the step of forming a compound of Formula (DXI) comprises:

[0380] reacting a compound of Formula (DXII) with a compound of Formula (DXIII):

[0381] PPh3* C 1-6 alkyl-Hal (DXIII), wherein

[0382] Hal is halogen,

[0383] to produce a compound of Formula (DXI).

[0384] In some embodiments, the compound of Formula (DXII) has the structure of Formula (DXIIa):

[0385]

[0386] In some embodiments, the compound of Formula (DXIII) has the structure of Formula (DXIIIa):

[0387] PPh3*MeBr (DXIIIa).

[0388] In some embodiments, the process for preparing a compound of Formula (DI) can comprise the steps of: providing a compound of Formula (DXIV) having the structure of:

[0389] and forming a compound of Formula (DXII) from the compound of Formula (DXIV).

[0390] In some embodiments, the step of forming a compound of Formula (DXII) comprises:

[0391] reacting a compound of Formula (DXIV) with a compound of Formula (DXV):

[0392]

[0393] wherein

[0394] LG”’ is a suitable leaving group,

[0395] to produce a compound of Formula (DXII).

[0396] In some embodiments, LG”’ is selected from the group consisting of halogen, OTf, OMs, and OTs.

[0397] In some embodiments, the compound of Formula (DXIV) has the structure of Formula (DXIVa):

[0398]

[0399] In some embodiments, the process for preparing a compound of Formula (DI) can comprise the steps of: providing a compound of Formula (DXVI) having the structure of:

[0400] and

[0401] wherein

[0402] LG* is a suitable leaving group,

[0403] forming a compound of Formula (DX) from the compound of Formula (DXVI).

[0404] In some embodiments, LG* is selected from the group consisting of halogen, OTf, OMs, and OTs.

[0405] In some embodiments, the step of forming a compound of Formula (DX) comprises:

[0406] reacting a compound of Formula (DXVI) with a compound of Formula (DXVII):

[0407]

[0408] to produce a compound of Formula (DX).

[0409] In some embodiments, the compound of Formula (DXVI) has the structure of Formula (DXVIc):

[0410]

[0411] In some embodiments, the process for preparing a compound of Formula (DI) can comprise the steps of: providing a compound of Formula (DXVIII) having the structure:

[0412] and

[0413] forming a compound of Formula (DXVI) from the compound of Formula (DXVIII).

[0414] In some embodiments, the step of forming a compound of Formula (DXVI) comprises:

[0415] reacting a compound of Formula (DXVIII) with a compound of Formula (DXIXa) or Formula (DXIXb):

[0416] LG*-X (DXIXa) or LG*2O (DXIXb),

[0417] wherein

[0418] X is OSu, Cl, or Br;

[0419] to produce a compound of Formula (DXVI).

[0420] In some embodiments, the compound of Formula (DXIXa) or Formula (DXIXb) is selected from the group consisting of TfCl, Tf20, MsCl, and Ms20.

[0421] In another aspect, provided herein is a process for preparing a compound of Formula (DII) or a salt thereof:

[0422]

[0423] wherein:

[0424] q is an integer from 0 to 10,

[0425] R 1d is C 1-6 alkyl; and

[0426] PG 6 is a suitable protecting group,

[0427] The process comprises:

[0428] (a) providing a compound of Formula (DXXI) having the structure:

[0429] and

[0430] (b) forming a compound of Formula (DII) from the compound of Formula (DXXI).

[0431] In some embodiments, PG 6 is selected from the group consisting of allyloxycarbonyl (Alloc), benzyloxycarbonyl (Cbz), tert-butyloxycarbonyl (Boc), and 9-fluorenylmethoxycarbonyl (Fmoc).

[0432] In some embodiments, the compound of Formula (DXXI) is treated with a compound .

[0433] In some embodiments, the compound of Formula (DXXI) is treated with a catalyst. In some embodiments, the catalyst is Pd(dppf)Cl2-DCM.

[0434] In some embodiments, the compound of Formula (DXXI) is treated with a base. In some embodiments, the base is K2CO3.

[0435] In some embodiments, the preparation is carried out in a solvent mixture comprising H2O / iPrOH.

[0436] In some embodiments, the compound of Formula (DII) has the structure of Formula (DIIa) or Formula (DIIb):

[0437]

[0438] In some embodiments, the compound of Formula (DXXI) is formed by exposing to .

[0439] In some embodiments, the compound of Formula (DXXI) is formed in the presence of PPh3.

[0440] In some embodiments, the compound of Formula (DXXI) is formed in the presence of DIAD.

[0441] In some embodiments, the compound of Formula (DXXI) is formed in the presence of molecular sieves.

[0442] In some embodiments, the compound of Formula (DXXI) is formed in an organic solvent. In some embodiments, the organic solvent is THF.

[0443] In some embodiments, is by Formed by exposure to PBin2.

[0444] In some embodiments, Formed in the presence of Pd(OAc)2.

[0445] In some embodiments, Formed in the presence of PCy3.

[0446] In some embodiments, Formed in the presence of KOAc.

[0447] In some embodiments, Formed in the presence of dioxane.

[0448] In some embodiments, Formed at elevated temperatures over a period of time.

[0449] In some embodiments, is by Exposure to Bu4N + Br3 - And formed.

[0450] In some embodiments, Formed in an organic solvent. In some embodiments, the organic solvent is a mixture of DCM and methanol.

[0451] In a further aspect, provided herein is a method for preparing a compound of formula (DII) or a salt thereof:

[0452]

[0453] in:

[0454] q is an integer from 0 to 10,

[0455] R 1d It is C 1-6 alkyl; and

[0456] PG 6 is a suitable protecting group,

[0457] The process comprises:

[0458] (a) providing a compound of formula (DIV) or a salt thereof having the following structure:

[0459] and

[0460] (b) forming a compound of Formula (DII) from a compound of Formula (DIV).

[0461] In some embodiments, PG 6 may be selected from the group consisting of allyloxycarbonyl (Alloc), benzyloxycarbonyl (Cbz), tert-butyloxycarbonyl (Boc), and 9-fluorenylmethoxycarbonyl (Fmoc).

[0462] In some embodiments, the compound of Formula (DII) has the structure of Formula (DIIa) or Formula (DIIb):

[0463]

[0464]

[0465] In some embodiments, the compound of Formula (DIV) has the structure of Formula (DIVa) or Formula (DIVb):

[0466]

[0467] In some embodiments, the process of forming a compound of Formula (DII) comprises:

[0468] reacting a compound of Formula (DIV) with a compound of Formula (DVa) or Formula (DVb):

[0469] PG 6 - X (DVa) or PG 6 2O (DVb),

[0470] wherein

[0471] X is OSu, OTf, Cl, or Br;

[0472] to produce a compound of Formula (DII).

[0473] In some embodiments, the compound of Formula (DVa) or Formula (DVb) is selected from the group consisting of AllocCl, Alloc2O, Cbz2O, CbzCl, FmocCl, FmocOSu, and Boc2O.

[0474] In some embodiments, the process for preparing a compound of Formula (DII) can comprise the following steps:

[0475] providing a compound of Formula (DXVI) having the structure:

[0476]

[0477] wherein

[0478] PG is a suitable protecting group; and 8 is a suitable protecting group; and

[0479] forming a compound of Formula (DIV) from a compound of Formula (DXVI).

[0480] In some embodiments, PG is selected from the group consisting of allyloxycarbonyl (Alloc), benzyloxycarbonyl (Cbz), tert-butyloxycarbonyl (Boc), and 9-fluorenylmethoxycarbonyl (Fmoc). 8 is selected from the group consisting of allyloxycarbonyl (Alloc), benzyloxycarbonyl (Cbz), tert-butyloxycarbonyl (Boc), and 9-fluorenylmethoxycarbonyl (Fmoc).

[0481] In some embodiments, the step of forming a compound of Formula (DIV) comprises reacting a compound of Formula (DXVI) with a protecting group-removing agent to produce a compound of Formula (DIV). In some embodiments, the protecting group-removing agent is selected from the group consisting of HC1, Pd(PPh)3, PhSiH3, H2, piperidine, and trifluoroacetic acid (TFA).

[0482] In some embodiments, the compound of Formula (DXVI) has the structure of Formula (DXVIa) or Formula (DXVIb):

[0483]

[0484] In some embodiments, the process for preparing a compound of Formula (DII) can comprise the following steps:

[0485] providing a compound of Formula (DXVII) having the structure:

[0486]

[0487] wherein

[0488] LG’ is a suitable leaving group; and

[0489] forming a compound of Formula (DXVI) from a compound of Formula (DXVII).

[0490] In some embodiments, the step of forming a compound of Formula (DXVI) comprises reacting a compound of Formula (DXVII) with an azido group-introducing agent to produce a compound of Formula (DXVI). In some embodiments, the azido group-introducing agent is selected from the group consisting of NaN3, TMSN3, (PhO)2P(O)N3, Zn(N3)2*2Py, and n-Bu4NN3.

[0491] In some embodiments, LG’ is selected from the group consisting of halogen, OTf, OMs, and OTs.

[0492] In some embodiments, the compound of Formula (DXVII) has the structure of Formula (DXVIIa) or Formula (DXVIIb):

[0493]

[0494] In some embodiments, the process for preparing a compound of Formula (DII) can comprise the steps of:

[0495] providing a compound of Formula (DIX) having the structure:

[0496]

[0497] wherein

[0498] LG” is a suitable leaving group; and

[0499] forming a compound of Formula (DXVII) from the compound of Formula (DIX).

[0500] In some embodiments, LG” is selected from the group consisting of halogen, OTf, OMs, and OTs.

[0501] In some embodiments, the step of forming a compound of Formula (DXVII) comprises:

[0502] reacting a compound of Formula (DIX) with a compound of Formula (DXVIII):

[0503]

[0504] to produce a compound of Formula (DXVII).

[0505] In some embodiments, the compound of Formula (DIX) has the structure of Formula (DIXa):

[0506]

[0507] In some embodiments, the compound of Formula (DXVIII) has the structure of Formula (DXVIIIa):

[0508] In some embodiments, the process for preparing a compound of Formula (DII) can comprise the steps of: providing a compound of Formula (DXIX) having the structure:

[0509] and forming a compound of Formula (DIX) from the compound of Formula (DXIX).

[0510] In some embodiments, the step of forming the compound of formula (DIX) comprises:

[0511] reacting the compound of formula (DXIX) with a compound of formula (DXV):

[0512]

[0513] wherein

[0514] LG”’ is a suitable leaving group,

[0515] to produce the compound of formula (DXIX).

[0516] In some embodiments, LG”’ is selected from the group consisting of halogen, OTf, OMs, and OTs.

[0517] In some embodiments, the compound of formula (DXIX) has the structure of formula (DXIXa):

[0518]

[0519] In some embodiments, the compound of formula (DXV) has the structure of formula (DXVa):

[0520]

[0521] In some embodiments, the process for preparing the compound of formula (DII) can comprise the steps of:

[0522] providing a compound of formula (DXX) having the structure:

[0523] and

[0524] forming the compound of formula (DXIX) from the compound of formula (DXX).

[0525] In some embodiments, the step of forming the compound of formula (DXIX) comprises:

[0526] reacting the compound of formula (DXX) with a LG” introducing agent to produce the compound of formula (DXIX).

[0527] In some embodiments, the LG” introducing agent is NBu4Br3.

[0528] In some embodiments, the compound of formula (DXX) has the structure of formula (DXXa):

[0529]

[0530] In another aspect, provided herein is a pharmaceutical composition comprising a peptidomimetic or compound of any embodiment described herein.

[0531] In another aspect, provided herein is a pharmaceutical dosage form comprising a peptidomimetic or compound of any embodiment described herein.

[0532] In another aspect, provided herein is a method of selectively targeting GLP1R on a cell surface with a peptidomimetic or compound of any embodiment described herein. In some embodiments, the cell is a mammalian cell. In some embodiments, the cell is a human cell. In some embodiments, the cell is a pancreatic cell, a brain cell, a heart cell, a vascular tissue cell, a kidney cell, an adipose tissue cell, a liver cell, or a muscle cell.

[0533] In another aspect, provided herein is a method of enhancing GLP1R activity in an individual in need thereof, the method comprising administering to the individual an effective amount of a peptidomimetic or compound of any embodiment described herein, a composition described herein, or a dosage form described herein.

[0534] In another aspect, provided herein is a method of reducing blood glucose levels in an individual in need thereof, the method comprising administering to the individual an effective amount of a peptidomimetic or compound of any embodiment described herein, a composition described herein, or a dosage form described herein.

[0535] In another aspect, provided herein is a method of reducing body weight in an individual in need thereof, the method comprising administering to the individual an effective amount of a peptidomimetic or compound of any embodiment described herein, a composition described herein, or a dosage form described herein.

[0536] In another aspect, provided herein is a method of treating a GLP1R-related condition in an individual in need thereof, the method comprising administering to the individual an effective amount of a peptidomimetic or compound of any embodiment described herein, a composition described herein, or a dosage form described herein. In some embodiments, the GLP1R-related condition is type II diabetes, obesity, liver disease, coronary artery disease, or kidney disease. In some embodiments, the GLP1R-related condition is type II diabetes and / or obesity.

[0537] In various embodiments of any of the methods described herein, the compound, composition, or dosage form of the disclosure is administered subcutaneously, intravenously, intradermally, intraperitoneally, or intramuscularly.

[0538] These and other aspects of the present disclosure will become evident to those skilled in the art after review of the following detailed description of the disclosure, including the accompanying claims. BRIEF DESCRIPTION OF DRAWINGS

[0540] The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee.

[0541] Figure 1 A liquid phase peptide support (LPPS) method for preparing M1 is shown.

[0542] Figure 2 Synthesis of Tag 1 - M2 is shown.

[0543] Figure 3 Synthesis of Tag 2 - M2 is shown.

[0544] Figure 4 Synthesis of Tag 3 - M2 is shown.

[0545] Figure 5 Synthesis of Tag 4 - M2 is shown.

[0546] Figure 6 Synthesis of Tag 5 - M1 is shown.

[0547] Figure 7 Synthesis of Tag 5 - M3 is shown.

[0548] Figure 8 Some examples of known LPPS tags are shown.

[0549] Figure 9 SPPS for peptide extension is shown.

[0550] Figures 10A-10B Table 1 is a summary showing the procedures and conditions used to prepare M1 using Tag 5.

[0551] Figures 11A-11B Table 2 is a summary showing the extension conditions and results for preparing M1 using Tag 5.

[0552] Figure 12 Table 3 is a summary showing the extension conditions and results for preparing M3 using Tag 5.

[0553] Figure 13 Table 4 is a summary showing a comparison of five approaches for preparing AA2 and AA2 + linker.

[0554] Figure 14 Synthesis of AA2 using Approach 1 is shown.

[0555] Figure 15 Alternative processes for preparing AA2 and AA2 + linker are shown.

[0556] DETAILED DESCRIPTION

[0557] In some aspects, the present disclosure provides compounds useful as carriers for liquid phase organic synthesis. Several new LPPS tags (tags 2 - tag 5 in Figure 1 were designed by a) increasing the hydrophobicity of the tags to solidify the TCP components to remove residual reactants and reagents and impurities in solution to ease isolation and purification; and b) introducing 2,4-dimethoxy-benzyl amide moiety (e.g., in tag 5) to ease C-N bond cleavage to make GLP1 peptidomimetics on new tags after Fmoc / tBu C→N synthesis.

[0558] The new liquid phase peptide carrier (LPPS) tags were designed to combine the advantages of solid phase peptide carrier (SPPS) synthesis, allowing the use of fewer molar equivalents of reactants and reagents to grow the peptide chain from the extension mixture and classic solution peptide synthesis (CSPS) to isolate the solid-supported peptide. The increased hydrophobicity of the LPPS tags allows i) more peptide extension steps on the soluble tags in the extension solvent and ii) precipitation of the tagged carrying / growing peptide in polar solvents. Tag 5 with six long alkyl chains allows LPPS of hydrophilic GLP1 agonists using fewer molar equivalents of reactants and reagents compared to SPPS synthesis.

[0559] Detailed embodiments of the present disclosure are disclosed herein; however, it is understood that the disclosed embodiments are merely illustrative of the present disclosure which can take many forms. Furthermore, each example given in connection with the various embodiments of the present disclosure is intended to be illustrative only, and is not intended to limit the disclosure in any way. Thus, the particular structure and function disclosed herein are not to be interpreted as limiting but merely as a representative basis for teaching one skilled in the art to utilize the present disclosure in a variety of ways.

[0560] Definitions

[0561] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs.

[0562] As used in this specification and the appended claims, the singular forms“a,”“an,” and“the” include plural referents unless the content clearly dictates otherwise. Thus, for example, reference to“a method” includes one or more methods, and / or steps of the type described herein and / or equivalents thereof as would be apparent to one skilled in the art having the benefit of this disclosure, unless otherwise indicated.

[0563] “Subject” or“patient” or“individual” or“animal” as used herein refers to humans, veterinary animals (e.g., cats, dogs, cows, horses, sheep, pigs, etc.) and experimental animal models of disease (e.g., mice, rats). In preferred embodiments, the subject is a human.

[0564] The phrase "pharmaceutically acceptable salt" as used in connection with the compositions of the present disclosure refers to any salt suitable for administration to a patient. Suitable salts include, but are not limited to, those disclosed in Berge et al., "Pharmaceutical Salts", J. Pharm. Sci., 1977, 66: 1, which is incorporated herein by reference. Examples of salts include, but are not limited to, salts of an acid source, salts of a base source, organic salts, inorganic salts, amine salts, and alkali or alkaline earth metal salts, including but not limited to calcium salts, magnesium salts, potassium salts, sodium salts, salts of hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, acetic acid, propionic acid, glycolic acid, pyruvic acid, oxalic acid, maleic acid, malonic acid, succinic acid, fumaric acid, tartaric acid, citric acid, benzoic acid, cinnamic acid, mandelic acid, methanesulfonic acid, ethanesulfonic acid, p-toluenesulfonic acid, salicylic acid, and the like.

[0565] Ranges can be expressed herein as from "about" or "approximately" one particular value and / or to "about" or "approximately" another particular value. When such a range is expressed, another embodiment includes from the one particular value and / or to the other particular value.

[0566] "Comprising" or "containing" or "including" means that at least the named compound, element, particle, or method step is present in the composition or article or method, but does not exclude the presence of other compounds, materials, particles, or method steps, even if the other such compounds, material, particles, or method steps have the same function as does the named compound, material, particle, or method step.

[0567] Compounds of the present disclosure, such as tags, include compounds generally described herein and are further illustrated by the classes, subclasses, and species disclosed herein. As used herein, the following definitions shall apply unless otherwise indicated. For purposes of the present disclosure, the chemical elements are identified in accordance with the Periodic Table of the Elements, CAS version, Handbook of Chemistry and Physics, 75thEd. In particular, the atomic number of an element provides the number of protons in its nucleus, and the number of electrons it has (the number of electrons being equal to the number of protons, unless it has an electric charge). Identical elements are not combined unless explicitly indicated as such. The practice of organic synthesis is described in "Organic Chemistry", Thomas Sorrell, University Science Books, Sausalito: 1999, and "March's Advanced Organic Chemistry", 5thEd., Ed.: Smith, M.B. and March, J., John Wiley & Sons, New York: 2001, the entire contents of which are incorporated herein by reference.

[0568] As used herein, the term "alkyl" is given its ordinary meaning in the art and can include saturated aliphatic groups, including straight-chain alkyl groups and branched-chain alkyl groups, cycloalkyl (alicyclic) groups, alkyl substituted cycloalkyl groups, and cycloalkyl substituted alkyl groups. In certain embodiments, straight-chain or branched-chain alkyl groups have from about 1 to 30 carbon atoms in their backbone (e.g., C1-C30), and optionally, from about 1 to 10 carbon atoms or from about 1 to 6 carbon atoms. In some embodiments, cycloalkyl rings in their structure have from about 3 to 10 carbon atoms in their ring structure, wherein such rings are either monocyclic or bicyclic, and optionally have about 5, 6, or 7 carbon atoms in their ring structure. In some embodiments, alkyl groups can be lower alkyl groups, wherein lower alkyl groups contain from 1 to 5 carbon atoms (e.g., straight-chain lower alkyl groups are C1-C5). 30 , branched-chain C2-C 20 ), and optionally, from about 1 to 10 carbon atoms or from about 1 to 6 carbon atoms. In some embodiments, cycloalkyl rings in their structure have from about 3 to 10 carbon atoms in their ring structure, wherein such rings are either monocyclic or bicyclic, and optionally have about 5, 6, or 7 carbon atoms in their ring structure. In some embodiments, alkyl groups can be lower alkyl groups, wherein lower alkyl groups contain from 1 to 5 carbon atoms (e.g., straight-chain lower alkyl groups are C1-C5).

[0569] The term "halogen" means F, Cl, Br, or I; the term "halide" refers to a halogen group or substituent, i.e., -F, -Cl, -Br, or -I.

[0570] As described herein, the compounds of the present disclosure (e.g., tags) can comprise “optionally substituted” moieties. In general, the term “substituted”, whether preceded by the term “optionally” or not, refers to the replacement of one or more hydrogens on a specified moiety with a suitable substituent. For example, up to three H atoms on each residue are replaced with alkyl, halo, haloalkyl, hydroxy, lower alkoxy, carboxy, carboalkoxy (also referred to as alkoxycarbonyl), formylamino (also referred to as alkylaminocarbonyl), cyano, carbonyl, nitro, amino, alkylamino, dialkylamino, mercapto, alkylthio, sulfoxide, sulfone, acylamino, amidino, phenyl, benzyl, heteroaryl, phenoxy, benzyloxy, or heteroaryloxy. Unless otherwise indicated, an “optionally substituted” group can have a suitable substituent at each substitutable position of the group, and when more than one position in any given structure can be substituted with more than one substituent selected from a specified group, the substituent can be either the same or different at every position. Combinations of substituents that are contemplated by the present disclosure are preferably those that result in the formation of stable compounds or chemically feasible compounds.

[0571] The term “stable”, as used herein in connection with compounds, refers to compounds that are not substantially altered when subjected to conditions that can

[0572] Unless otherwise specified, structures depicted herein are also meant to include all isomeric (e.g., enantiomeric, diastereomeric, and geometric (or conformational)) forms of the structure; for example, the R and S configurations for each asymmetric center, (Z) and (E) double bond isomers, and (Z) and (E) conformational isomers. Therefore, single stereochemical isomers, as well as enantiomeric, diastereomeric, and geometric (or conformational) mixtures of the present compounds are within the scope of the disclosure.

[0573] Unless otherwise specified, cyclic adducts, such as the products of cycloaddition reactions (e.g., azide-alkyne cycloaddition reactions or Diels-Alder reactions) depicted herein include all regioisomers, i.e., structural isomers that differ only in the position of functional groups or substituents. By way of example, the following structures represent triazole regioisomers that differ only in the position of substituents on the following triazole ring:

[0574] The triazole regioisomers can also be represented by the following structures:

[0575] Unless otherwise specified, all tautomeric forms of the compounds of the present disclosure are within the scope of the present disclosure.

[0576] In addition, unless otherwise indicated, structures depicted herein are also meant to include compounds which differ only in the presence of one or more isotopically enriched atoms. For example, compounds having the present structures except for the replacement of a hydrogen by a deuterium or tritium, or the replacement of a 11 C- or 13 C- or 14 C-enriched carbon in place of carbon, the compounds having the present structures are within the scope of the disclosure.

[0577] It should also be understood that reference to one or more method steps does not preclude the presence of additional method steps or intervening method steps between those steps expressly identified. Similarly, it should be understood that reference to one or more components in an apparatus or system does not preclude the presence of additional components or intervening components between those components expressly identified.

[0578] Unless otherwise stated, all crystalline forms of the compounds of the disclosure and salts thereof are within the scope of the disclosure. The compounds of the disclosure can be isolated in a variety of amorphous and crystalline forms, including but not limited to anhydrous, hydrated, non-solvated, or solvated forms. Exemplary hydrates include hemihydrates, monohydrates, dihydrates, and the like. In some embodiments, the compounds of the disclosure are anhydrous and non-solvated. “Anhydrous” means that the crystalline form of the compound does not substantially include bound water in the crystal lattice structure, i.e., the compound does not form a crystalline hydrate.

[0579] As used herein, “crystalline form” is intended to refer to a certain crystal lattice configuration of a crystalline substance. Different crystalline forms of the same substance typically have different crystalline lattices (e.g., unit cells), which result in different physical properties characteristic of each crystalline form. In some cases, different crystal lattice configurations have different water or solvent content. Different lattices can be identified by solid state characterization methods such as by X-ray powder diffraction (PXRD). Other characterization methods, such as differential scanning calorimetry (DSC), thermogravimetric analysis (TGA), dynamic vapor sorption (DVS), solid state NMR, and the like, also aid in identifying crystalline forms and in determining stability and solvent / water content.

[0580] Crystalline forms of a substance include both solvated (e.g., hydrated) and non-solvated (e.g., anhydrous) forms. Hydrated forms are crystalline forms that include water in the crystal lattice. Hydrated forms can be stoichiometric hydrates, in which water is present in the crystal lattice in a certain water / molecule ratio, such as hemihydrates, monohydrates, dihydrates, and the like. Hydrated forms can also be non-stoichiometric, in which the water content is variable and depends on external conditions, such as humidity.

[0581] In some embodiments, the compounds of the present disclosure are substantially isolated. By "substantially isolated" is meant that a specified compound is at least partially separated from the other materials with which it was associated in nature. For example, in some embodiments, the compounds of the present disclosure comprise less than about 50%, less than about 40%, less than about 30%, less than about 20%, less than about 15%, less than about 10%, less than about 5%, less than about 2.5%, less than about 1%, or less than about 0.5% of impurities. Impurities generally include anything that is not the substantially isolated compound, including, for example, other crystalline forms and other substances.

[0582] The term "GLP1R" refers to a glucagon-like peptide 1 receptor and includes a recombinant GLP1R protein or fragment thereof. GLP1R has a sequence of 463 residues. Donnelly, Br J Pharmacol, 166(1): 27-41 (2011). Glucagon-like peptide 1 (GLP1) is a 31 amino acid peptide hormone released from intestinal L cells after nutrient consumption. Binding of GLP1 to GLP1R potentiates glucose-induced insulin secretion from pancreatic beta cells, increases insulin expression, suppresses beta cell apoptosis, promotes beta cell neogenesis, reduces glucagon secretion, delays gastric emptying, promotes satiety and increases peripheral glucose disposal.

[0583] Label

[0584] In accordance with the foregoing and other objects, the present disclosure provides compounds and precursors and intermediates thereof, which can be used as labels for the preparation of GLP1 peptidomimetics. The present disclosure also provides methods of preparing these compounds and precursors and intermediates thereof.

[0585] Also provided in the present disclosure are GLP1 peptidomimetics prepared using the labels described herein and pharmaceutical compositions comprising GLP1 peptidomimetics, as well as methods for treating certain diseases in a subject in need of such treatment.

[0586] In one aspect, the present disclosure provides a compound having the structure of Formula (I):

[0587]

[0588] wherein:

[0589] m and n are independently an integer from 1 to 5, and (m+n) > 3;

[0590] R 1 is C 1-5 alkyl;

[0591] R 2 is, at each occurrence, independently optionally substituted C 10-30 alkyl; and

[0592] R 3 independently at each occurrence is optionally substituted C 10-30 alkyl,

[0593] or a salt thereof.

[0594] According to the present disclosure, m can be an integer from 1 to 5, and n can be an integer from 1 to 5, with the proviso that (m+n) > 3. For example, m can be 1, 2, 3, 4, or 5, and n can be 1, 2, 3, 4, or 5.

[0595] In at least one embodiment, m is 3 and n is 3.

[0596] According to the present disclosure, R 1 may be methyl, ethyl, propyl, butyl, or pentyl. For example, R 1 may be n-propyl, i-propyl, n-butyl, i-butyl, t-butyl, or n-pentyl. In at least one embodiment, R 1 is methyl.

[0597] According to the present disclosure, R 2 independently at each occurrence is optionally substituted C 10-30 alkyl. For example, C 10-30 alkyl can be optionally substituted from 1 to 10 times with a substituent selected from the group consisting of halogen, hydroxyl, lower alkoxy, carboxyl, carboalkoxy (also known as alkoxycarbonyl), formylamino (also known as alkylaminocarbonyl), cyano, nitro, amino, alkylamino, dialkylamino, mercapto, alkylthio, phenyl, benzyl, heteroaryl, phenoxy, or benzyloxy. For example, C 10-30 alkyl can be optionally substituted 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 times with any of the above substituents, wherein each substituent is independently selected from halogen, hydroxyl, lower alkoxy, carboxyl, carboalkoxy, formylamino, cyano, nitro, amino, alkylamino, dialkylamino, mercapto, alkylthio, phenyl, benzyl, heteroaryl, phenoxy, or benzyloxy.

[0598] According to the present disclosure, R 3 independently at each occurrence is optionally substituted C 10-30 alkyl. For example, C 10-30 alkyl can be optionally substituted from 1 to 10 times with a substituent selected from the group consisting of halogen, hydroxyl, lower alkoxy, carboxyl, carboalkoxy (also known as alkoxycarbonyl), formylamino (also known as alkylaminocarbonyl), cyano, nitro, amino, alkylamino, dialkylamino, mercapto, alkylthio, phenyl, benzyl, heteroaryl, phenoxy, or benzyloxy. For example, C 10-30The alkyl group can be optionally substituted one, two, three, four, five, six, seven, eight, nine, or ten times with any of the above substituents, wherein each substituent is independently selected from halogen, hydroxyl, lower alkoxy, carboxyl, carbon alkoxy, formamido, cyano, nitro, amino, alkylamino, dialkylamino, sulfhydryl, alkylthio, phenyl, benzyl, heteroaryl, phenoxy, or benzyloxy.

[0599] In at least one embodiment, R 2 is, at each occurrence, independently optionally substituted C 18-26 alkyl, and R 3 is, at each occurrence, independently optionally substituted C 18-26 alkyl. For example, each R 2 is, at each occurrence, independently optionally substituted C 22 alkyl, and each R 3 is, at each occurrence, independently optionally substituted C 22 alkyl.

[0600] In one aspect, provided herein is a compound having the structure of Formula (Ia):

[0601]

[0602] or a salt thereof.

[0603] In another aspect, the present disclosure provides a process for preparing a compound of Formula (I), or a salt thereof:

[0604]

[0605] wherein:

[0606] m and n are independently integers from 1 to 5, and (m+n) > 3;

[0607] R 1 is C 1-5 alkyl;

[0608] R 2 is, at each occurrence, independently optionally substituted C 10-30 alkyl; and

[0609] R 3 is, at each occurrence, independently optionally substituted C 10-30 alkyl. The process comprises:

[0610] (a) providing a compound of Formula (II) having the structure:

[0611]

[0612] wherein:

[0613] PG is a suitable protecting group; and

[0614] (b) forming a compound of Formula (I) from a compound of Formula (II).

[0615] According to the present disclosure, PG is any suitable protecting group that can be used to protect an amino group. Suitable protecting groups that can be used are known to those skilled in the art. A detailed description of these groups and their selection and chemistry are contained in “The Peptides, Volume 3”, Gross and Meinenhofer, Eds., Academic Press, New York, 1981, which is hereby incorporated by reference in its entirety. For example, PG can be selected from the group consisting of allyloxycarbonyl (Alloc), benzyloxycarbonyl (Cbz), tert-butyloxycarbonyl (Boc), and 9-fluorenylmethoxycarbonyl (Fmoc). In some embodiments, PG is allyloxycarbonyl (Alloc).

[0616] In some embodiments, the compound of Formula (II) has the structure of Formula (Ila):

[0617]

[0618] In some embodiments, step (b) of forming a compound of Formula (I) can comprise reacting the compound of Formula (II) with a protecting group removing agent to produce the compound of Formula (I).

[0619] According to the present disclosure, any suitable protecting group removing agent can be used to remove the protecting group PG from the amino group of the compound of Formula (II). Suitable protecting group removing agents that can be used to remove a particular protecting group are known to those skilled in the art. A detailed description of these groups and their selection and chemistry are contained in “The Peptides, Volume 3”, Gross and Meinenhofer, Eds., Academic Press, New York, 1981, which is hereby incorporated by reference in its entirety. For example, the protecting group removing agent is selected from the group consisting of Pd(PPh)3, PhSiH3, H2, piperidine, and trifluoroacetic acid (TFA).

[0620] In some embodiments, the process for preparing a compound of Formula (I) can comprise the following steps:

[0621] Providing a compound of Formula (III) having the structure:

[0622]

[0623] wherein

[0624] PG is a suitable protecting group; 1 is a suitable protecting group;

[0625] PG is a suitable protecting group; 2 is a suitable protecting group; and

[0626] forming a compound of Formula (II) from a compound of Formula (III) prior to step (a).

[0627] According to the present disclosure, PG 1 is any suitable protecting group that can be used to protect a hydroxyl group. Suitable protecting groups that can be used are known to those skilled in the art and can be selected from the protecting groups described in “Handbook of Reagents for Organic Synthesis. Activating Reagents and Protecting Groups,” Pearson and Roush, Eds., John Wiley & Sons, 2005, which is hereby incorporated by reference in its entirety. For example, PG 1 may be selected from the group consisting of tert-butyldimethylsilyl (TBS), trimethylsilyl (TMS), triethylsilyl (TES), tert-butyldiphenylsilyl (TBDPS), and triisopropylsilyl (TIPS).

[0628] According to the present disclosure, PG 2 is any suitable protecting group that can be used to protect a hydroxyl group. Suitable protecting groups that can be used are known to those skilled in the art and can be selected from the protecting groups described in “Handbook of Reagents for Organic Synthesis. Activating Reagents and Protecting Groups,” Pearson and Roush, Eds., John Wiley & Sons, 2005, which is hereby incorporated by reference in its entirety. For example, PG 2 may be selected from the group consisting of tert-butyldimethylsilyl (TBS), trimethylsilyl (TMS), triethylsilyl (TES), tert-butyldiphenylsilyl (TBDPS), and triisopropylsilyl (TIPS).

[0629] According to the present disclosure, each PG 1 and each PG 2 may be the same or different. In some embodiments, PG 1 is the same as PG 2groups are the same. For example, if m is 3 and n is 3, then each of the three PG 1 groups is the same, and each of the three PG 2 groups is the same and PG 1 = PG 2 = PG 1 = PG 2 = PG 1 = PG 2 = PG 1 groups (PG 11 , PG 12 , and PG 13 ) are the same (PG 1 = PG 11 = PG 12 = PG 13 ), and each of the three PG 2 groups (PG 21 , PG 22 , and PG 23 ) is the same (PG 2 = PG 21 = PG 22 = PG 23 ), but PG 1 is different from PG 2 . In some embodiments, PG 1 is TBS, and PG 2 is TIPS. Alternatively, each PG 1 and each PG 2 can be different from each other. For example, if m is 3 and n is 3, then each of the three PG 1 groups is different from each other (PG 11 is different from PG 12 , PG 12 is different from PG 13 , and PG 11 is different from PG 13 ), and each of the three PG 2 groups is different from each other (PG 21 is different from PG 22 , PG 22 is different from PG 23 , and PG 21 is different from PG 23 ), and PG 11 , PG 12 , PG 13 , PG 21 , PG22 and PG 23 They are all different.

[0630] In some embodiments, the compound of Formula (III) has the structure of Formula (IIIa):

[0631]

[0632] In some embodiments, the step of forming the compound of formula (II) prior to step (a) comprises:

[0633] reacting the compound of formula (III) with a protecting group removing agent to obtain a deprotected intermediate;

[0634] Said intermediate is reacted with at least one compound of formula (IV):

[0635] R-LG(IV),

[0636] in

[0637] R is optionally substituted C 10-30 alkyl; and

[0638] LG is a suitable leaving group;

[0639] to produce the compound of formula (II).

[0640] According to the present disclosure, any suitable protecting group removal agent can be used to remove the protecting group PG from the hydroxyl group of the compound of formula (III) 1 and PG 2 Suitable protecting group removal agents that can be used to remove a particular protecting group are known to those skilled in the art and can be selected from the protecting group removal agents described in "Handbook of Reagents for Organic Synthesis. Activating Reagents and Protecting Groups," Pearson and Roush, eds., John Wiley & Sons, 2005, which is hereby incorporated by reference in its entirety. For example, the protecting group removal agent can be selected from the group consisting of tetrabutylammonium fluoride (TBAF), NH4F, SiF4, AcOH, HCl, LiAlH4, and K2CO3.

[0641] In some embodiments, if PG 1 and PG 2 In this case, one protecting group removing agent can be used to selectively remove all PG present in the compound of formula (III).1 groups. The formed intermediate can then be reacted with a compound of Formula (IVa) of a first compound of Formula (IV). Thereafter, another protecting group remover can be used to selectively remove all PG 2 groups, and then another compound of Formula (IV) of a compound of Formula (IVb) can be used to modify the deprotected hydroxyl group. In this case, the compound of Formula (IVa) can be different from the compound of Formula (IVb).

[0642] According to the present disclosure, LG is a suitable leaving group. Suitable leaving groups that can be used are known to those skilled in the art. For example, LG can be selected from the group consisting of halogen, OTf, OMs, and OTs.

[0643] In some embodiments, a process for preparing a compound of Formula (I) can comprise the steps of:

[0644] providing a compound of Formula (V) having the structure:

[0645] and

[0646] forming a compound of Formula (III) from the compound of Formula (V).

[0647] In some embodiments, the step of forming a compound of Formula (III) can comprise:

[0648] reacting a compound of Formula (V) with a compound of Formula (Via) or Formula (VIb):

[0649] PG-X (Via) or PG2O (VIb),

[0650] wherein

[0651] X is Cl or Br;

[0652] to produce a compound of Formula (III).

[0653] According to the present disclosure, a suitable compound of Formula (Via) or Formula (VIb) can be selected from, but not limited to, the group consisting of: AllocCl, Alloc2O, Cbz2O, CbzCl, FmocCl, and Boc2O.

[0654] In some embodiments, a process for preparing a compound of Formula (I) can comprise the steps of:

[0655] providing a compound of Formula (VII) having the structure:

[0656]

[0657] (VII) wherein PG 3 is a suitable protecting group; and

[0658] forming a compound of Formula (V) from a compound of Formula (VII).

[0659] According to the present disclosure, PG 3 is any suitable protecting group that can be used to protect an amino group. Suitable protecting groups that can be used are known to those skilled in the art. A detailed description of these groups and their selection and chemistry are contained in "The Peptides, Volume 3", Gross and Meinenhofer, Eds., Academic Press, New York, 1981, which is hereby incorporated by reference in its entirety. For example, PG 3 may be selected from the group consisting of allyloxycarbonyl (Alloc), benzyloxycarbonyl (Cbz), tert-butyloxycarbonyl (Boc), and 9-fluorenylmethoxycarbonyl (Fmoc). In some embodiments, PG 3 is 9-fluorenylmethoxycarbonyl (Fmoc).

[0660] In some embodiments, the compound of Formula (VII) has the following structure:

[0661]

[0662] In some embodiments, the process for preparing a compound of Formula (V) comprises reacting a compound of Formula (VII) with a protecting group removing agent to produce a compound of Formula (V).

[0663] According to the present disclosure, any suitable protecting group removing agent can be used to remove the protecting group PG 3 from the amino group of a compound of Formula (VII). Suitable protecting group removing agents that can be used to remove a particular protecting group are known to those skilled in the art. A detailed description of these groups and their selection and chemistry are contained in "The Peptides, Volume 3", Gross and Meinenhofer, Eds., Academic Press, New York, 1981, which is hereby incorporated by reference in its entirety. For example, the protecting group removing agent is selected from the group consisting of Pd(PPh)3, PhSiH3, H2, piperidine, and trifluoroacetic acid (TFA).

[0664] In some embodiments, the process for preparing a compound of Formula (I) can comprise the following steps:

[0665] providing a compound of Formula (VIII) having the following structure:

[0666] and

[0667] forming a compound of Formula (VII) from a compound of Formula (VIII).

[0668] In some embodiments, the process for forming a compound of Formula (VII) can comprise reacting a compound of Formula (VIII) with a compound of Formula (IX) having the structure:

[0669]

[0670] to produce a compound of Formula (VII).

[0671] In one embodiment, the compound of Formula (VIII) has the structure:

[0672]

[0673] In another embodiment, the compound of Formula (IX) has the structure:

[0674]

[0675] In some embodiments, the process for preparing a compound of Formula (I) can comprise the steps of:

[0676] providing a compound of Formula (X) having the structure:

[0677]

[0678] wherein PG 4 is a suitable protecting group; and

[0679] forming a compound of Formula (VIII) from a compound of Formula (X).

[0680] According to the present disclosure, PG 4 is any suitable protecting group that can be used to protect an amino group. Suitable protecting groups that can be used are known to those skilled in the art. A detailed description of these groups and their selection and chemistry are contained in "The Peptides, Volume 3", Gross and Meinenhofer, Eds., Academic Press, New York, 1981, which is hereby incorporated by reference in its entirety. For example, PG 4 may be selected from the group consisting of allyl, allyloxycarbonyl (Alloc), benzyloxycarbonyl (Cbz), tert-butyloxycarbonyl (Boc), and 9-fluorenylmethyloxycarbonyl (Fmoc). In some embodiments, PG 4 is allyl.

[0681] In an embodiment, the compound of Formula (X) has the following structure:

[0682]

[0683] In some embodiments, the process of forming the compound of Formula (VIII) comprises reacting the compound of Formula (X) with a protecting group removing agent.

[0684] According to the present disclosure, any suitable protecting group removing agent can be used to remove the protecting group PG 4 from the amino group of the compound of Formula (X). Suitable protecting group removing agents that can be used to remove particular protecting groups are known to those skilled in the art. A detailed description of these groups, their selection, and chemistry is contained in "The Peptides, Volume 3", Gross and Meinenhofer, Eds., Academic Press, New York, 1981, which is hereby incorporated by reference in its entirety. For example, the protecting group removing agent is selected from the group consisting of Pd / K2CO3, 1,3-dimethylbarbituric acid / Pd(PPh3)4, Pd(PPh)3, PhSiH3, H2, piperidine, and trifluoroacetic acid (TFA).

[0685] In some embodiments, the process for preparing the compound of Formula (I) can comprise the following steps:

[0686] providing a compound of Formula (XI) having the following structure:

[0687] and

[0688] forming the compound of Formula (X) from the compound of Formula (XI).

[0689] In an embodiment, the compound of Formula (XI) has the following structure:

[0690]

[0691] In some embodiments, the process of forming the compound of Formula (X) comprises:

[0692] reacting the compound of Formula (XI) with a compound of Formula (XII) or a salt thereof:

[0693]

[0694] to produce the compound of Formula (X).

[0695] In some embodiments, the process for preparing the compound of Formula (I) can comprise the following steps:

[0696] A compound of Formula (XIII) having the structure:

[0697] and

[0698] forming a compound of Formula (XI) from a compound of Formula (XIII).

[0699] In one embodiment, the compound of Formula (XIII) has the structure:

[0700]

[0701] In some embodiments, the process for forming a compound of Formula (XI) comprises reacting a compound of Formula (XIII) with an oxidizing agent to produce a compound of Formula (XI).

[0702] According to the present disclosure, any suitable oxidizing agent that can convert a primary alcohol to an aldehyde group can be used. Suitable oxidizing agents that can be used are known to one of skill in the art. For example, the oxidizing agent can be selected from the group consisting of pyridinium chlorochromate (PCC), pyridinium dichromate (PDC), silver carbonate (Ag2CO3), tetra-n-propylammonium perruthenate (TPAP), and Dess-Martin periodinane (DMP).

[0703] In some embodiments, the process for preparing a compound of Formula (I) can comprise the steps of:

[0704] providing a compound of Formula (XIV) having the structure:

[0705]

[0706] wherein

[0707] Y is -OC 1-6 alkyl; and

[0708] forming a compound of Formula (XIII) from a compound of Formula (XIV).

[0709] In one embodiment, the compound of Formula (XIV) has the structure:

[0710]

[0711] In some embodiments, the process for forming a compound of Formula (XIII) comprises reacting a compound of Formula (XIV) with a reducing agent.

[0712] According to the present disclosure, any suitable reducing agent that can convert an ester to a primary alcohol group can be used. Suitable reducing agents that can be used are known to those skilled in the art. For example, the reducing agent is selected from the group consisting of LiAlH4, DIBALH, and LiBH4.

[0713] In some embodiments, the process for preparing the compound of Formula (I) can comprise the following steps:

[0714] providing a compound of Formula (XV) having the following structure:

[0715] and

[0716] forming the compound of Formula (XIV) from the compound of Formula (XV).

[0717] In one embodiment, the compound of Formula (XV) has the following structure:

[0718]

[0719] In some embodiments, the process of forming the compound of Formula (XIV) comprises reacting the compound of Formula (XV) with a protecting group introducing agent to produce the compound of Formula (XIV).

[0720] According to the present disclosure, any suitable protecting group introducing agent can be used to protect the hydroxyl group in the compound of Formula (XV). Suitable protecting group introducing agents that can be used are known to those skilled in the art. In some embodiments, the protecting group introducing agent is selected from the group consisting of TBSCl, TBSOTf, TMSCl, TMSOTf, TESCl, TESOTf, TBDPSCl, TBDPSOTf, TIPSCl, and TIPSOTf.

[0721] In another aspect, the present disclosure provides a product prepared according to any of the methods described in the present disclosure.

[0722] In one embodiment, the product is a compound of Formula (I):

[0723]

[0724] wherein:

[0725] m and n are independently an integer from 1 to 5, and (m+n) > 3;

[0726] R 1 is C 1-5 alkyl;

[0727] R 2 is independently at each occurrence optionally substituted C10-30 alkyl; and

[0728] R 3 is independently at each occurrence an optionally substituted C 10-30 alkyl,

[0729] or a salt thereof.

[0730] In another embodiment, the product is a compound of formula (Ia):

[0731]

[0732] or a salt thereof.

[0733] In another aspect, the present disclosure provides a method for preparing a peptide or peptidomimetic using a compound of formula (I) or a salt thereof:

[0734]

[0735] in:

[0736] m and n are independently integers from 1 to 5, and (m+n)≥3;

[0737] R 1 It is C 1-5 alkyl;

[0738] R 2 is independently at each occurrence an optionally substituted C 10-30 alkyl; and

[0739] R 3 is independently at each occurrence an optionally substituted C 10-30 alkyl.

[0740] In at least one embodiment, the compound of formula (I) is used as a carrier or tag for the preparation of a peptide or peptidomimetic.

[0741] In one embodiment, the peptidomimetic is a compound of the formula:

[0742] or a salt thereof.

[0743] In one embodiment, the peptidomimetic is a compound of the formula:

[0744] or a salt thereof.

[0745] In another aspect, the present disclosure provides compounds prepared using a compound of formula (I) or a salt thereof:

[0746]

[0747] in:

[0748] m and n are independently an integer from 1 to 5, and (m+n) > 3;

[0749] R 1 is C 1-5 alkyl;

[0750] R 2 is, at each occurrence, independently selected from optionally substituted C 10-30 alkyl; and

[0751] R 3 is, at each occurrence, independently selected from optionally substituted C 10-30 alkyl.

[0752] In one embodiment, the compound has the formula:

[0753] or a salt thereof.

[0754] In one embodiment, the compound has the formula:

[0755] or a salt thereof.

[0756] The present disclosure provides a compound having the structure of Formula (AI):

[0757]

[0758] wherein:

[0759] m and n are independently an integer from 0 to 5;

[0760] R 1a is C 1-5 alkyl;

[0761] R 2a is, at each occurrence, independently optionally substituted C 10-30 alkyl; and

[0762] R 3a is, at each occurrence, independently optionally substituted C 10-30 alkyl,

[0763] or a salt thereof.

[0764] According to the present disclosure, m can be an integer from 0 to 5, and n can be an integer from 0 to 5. For example, m can be 0, 1, 2, 3, 4, or 5, and n can be 0, 1, 2, 3, 4, or 5.

[0765] According to the present disclosure, R 1a may be methyl, ethyl, propyl, butyl, or pentyl. For example, R1 may be n-propyl, i-propyl, n-butyl, i-butyl, t-butyl, or n-pentyl. In at least one embodiment, R 1a is methyl.

[0766] According to the present disclosure, R 2a independently at each occurrence is optionally substituted C 10-30 alkyl. For example, C 10-30 alkyl can be optionally substituted one to ten times with a substituent selected from halogen, hydroxy, lower alkoxy, carboxy, carboalkoxy (also known as alkoxycarbonyl), formylamino (also known as alkylaminocarbonyl), cyano, nitro, amino, alkylamino, dialkylamino, mercapto, alkylthio, phenyl, benzyl, heteroaryl, phenoxy, or benzyloxy. For example, C 10-30 alkyl can be optionally substituted one, two, three, four, five, six, seven, eight, nine, or ten times with any of the above substituents, wherein each substituent is independently selected from halogen, hydroxy, lower alkoxy, carboxy, carboalkoxy, formylamino, cyano, nitro, amino, alkylamino, dialkylamino, mercapto, alkylthio, phenyl, benzyl, heteroaryl, phenoxy, or benzyloxy.

[0767] According to the present disclosure, R 3a independently at each occurrence is optionally substituted C 10-30 alkyl. For example, C 10-30 alkyl can be optionally substituted one to ten times with a substituent selected from halogen, hydroxy, lower alkoxy, carboxy, carboalkoxy (also known as alkoxycarbonyl), formylamino (also known as alkylaminocarbonyl), cyano, nitro, amino, alkylamino, dialkylamino, mercapto, alkylthio, phenyl, benzyl, heteroaryl, phenoxy, or benzyloxy. For example, C 10-30 alkyl can be optionally substituted one, two, three, four, five, six, seven, eight, nine, or ten times with any of the above substituents, wherein each substituent is independently selected from halogen, hydroxy, lower alkoxy, carboxy, carboalkoxy, formylamino, cyano, nitro, amino, alkylamino, dialkylamino, mercapto, alkylthio, phenyl, benzyl, heteroaryl, phenoxy, or benzyloxy.

[0768] In at least one embodiment, R 2a independently at each occurrence is optionally substituted C 18-26 alkyl, and R 3a independently at each occurrence is optionally substituted C 18-26 alkyl. For example, each R 2a independently is optionally substituted C 22 alkyl, and each R 3aindependently is optionally substituted C 22 alkyl.

[0769] The present disclosure provides a compound having the structure of Formula (BI):

[0770]

[0771] wherein:

[0772] m is an integer from 1 to 5;

[0773] R 1b is C 1-5 alkyl; and

[0774] R 2b independently at each occurrence is optionally substituted C 10-30 alkyl;

[0775] or a salt thereof.

[0776] According to the present disclosure, m can be an integer from 1 to 5. For example, m can be 1, 2, 3, 4, or 5.

[0777] According to the present disclosure, R 1b may be methyl, ethyl, propyl, butyl, or pentyl. For example, R 1b may be n-propyl, isopropyl, n-butyl, isobutyl, t-butyl, or n-pentyl. In at least one embodiment, R 1b is methyl.

[0778] According to the present disclosure, R 2b independently at each occurrence is optionally substituted C 10-30 alkyl. For example, C 10-30 alkyl can be optionally substituted from 1 to 10 times with a substituent selected from halogen, hydroxyl, lower alkoxy, carboxyl, carboalkoxy (also known as alkoxycarbonyl), formylamino (also known as alkylaminocarbonyl), cyano, nitro, amino, alkylamino, dialkylamino, mercapto, alkylthio, phenyl, benzyl, heteroaryl, phenoxy, or benzyloxy. For example, C 10-30 alkyl can be optionally substituted 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 times with any of the above substituents, wherein each substituent is independently selected from halogen, hydroxyl, lower alkoxy, carboxyl, carboalkoxy, formylamino, cyano, nitro, amino, alkylamino, dialkylamino, mercapto, alkylthio, phenyl, benzyl, heteroaryl, phenoxy, or benzyloxy.

[0779] In at least one embodiment, R 2b independently at each occurrence is optionally substituted C 18-26alkyl. For example, each R 2b independently is optionally substituted C 22 alkyl.

[0780] The present disclosure provides a compound having the structure of Formula (CI):

[0781]

[0782] wherein:

[0783] m and n are independently an integer from 1 to 5, and (m+n) > 3;

[0784] R 2c independently at each occurrence is optionally substituted C 10-30 alkyl; and

[0785] R 3c independently at each occurrence is optionally substituted C 10-30 alkyl,

[0786] or a salt thereof.

[0787] According to the present disclosure, m can be an integer from 1 to 5, and n can be an integer from 1 to 5, provided that (m+n) > 3. For example, m can be 1, 2, 3, 4, or 5, and n can be 1, 2, 3, 4, or 5.

[0788] According to the present disclosure, R 2c independently at each occurrence is optionally substituted C 10-30 alkyl. For example, C 10-30 The C1-C6 alkyl can be optionally substituted from 1 to 10 times with a substituent selected from halogen, hydroxyl, lower alkoxy, carboxyl, carboalkoxy (also known as alkoxycarbonyl), formylamino (also known as alkylaminocarbonyl), cyano, nitro, amino, alkylamino, dialkylamino, mercapto, alkylthio, phenyl, benzyl, heteroaryl, phenoxy, or benzyloxy. For example, the C1-C6 alkyl can be optionally substituted from 1 to 10 times with a substituent selected from halogen, hydroxyl, lower alkoxy, carboxyl, carboalkoxy, formylamino, cyano, nitro, amino, alkylamino, dialkylamino, mercapto, alkylthio, phenyl, benzyl, heteroaryl, phenoxy, or benzyloxy. 10-30 The C1-C6 alkyl can be optionally substituted from 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 times with any of the above substituents, wherein each substituent is independently selected from halogen, hydroxyl, lower alkoxy, carboxyl, carboalkoxy, formylamino, cyano, nitro, amino, alkylamino, dialkylamino, mercapto, alkylthio, phenyl, benzyl, heteroaryl, phenoxy, or benzyloxy.

[0789] According to the present disclosure, R 3c independently at each occurrence is optionally substituted C 10-30 alkyl. For example, C 10-30The alkyl group can be optionally substituted from 1 to 10 times with a substituent selected from halogen, hydroxy, lower alkoxy, carboxy, carboalkoxy, carboxamido, cyano, nitro, amino, alkylamino, dialkylamino, mercapto, alkylthio, phenyl, benzyl, heteroaryl, phenoxy, or benzyloxy. For example, C 10-30 The alkyl group can be optionally substituted 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 times with any of the above substituents, wherein each substituent is independently selected from halogen, hydroxy, lower alkoxy, carboxy, carboalkoxy, carboxamido, cyano, nitro, amino, alkylamino, dialkylamino, mercapto, alkylthio, phenyl, benzyl, heteroaryl, phenoxy, or benzyloxy.

[0790] In at least one embodiment, R 2c is, independently at each occurrence, optionally substituted C 18-26 alkyl, and R 3c is, independently at each occurrence, optionally substituted C 18-26 alkyl. For example, each R 2c is, independently at each occurrence, optionally substituted C 22 alkyl, and each R 3c is, independently at each occurrence, optionally substituted C 22 alkyl.

[0791] Methods of making a peptide or peptidomimetic using tags

[0792] The present disclosure provides a method of making a peptide or peptidomimetic, the method comprising the steps of:

[0793] (a) providing a compound of Formula (I) or a salt thereof:

[0794]

[0795] wherein:

[0796] m and n are independently integers from 1 to 5, and (m+n) > 3;

[0797] R 1 is C 1-5 alkyl;

[0798] R 2 is, independently at each occurrence, selected from optionally substituted C 10-30 alkyl; and

[0799] R 3 is, independently at each occurrence, selected from optionally substituted C 10-30 alkyl;

[0800] (b) providing a first amino acid (AA1); and

[0801] (c) coupling the first amino acid (AA1) to the compound of Formula (I) to form a peptide bond between the first amino acid (AA1) and the compound of Formula (I).

[0802] The term "amino acid" refers to a molecule comprising both an amino group and a carboxyl group. Amino acids include a-amino acids and β-amino acids, the structures of which are depicted below. In certain embodiments, the amino acid is an a amino acid.

[0803]

[0804] Suitable amino acids that can be used in accordance with the present disclosure include, but are not limited to, i) natural a-amino acids such as the D- and L-isomers of the 20 common naturally occurring a-amino acids found in peptides and proteins: alanine, arginine, asparagine, aspartic acid, cysteine, glutamic acid, glutamine, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, valine, and derivatives thereof; ii) unnatural a-amino acids; iii) natural β-amino acids (e.g., β-alanine), and iv) unnatural β-amino acids.

[0805] In one embodiment, step (c) of coupling AA1 to the compound of Formula (I) comprises coupling a carboxylic acid moiety of AA1 to an amino moiety of the compound of Formula (I).

[0806] In some embodiments, the first amino acid (AA1) comprises a protected amino group.

[0807] In one embodiment, the first amino acid (AA1) is

[0808] In some embodiments, the method of making a peptide or peptidomimetic can comprise a step of activating a chemical group on the first amino acid (AA1) prior to step (c) to prepare the first amino acid (AA1) for coupling to the compound of Formula (I).

[0809] In some embodiments, the method of making a peptide or peptidomimetic can comprise the following steps:

[0810] (d) removing the protecting group from the first amino acid (AA1);

[0811] (e) providing a second amino acid (AA2); and

[0812] (f) coupling the second amino acid (AA2) to the first amino acid (I-AA1) to form a peptide bond between the second amino acid (AA2) and the first amino acid (I-AA1-AA2).

[0813] In one embodiment, step (f) comprises coupling a carboxylic acid moiety of AA2 to an amino moiety of AA1.

[0814] In one embodiment, the second amino acid (AA2) is

[0815] In some embodiments, the method of making a peptide or peptidomimetic can comprise the following steps:

[0816] (g) removing the protecting group from the second amino acid (AA2);

[0817] (h) providing a third amino acid (AA3); and

[0818] (i) coupling the third amino acid (AA3) to the second amino acid to form a peptide bond between the third amino acid (AA3) and the second amino acid (I-AA1-AA2-AA3).

[0819] In one embodiment, step (i) comprises coupling a carboxylic acid moiety of AA3 to an amino moiety of AA2.

[0820] In one embodiment, the third amino acid (AA3) is

[0821] In some embodiments, the method of making a peptide or peptidomimetic can comprise the following steps:

[0822] (j) removing the protecting group from the third amino acid (AA3);

[0823] (k) providing a fourth amino acid (AA4); and

[0824] (l) coupling the fourth amino acid (AA4) to the third amino acid to form a peptide bond between the fourth amino acid (AA4) and the third amino acid (I-AA1-AA2-AA3-AA4).

[0825] In one embodiment, step (l) comprises coupling a carboxylic acid moiety of AA4 to an amino moiety of AA3.

[0826] In one embodiment, the fourth amino acid (AA4) is

[0827] In some embodiments, the method of making a peptide or peptidomimetic can comprise the following steps:

[0828] (m) removing the protecting group from the fourth amino acid (AA4);

[0829] (n) providing a fifth amino acid (AA5); and

[0830] (o) coupling the fifth amino acid (AA5) to the fourth amino acid to form a peptide bond between the fifth amino acid (AA5) and the fourth amino acid (I-AA1-AA2-AA3-AA4-AA5).

[0831] In one embodiment, step (o) comprises coupling a carboxylic acid moiety of AA5 to an amino moiety of AA4.

[0832] In one embodiment, the fifth amino acid (AA5) is

[0833] In some embodiments, the method of making a peptide or peptidomimetic can comprise the following steps:

[0834] (p) removing the protecting group from the fifth amino acid (AA5);

[0835] (q) providing a sixth amino acid (AA6); and

[0836] (r) coupling the sixth amino acid (AA6) to the fifth amino acid to form a peptide bond between the sixth amino acid (AA6) and the fifth amino acid (I-AA1-AA2-AA3-AA4-AA5-AA6).

[0837] In one embodiment, step (r) comprises coupling a carboxylic acid moiety of AA6 to an amino moiety of AA5.

[0838] In one embodiment, the sixth amino acid (AA6) is

[0839] In some embodiments, the method of making a peptide or peptidomimetic can comprise the following steps:

[0840] (s) removing the protecting group from the sixth amino acid (AA6);

[0841] (t) providing a seventh amino acid (AA7); and

[0842] (u) coupling the seventh amino acid (AA7) to the sixth amino acid to form a peptide bond between the seventh amino acid (AA7) and the sixth amino acid (I-AA1-AA2-AA3-AA4-AA5-AA6-AA7).

[0843] In one embodiment, step (u) comprises coupling a carboxylic acid moiety of AA7 to an amino moiety of AA6.

[0844] In one embodiment, the seventh amino acid (AA7) is

[0845] In some embodiments, the method of making a peptide or peptidomimetic can comprise the following steps:

[0846] (v) removing the protecting group from the seventh amino acid (AA7);

[0847] (w) providing an eighth amino acid (AA8); and

[0848] (x) coupling the eighth amino acid (AA8) to the seventh amino acid to form a peptide bond between the eighth amino acid (AA8) and the seventh amino acid (I-AA1-AA2-AA3-AA4-AA5-AA6-AA7-AA8).

[0849] In one embodiment, step (x) comprises coupling a carboxylic acid moiety of AA8 to an amino moiety of AA7.

[0850] In one embodiment, the eighth amino acid (AA8) is

[0851] In some embodiments, the method of making a peptide or peptidomimetic can comprise the following steps:

[0852] (y) removing the protecting group from the eighth amino acid (AA8);

[0853] (z) providing a ninth amino acid (AA9); and

[0854] (aa) coupling the ninth amino acid (AA9) to the eighth amino acid to form a peptide bond between the ninth amino acid (AA9) and the eighth amino acid (I-AA1-AA2-AA3-AA4-AA5-AA6-AA7-AA8-AA9).

[0855] In one embodiment, step (aa) comprises coupling a carboxylic acid moiety of AA9 to an amino moiety of AA8.

[0856] In one embodiment, the ninth amino acid (AA9) is

[0857] In some embodiments, the method of making a peptide or peptidomimetic can comprise the following steps:

[0858] (bb) removing the protecting group from the ninth amino acid (AA9);

[0859] (cc) providing a tenth amino acid (AA10); and

[0860] (dd) coupling the tenth amino acid (AA10) to the ninth amino acid to form a peptide bond between the tenth amino acid (AA10) and the ninth amino acid (I-AA1-AA2-AA3-AA4-AA5-AA6-AA7-AA8-AA9-AA10).

[0861] In one embodiment, step (dd) comprises coupling a carboxylic acid moiety of AA10 to an amino moiety of AA9.

[0862] In one embodiment, the tenth amino acid (AA10) is

[0863] In some embodiments, the method of making a peptide or peptoid can comprise the following steps:

[0864] repeating the following steps: (i) removing a protecting group from an amino acid (AA n );

[0865] (ii) providing a subsequent amino acid (AA n+1 ); and

[0866] (iii) coupling the subsequent amino acid (AA n+1 ) to the amino acid (AA n ) to form a peptide bond between the subsequent amino acid (AA n+1 ) and the amino acid (AA n ),

[0867] wherein the repeating can be performed from 1 to 100 times.

[0868] In some embodiments, the method of making a peptide or peptoid can comprise the following steps:

[0869] (ee) removing a protecting group from the tenth amino acid (AA10);

[0870] (ff) providing a compound of Formula (B):

[0871]

[0872] wherein x is an integer from 1 to 15; and

[0873] (gg) coupling the compound of Formula (B) to one of the amino acids in the peptide or peptoid to form a triazole ring.

[0874] In one embodiment, the compound of Formula (B) has the following formula:

[0875]

[0876] In some embodiments of the present disclosure, one or more peptides can be attached to an amino acid (AAn For example, in some embodiments, the method of preparing a peptide or peptoid can comprise the steps of:

[0877] repeating the steps of (i) removing a protecting group from an amino acid (AA n );

[0878] (ii) providing a peptide (AA m1 -AA mp ), wherein p is from 2 to 20; and

[0879] (iii) coupling an amino group on the peptide (AA m1 -AA mp ) with an amino acid (AA n ) to form a peptide bond between the amino acid on the peptide (AA m1 -AA mp ) and the amino acid (AA n ),

[0880] wherein the repeating can be performed from 1 to 100 times.

[0881] According to the present disclosure, in the peptide (AA m1 -AA mp ), m can be 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20. In some embodiments, m is 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15. In some embodiments, m is 2, 3, 4, 5, 6, 7, 8, 9, or 10.

[0882] According to the present disclosure, the peptide (AA m1 -AA mp ) can be a dipeptide, a tripeptide, a tetrapeptide, a pentapeptide, a hexapeptide, a heptapeptide, an octapeptide, a nonapeptide, or a decapeptide.

[0883] The peptide can include all D-amino acids, all L-amino acids, or a mixture of L-amino acids and D-amino acids. In preferred embodiments, the peptide includes only D-amino acids or a mixture of D-amino acids and L-amino acids, wherein the D-amino acid content is greater than 50%, 60%, 70%, 80%, 90%, or 95%.

[0884] During synthesis, non-participating carboxylic acids or amines on the reactive set of amino acids or peptide fragments can be protected by suitable protecting groups (PG), which can be selectively removed at a later time, if desired. A detailed description of these groups and their selection and chemistry is contained in "The Peptides, Volume 3", Gross and Meinenhofer, Eds., Academic Press, New York, 1981, which is hereby incorporated by reference in its entirety.

[0885] In some embodiments, the method of preparing a peptide or peptidomimetic according to any of the above embodiments further comprises:

[0886] (hh) cleaving the peptide bond between the first amino acid (AA1) and the compound of Formula (I) to obtain the peptide or peptidomimetic.

[0887] According to the present disclosure, each of the first amino acid (AA1), the second amino acid (AA2), the third amino acid (AA3), the fourth amino acid (AA4), the fifth amino acid (AA5), the sixth amino acid (AA6), the seventh amino acid (AA7), the eighth amino acid (AA8), the ninth amino acid (AA9), the tenth amino acid (AA10), and subsequent amino acids (AA n+1 ) can be independently selected from the group consisting of:

[0888]

[0889]

[0890] wherein PG 5 is a suitable protecting group.

[0891] According to the present disclosure, any suitable protecting group that can be used to protect an amino group in an amino acid can be used as PG 5 . Suitable protecting groups that can be used are known to those skilled in the art. A detailed description of these groups and their selection and chemistry is contained in "The Peptides, Volume 3", Gross and Meinenhofer, Eds., Academic Press, New York, 1981, which is hereby incorporated by reference in its entirety. For example, PG 5 may be independently selected at each occurrence from the group consisting of triphenylmethyl (Trt), allyloxycarbonyl (Alloc), benzyloxycarbonyl (Cbz), tert-butyloxycarbonyl (Boc), and 9-fluorenylmethyloxycarbonyl (Fmoc).

[0892] According to the present disclosure, each of the first amino acid (AA1), the second amino acid (AA2), the third amino acid (AA3), the fourth amino acid (AA4), the fifth amino acid (AA5), the sixth amino acid (AA6), the seventh amino acid (AA7), the eighth amino acid (AA8), the ninth amino acid (AA9), the tenth amino acid (AA10), and subsequent amino acids (AA n+1 ) can be independently selected from the group consisting of:

[0893]

[0894] In an embodiment, the peptidomimetic has the formula:

[0895] or a salt thereof.

[0896] In an embodiment, the peptidomimetic has the formula:

[0897] or a salt thereof.

[0898] According to the present disclosure, the amino acids that can be used to make the peptides and peptidomimetics can be both natural and unnatural amino acids.

[0899] The present disclosure provides a method for making a compound of formula (DI) or a salt thereof:

[0900]

[0901] wherein:

[0902] k is an integer from 1 to 15;

[0903] q is an integer from 0 to 10,

[0904] R 1d is C 1-6 alkyl;

[0905] PG 6 is a suitable protecting group; and

[0906] PG 7 is a suitable protecting group. The method comprises:

[0907] (a) providing a compound of formula (DII) having the structure:

[0908] and

[0909] (b) forming the compound of formula (DI) from the compound of formula (DII).

[0910] According to the present disclosure, PG 6 is any suitable protecting group that can be used to protect an amino group. Suitable protecting groups that can be used are known to those skilled in the art. A detailed description of these groups, their selection and chemistry, is contained in "The Peptides, Volume 3", Gross and Meinenhofer, Eds., Academic Press, New York, 1981, which is hereby incorporated by reference in its entirety. For example, PG 6 may be selected from the group consisting of allyloxycarbonyl (Alloc), benzyloxycarbonyl (Cbz), tert-butyloxycarbonyl (Boc), and 9-fluorenylmethyloxycarbonyl (Fmoc). In some embodiments, PG 6 is 9-fluorenylmethyloxycarbonyl (Fmoc).

[0911] According to the present disclosure, PG 7 is any suitable protecting group that can be used to protect an amino group. Suitable protecting groups that can be used are known to those skilled in the art. A detailed description of these groups, their selection and chemistry, is contained in "The Peptides, Volume 3", Gross and Meinenhofer, Eds., Academic Press, New York, 1981, which is hereby incorporated by reference in its entirety. For example, PG 7 may be selected from the group consisting of allyloxycarbonyl (Alloc), benzyloxycarbonyl (Cbz), tert-butyloxycarbonyl (Boc), and 9-fluorenylmethyloxycarbonyl (Fmoc). In some embodiments, PG 7 is tert-butyloxycarbonyl (Boc).

[0912] In one embodiment, the compound of formula (DI) has the structure of formula (DIa) or formula (DIb):

[0913]

[0914]

[0915] In another embodiment, the compound of formula (DII) has the structure of formula (DIIa) or formula (DIIb):

[0916]

[0917] In some embodiments, step (b) of forming the compound of formula (DI) comprises:

[0918] reacting the compound of formula (DII) with a compound of formula (DIII):

[0919]

[0920] to produce a compound of Formula (II).

[0921] In an embodiment, the compound of Formula (DIII) has the structure of Formula (DIIIa):

[0922]

[0923] In some embodiments, the process for preparing a compound of Formula (DI) can comprise the step of providing a compound of Formula (DIV) having the structure:

[0924] and

[0925] forming a compound of Formula (DII) from a compound of Formula (DIV) prior to step (a).

[0926] In an embodiment, the compound of Formula (DIV) has the structure of Formula (DIVa) or Formula (DIVb):

[0927]

[0928] In some embodiments, the step of forming a compound of Formula (DII) comprises:

[0929] reacting a compound of Formula (DIV) with a compound of Formula (DVa) or Formula (DVb):

[0930] PG 6 - X (DVa) or PG 6 2O (DVb),

[0931] wherein

[0932] X is OSu, OTf, CI, or Br;

[0933] to produce a compound of Formula (DII).

[0934] According to the present disclosure, the compound of Formula (DVa) or Formula (DVb) can be selected from the group consisting of AllocCI, Alloc20, Cbz20, CbzCI, FmocCI, FmocOSu, and Boc20.

[0935] In some embodiments, the process for preparing a compound of Formula (DI) can comprise the step of:

[0936] providing a compound of Formula (DVI) having the structure:

[0937] and

[0938] forming a compound of Formula (DIV) from a compound of Formula (DVI).

[0939] In some embodiments, the step of forming a compound of Formula (DIV) comprises:

[0940] reacting the compound of Formula (DVI) with a protecting group-removing reagent to produce a compound of Formula (DIV).

[0941] According to the present disclosure, any suitable protecting group-removing reagent can be used to remove the protecting group PG 8 from the amino group of the compound of Formula (DVI). Suitable protecting group-removing reagents that can be used to remove particular protecting groups are known to those skilled in the art. A detailed description of these groups, their selection, and chemistry is contained in “The Peptides, Volume 3”, Gross and Meinenhofer, Eds., Academic Press, New York, 1981, which is hereby incorporated by reference in its entirety. For example, the protecting group-removing reagent can be selected from the group consisting of Pd(PPh)3, PhSiH3, H2, HC1, piperidine, and trifluoroacetic acid (TFA).

[0942] In one embodiment, the compound of Formula (DVI) has the structure of Formula (DVIa) or Formula (DVIb):

[0943]

[0944] In some embodiments, the process for preparing a compound of Formula (DI) can comprise the steps of:

[0945] providing a compound of Formula (DVII) having the structure:

[0946] and

[0947] forming a compound of Formula (DVI) from a compound of Formula (DVII).

[0948] In some embodiments, the step of forming a compound of Formula (DVI) comprises:

[0949] reacting the compound of Formula (DVII) with a base to produce a compound of Formula (DVI).

[0950] According to the present disclosure, any suitable base can be used to hydrolyze the methyl ester group in the compound of Formula (DVII). In some embodiments, the base is selected from the group consisting of LiOH, NaOH, and KOH.

[0951] In an embodiment, the compound of Formula (DVII) has the structure of Formula (DVIIa) or Formula (DVIIb):

[0952]

[0953] In some embodiments, the process for preparing a compound of Formula (DI) can comprise the following steps:

[0954] providing a compound of Formula (DIX) having the structure:

[0955]

[0956] wherein

[0957] LG’ is a suitable leaving group; and

[0958] forming a compound of Formula (DVII) from the compound of Formula (DVIII).

[0959] In some embodiments, the step of forming a compound of Formula (DVII) comprises:

[0960] reacting the compound of Formula (DVIII) with an azido group introducing agent to produce a compound of Formula (DVII).

[0961] Azido group introducing agents that can be used to practice the above embodiments are known to those skilled in the art. In an embodiment, the azido group introducing agent is selected from the group consisting of NaN3, TMSN3, (PhO)2P(O)N3, Zn(N3)2*2Py, and n-Bu4NN3.

[0962] In an embodiment, LG’ is selected from the group consisting of halogen, OTf, OMs, and OTs.

[0963] In another embodiment, the compound of Formula (DVIII) has the structure of Formula (DVIIIa) or Formula (DVIIIb):

[0964]

[0965] In some embodiments, the process for preparing a compound of Formula (DI) can comprise the following steps:

[0966] providing a compound of Formula (DIX) having the structure:

[0967]

[0968] wherein

[0969] LG” is a suitable leaving group; and

[0970] forming a compound of formula (DVIII) from a compound of formula (DIX).

[0971] In an embodiment, LG” is selected from the group consisting of halogen, OTf, OMs, and OTs.

[0972] In an embodiment, the step of forming a compound of formula (DVIII) comprises:

[0973] reacting a compound of formula (DIX) with a compound of formula (DX):

[0974]

[0975] wherein

[0976] PG 8 is a suitable protecting group;

[0977] to produce a compound of formula (DVII).

[0978] According to the present disclosure, PG 8 is any suitable protecting group that can be used to protect an amino group. Suitable protecting groups that can be used are known to those skilled in the art. A detailed description of these groups and their selection and chemistry are contained in “The Peptides, Volume 3”, Gross and Meinenhofer, Eds., Academic Press, New York, 1981, which is hereby incorporated by reference in its entirety. For example, PG 8 may be selected from the group consisting of allyloxycarbonyl (Alloc), benzyloxycarbonyl (Cbz), tert-butyloxycarbonyl (Boc), and 9-fluorenylmethoxycarbonyl (Fmoc). In some embodiments, PG 8 is tert-butyloxycarbonyl (Boc).

[0979] In another embodiment, the compound of formula (DIX) has the structure of formula (DIXa):

[0980]

[0981] In yet another embodiment, the compound of formula (DX) has the structure of formula (DXa):

[0982]

[0983] In some embodiments, the process for preparing a compound of formula (DI) can comprise the following steps:

[0984] providing a compound of formula (DXI) having the structure:

[0985]

[0986] wherein

[0987] R 2d is C 1-6 alkylene; and

[0988] forming the compound of Formula (DIX) from the compound of Formula (DXI).

[0989] In some embodiments, the step of forming the compound of Formula (DIX) comprises reacting the compound of Formula (DXI) with a reducing agent to produce the compound of Formula (DIX).

[0990] According to the present disclosure, any reducing agent capable of reducing an olefinic double bond can be used to prepare the compound of Formula (DIX). In some embodiments, the reducing agent is selected from the group consisting of H2, NH4HCO2, NABH(OAc)3, and LiAlH4.

[0991] In one embodiment, the compound of Formula (DXI) has the structure of Formula (DXIa):

[0992]

[0993] In some embodiments, the process for preparing the compound of Formula (DI) can comprise the following steps:

[0994] providing a compound of Formula (DXII) having the structure:

[0995] and

[0996] forming the compound of Formula (DIX) from the compound of Formula (DXII).

[0997] In some embodiments, the step of forming the compound of Formula (DXI) comprises:

[0998] reacting the compound of Formula (DXII) with a compound of Formula (DXIII):

[0999] PPh3* C 1-6 alkyl-Hal (DXIII),

[1000] wherein

[1001] Hal is halogen,

[1002] to produce the compound of Formula (DXI).

[1003] In one embodiment, the compound of Formula (DXII) has the structure of Formula (DXIIa):

[1004]

[1005] In another embodiment, the compound of formula (DXIII) has the structure of formula (DXIIIa):

[1006] In some embodiments, the process for preparing a compound of formula (DI) can comprise the steps of: providing a compound of formula (DXIV) having the structure of:

[1007] and forming a compound of formula (DXII) from the compound of formula (DXIV).

[1008] In some embodiments, the step of forming a compound of formula (DXII) comprises:

[1009] reacting a compound of formula (DXIV) with a compound of formula (DXV):

[1010]

[1011] wherein

[1012] LG”’ is a suitable leaving group,

[1013] to produce a compound of formula (DXII).

[1014] In one embodiment, LG”’ is selected from the group consisting of halogen, OTf, OMs, and OTs. In another embodiment, the compound of formula (DXIV) has the structure of formula (DXIVa):

[1015]

[1016] In some embodiments, the process for preparing a compound of formula (DI) can comprise the steps of: providing a compound of formula (DXVI) having the structure of:

[1017] and

[1018] wherein

[1019] LG* is a suitable leaving group, forming a compound of formula (DX) from the compound of formula (DXVI).

[1020] In one embodiment, LG* is selected from the group consisting of halogen, OTf, OMs, and OTs.

[1021] In some embodiments, the step of forming a compound of formula (DX) comprises:

[1022] reacting a compound of Formula (DXVI) with a compound of Formula (DXVII):

[1023]

[1024] to produce a compound of Formula (DX).

[1025] In an embodiment, the compound of Formula (DXVI) has the structure of Formula (DXVIc):

[1026]

[1027] In some embodiments, the process for preparing a compound of Formula (DI) can comprise the steps of: providing a compound of Formula (DXVIII) having the structure:

[1028] and forming a compound of Formula (DXVI) from the compound of Formula (DXVIII).

[1029] In some embodiments, the step of forming a compound of Formula (DXVI) comprises:

[1030] reacting a compound of Formula (DXVIII) with a compound of Formula (DXIXa) or Formula (DXIXb):

[1031] wherein

[1032] X is OSu, Cl, or Br;

[1033] to produce a compound of Formula (DXVI).

[1034] In an embodiment, the compound of Formula (DXIXa) or Formula (DXIXb) is selected from the group consisting of TfCl, Tf20, MsCl, and Ms20.

[1035] The present disclosure provides a process for preparing a compound of Formula (DII) or a salt thereof:

[1036]

[1037] wherein:

[1038] q is an integer from 0 to 10,

[1039] R 1d is C 1-6 alkyl; and

[1040] PG 6 is a suitable protecting group,

[1041] the process comprising:

[1042] (a) providing a compound of Formula (DXXI) having the structure:

[1043] and

[1044] (b) forming a compound of Formula (DII) from the compound of Formula (DXXI).

[1045] In some embodiments, PG 6 is selected from the group consisting of allyloxycarbonyl (Alloc), benzyloxycarbonyl (Cbz), tert-butyloxycarbonyl (Boc), and 9-fluorenylmethyloxycarbonyl (Fmoc).

[1046] In some embodiments, the compound of Formula (DXXI) is treated with a compound .

[1047] In some embodiments, the compound of Formula (DXXI) is treated with a catalyst. In some embodiments, the catalyst is Pd(dppf)Cl2-DCM.

[1048] In some embodiments, the compound of Formula (DXXI) is treated with a base. In some embodiments, the base is K2CO3.

[1049] In some embodiments, the preparation is carried out in a solvent mixture comprising H2O / iPrOH.

[1050] In some embodiments, the compound of Formula (DII) has the structure of Formula (DIIa) or Formula (DIIb):

[1051]

[1052] In some embodiments, the compound of Formula (DXXI) is formed by exposing to .

[1053] In some embodiments, the compound of Formula (DXXI) is formed in the presence of PPh3.

[1054] In some embodiments, the compound of Formula (DXXI) is formed in the presence of DIAD.

[1055] In some embodiments, the compound of Formula (DXXI) is formed in the presence of molecular sieves.

[1056] In some embodiments, the compound of Formula (DXXI) is formed in an organic solvent. In some embodiments, the organic solvent is THF.

[1057] In some embodiments, is formed by exposing to PBin2.

[1058] In some embodiments, is formed in the presence of Pd(OAc)2.

[1059] In some embodiments, is formed in the presence of PCy3.

[1060] In some embodiments, is formed in the presence of KOAc.

[1061] In some embodiments, is formed in the presence of dioxane.

[1062] In some embodiments, is formed at an elevated temperature for a period of time.

[1063] In some embodiments, is formed by exposing to Bu4N + Br3 - .

[1064] In some embodiments, is formed in an organic solvent. In some embodiments, the organic solvent is a mixture of DCM and methanol.

[1065] The present disclosure provides a method for preparing a compound of formula (DII), or a salt thereof:

[1066]

[1067] wherein:

[1068] q is an integer from 0 to 10,

[1069] R 1d is C 1-6 alkyl; and

[1070] PG 6 is a suitable protecting group,

[1071] The process comprises:

[1072] (a) providing a compound of formula (DIV), or a salt thereof, having the structure:

[1073] and

[1074] (b) forming a compound of Formula (DII) from a compound of Formula (DIV).

[1075] According to the present disclosure, PG 6 is any suitable protecting group that can be used to protect an amino group. Suitable protecting groups that can be used are known to those skilled in the art. A detailed description of these groups and their selection and chemistry are contained in "The Peptides, Volume 3", Gross and Meinenhofer, Eds., Academic Press, New York, 1981, which is hereby incorporated by reference in its entirety. For example, PG 6 may be selected from the group consisting of allyloxycarbonyl (Alloc), benzyloxycarbonyl (Cbz), tert-butyloxycarbonyl (Boc), and 9-fluorenylmethoxycarbonyl (Fmoc). In some embodiments, PG 6 is 9-fluorenylmethoxycarbonyl (Fmoc).

[1076] In one embodiment, the compound of Formula (DII) has the structure of Formula (DIIa) or Formula (DIIb):

[1077]

[1078] In another embodiment, the compound of Formula (DIV) has the structure of Formula (DIVa) or Formula (DIVb):

[1079]

[1080]

[1081] In some embodiments, the process of forming the compound of Formula (DII) comprises:

[1082] reacting a compound of Formula (DIV) with a compound of Formula (DVa) or Formula (DVb):

[1083] PG 6 - X (DVa) or PG 6 2 0 (DVb),

[1084] wherein

[1085] X is OSu, OTf, CI, or Br;

[1086] to produce the compound of Formula (DII).

[1087] According to the present disclosure, the compound of Formula (DVa) or Formula (DVb) is selected from the group consisting of AllocCl, Alloc20, Cbz20, CbzCl, FmocCl, FmocOSu, and Boc20.

[1088] In some embodiments, the process for preparing the compound of Formula (DII) can comprise the following steps:

[1089] A compound of Formula (DXVI) having the following structure is provided:

[1090]

[1091] wherein

[1092] PG 8 is a suitable protecting group; and

[1093] forming a compound of Formula (DIV) from the compound of Formula (DXVI).

[1094] According to the present disclosure, PG 8 is any suitable protecting group that can be used to protect an amino group. Suitable protecting groups that can be used are known to those skilled in the art. A detailed description of these groups, their selection, and chemistry is contained in "The Peptides, Volume 3", Gross and Meinenhofer, Eds., Academic Press, New York, 1981, which is hereby incorporated by reference in its entirety. For example, PG 8 may be selected from the group consisting of allyloxycarbonyl (Alloc), benzyloxycarbonyl (Cbz), tert-butyloxycarbonyl (Boc), and 9-fluorenylmethoxycarbonyl (Fmoc). In some embodiments, PG 8 is tert-butyloxycarbonyl (Boc).

[1095] In some embodiments, the step of forming the compound of Formula (DIV) comprises reacting the compound of Formula (DXVI) with a protecting group removing agent to produce the compound of Formula (DIV).

[1096] According to the present disclosure, any suitable protecting group removing agent can be used to remove the protecting group PG 8Suitable protecting group removing agents that can be used to remove particular protecting groups are known to those skilled in the art. A detailed description of these groups, their selection, and chemistry is contained in "The Peptides, Volume 3", Gross and Meinenhofer, Eds., Academic Press, New York, 1981, which is hereby incorporated by reference in its entirety. In some embodiments, the protecting group removing agent is selected from the group consisting of HC1, Pd(PPh)3, PhSiH3, H2, piperidine, and trifluoroacetic acid (TFA).

[1097] In one embodiment, the compound of formula (DXVI) has the structure of formula (DXVIa) or formula (DXVIb):

[1098]

[1099] In some embodiments, the process for preparing the compound of formula (DII) can comprise the following steps:

[1100] providing a compound of formula (DXVII) having the structure:

[1101]

[1102] wherein

[1103] LG' is a suitable leaving group; and

[1104] forming the compound of formula (DXVI) from the compound of formula (DXVII).

[1105] In some embodiments, the step of forming the compound of formula (DXVI) comprises reacting the compound of formula (DXVII) with an azido group introducing agent to produce the compound of formula (DXVI).

[1106] In some embodiments, the azido group introducing agent is selected from the group consisting of NaN3, TMSN3, (PhO)2P(O)N3, Zn(N3)2*2Py, and n-Bu4NN3.

[1107] In some embodiments, LG' is selected from the group consisting of halogen, OTf, OMs, and OTs.

[1108] In one embodiment, the compound of formula (DXVII) has the structure of formula (DXVIIa) or formula (DXVIIb):

[1109]

[1110] In some embodiments, the process for preparing a compound of Formula (DII) can comprise the steps of:

[1111] A compound of Formula (DIX) having the structure:

[1112]

[1113] wherein

[1114] LG" is a suitable leaving group; and

[1115] forming a compound of Formula (DXVII) from the compound of Formula (DIX).

[1116] In some embodiments, LG" is selected from the group consisting of halogen, OTf, OMs, and OTs.

[1117] In some embodiments, the step of forming a compound of Formula (DXVII) comprises:

[1118] reacting a compound of Formula (DIX) with a compound of Formula (DXVIII):

[1119]

[1120] to produce a compound of Formula (DXVII).

[1121] In one embodiment, the compound of Formula (DIX) has the structure of Formula (DIXa):

[1122]

[1123] In another embodiment, the compound of Formula (DXVIII) has the structure of Formula (DXVIIIa):

[1124]

[1125] In some embodiments, the process for preparing a compound of Formula (DII) can comprise the steps of providing a compound of Formula (DXIX) having the structure:

[1126] and forming a compound of Formula (DIX) from the compound of Formula (DXIX).

[1127] In some embodiments,

[1128] the step of forming a compound of Formula (DIX) comprises:

[1129] reacting a compound of Formula (DXIX) with a compound of Formula (DXV):

[1130]

[1131] wherein

[1132] LG'" is a suitable leaving group,

[1133] to produce a compound of Formula (DXIX).

[1134] In one embodiment, LG'" is selected from the group consisting of halogen, OTf, OMs, and OTs.

[1135] In another embodiment, the compound of Formula (DXIX) has the structure of Formula (DXIXa):

[1136]

[1137] In another embodiment, the compound of Formula (DXV) has the structure of Formula (DXVa):

[1138]

[1139] In some embodiments, the process for preparing a compound of Formula (DII) can comprise the following steps:

[1140] providing a compound of Formula (DXX) having the structure:

[1141] and

[1142] forming a compound of Formula (DXIX) from the compound of Formula (DXX).

[1143] In some embodiments, the step of forming a compound of Formula (DXIX) comprises:

[1144] reacting the compound of Formula (DXX) with a LG" introduction agent to produce a compound of Formula (DXIX).

[1145] In one embodiment, the LG" introduction agent is NBu4Br3.

[1146] In another embodiment, the compound of Formula (DXX) has the structure of Formula (DXXa):

[1147]

[1148] Therapeutic formulations, administration, and uses

[1149] The present disclosure provides pharmaceutical compositions comprising a peptidomimetic or compound of the present disclosure.

[1150] The compositions of the present disclosure can be formulated with suitable carriers, excipients, and other agents that provide improved transfer, delivery, tolerability, and the like. Many suitable formulations can be found in the pharmacopoeia known to all pharmaceutical chemists: Remington's Pharmaceutical Sciences, Mack Publishing Company, Easton, PA. These formulations include, for example, powders, pastes, ointments, jellies, waxes, oils, lipids, lipid (cationic or anionic)-containing vesicles (such as LIPOFECTIN TM , Life Technologies, Carlsbad, CA), DNA conjugates, anhydrous absorption pastes, oil-in-water and water-in-oil emulsions, emulsion carbowax (polyethylene glycol of various molecular weights), semisolid gels, and semisolid mixtures containing carbowax. See also Powell et al. "Compendium of excipients for parenteral formulations" PDA (1998) J Pharm Sci Technol 52:238-311.

[1151] The dosage of the peptidomimetic or compound administered to the patient can vary according to the patient's age and size, target disease, condition, route of administration, etc. Suitable dosages are generally calculated based on body weight or body surface area. When the peptidomimetic or compound of the present disclosure is used for therapeutic purposes in adult patients, it is generally advantageous to administer the peptidomimetic or compound of the present disclosure intravenously in a single dose of about 0.01 mg / kg body weight to about 20 mg / kg body weight, more preferably about 0.02 mg / kg body weight to about 7 mg / kg body weight, about 0.03 mg / kg body weight to about 5 mg / kg body weight, or about 0.05 mg / kg body weight to about 3 mg / kg body weight. Depending on the severity of the condition, the frequency and duration of treatment can be adjusted. The effective dose and schedule for administering the peptidomimetic or compound can be determined empirically; for example, patient progress can be monitored by regular assessments, and the dosage can be adjusted accordingly. In addition, interspecies scaling of dosage can be performed using methods well known in the art (e.g., Mordenti et al., 1991, Pharmaceut. Res. 8: 1351).

[1152] A variety of delivery systems are known and can be used to administer the pharmaceutical compositions of the present disclosure, e.g., encapsulation in liposomes, microparticles, microcapsules, recombinant cells capable of expressing mutant viruses, receptor mediated endocytosis (see, e.g., Wu et al., 1987, J. Biol. Chem. 262:4429-4432). Methods of introduction include, but are not limited to, intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal, epidural, and oral routes. The composition can be administered by any convenient route, for example, by infusion or bolus injection, by absorption through epithelial or mucocutaneous linings (e.g., the lining of the mouth, rectum, etc.), and can be administered together with other biologically active agents. Administration can be systemic or local.

[1153] The pharmaceutical compositions of the present disclosure can be delivered subcutaneously or intravenously by standard needle and syringe. In addition, with respect to subcutaneous delivery, pen delivery devices are readily applicable in delivering the pharmaceutical compositions of the present disclosure. Such pen delivery devices can be reusable or disposable. Reusable pen delivery devices generally utilize a replaceable cartridge containing the pharmaceutical composition. After all of the pharmaceutical composition within the cartridge has been administered and the cartridge is empty, the empty cartridge can be readily discarded and replaced with a new cartridge containing the pharmaceutical composition. The pen delivery device can then be reused. In disposable pen delivery devices, there is no replaceable cartridge. Rather, the disposable pen delivery device comes prefilled with the pharmaceutical composition held in a reservoir within the device. After the pharmaceutical composition in the reservoir is depleted, the entire device is discarded.

[1154] Many reusable pen and auto-injector delivery devices are readily applicable in subcutaneously delivering the pharmaceutical compositions of the present disclosure. Examples include, but are not limited to, AUTOPEN® (Owen Mumford, Inc., Woodstock, UK), DISETRONIC® TM pen (Disetronic Medical Systems, Bergdorf, Switzerland), HUMALOG MIX 75 / 25 TM pen (Disetronic Medical Systems, Bergdorf, Switzerland), HUMALOG MIX 75 / 25 TM pen (Disetronic Medical Systems, Bergdorf, Switzerland), HUMALOG MIX 75 / 25 TM pen (Disetronic Medical Systems, Bergdorf, Switzerland), HUMALOG MIX 75 / 25 TM pen (Disetronic Medical Systems, Bergdorf, Switzerland), HUMALOG MIX 75 / 25 TM I, II, and III (Novo Nordisk, Copenhagen, Denmark), NOVOPEN JUNIOR TMBD DIAL HEME® pen (Becton Dickinson, Franklin Lakes, NJ), OPTIPEN TM pen (Becton Dickinson, Franklin Lakes, NJ), OPTIPEN TM , OPTIPEN PRO TM , OPTIPEN STARLET TM , and OPTICLIK TM (sanofi-aventis, Frankfurt, Germany). Examples of disposable pen delivery devices that have application in the subcutaneous delivery of the pharmaceutical compositions of the present disclosure include, but are not limited to, SOLOSTAR TM pen (sanofi-aventis), FLEXPEN TM (Novo Nordisk), and KWIKPEN TM (Eli Lilly), SURECLICK™ autoinjector (Amgen, Thousand Oaks, CA), PENLETTM (Haselmeier, Stuttgart, Germany), EPIPEN (Dey, L.P.), and HUMIRATM pen (Abbott Labs, Abbott Park IL).

[1155] In certain instances, the pharmaceutical composition can be delivered in a controlled release system. In one embodiment, a pump can be used (see, Langer, supra; Sefton, 1987, CRC Crit. Ref. Biomed. Eng. 14:201). In another embodiment, polymeric materials can be used; see, Medical Applications of Controlled Release, Langer and Wise (eds.), 1974, CRC Pres., Boca Raton, Florida. In yet another embodiment, a controlled release system can be placed in proximity of the composition's target, thus requiring only a fraction of the systemic dose (see, e.g., Goodson, 1984, Medical Applications of Controlled Release, supra, vol. 2, pp. 115-138). Other controlled release systems are discussed in the review by Langer, 1990, Science 249:1527-1533.

[1156] The injectable preparations can include dosage forms for intravenous, subcutaneous, intracutaneous and intramuscular injection, drip infusion, etc. These injectable preparations can be prepared by publicly known methods. For example, the injectable preparations can be prepared by, for example, dissolving, suspending or emulsifying the above-described antibody or salt thereof in a sterile aqueous medium or an oily medium conventionally used for injections. As the aqueous medium for injections, there are, for example, physiological saline, isotonic solutions containing glucose and other auxiliary agents, etc., which can be used in combination with an appropriate solubilizing agent such as an alcohol (e.g., ethanol), a polyol (e.g., propylene glycol, polyethylene glycol), a nonionic surfactant [e.g., polysorbate 80, HCO-50 (polyoxyethylene (50 mol) adduct of hydrogenated castor oil)], etc. As the oily medium, there are employed, for example, sesame oil, soybean oil, etc., which can be used in combination with a solubilizing agent such as benzyl benzoate, benzyl alcohol, etc. The injection thus prepared is preferably filled in a suitable ampoule.

[1157] Advantageously, the pharmaceutical compositions described above for oral or parenteral use are prepared into dosage forms in unit dose suited to fit a dose of the active ingredient. Such dosage forms in unit dose include, for example, tablets, pills, capsules, injections (ampoules), suppositories, etc. In the unit dose, the amount of the aforementioned antibody contained is usually about 5 mg to about 500 mg per dosage form; particularly in the injection form, it is preferred that the aforementioned antibody is contained in about 5 mg to about 100 mg and for other dosage forms in about 10 mg to about 250 mg.

[1158] Therapeutic uses of peptidomimetics or compounds

[1159] In another aspect, the peptidomimetics or compounds (e.g., GLP1 peptidomimetics) disclosed herein are particularly useful for treating, preventing and / or ameliorating a disease, disorder or condition in need of such treatment.

[1160] In one aspect, the disclosure provides a method of treating a condition in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of a peptidomimetic or compound (e.g., GLP1 peptidomimetic) according to the disclosure, or a composition comprising any compound according to the disclosure.

[1161] In some embodiments, the peptidomimetics or compounds (e.g., GLP1 peptidomimetics) disclosed herein are useful for treating any disease or disorder in which stimulation, activation and / or targeting of GLP1R would be beneficial. In particular, the compounds of the disclosure can be used to treat, prevent and / or ameliorate any disease or disorder associated with or mediated by GLP1R expression or activity.

[1162] In some embodiments, the peptidomimetic or compound disclosed herein (e.g., a GLP1 peptidomimetic) can be used to treat a GLP1R-related condition. In some embodiments, the GLP1R-related condition is type 1 or type 2 diabetes. The administered peptidomimetic or compound (e.g., a GLP1 peptidomimetic) can result in at least one of the following: inducing insulin secretion, suppressing glucagon release, lowering blood glucose, improving glycemic control, promoting islet neogenesis, and delaying gastric emptying or enhancing glucose-tolerant islets.

[1163] In some embodiments, the GLP1R-related condition is a neurodegenerative disorder, a cognitive disorder, a memory disorder, or a learning disorder. The neurodegenerative disorder can be, for example, dementia, senile dementia, mild cognitive impairment, Alzheimer’s disease-related dementia, Huntington’s chorea, tardive dyskinesia, hyperkinesia, mania, Morbus Parkinson, steel-Richard syndrome, Down’s syndrome, myasthenia gravis, neural trauma, brain trauma, vascular amyloidosis, cerebral hemorrhage I with amyloidosis, encephalitis, Friedrich’s ataxia, acute confusional disorder, amyotrophic lateral sclerosis, glaucoma, and Alzheimer’s disease.

[1164] In some embodiments, the GLP1R-related condition is a liver disease. The liver disease can be, for example, non-alcoholic fatty liver disease (NAFLD), fatty liver, non-alcoholic steatohepatitis (NASH), and cirrhosis.

[1165] In some embodiments, the GLP1R-related condition is a coronary artery disease. The coronary artery disease can be, for example, cardiomyopathy and myocardial infarction.

[1166] In some embodiments, the GLP1R-related condition is a kidney disease. The kidney disease can be, for example, hypertension or chronic renal failure.

[1167] In some embodiments, the GLP1R-related condition is an eating disorder. The eating disorder can be, for example, binge eating disorder.

[1168] Without wishing to be bound by theory, the peptidomimetics or compounds disclosed herein (e.g., GLP1 peptidomimetics) can be used to attenuate the role of apoptosis-mediated central nervous system degenerative diseases such as Alzheimer's disease, Creutzfeld-Jakob Disease, and bovine spongiform encephalopathy, chronic fatigue syndrome, and other prion-mediated apoptotic neurological diseases (see, e.g., Perry and Greig (2004) Current Drug Targets 6:565-571). Administration of the peptidomimetics or compounds disclosed herein (e.g., GLP1 peptidomimetics) can also result in down-regulation of βAPP and thereby ameliorate Aβ monomer or oligomer-mediated pathology associated with Alzheimer's disease (see, e.g., Perry et al. (2003) Journal of Neuroscience Research 72:603-612).

[1169] It is also contemplated that the peptidomimetics or compounds disclosed herein (e.g., GLP1 peptidomimetics) can be used to enhance learning and memory, for example, by enhancing neuronal plasticity and promoting cell differentiation (see, During et al. (2003) Nature Medicine 9:1173-1179). In addition, the compounds disclosed herein (e.g., antibody-drug conjugates, linker-payloads, and / or payloads) can also be used to preserve dopamine neurons and motor function in Morbus Parkinson (see, e.g., Greig et al. (2005) Abstract 897.6, Society for Neuroscience, Washington, D.C.).

[1170] In some embodiments, the peptidomimetics or compounds disclosed herein (e.g., GLP1 peptidomimetics) can also be used to treat metabolic disorders. The metabolic disorder can be, for example, obesity, dyslipidemia, metabolic syndrome X, and conditions resulting from pancreatic insufficiency.

[1171] Additional diseases that can be treated by the compounds of the present disclosure (e.g., antibody-drug conjugates, linker-payloads, and / or payloads) include autoimmune diseases, particularly diseases associated with inflammation, including but not limited to autoimmune diabetes, adult-onset diabetes, morbid obesity, metabolic syndrome X, and dyslipidemia. For example, an anti-GLP1R antibody-drug conjugate can be used as a growth factor to promote islet growth in patients with autoimmune diabetes. The compounds described herein (e.g., antibody-drug conjugates, linker-payloads, and / or payloads) can also be used to treat congestive heart failure.

[1172] In one aspect, the present disclosure provides methods of selectively targeting an antigen (e.g., GLP1R) on the surface of a cell with a peptidomimetic or compound disclosed herein. In one embodiment, the method of selectively targeting an antigen (e.g., GLP1R) on the surface of a cell with a compound comprises linking the compound to a targeting antibody. In one embodiment, the cell is a mammalian cell. In one embodiment, the cell is a human cell. In one embodiment, the cell is a pancreatic cell or a brain cell. Examples

[1173] The following examples illustrate specific aspects of the present specification. The examples are not to be construed as limiting, as the examples merely provide a specific understanding and practice of the embodiments and their various aspects.

[1174] Abbreviations used in the examples and throughout the specification are as follows:

[1175]

[1176]

[1177]

[1178]

[1179]

[1180] Example 1. General Methods

[1181] Unless otherwise noted, all anhydrous reactions were carried out under an argon atmosphere with a stir bar or magnetic stirring in an oven-dried glassware. Rink amide linkers were sourced from GL Biochem. All other reagents and anhydrous solvents were purchased from commercial sources and used without any further purification unless otherwise noted. Reactions were monitored using thin layer chromatography (TLC) analysis on pre-coated silica gel 60 F254 plates, where possible, and the plates were visualized by short wave UV irradiation (254 nm; where applicable), staining with PMA (5% wt / v in EtOH) or ninhydrin (5% wt / v in EtOH), and then charring at ~200 °C, unless otherwise noted. Column chromatography (silica gel 60, 40 mm) was performed in air and under pressure (0.1-0.3 bar), unless otherwise noted. Bulk solvents for flash chromatography (EtOAc, hexanes, THF, DCM) were distilled before use. IR spectra were recorded with an FT-IR spectrometer (Thermo NICOLET iS10) using a diamond tip sampling, and are reported in wavenumbers (cm-1), unless otherwise noted. -1) reports. Residual solvent reports were recorded using GC (SHIMADZU-GC2030 & HS-20). Water content was recorded on a Mettler toledo C30s Karl Fischer reaction instrument. Melting point (MP) was measured via DSC (Differential Scanning Calorimeter, DSC 214). Inorganic salt reports were provided by Thermo ICS-6000 Ion Chromatography Detector. Purity of >90% of the synthetic intermediates after chromatographic purification was judged by analysis of1H NMR spectra. Purity of the final compounds was >95% after trituration with MeOH and MeCN (Honeywell, HPLC grade) (NMR analysis).

[1182] General NMR methods and instrumentation

[1183] 1 H NMR and13C: were recorded on a Bruker Avance 400 (400 MHz, 1 H, 100 MHz, 13 C) spectrometer 1 HNMR spectra. Chemical shift values (δ) are reported in ppm relative to residual chloroform (for1H, δ 7.26 ppm; for13C, δ 78.0 ppm). Multiplicity is indicated by s (singlet), d (doublet), t (triplet), q (quartet), p (pentet), h (heptet), dd - doublet of doublets, dt - doublet of triplets, dq - doublet of quartets, m (multiplet), and br (broad). 1 H, δ 7.26 ppm; for 13 C, δ 78.0 ppm). Multiplicity is indicated by s (singlet), d (doublet), t (triplet), q (quartet), p (pentet), h (heptet), dd - doublet of doublets, dt - doublet of triplets, dq - doublet of quartets, m (multiplet), and br (broad). 1 H, and 13 Identification of1H, 1 H, 13 C, DEPT, COSY, HMBC, HMQC, and NOESY experiments. Coupling constants (J) are reported in Hertz (Hz).

[1184] Example 2. General LC-MS method and instrument

[1185] Method 1

[1186] This method was used to analyze intermediates 1, 2, 3, 4, 5, 6, and 7 of tag 5. Instrument: Agilent 1200+6110MS (Table 1). Chromatography was performed using an XBridge Protein BEH C4 300A 2.5 μm 4.6×50 mm column. Diode array (DAD) and evaporative light scattering (ELSD) were used as detection methods. The MS mode was positive electrospray ionization. The MS range was 50-1500. Mobile phase A was 0.04% TFA in water, and mobile phase B was 0.02% TFA in HPLC grade ACN. The gradient was 50%-100% B in 6 min, 50% B in 0.00 min, 50%-100% B from 0.00 min to 4.00 min, with a hold at 100% B for 1.5 min, and 100%-50% B from 5.51 min to 6.00 min, with a hold at 50% B for 0.49 min. The flow rate was 1.50 mL / min.

[1187] Table 1. Conditions for LC-MS Method 1

[1188]

[1189]

[1190] Method 2

[1191] This method was used to analyze intermediate 8 of tag 5. Instrument: Waters H Class & SQD2 (Table 2). The gradient was 50%-100% B in 3.40 min, maintained at 100% B from 3.40 min to 3.85 min, 100%-5% B in 0.01 min, and then maintained at 5% for 0.64 min. The flow rate was 0.80 ml / min. Mobile phase A was H2O+10 mM NH4HCO3, and mobile phase B was HPLC grade THF. The column used for chromatography was a 2.1*50 mm Xbridge Shield RPC18 column (5 μm particles). Diode array (DAD) detection and positive electrospray ionization were used as detection methods. The MS range was 100 Da-3000 Da.

[1192] Table 2. Conditions for LC-MS Method 2

[1193]

[1194] Method 3

[1195] This method was used to analyze intermediate 9 of Tag 5. Instrument: Waters H Class & SQD2 (Table 3). The gradient was 70%-100% B in 16.00 min and held at 100% B for 4.00 min, 100%-70% B in 0.01 min, and then held at 70% for 3 min with a flow rate of 1.0 ml / min. The mobile phase A was 10 mM ammonium bicarbonate and the mobile phase B was HPLC grade THF. The column used for the chromatography was an Xbridge C18 4.6*150 mm column (3.5 pm particles). Diode array (DAD) and positive electrospray ionization were used as detection methods. The MS range was 100 Da-3000 Da.

[1196] Table 3. Conditions for LC-MS method 3

[1197]

[1198] Method 4

[1199] This method was used to analyze Tag 1-TCP (Tag 5-1AA). LC-MS conditions: mobile phase: 1.5 mL / 4 L of TFA in water (solvent A) and 0.75 mL / 4 L of TFA in ACN (solvent B) using an elution gradient of 50%-100% (solvent B) in 1.35 min and held at 100% for 0.9 min with a flow rate of 0.8 ml / min; column: Xtimate C18 2.1*30 mm, 3 pm.

[1200] Method 5

[1201] This method was used to analyze Tag 1-2AA, Tag 1-3AA, Tag 1-4AA, Tag 1-5AA, Tag 1-6AA, Tag 1-7AA, Tag 1-10AA. LC-MS conditions: mobile phase: 1.5 mL / 4 L of TFA in water (solvent A) and 0.75 mL / 4 L of TFA in ACN (solvent B) using a gradient of 10%-80% (solvent B) in 6 min and held at 80% for 0.5 min with a flow rate of 0.8 ml / min. Column: X timate C18 2.1*30 mm, 3 pm.

[1202] Method 6

[1203] This method was used for the analysis of M2. LC-MS conditions: Reversed phase LCMS was performed using a Chromolith Flash RP-C18 25-3mm column eluting with a gradient of 10% to 80% of ACN containing 0.02% TFA (solvent B) and water containing 0.04% TFA (solvent A) at a flow rate of 1.5 mL / min.

[1204] Method 7

[1205] This method was used for the analysis of Tag 5-2AA, Tag 5-3AA, Tag 5-4AA, Tag 5-5AA, Tag 5-6AA, Tag 5-7AA, Tag 5-8AA, Tag 5-9AA and Tag 5-10AA. LCMS conditions (BF): Mobile phase: 1.5 mL / 4 L of TFA in water (solvent A) and 0.75 mL / 4 L of TFA in ACN (solvent B), using an elution gradient of 10%-80% (solvent B) in 6 min and hold at 80% for 0.5 min at a flow rate of 0.8 ml / min; Column: Xtimate C18 2.1*30 mm 3 pm; Wavelength: UV 220 nm, 254 nm; Column temperature: 50 °C; MS ionization: ESI.

[1206] Method 8

[1207] This method was used for the analysis of Tag 5-2AA, Tag 5-3AA, Tag 5-4AA, Tag 5-5AA, Tag 5-6AA, Tag 5-7AA, Tag 5-8AA, Tag 5-9AA and Tag 5-10AA. LCMS conditions (BF): Mobile phase: 1.5 mL / 4 L of TFA in water (solvent A) and 0.75 mL / 4 L of TFA in ACN (solvent B), using an elution gradient of 10%-80% (solvent B) in 6 min and hold at 80% for 0.5 min at a flow rate of 0.8 ml / min; Column: Xtimate C18 2.1*30 mm 3 pm; Wavelength: UV 220 nm, 254 nm; Column temperature: 50 °C; MS ionization: ESI.

[1208] Method 9

[1209] This method was used to analyze Label 5-2AA, Label 5-3AA, Label 5-4AA, Label 5-5AA, Label 5-6AA, Label 5-7AA, Label 5-8AA, Label 5-9AA and Label 5-10AA. LCMS conditions (BV): Mobile phase: 1.5 mL / 4 L of TFA in water (solvent A) and 0.75 mL / 4 L of TFA in ACN (solvent B), using an elution gradient of 10-80% (solvent B) in 6 min and hold at 80% for 0.5 min, flow rate of 0.8 ml / min; Column: NanoChrom ChromCore 120 C18 3μm, 3.0*30 mm; Wavelength: UV 220 nm; Column temperature: 50 °C; MS ionization: ESI.

[1210] Method 10

[1211] This method was used to analyze Label 5-2AA, Label 5-3AA, Label 5-4AA, Label 5-5AA, Label 5-6AA, Label 5-7AA, Label 5-8AA, Label 5-9AA and Label 5-10AA. LCMS conditions (BK): Instrument and column: Xtimate 3μm, C18, 2.1*30 mm S / N3U411701820, Mobile phase: 1.5 mL / 4 L of TFA in water (solvent A) and 0.75 mL / 4 L of TFA in ACN (solvent B), using a gradient of 10-80% (solvent B) in 6 min and hold at 80% for 0.5 min, flow rate of 0.8 ml / min. ESI source, positive ion mode; Wavelength 220 nm and 254 nm, oven temperature 50 °C.

[1212] Method 11

[1213] This method was used to analyze Label 4-10AA. HPLC Method A: Column: YMC-Pack ODS-A 150*4.6 mm, 5μm; 2.75 mL / 4 L of TFA in water (solvent A) and 2.5 mL / 4 L of TFA in ACN (solvent B), using an elution gradient of 10-80% (solvent B) in 10 min and hold at 80% for 5 min, flow rate of 1.5 ml / min.

[1214] Method 12

[1215] This method was used to analyze Tag 4-10AA. LCMS conditions: Reversed phase LCMS using a Chromolith Flash RP-C18 25-3 mm column eluted with a gradient of 10% to 80% ACN containing 0.02% TFA (solvent B) and water containing 0.04% TFA (solvent A) at a flow rate of 1.5 mL / min.

[1216] Method 13

[1217] This method was used to analyze intermediate AA10-AA9-AA8-OH. LC-MS conditions: Mobile phase: 1.5 mL / 4 L of TFA in water (solvent A) and 0.75 mL / 4 L of TFA in ACN (solvent B), using an elution gradient of 5%-95% (solvent B) in 0.7 min and hold at 95% for 0.4 min at a flow rate of 1.5 mL / min; Column: Agilent Pursuit 5 C18 20*2.0 mm.

[1218] Method 14

[1219] This method was used to analyze DMB-AA9. Reversed phase HPLC was performed under the following conditions: using Ultimate XB-C18, 3 pm, 3.0*50 mm column, mobile phase: 1.0% ACN in water (0.1% TFA) to 5% ACN in water (0.1% TFA) in 1 min; then from 5% ACN in water (0.1% TFA) to 100% ACN (0.1% TFA) in 5 min; hold at 100% ACN (0.1% TFA) for 2 min; back to 1.0% ACN in water (0.1% TFA) at 8.01 min and hold for two minutes. Flow: 1.2 ml / min.

[1220] Method 15

[1221] This method was used to analyze M2. HPLC conditions: Mobile phase: 1.0% ACN in water (0.1% TFA) to 5% ACN in water (0.1% TFA) in 1 min; then from 5% ACN in water (0.1% TFA) to 100% ACN (0.1% TFA) in 5 min; hold at 100% ACN (0.1% TFA) for 2 min; back to 1.0% ACN in water (0.1% TFA) at 8.01 min and hold for two minutes. Flow: 1.2 ml / min. Column: Ultimate XB-C18, 3 pm, 3.0*50 mm.

[1222] ELSD LCMS conditions

[1223] Mobile phase: 10 mM NH4OAc in water (solvent A) and ACN (solvent B) using an elution gradient of 10-80% (solvent B) in 5 min, 80% hold for 1.95 min; 80% B changed to 10% B at 7 min, 10% hold for 4 min, flow rate 0.8 mL / min. Column: Shim-pack Scepter C18-120 1.9 pm, 2.1*100 mm.

[1224] Example 3. Synthesis of DMB-AA9

[1225] The following Scheme 1 depicts the synthesis of DMB-AA9:

[1226] Scheme 1

[1227]

[1228] Conditions: 1) (2,4-dimethoxyphenyl)methanamine (1.2 eq), HATU (1.0 eq), DIPEA (3 eq), DMF (100 mL), 20 °C, 2 h, 84% yield; 2) (a) PCl5 (1.5 eq), TMSN3 (4 eq), pyridine (8 V), 0 °C, 4 h; (b) MeOH (4 eq), 0 °C, 2 h, 81% yield; 3) (a) NaOH (3 eq), THF / H2O (2:1, 10 mL), 20 °C, 2 h; (b) FmocOSu (1 eq), aq. NaHC03, 20 °C, 2 h; 40.5% yield.

[1229] Step 1: Synthesis of methyl (2S)-4-[(2,4-dimethoxyphenyl)methylamino]-2-(9H-fluoren-9- ylmethoxycarbonylamino)-4-oxo-butyrate (DMB-AA9-2)

[1230]

[1231] To a solution of (3S)-3-(9H-fluoren-9-ylmethoxycarbonylamino)-4- methoxy-4-oxo-butyric acid (DMB-AA9-1, 90 g, 243.66 mmol, 1 eq) in DMF (200 mL) was added HATU (101.91 g, 268.03 mmol, 1.1 eq) and DIPEA (62.98 g, 487.32 mmol, 84.88 mL, 2 eq), and the mixture was stirred at 20 °C for 30 min. Then (2,4-dimethoxyphenyl)methanamine (44.82 g, 268.03 mmol, 40.37 mL, 1.1 eq) was added, and the mixture was stirred at 20 °C for 1 h. Most of the reactants were consumed on TLC (PE / EtOAc = 2 / 1), and the desired compound was detected according to LCMS. The reaction mixture was poured into DCM (1500 mL), washed with H2O (900 mL) and saturated citric acid (100 mL). The separated organic layer was washed with brine (1000 mL), dried over Na2SO4and filtered. Then it was concentrated under reduced pressure to give the crude product as a light yellow solid. The crude product was triturated with EtOAc (600 mL) for 12 h and filtered to give the target compound. The desired compound DMB-AA9-2 (109 g, 197.59 mmol, 81.09% yield, 94% purity) was obtained as a white solid. ESI-MS (Method 10): RT = 3.896 min, m / z calcd 519.2 [M+H], found 519.1 [M+H]. 29 H 30 O7N2H, mass calcd 519.2, m / z found 519.1 [M+H] + . 1 H NMR (400 MHz, Chloroform-d) δ = 7.76 (d, J = 7.5 Hz, 2H), 7.61 (br dd, J = 3.6, 6.9 Hz, 2H), 7.44 - 7.37 (m, 2H), 7.36 - 7.28 (m, 2H), 7.15 (d, J = 8.3 Hz, 1H), 6.48 - 6.38 (m, 2H), 6.15 (br s, 2H), 4.60 (br s, 1H), 4.44 - 4.26 (m, 4H), 4.26 - 4.19 (m, 1H), 3.84 - 3.72 (m, 9H), 2.97 (br dd, J = 4.4, 15.7 Hz, 1H), 2.73 (br dd, J = 3.9, 15.7 Hz, 1H).

[1232] Step 2: Synthesis of methyl (2S)-3-[1-[(2,4-dimethoxyphenyl)methyl]tetrazol-5-yl]-2- (9H-fluoren-9-ylmethoxycarbonylamino)propanoate (DMB-AA9-3)

[1233]

[1234] To a suspension of compound DMB-AA9-2 (100 g, 192.84 mmol, 1 eq) in pyridine (800 mL) was added TMSN3 (88.87 g, 771.37 mmol, 101.45 mL, 4 eq) followed by portionwise addition of PCI5 (60.24 g, 289.26 mmol, 1.5 eq). The temperature was maintained below 0 °C by using ice-salt water bath. The reaction mixture was stirred at 0 °C for 5 h. LCMS showed most of the reactant was consumed. Then MeOH (24.71 g, 771.37 mmol, 31.21 mL, 4 eq) was added dropwise and the reaction mixture was stirred at 0 °C for 1 h. TLC (PE / EtOAc = 2 / 1) and LCMS showed most of the reactant was consumed and the desired compound was detected as the major peak. The reaction was quenched with MeOH (20 mL) and saturated aqueous NaHC03solution (1000 mL x 2) and extracted with EtOAc (1000 mL x 3). The aqueous layer was quenched with 8-10% NaClO (1000 mL). The combined organic layers were concentrated under reduced pressure to give a residue which was re-dissolved in EtOAc (2000 mL) and washed with saturated citric acid (500 mL x 2) and brine (1000 mL), dried over Na2S04and concentrated under reduced pressure to give a residue. The crude product was used directly in the next step without any further purification. The desired compound DMB-AA9-3 (97 g, 156.86 mmol, 81.34% yield, 87.9% purity) was obtained as a yellow oil. LC-MS: RT = 4.310 min, m / z found 544.2 [M+H] for C 29 H 29 N5O6H, mass calcd 544.2 [M+H] + , m / z found 544.2 [M+H] + , Method 10. 1 H NMR (400 MHz, Chloroform-d) δ = 7.68 (d J = 7.6 Hz, 2H), 7.48 (br d, J = 7.4 Hz, 2H), 7.37 - 7.25 (m, 3H), 7.25 - 7.20 (m, 2H), 7.09 (d, J = 8.3 Hz, 1H), 6.43 - 6.31 (m, 2H), 6.09 (br d, J = 8.5 Hz, 1H), 4.90 - 4.75 (m, 1H), 4.36 - 4.22 (m, 2H), 4.20 - 4.10 (m, 1H), 3.71 - 3.63 (m, 9H), 3.54 - 3.41 (m, 1H), 3.26 (dd, J = 4.6, 16.3 Hz, 1H) ppm.

[1235] Step 3: Synthesis of (2S)-3-[1-[(2,4-dimethoxyphenyl)methyl]tetrazol-5-yl]-2-(9H-fluoren-9-ylmethoxycarbonylamino)propanoic acid (DMB-AA9)

[1236]

[1237] To compound DMB-AA9-3 (95g, 174.77mmol, 1 equivalent) in THF (600mL) and H o (100mL) solution add NaOH (20.97g, 524.31mmol, 3 equivalents), and reactant mixture is stirred at 20 DEG C for 2 hours.LCMS shows that reactant is consumed.Use citric acid by the pH adjustment of reactant mixture to pH 7-8, and by mixture H o (200mL) dilution, and then extract with TBME (300mL×2).To aqueous layer add the NaHCO in THF (600mL) (44.05g, 524.31mmol, 20.39mL, 3 equivalents) and FmocOSu (58.96g, 174.77mmol, 1 equivalent), then reactant mixture is stirred at 10 DEG C for 12 hours.LCMS shows the desired compound detected as main peak. The reaction was concentrated under reduced pressure to remove THF, then poured into saturated NaHCO (500 mL) and H2O (300 mL), and extracted with TBME (500 mL × 3). The aqueous layer was extracted with EtOAc / MeOH (10 / 1, 800 mL × 3). The combined EtOAc layers were washed with saturated NaHCO (200 mL) and brine (200 mL), then dried over Na2SO4 and concentrated under reduced pressure to give a residue. The crude product was ground at 20 ° C for 12 h with TBME: ACN: EtOAc (10: 1: 1, 250 mL), and filtered to give a light yellow solid. Compound DMB-AA9 (38 g, 70.90 mmol, 40.57% yield, 98.8% purity) was obtained as a light yellow solid. LC-MS: RT = 3.207 min, for C 28 H 27 N5O6H, mass calculated 530.2 [M+H] + , m / z found 530.2 [M+H] + , Method 7. HPLC purity: 98.84% after trituration (Method 14 for analysis of DMB-AA9). 1H NMR (400 MHz, Methanol-d4) δ = 7.77 (d, J = 7.5 Hz, 2H), 7.62 (br d, J = 7.3 Hz, 2H), 7.40 - 7.16 (m, 5H), 6.56 - 6.43 (m, 2H), 5.52 - 5.37 (m, 2H), 4.45 (dd, J = 5.0, 7.5 Hz, 1H), 4.35 - 4.08 (m, 3H), 3.75 (s, 6H), 3.55 (dd, J = 4.9, 15.2 Hz, 1H), 3.47 - 3.35 (m, 1H) ppm. SFC ee = 98.53 - 1.47 = 97.06%.

[1238] Example 4. Synthesis of (LPPS) M2 on Tag 1

[1239] 4.1 Synthesis of Tag 1

[1240] The synthetic route to prepare Tag 1 is shown in Scheme 2.

[1241] Scheme 2

[1242]

[1243] Reagents and conditions: a) 1-bromodocosane (2.2 eq), K2CO3(6 eq), DMF / THF, 90 °C, 12 h, 99%; (b) NaBH4(5 eq), EtOH (1 V), THF (10 V), 70 °C, 12 h, 96%; c) FmocNH2(2 eq), MeSO3H (0.3 eq), toluene / THF, 110 °C, 36 h, 82%; d) 20% piperidine / THF (10 V), 20 °C, 2 h, 92%. Overall yield: 71.6% over 4 steps. Overall yield: 71.6% over 4 steps.

[1244] This synthesis is reported in U.S. Patent Application Publication No. 2012 / 0296074 to Hirai et al. Alkylation of Tag 1-1 with 1-bromodocosane produced Tag 1-2, which was reduced by sodium borohydride to form the alcohol Tag 1-3. Compound Tag 1-4 was obtained from Tag 1-3 via nucleophilic substitution of the 9-fluorenylmethyl carbamate using methanesulfonic acid as a catalyst. The reaction yield was slightly improved (82% versus 60%) by using a co-solvent (toluene / THF) instead of toluene. Deprotection of the Fmoc in Tag 1-4 was performed using piperidine / THF and provided Tag 1 in 72% overall yield. U.S. Patent Application Publication No. 2012 / 0296074 to Hirai et al. reports a Fmoc deprotection yield of 45% for Tag 1-4 using DBU / DCM.

[1245] Step a: Synthesis of bis(4-dodecyloxyphenyl)methanone (Tag 1-2)

[1246]

[1247] To a mixture of compound Tag 1-1 (10 g, 46.68 mmol, 1.0 eq) and 1- bromododecane (40.00 g, 102.70 mmol, 2.2 eq) in DMF (200 mL) and THF (40 mL) was added K2CO3(38.71 g, 280.09 mmol, 6 eq), and the mixture was stirred at 90 °C for 12 h. The reactants were consumed and a new spot with lower polarity was formed on TLC (PE / EtOAc = 2 / 1). The reaction mixture was poured into water (1000 mL), and the mixture was stirred for 1 h, then the precipitated solid was collected by filtration and slurry-washed with water (200 mL x 2). The obtained solid was triturated with acetone (500 mL) and collected by filtration with slurry-washing in acetone. After filtration, the obtained solid was dried under reduced pressure to give Tag 1-2 (38.8 g, 46.67 mmol, 99.97% yield) as a white solid.

[1248] Compound Tag 1-2 (C 57 H 98 O3, MW: 830.75): 1 H NMR (400 MHz, CDC13) δ = 7.78 (d, J = 8.8 Hz, 4H), 6.95 (d, J = 8.8 Hz, 4H), 4.04 (t, J = 6.5 Hz, 4H), 1.86 - 1.78 (m, 4H), 1.51 - 1.43 (m, 4H), 1.26 (s, 72H), 0.89 (t, J = 6.8 Hz, 6H) ppm.

[1249] Step b: Synthesis of bis(4-dodecyloxyphenyl)methanol (Tag 1-3)

[1250]

[1251] A solution of compound Label 1-2 (35 g, 42.10 mmol, 1 equiv) in THF (500 mL) and EtOH (25 mL) was heated to 70 °C. Then NaBH4(7.96 g, 210.49 mmol, 5.0 equiv) was added slowly and the mixture was stirred at the same temperature for 20 h. TLC (PE / EtOAc = 3 / 1) showed that the reactants were completely consumed and a new spot was detected. The reaction mixture was ice-cooled and 1 N hydrochloric acid (80 mL) was added dropwise. THF was evaporated, water (450 mL) was added and 1 N hydrochloric acid was added to pH 5-7. The slurry was filtered and the obtained crystals were washed with water and methanol to give the product Label 1-3 (34 g, 40.80 mmol, 96.91% yield) as a white solid.

[1252] Compound Label 1-3 (C 57 H 100 O3, MW: 832.77): 1 H NMR (400 MHz, Chloroform-d) δ = 7.29 - 7.25 (m, 4H), 6.88 - 6.84 (m, 4H), 5.77 (d, J = 2.8 Hz, 1H), 3.94 (t, J = 6.7 Hz, 4H), 2.09 (d, J = 3.3 Hz, 1H), 1.81 - 1.73 (m, 4H), 1.48 - 1.41 (m, 4H), 1.35 - 1.26 (m, 72H), 0.91 - 0.87 (m, 6H) ppm.

[1253] Step c: Synthesis of 9H-fluoren-9-ylmethyl N-[bis(4-dodeca- n- yloxyphenyl)methyl]carbamate (Label 1-4)

[1254]

[1255] To a mixture of Tag 1-3 (10 g, 12.00 mmol, 1 equiv) and 9H-fluoren-9-ylmethyl carbamate (FmocNH2, 5.74 g, 24.00 mmol, 2.0 equiv) in toluene (200 mL) was added MsOH (345.97 mg, 3.60 mmol, 256.27 μί, 0.3 equiv) and the mixture was stirred at 110 °C for 36 h. Most of the reactant was consumed and a new spot was formed on TLC (PE / EtOAc = 10 / 1). The reaction mixture was poured into 5% NaHC03(30 mL) and the mixture was stirred for 1 h, then the precipitated solid was collected by filtration and washed with water (50 mL). The obtained solid was triturated with ACN (200 mL) and the solid was collected by filtration and slurry washed in methanol. After filtration, the obtained solid was dried under reduced pressure to give Tag 1-4 (11 g, 9.91 mmol, 82.58% yield, 95% purity) as off-white solid.

[1256] Compound Tag 1-4 (C 72 H 111 NO4, MW: 1053.85): 1 H NMR (400 MHz, Chloroform-d) δ = 9.89 (s, 1H), 7.78 (br d, J = 8.4 Hz, 2H), 7.61 (br d, J = 7.4 Hz, 2H), 7.44 - 7.31 (m, 4H), 7.14 - 7.10 (m, 1H), 7.12 (d, J = 8.6 Hz, 2H), 6.98 (dd, J = 8.7, 19.2 Hz, 1H), 6.88 - 6.75 (m, 4H), 5.87 (br s, 1H), 4.43 (d, J = 7.0 Hz, 1H), 4.28 - 4.21 (m, 1H), 4.04 (dt, J = 2.9, 6.5 Hz, 1H), 3.99 - 3.85 (m, 4H), 1.82 - 1.74 (m, 4H), 1.59 (br s, 4H), 1.27 (s, 72H), 0.91 - 0.89 (m, 6H) ppm.

[1257] Step d: Synthesis of Bis(4-dodecaoxylphenyl)methanamine (Tag 1-5)

[1258]

[1259] To a solution of Tag 1-4 (11 g, 10.43 mmol, 1 eq) in THF (80 mL) was added piperidine (20 mL). The mixture was then stirred at 20 °C for 2 h. TLC (PE / EtOAc = 3 / 1) showed the reactant was consumed completely and one new spot was detected. To the reaction solution was added ACN (400 mL), and the resulting suspension was filtered and washed with ACN (100 mL x 2) to give off-white solid. The crude product Tag 1-5 (8 g, 9.61 mmol, 92.14% yield) was obtained as a white solid, which was dried under reduced pressure and used in the synthesis of Tag 1-M2 below.

[1260] Compound Tag 1-5 (C 57 H 101 NO4, MW: 831.78): 1 H NMR (400 MHz, Chloroform-d) δ = 7.29 (s, 4H), 6.86 (d, J = 8.6 Hz, 4H), 5.15 (s, 1H), 3.95 (t, J = 6.6 Hz, 4H), 1.80 - 1.76 (m, 4H), 1.49 - 1.43 (m, 4H), 1.29 (s, 72H), 0.91 (t, J = 6.8 Hz, 6H) ppm.

[1261] 4.2 Synthesis of Tag 1-M2

[1262] Figure 2 Table 4 summarizes the synthesis of M2 using Tag 1.

[1263] Table 4.

[1264]

[1265] a Tag 1-8AA could not be isolated via precipitation in ACN, so Tag 1-10AA was obtained from a coupling reaction of Tag 1-7AA with AA10-AA9-AA8 using 1.2 eq of HATU (instead of HOAt), followed by cleavage of Tag 1 to produce M2

[1266] b eq: molar equivalent of reactant

[1267] c yield: crude yield

[1268] d V: volume (mL) relative to the mass of tag (g)

[1269] e In Step 8, HATU was used instead of coupling reagent (DIC)

[1270] General procedure A for amide coupling reaction to produce N-Fmoc-tag 1-n AA

[1271] To a solution of tag 1 or tag 1-(n-1) AA (1 eq) and AAn (1.5 eq) in DCM (20 V) was added DIC (1.5 eq) and HOAt (1.5 eq). The solution was stirred at 20 °C for 2 h. TLC (PE / EtOAc = 1 / 1) showed the reactants were completely consumed and a new spot was detected, which was further confirmed by LC-MS after a small amount of cleavage. To the reaction solution was added ACN (80 V), and the resulting suspension was filtered and washed with MeOH (20 V x 2) to give off-white solid. The crude product was dried under reduced pressure, and N-Fmoc-tag 1-(n) AA was obtained as a white solid and used directly for the next step.

[1272] General procedure B for Fmoc deprotection to give tag 1-n AA

[1273] A solution of N-Fmoc-tag 1-(n) AA (1 eq) in 20% piperidine in THF (20 V) was stirred at 20 °C for 1 h. TLC (PE / EtOAc = 1 / 1) showed the reactants were completely consumed and the desired spot was detected. To the reaction solution was added ACN (80 V), and the resulting suspension was filtered and washed with ACN (20 V x 2) to give off-white solid. The crude product tag 1-n AA was dried under reduced pressure and used directly for the next step.

[1274] Step 1: Synthesis of (2S)-2-amino-N-[bis(4-dodecyloxyphenyl)methyl]-5-(3,5- dimethylphenyl)pentanamide (tag 1-1 AA)

[1275]

[1276] To a solution of Tag 1 (6 g, 7.21 mmol, 1 eq) and AA1 (4.80 g, 10.81 mmol, 1.5 eq) in DCM (100 mL) was added DIC (1.18 g, 9.37 mmol, 1.45 mL, 1.3 eq) and HOAt (1.28 g, 9.37 mmol, 1.31 mL, 1.3 eq). The solution was stirred at 20 °C for 2 h. TLC (PE / EtOAc = 1 / 1) showed the reactants were consumed completely and a new spot was detected, which was further confirmed by testing the crude product. To the reaction solution was added ACN (400 mL), and the resulting suspension was filtered and washed with MeOH (100 mL x 2) to give off-white solid. The crude product was dried under reduced pressure, and N-Fmoc-Tag 1-1AA (8 g, 6.36 mmol, 88.23% yield) was obtained as a white solid and used directly for the next step. LCMS (ESI): RT = 4.266 min, for C 28 H 31 N2O3 + , mass calcd 443.23, m / z found 443.20 [M-Tag-1+H] + ; (Tag 1 = C 57 H 99 O2, MS = 815.76).

[1277] To a solution of N-Fmoc-Tag 1-1AA (8 g, 6.36 mmol, 1 eq) in THF (80 mL) was added piperidine (20 mL). The mixture was then stirred at 20 °C for 1 hour. TLC (PE / EtOAc = 1 / 1) showed the reactants were consumed completely and the desired spot was detected. To the reaction solution was added ACN (400 mL), and the resulting suspension was filtered and washed with ACN (100 mL x 2) to give off-white solid. The crude product Tag 1-1AA as a white solid was dried under reduced pressure and used directly for the next step.

[1278] Step 2: Synthesis of (2S)-2-[[(2S)-2-amino-3-[4-[4-(4-azidobutoxy)-2- ethyl-phenyl]phenyl]propanoyl]amino]-N-[bis(4-dodecyloxyphenyl)methyl]-5- (3,5-dimethylphenyl)pentanamide (Tag 1-2AA)

[1279]

[1280] The reaction was performed according to the general procedure A for amide coupling using Tag 1-1 AA (7 g, 6.76 mmol). The crude product N-Fmoc-Tag 1-2 AA (11 g, white solid) was dried and used directly for the next step. LCMS (ESI): RT = 5.759 min, for C 49 H 55 N6O5 + , mass calcd 807.42, m / z found 807.40 [M-Tag-1 + H] + ; (Tag 1 = C 57 H 99 O2, MS = 815.76).

[1281] The deprotection reaction was performed according to the general procedure B for Fmoc deprotection. Tag 1-2 AA was obtained as a white solid (8 g, 5.71 mmol, 84.27% yield). LCMS (ESI): RT = 3.568 min, for C 34 H 45 N6O3 + , mass calcd 585.35, m / z found 585.50 [M-Tag-1 + H] + ; (Tag 1 = C 57 H 99 O2, MS = 815.76).

[1282] Step 3: Synthesis of tert-butyl (3S)-3-amino-4-[[(1S)-1-[[4-[4-(4-azidobutoxy)-2- ethyl-phenyl]phenyl]methyl]-2-[[(1S)-1-[bis(4-dodecyloxyphenyl)methylaminocarbonyl]- 4-(3,5-dimethylphenyl)butyl]amino]-2-oxo-ethyl]amino]-4-oxo-butanoate (Tag 1-3 AA)

[1283]

[1284] The reaction was performed according to the general procedure A for amide coupling using Tag 1-2 AA (7 g, 5.00 mmol). N-Fmoc-Tag 1-3 AA was obtained as a yellow solid (8.9 g, 4.96 mmol, 99.25% yield). LCMS (ESI): RT = 5.388 min, for C 53 H 60 N7O8 + , mass calcd 922.44, m / z found 922.50 [M-Tag-1 + H] + ; (Tag 1 = C 57 H 99 O2, MS = 815.76).

[1285] The deprotection reaction was performed according to the general procedure B for Fmoc deprotection. Tag 1-3AA was obtained as a white solid (6.5 g, 4.14 mmol, 83.36% yield). LCMS (ESI): RT = 4.294 min, for C 38 H 50 N7O6 + , mass calcd 700.37, m / z found 700.50 [M-Tag-1 + H] + ; (Tag 1 = C 57 H 99 O2, MS = 815.76).

[1286] Step 4: Synthesis of tert-butyl (3S)-3-[[(2S)-2-amino-3-tert-butoxy- propionyl]amino]-4-[[(1S)-1-[[4-[4-(4-azidobutoxy)-2-ethyl- phenyl]phenyl]methyl]-2-[[(1S)-1-[bis(4-dodecyloxyphenyl)methyl- aminocarbonyl]-4-(3,5-dimethylphenyl)butyl]amino]-2-oxo- ethyl]amino]-4-oxo-butanoate (Tag 1-4AA)

[1287]

[1288] The reaction was performed according to the general procedure A for amide coupling using Tag 1-3AA (6.5 g, 4.14 mmol, 1 eq). N-Fmoc-Tag 1-4AA was obtained as a yellow solid (8 g, 4.13 mmol, 99.86% yield). LCMS (ESI): RT = 5.198 min, for C 56 H 65 N8O 10 + , mass calcd 1009.47, m / z found 1009.60 [M-Tag-1 + H] + ; (Tag 1 = C 57 H 99 O2, MS = 815.76).

[1289] The deprotection reaction was performed according to the general procedure B for Fmoc deprotection. Tag 1-4AA was obtained as a white solid (6.6 g, 3.85 mmol, 93.19% yield). LCMS (ESI): RT = 3.540 min, for C 41 H 55 N8O8 + , mass calcd 787.41, m / z found 787.50 [M-Tag-1 + H] +; (Tag 1 = C 57 H 99 O2, MS = 815.76).

[1290] Step 5: Synthesis of tert-butyl (3S)-3-[[(2S)-2-[[(2S,3R)-2-amino-3-tert- butoxy-butyryl]amino]-3-tert-butoxy-propionyl]amino]-4-[[(1S)-1-[[4-[4-(4- azidobutoxy)-2-ethyl-phenyl]phenyl]methyl]-2-[[(1S)-1-[bis(4-dodecyloxyphenyl) methylaminocarbamoyl]-4-(3,5-dimethylphenyl)butyl]amino]-2-oxo-ethyl]amino]- 4-oxo-butyrate (Tag 1-5AA)

[1291]

[1292] The reaction was performed according to the general procedure A for amide coupling using Tag 1-4AA (6.60 g, 3.85 mmol, 1 eq). N-Fmoc-Tag 1-5AA was obtained as a white solid (8.0 g, 3.82 mmol, 99.25% yield). LCMS (ESI): RT = 5.024 min for C 60 H 72 N9O 12 + Mass calcd. 1110.52, m / z found 1110.60 [M-Tag-1 + H] + ; (Tag 1 = C 57 H 99 O2, MS = 815.76).

[1293] The deprotection reaction was performed according to the general procedure B for Fmoc deprotection. Tag 1-5AA was obtained as a white solid (7.0 g, 3.74 mmol, 97.89% yield) which was used directly for the next step. LCMS (ESI): RT = 3.542 min for C 45 H 62 N9O 10 + Mass calcd. 888.45, m / z found 888.50 [M-Tag-1 + H] + ; (Tag 1 = C 57 H 99 O2, MS = 815.76).

[1294] Step 6: Synthesis of tert-butyl (3S)-3-[[(2S)-2-[[(2S,3R)-2-[[2-amino-3-(2- fluorophenyl)-2-methyl-propanoyl]amino]-3-tert-butoxy-butyryl]amino]-3-tert- butoxy-propionyl]amino]-4-[[(1S)-1-[[4-[4-(4-azidobutoxy)-2-ethyl-phenyl]phenyl]methyl]- 2-[[(1S)-1-[bis(4-dodecaunoxylphenyl)methylaminocarbonyl]-4-(3,5-dimethylphenyl)butyl] amino]-2-oxo-ethyl]amino]-4-oxo-butyrate (Tag 1-6AA)

[1295]

[1296] The reaction was performed according to the general procedure A for amide coupling using Tag 1-5AA (7.00 g, 3.74 mmol, 1 equiv). N-Fmoc Tag 1-6AA was obtained as a white solid (7.3 g, 3.21 mmol, 85.87% yield). LCMS (ESI): RT = 5.024 min for C 70 H 82 FN 10 O 13 + , mass calcd. 1289.60, m / z found 1289.60 [M-Tag-1+H] + ; (Tag 1 = C 57 H 99 O2, MS = 815.76).

[1297] The deprotection reaction was performed according to the general procedure B for Fmoc deprotection. Tag 1-6AA was obtained as a white solid (6.5 g, 3.17 mmol, 98.69% yield). LCMS (ESI): RT = 3.699 min for C 55 H 72 FN 10 O 11 + , mass calcd. 1067.53, m / z found 1067.60 [M-Tag-1-3 t Bu + 4H] + ; (Tag-1 = C 57 H 99 O2, MS = 815.76).

[1298] Step 7: Synthesis of tert-butyl (3S)-3-[[(2S)-2-[[(2S,3R)-2-[[2-[[(2S,3R)-2-amino-3-tert- butoxy-butyryl]amino]-3-(2-fluoro-phenyl)-2-methyl-propionyl]amino]-3-tert- butoxy-butyryl]amino]-3-tert-butoxy-propionyl]amino]-4-[[(1S)-1-[[4-[4-(4- azidobutoxy)-2-ethyl-phenyl]phenyl]methyl]-2-[[(1S)-1-[bis(4-dodecyloxyphenyl) methylaminocarbonyl]-4-(3,5-dimethylphenyl)butyl]amino]-2-oxo-ethyl]amino]-4- oxo-butyric acid (Tag 1-7AA)

[1299]

[1300] To a solution of Tag 1-6AA (6.5 g, 3.17 mmol, 1 eq) and AA7 (6.30 g, 15.85 mmol, 5.0 eq) in DCM (100 mL) was added DIC (1.80 g, 14.26 mmol, 2.21 mL, 4.5 eq) and HOAt (1.94 g, 14.26 mmol, 2.00 mL, 4.5 eq). The solution was then stirred at 20 °C for 2 h. TLC (PE / EtOAc = 1 / 1) showed the reactants were completely consumed and a new spot was detected. ACN (400 mL) was added to the reaction solution but only a little solid was formed. H2O (50 mL) was added and the resulting suspension was filtered and then washed with ACN (200 mL x 2) to give off-white solid. (Note: The filtration was very slow due to tiny solid particles.) N-Fmoc-Tag 1-7AA (6.6 g, 2.72 mmol, 85.69% yield) was obtained as a white solid which was used directly for the next step. LCMS (ESI): RT = 5.437 min, for C 74 H 89 FN 11 O 15 + , mass calcd 1390.46, m / z found 1390.70 [M-Tag-1+H] + ; (Tag 1 = C 57 H 99 O2, MS = 815.76).

[1301] To a solution of N-Fmoc-tag 1-7 AA (7.80 g, 3.21 mmol, 1 eq) in THF (80 mL) was added piperidine (20 mL). The solution was then stirred at 20 °C for 2 h. TLC (PE / EtOAc = 1 / 1) showed the reactant was completely consumed and a new spot was detected. The reaction mixture was added to ACN / H2O = 1 / 8 (600 mL) and the resulting suspension was filtered. (Note: The filtration was very slow due to tiny solid particles.) The filter cake was triturated with THF / ACN = 1 / 12 (500 mL) and filtered. The filter cake was then washed with ACN (50 mL x 3) to give the crude product. Tag 1-7 AA (5.0 g, 2.26 mmol, 70.54% yield) was obtained as a white solid, which was used directly for the next step. LCMS (ESI): RT = 3.550 min, m / z calculated 888.45, found 888.50 [M-tag-1+H] 45 H 62 N9O 10 + , mass calcd 888.45, m / z found 888.50 [M-tag-1+H] + ; (Tag 1 = C 57 H 99 O2, MS = 815.76).

[1302] Step 8: Synthesis of (3S)-4-[[(1S)-1-[[4-[4-(4-azidobutoxy)-2-ethyl- phenyl]phenyl]methyl]-2-[[(1S)-1-aminocarbonyl-4-(3,5-dimethylphenyl)butyl]amino]-2- oxo-ethyl]amino]-3-[[(2S)-2-[[(2S,3R)-2-[[3-(2-fluorophenyl)-2-[[(2S,3R)-3-hydroxy-2- [[2-[[(2S)-2-[[3-[2-(1H-imidazol-5-yl)ethylamino]-2,2-dimethyl-3-oxo-propionyl]amino]- 3-(1H-tetrazol-5-yl)propionyl]amino]acetyl]amino]butyryl]amino]-2-methyl-propionyl] amino]-3-hydroxy-butyryl]amino]3-hydroxy-propionyl]amino]-4-oxo-butyric acid (Tag 1-M2)

[1303]

[1304] To a solution of Tag 1-7AA (300 mg, 135.86 pmol, 1 eq) and AA10-AA9-AA8-OH (116.11 mg, 142.66 pmol, 1.05 eq) in DMF (0.5 mL) and DCM (1.5 mL) was added HATU (61.99 mg, 163.03 pmol, 1.2 eq). The mixture was then stirred at 20 °C for 2 h. TLC (PE / EtOAc = 1 / 1) showed the reactants were consumed completely and a new spot was detected. The reaction mixture was quenched with water (50 mL) and extracted with DCM (80 mL*2). The combined organic layers were washed with brine (30 mL*2), dried over Na2S04, filtered, and concentrated under reduced pressure to give compound Tag 1-10AA (408 mg, crude). LCMS (ESI): RT = 3.94 min for C 75 H 101 FN 20 O 17 ; m / z calculated 786.375 [M + H] + ; m / z found 786.8; (Tag 1 = C 57 H 99 02, MS = 815.76). LC-MS conditions: Method 7.

[1305] The crude Tag 1-10AA was subjected to acidic cleavage by using TFA mixture (10 mL, TFA:Tips:H20 = 95:2.5:2.5) for 1.5 hours. The reaction mixture was filtered, and the filtrate was concentrated to give a residue, which was purified by preparative HPLC (TFA; mobile phase: [water (TFA)-ACN] B%: 28%-58%, 12 min condition). M2 was obtained as a white solid (40 mg, 25.45 pmol, 18.73% yield). LCMS: RT = 3.942 min for C 75 H 101 FN 20 O 17 ; m / z calculated 786.375 [M + H] + ; m / z found 786.8. LC-MS conditions: Method 7.

[1306] HPLC: RT = 9.66 min, 92.86% purity. HPLC Method A: Column: YMC-Pack ODS-A 150*4.6 mm, 5 pm; 2.75 mL / 4 L of TFA in water (solvent A) and 2.5 mL / 4 L of TFA in ACN (solvent B) using an elution gradient of 10%-80% (solvent B) in 10 min and 80% for 5 min hold with a flow rate of 1.5 ml / min.

[1307] 2-[[(2S)-3-[1-[(2,4-dimethoxyphenyl)methyl]tetrazol-5-yl]-2-[[2,2-dimethyl-3-oxo-3-[2-(3-tritylimidazol-4-yl)ethylamino]propanoyl]amino]propanoyl]amino]acetic acid (AA10-AA9-AA8-OH) was synthesized

[1308]

[1309] To a solution of DMB-AA9 (3.23 g, 6.10 mmol, 2 eq) and DIEA (1.58 g, 12.20 mmol, 2.12 mL, 4 eq) in DMF (20 mL) was added HATU (2.32 g, 6.10 mmol, 2 eq) in one portion at 20 °C. The mixture was stirred at 20 °C for 0.2 h. Compound 21 (2.82 g, 3.05 mmol, 37.94% purity, 1 eq) was added and the mixture was stirred at 20 °C for 2 h. After completion, the mixture was filtered and the collected resin was washed with DMF (25 mL x 3), DCM (25 mL x 3) to give the crude product Fmoc-22 (4.38 g, 3.05 mmol, 99.93% yield, 60% purity) as a light yellow solid on chlorotrityl chloride (CTC) resin. LCMS (ESI): RT = 0.942 min for C 30 H 30 N6O7Na, m / z calcd 609.22, [M+Na] + ; m / z found 609.1; LC-MS conditions: Method 13.

[1310] To a solution of Fmoc-22 (4.38 g, 3.05 mmol, 60% purity, 1 eq) in DMF (32 mL) was added piperidine (8 mL) in one portion at 20 °C. The mixture was stirred at 20 °C for 1 h. The mixture was filtered and the collected resin was washed with DMF (25 mL x 3) and DCM (25 mL x 3) to give the crude product 22 (3.7 g, 3.05 mmol, 99.96% yield, 52.7% purity) as a light yellow solid on CTC resin. LCMS (ESI): RT = 0.716 min for C 15 H 20 N6O5Na, m / z calcd 387.15 [M+Na] + ; m / z found 387.1; LC-MS conditions: Method 13.

[1311] At 20 ° C, HATU (2.32 g, 6.09 mmol, 2 equivalents) was added to a solution of AA10 (2.85 g, 6.09 mmol, 2 equivalents) and DIEA (1.57 g, 12.18 mmol, 2.12 mL, 4 equivalents) in DMF (20 mL) in one portion. The mixture was stirred at 20 ° C for 0.2 hours. Compound 22 (3.7 g, 3.05 mmol, 52.7% purity, 1 equivalent) was added and the mixture was stirred at 20 ° C for 2 hours. After completion, the reaction mixture was subjected to acidic cleavage for 2 hours by using an AcOH mixture (20 mL, AcOH: TFE: DCM = 1: 1: 8). The mixture was then filtered and the collected resin was washed with DCM (5 mL × 3). The filtrate was washed with H2O (10 mL × 3) and the combined aqueous layer was extracted with DCM (10 mL × 2). The combined organic layers were dried over Na2SO4, filtered and concentrated under reduced pressure to give a residue which was purified by C-18 reverse phase chromatography ( Purification was performed on a C-18 column with a gradient of 0% to 60% ACN / H2O (0.1% AcOH) at 40 mL / min over 30 min, a total volume of 1.2 L. After lyophilization, AA10-AA9-AA8-OH (260 mg, 287.51 μmol, 9.44% yield, 90% purity) was obtained as an off-white solid. LCMS (ESI): RT = 3.050 min, for C 44 H 48 N9O7, m / z calculated 814.36 [M+H] + ; m / z found 814.4; LC-MS conditions: Method 13.

[1312] Example 5. Synthesis of (LPPS) M2 on Tag 2

[1313] 5.1 Synthesis of Tag 2

[1314] The synthetic route to prepare Tag 2 is shown in Scheme 3 below.

[1315] Option 3

[1316]

[1317] Reagents and conditions: a) C 22 H 45Br (2.2 eq), K2CO3 (6 eq), DMF / THF, 90 °C, 12 h, 91%; b) (4-methoxyphenyl)magnesium bromide (1 M, 3 eq), THF, 70 °C, 12 h, 91%; c) FmocNH2 (2 eq), MsOH (0.3 eq), THF, 70 °C, 12 h, 86%; d) 20% piperidine in THF, 20 °C, 2 h, 96%. Overall yield: 75.8%

[1318] Treatment of 2,4-dihydroxybenzaldehyde (Tag 2-1) with 2.2 eq of 1-bromodocosane in the presence of potassium carbonate in DMF / THF provided the bis-C22-alkyl substituted compound (Tag 2-2). Grignard reaction of Tag 2-2 with Grignard reagent (Tag 2-2a), followed by nucleophilic substitution of alcohol (Tag 2-3) with 9-fluorenylmethyl carbamate yielded Tag 2-4. Tag 2 was obtained from Tag 2-4 by common Fmoc deprotection.

[1319] Both alcohol Tag (Tag 2-3) and amine Tag (Tag 2) are new Tags designed for the manufacture of short peptide acids and amides. The two hydrophobic C22-alkyl chains in Tag 2-3 and Tag 2 retain their TCP hydrophobicity, which they can precipitate out in polar solvents during the synthesis process.

[1320] Step a: synthesis of 2,4-di(docosyloxy)benzaldehyde (Tag 2-2)

[1321]

[1322] To a mixture of 2,4-dihydroxybenzaldehyde (Tag 2-1, 5 g, 36.20 mmol, 1 eq) and 1-bromodocosane (31.02 g, 79.64 mmol, 2.2 eq) in DMF (100 mL) and THF (20 mL) was added K2CO3 (30.02 g, 217.20 mmol, 6 eq) and stirred at 90 °C for 12 hours. Most of the reactants were consumed and a new spot was formed on TLC (PE / EtOAc = 5 / 1). The reaction mixture was poured into water (1000 mL) and the mixture was stirred for 1 hour, then the precipitated solid was collected by filtration. The obtained solid was triturated with acetone (1000 mL), collected by filtration and slurry washed in methanol, and dried under reduced pressure to give the desired compound Tag 2-2 (27 g, crude) as off-white solid. 1HNMR (400 MHz, chloroform-d) δ = 10.34 (s, 1H), 7.80 (d, J = 8.8 Hz, 1H), 6.52 (dd, J = 1.5, 8.8 Hz, 1H), 6.43 (d, J = 1.9 Hz, 1H), 4.08 - 3.97 (m, 4H), 1.89 - 1.73 (m, 4H), 1.52 - 1.44 (m, 4H), 1.26 (s, 72H), 0.89 (t, J = 6.7 Hz, 6H) ppm.

[1323] Step b: Synthesis of (2,4-bis(dodecyloxy)phenyl)(4-methoxyphenyl)methanol (Tag 2-3)

[1324]

[1325] To a solution of Tag 2-2 (31 g, 41.04 mmol, 1 eq) in THF (300 mL) was added bromo-(4-methoxyphenyl)magnesium (Tag 2-2a, 1 M, 123.13 mL, 3 eq) and the reaction mixture was stirred at 70 °C for 12 h. TLC (PE:EtOAc = 10:1) showed the reaction was complete. After cooling to room temperature, the reaction was added with 1 N hydrochloric acid (100 mL) and THF (100 mL). The separated organic layer was washed with brine (50 mL x 2), dried over Na2S04, filtered and concentrated under reduced pressure to give a residue, which was triturated with ACN (150 mL) and MeOH (80 mL). The suspension was filtered, washed thoroughly with ACN (100 mL) and methanol (80 mL) to provide the product Tag 2-3 (36 g, 37.53 mmol, 91.43% yield, 90% purity) as a light yellow solid. 1 H NMR (400 MHz, chloroform-d) δ = 7.30 - 7.27 (m, 2H), 7.17 - 7.00 (m, 1H), 6.90 - 6.77 (m, 2H), 6.49 - 6.32 (m, 2H), 5.98 - 5.49 (m, 1H), 3.99 - 3.85 (m, 4H), 3.85 - 3.70 (m, 4H), 3.53 - 3.35 (m, 2H), 1.80 - 1.68 (m, 4H), 1.43 (br d, J = 5.0 Hz, 4H), 1.26 (s, 72H), 0.89 (t, J = 6.7 Hz, 6H) ppm.

[1326] Step c: Synthesis of (9H-fluoren-9-yl)methyl ((2,4-bis(dodecyloxy)phenyl)(4- methoxyphenyl)methyl)carbamate (Tag 2-4)

[1327]

[1328] To a solution of 9H-fluoren-9-ylmethyl carbamate (10.77 g, 45.03 mmol, 1.2 eq) in THF (300 mL) was added Tag 2-3 (36 g, 37.53 mmol, 90% purity, 1.0 eq) and MsOH (1.08 g, 11.26 mmol, 801.41 μί, 0.3 eq), then the reaction mixture was stirred at 70 °C for 12 h. TLC (PE:EtOAc = 10:1) showed the reaction was complete. After evaporation to remove most of the solvent, the residue was triturated with 5% aqueous NaHC03solution (10 mL), MeOH (80 mL) and ACN (150 mL). The suspension was filtered and washed thoroughly with ACN (120 mL) and methanol (80 mL) sequentially to give Tag 2-4 (39 g, 32.36 mmol, 86.24% yield, 90% purity) as a light yellow solid. 1 H NMR (400 MHz, Chloroform-d) δ = 7.85 - 7.69 (m, 2H), 7.68 - 7.50 (m, 2H), 7.46 - 7.36 (m, 2H), 7.36 - 7.28 (m, 2H), 7.14 (br d, J = 8.0 Hz, 2H), 6.81 (br d, J = 8.3 Hz, 2H), 6.50 - 6.42 (m, 2H), 6.04 - 5.89 (m, 1H), 4.47 - 4.35 (m, 2H), 4.25 (br t, J = 7.0 Hz, 1H), 3.97 - 3.91 (m, 2H), 3.86 - 3.71 (m, 5H), 3.42 - 3.19 (m, 1H), 1.80 - 1.64 (m, 4H), 1.48-1.36 (m, 4H), 1.1.32-1.1 (m, 72H), 0.95-0.82 (m, 6H) ppm.

[1329] Step d: Synthesis of (2,4-bis(dodecyloxy)phenyl)(4-methoxyphenyl)methanamine (Tag 2)

[1330]

[1331] To a solution of Tag 2-4 (37 g, 30.70 mmol, 90% purity, 1 eq) in THF (240 mL) was added piperidine (51.73 g, 607.56 mmol, 60 mL, 19.79 eq) and the reaction mixture was stirred at 20 °C for 2 h. TLC (PE:EtOAc = 10:1) showed the reaction was complete. The mixture was added to ACN (800 mL), the resulting suspension was filtered and washed thoroughly with ACN (200 mL) and methanol (100 mL) to give Tag 2 (30 g, 29.57 mmol, 96.31% yield, 85% purity) as a light yellow solid. LC-MS (Method 7): RT = 2.038 min for C 58 H 101 O3, m / z calcd 845.78 [M-NH3] + , found 845.8. 1 H NMR (400 MHz, Chloroform-d) δ = 7.29 (d, J = 8.5 Hz, 2H), 7.16 - 7.10 (m, 1H), 6.83 (d, J = 8.8 Hz, 2H), 6.45 - 6.38 (m, 2H), 5.37 (s, 1H), 3.95 - 3.85 (m, 4H), 3.79 (s, 3H), 1.77 - 1.71 (m, 4H), 1.48 - 1.38 (m, 4H), 1.27 (s, 72H), 0.89 (s, 6H) ppm.

[1332] 5.2 Synthesis of Tag 2-M2

[1333] Figure 3 Table 5 summarizes the synthesis of M2 using Tag 2.

[1334] Table 5.

[1335]

[1336] General Procedure C for amide coupling and Fmoc deprotection in one pot to give Tag 2-nAA

[1337] To a solution of Tag 2 or Tag 2-(n-l)AA (1 eq) and AAn (1.5 eq) and HOAt (1.5 eq) in DCM (20 V) was added DIC (1.5 eq) in one portion at 20 °C. The mixture was stirred at 20 °C for 12 h. After completion, prop-l-amine (2 eq) was added and the mixture was stirred at 20 °C for 0.5 h. Then 20% piperidine in THF (20 V) was added and the mixture was stirred at 20 °C for 1 h. TLC (PE:EtOAc = 3:1) showed the reaction was complete. The mixture was triturated with ACN / MeOH = 3 / 1 (40 V*2) to afford the product Tag 2-nAA as a light yellow solid, which was used directly in the next step without purification.

[1338] Step 1: Synthesis of (2S)-2-amino-N-((2,4-bis(dodecyloxy)phenyl)(4- methoxyphenyl)methyl)-5-(3,5-dimethylphenyl)pentanamide (Tag 2-lAA)

[1339]

[1340] To a solution of Tag 2 (6.5 g, 7.16 mmol, 95% purity, 1 eq), AA1 (4.76 g, 10.74 mmol, 1.5 eq) and HOAt (1.46 g, 10.74 mmol, 1.50 mL, 1.5 eq) in DCM (80 mL) was added DIC (1.36 g, 10.74 mmol, 1.66 mL, 1.5 eq) in one portion at 20 °C. The mixture was stirred at 20 °C for 12 h. After completion, prop-l-amine (846.45 mg, 14.32 mmol, 1.18 mL, 2 eq) was added and the mixture was stirred at 20 °C for 0.5 h. Then piperidine (16.65 g, 195.52 mmol, 19.31 mL, 27.31 eq) in THF (80 mL) was added and the mixture was stirred at 20 °C for 1 h. TLC (PE:EtOAc = 3:1) showed the reaction was complete. The mixture was triturated with ACN / MeOH = 3 / 1 (150 mL*2) to afford Tag 2-lAA (8.5 g, 6.78 mmol, 94.69% yield, 85% purity) as a light yellow solid, which was used directly in the next step without purification. LCMS (ESI): RT = 2.092 min for C 71 H 121 N2O4, m / z calcd 1065.92 [M+H] + ; m / z found 1065.9; LC-MS conditions: Method 7.

[1341] Step 2: Synthesis of (2S)-2-((S)-2-amino-3-(4'-(4-azidobutoxy)-2'- ethyl-[l,l'-biphenyl]-4-yl)propanoylamino)-N-((2,4-bis(dodecyloxy)phenyl)(4- methoxyphenyl)methyl)-5-(3,5-dimethylphenyl)pentanamide (Tag 2-2AA)

[1342]

[1343] The reaction was performed in one pot using Tag 2-1AA (3 g, 2.39 mmol, 85% purity, 1 eq) and AA2 (2.18 g, 3.61 mmol, 1.51 eq) according to the general procedure C for amide coupling and Fmoc deprotection. Crude Tag 2-2AA (3.6 g, 2.27 mmol, 94.68% yield, 90% purity) was obtained as a light yellow solid. LCMS (ESI): RT = 2.225 min, for C 92 H 144 N6O6, m / z calcd 1430.11, [M+H] + ; m / z found 1431.0; LC-MS conditions: Method 7.

[1344] Step 3: Synthesis of (3S)-tert-butyl 3-amino-4-(((2S)-3-(4'-(4-azidobutoxy)-2'- ethyl-[l,l'-biphenyl]-4-yl)-l-(((2S)-l-(((2,4-bis(dodecyloxy)phenyl)(4- methoxyphenyl)methyl)amino)-5-(3,5-dimethylphenyl)-l-oxopentan-2-yl)amino)- 1-oxopropan-2-yl)amino)-4-oxobutanoate (Tag 2-3AA)

[1345]

[1346] The reaction was performed in one pot using Tag 2-2AA (3.8 g, 2.39 mmol, 90% purity, 1 eq) and AA3 (1.48 g, 3.59 mmol, 1.5 eq) according to the general procedure C for amide coupling and Fmoc deprotection. Crude Tag 2-3AA (3.9 g, 2.19 mmol, 91.66% yield, 90% purity) was obtained as a light yellow solid. LCMS (ESI): RT = 3.660 min, for C 38 H 50 N7O6, m / z calcd 700.37 [M - anchor-tBu + 3H] + ; (anchor = C 58 H 101O3, MS = 845.8); m / z found 700.4; LC-MS conditions: Method 7. (Anchor = Tag 2).

[1347] Step 4: Synthesis of (3S)-tert-butyl 3-((S)-2-amino-3-(tert-butoxy)propanamido)-4- (((2S)-3-(4'-(4-azidobutoxy)-2'-ethyl-[l,l'-biphenyl]-4-yl)-l-(((2S)-l-(((2,4- bis(dodecyloxy)phenyl)(4-methoxyphenyl)methyl)amino)-5-(3,5- dimethylphenyl)-l-oxopentan-2-yl)amino)-l-oxopropan-2-yl)amino)-4- oxobutanoate (Tag 2-4AA)

[1348]

[1349] The reaction was performed according to the general procedure C for amide coupling and Fmoc deprotection in one pot using Tag 2-3AA (3.9 g, 2.19 mmol, 90% purity, 1 equiv) and AA4 (1.26 g, 3.29 mmol, 1.5 equiv). Crude Tag 2-4AA was obtained as a light yellow solid (3.8 g, 1.96 mmol, 89.44% yield, 90% purity). LCMS (ESI): RT = 3.540 min, for C 41 H 55 N8O8, m / z calcd 787.41 [M - Anchor - 2tBu + 4H] + ; (Anchor = C 58 H 101 O3, MS = 845.8); m / z found 787.5; LC-MS conditions: Method 7. (Anchor = Tag 2).

[1350] Step 5: Synthesis of (3S)-tert-butyl 3-((S)-2-(((2S,3R)-2-amino-3-(tert- butoxy)butanoyl)oxy)-3-(tert-butoxy)propanamido)-4-(((2S)-3-(4'-(4- azidobutoxy)-2'-ethyl-[l,l'-biphenyl]-4-yl)-l-(((2S)-l-(((2,4- bis(dodecyloxy)phenyl)(4-methoxyphenyl)methyl)amino)-5-(3,5- dimethylphenyl)-l-oxopentan-2-yl)amino)-l-oxopropan-2-yl)amino)-4- oxobutanoate (Tag 2-5AA)

[1351]

[1352] The reaction was carried out in one pot using Tag 2-4AA (3.8 g, 1.96 mmol, 90% purity, 1 eq) and AA5 (1.17 g, 2.94 mmol, 1.5 eq) according to the general procedure C for amide coupling and Fmoc deprotection. The crude Tag 2-5AA was obtained as a light yellow solid (3.1 g, 1.55 mmol, 78.99% yield, 95% purity). LCMS (ESI): RT = 3.510 min, for C 45 H 62 N9O 10 , m / z calcd 888.45 [M - Anchor - 3tBu + 5H] + ; (Anchor = C 58 H 101 O3, MS = 845.8); m / z found 888.5; LC-MS conditions: Method 7. (Anchor = Tag 2).

[1353] Step 6: Synthesis of (3S)-tert-butyl 3-((S)-2-((2S,3R)-2-((S)-2-amino-3-(2- fluorophenyl)-2-methylpropanamido)-3-(tert-butoxy)butanamido)-3-(tert- butoxy)propanamido)-4-(((2S)-3-(4'-(4-azidobutoxy)-2'-ethyl-[l,l'-biphenyl]-4-yl)- 1-(((2S)-l-(((2,4-bis(dodecyloxy)phenyl)(4-methoxyphenyl)methyl)amino)-5-(3,5- dimethylphenyl)-l-oxopentan-2-yl)amino)-l-oxopropan-2-yl)amino)-4- oxobutanoate (Tag 2-6AA)

[1354]

[1355] The reaction was carried out in one pot using Tag 2-5AA (1.5 g, 749.31 pmol, 95% purity, 1 eq) and AA6 (471.44 mg, 1.12 mmol, 1.5 eq) according to the general procedure C for amide coupling and Fmoc deprotection. Tag 2-6AA was obtained as a light yellow solid (1.36 g, 588.20 pmol, 78.50% yield, 90% purity). LCMS (ESI): RT = 3.726 min, for C 55 H 72 N 10 O 11 , m / z calcd 1067.53 [M - Anchor - 3tBu + 5H] + ; (Anchor = C 58 H 101O3, MS = 845.8); m / z found 1067.6; LC-MS conditions: Method 7. (Anchor = Tag 2).

[1356] Step 7: Synthesis of (4R,5S,8S,11S,14S,17S)-tert-butyl 5-amino-17-(((2S)-3-(4’-(4- azidobutoxy)-2’-ethyl-[1,1’-biphenyl]-4-yl)-1-(((2S)-1-(((2,4-bis(dodecyloxy)phenyl)(4- methoxyphenyl)methyl)amino)-1-oxo-5-(m-tolyl)pentan-2-yl)amino)-1-oxoprop-2- yl)aminoformyl)-11-(CAN-1-(tert-butoxy)ethyl)-14-(tert-butoxymethyl)-8-(2- fluorobenzyl)-2,2,4,8-tetramethyl-6,9,12,15-tetraoxo-3-oxa-7,10,13,16-tetraazanonadecan- 19-oate (Tag 2-7AA)

[1357]

[1358] The reaction was performed in one pot using Tag 2-6AA (0.7 g, 302.75 pmol, 90% purity, 1 equiv) and AA7 (360.99 mg, 908.25 pmol, 3 equiv) according to the general procedure C for amide coupling and Fmoc deprotection. Crude Tag 2-7AA (0.6 g, 241.27 pmol, 79.69% yield, 90% purity) was obtained as a light yellow solid. LCMS (ESI): RT = 3.977 min for C 59 H 79 FN 11 O 13 , m / z calculated 1168.58 [M-Anchor-4tBu+6H] + ; (Anchor = C 58 H 101 O3, MS = 845.8); m / z found 1168.5; LC-MS conditions: Method 7 (Anchor = Tag 2) (filtration was very slow and it was difficult to collect solid particles due to poor precipitation).

[1359] Step 8: Synthesis of (3S)-4-[[(1S)-1-[[4-[4-(4-azidobutoxy)-2-ethyl-phenyl]phenyl]methyl]-2-[[(1S)-1- aminocarbonyl-4-(3,5-dimethylphenyl)butyl]amino]-2-oxo-ethyl]amino]-3-[[(2S)-2-[[(2S,3R)-2-[[(2S)-3-(2- fluorophenyl)-2-[[(2S,3R)-3-hydroxy-2-[[2-[[(2S)-2-[[3-[2-(1H-imidazol-5-yl)ethylamino]-2,2-dimethyl-3- oxopropionyl]amino]-3-(1H-tetrazol-5-yl)propionyl]amino]acetyl]amino]butyryl]amino]-2-methyl- propionyl]amino]-3-hydroxy-butyryl]amino]3-hydroxy-propionyl]amino]-4-oxo-butyric acid (M2)

[1360]

[1361] To the solution of reactants AA10-AA9-AA8-OH (216.01 mg, 238.86 µmol, 90% purity, 1.1 eq) and Tag 2-7AA (540 mg, 217.14 µmol, 90% purity, 1 eq) in DMF (2 mL) and DCM (6 mL) was added HATU (99.08 mg, 260.57 µmol, 1.2 eq) in one portion at 20 °C. The mixture was stirred at 20 °C for 2 h. LCMS showed the reaction was completely converted. The mixture was subjected to acidic cleavage by using TFA mixture (14 mL, TFA: ethyl phenyl sulfide: H2O = 95:2.5:2.5) for 50 min. The reaction mixture was filtered, and the filtrate was quenched with water / ACN = 4 / 1 (150 mL) and extracted with PE (20 mL*2). The combined water layer was lyophilized to give a residue, which was purified by prep-HPLC (column: WelchXtimate C18 100*40mm*3µm; mobile phase: [water(TFA)-ACN]; B%: 28%-58%, 12 min condition) to provide the product M2 (50 mg, 31.58 µmol, 14.54% yield, 99.27% purity) as a white solid. LCMS (ESI): RT = 3.910 min for C 75 H 101 FN 20 O 17 m / z calcd 786.375 [M+H] +; m / z found 786.8; LC-MS conditions: Method 7. HPLC: RT = 4.86 min. HPLC conditions: Mobile Phase: 1.0% ACN in water (0.1% TFA) to 5% ACN in water (0.1% TFA) in 1 min; then 5% ACN in water (0.1% TFA) to 100% ACN (0.1% TFA) in 5 min; hold at 100% ACN (0.1% TFA) for 2 min; back to 1.0% ACN in water (0.1% TFA) at 8.01 min and hold for 2 min. Flow: 1.2 ml / min. Column: Ultimate XB-C18, 3 pm, 3.0*50 mm.

[1362] Example 6. Synthesis of (LPPS)M2 on Tag 3

[1363] 6.1 Synthesis of Tag 3

[1364] The synthetic route to prepare Tag 3 is shown in Scheme 4.

[1365] Scheme 4

[1366]

[1367] Reagents and conditions a) C 22 H 45 Br (2.2 eq), K2CO3 (6 eq), DMF / THF, 90 °C, 12 h, 99%; b) NH2OH.HCI (3 eq), Et3N (5 eq), toluene (20 mL), 100 °C, 12 h, 93%; c) DIBALH (3.0 eq), toluene (30 mL), 100 °C, 12 h, 91%; d) 2-[4-[(2,4-dimethoxyphenyl)-(9H-fluoren-9-ylmethoxycarbonylamino)methyl]phenoxy]acetic acid (1.5 eq), DIC (1.5 eq), HOAt (1.5 eq), DCM (2 mL), 20 °C, 12 h, 68%; e) 20% piperidine in THF, 20 °C, 2 h, 99%. Overall yield: 56.4%.

[1368] Double alkylation of 2,4-dihydroxybenzaldehyde with C22-alkyl bromide followed by treatment of tag 3-1 with same equivalent of hydroxylamine hydrochloride in presence of TEA in toluene provided the oxime product tag 3-2. Reduction of tag 3-2 with DIBAL to amine tag 3-3 and subsequent coupling with acid tag 3-3a provided amide tag 3-4. Reagent tag 3-3a (CAS: 145069-56-3, Rink amide linker), 4-[(2,4-dimethoxyphenyl)(fmoc-amino)methyl]phenoxyacetic acid is commonly used for the solid-phase synthesis of C-terminal primary amide-peptides. Finally, Fmoc deprotection of tag 3-4 provided tag 3.

[1369] Step a: synthesis of 2,4-di(dodecaoxy)benzaldehyde (tag 3-1)

[1370]

[1371] To a mixture of 2,4-dihydroxybenzaldehyde (2 g, 14.48 mmol, 1 eq) and 1- bromododecane (12 g, 30.81 mmol, 2.13 eq) in DMF (50 mL) was added K2CO3 (12.01 g, 86.88 mmol, 6 eq) and stirred at 90 °C for 12 h. Most of the reactants were consumed and a new spot was formed on TLC (PE / EtOAc = 5 / 1). After completion, the reaction mixture was poured into water (500 mL), the mixture was stirred for 1 h, and the precipitated solid was collected by filtration. The obtained solid was triturated with acetone (200 mL), and the solid was collected by filtration and washed with methanol in slurry. After filtration, the obtained solid was dried under reduced pressure to give the title compound as an off-white solid. The obtained solid was triturated with PE (100 mL) again, and the solid was collected by filtration. The desired compound tag 3-1 (10.9 g, 14.43 mmol, 99.67% yield, n / a purity) was obtained as an off-white solid. 1 H NMR (400 MHz, Chloroform-d) δ = 10.33 (s, 1H), 7.80 (d, J = 8.6 Hz, 1H), 6.52 (d, J = 8.8 Hz, 1H), 6.42 (d, J = 2.1 Hz, 1H), 4.08 - 3.97 (m, 4H), 1.89 - 1.69 (m, 4H), 1.52 - 1.42 (m, 4H), 1.26 (s, 72H), 0.89 (t, J = 6.8 Hz, 6H) ppm.

[1372] Step b: synthesis of (1E)-2,4-di(dodecaoxy)benzaldehyde oxime (tag 3-2)

[1373]

[1374] To a mixture of Label 3-1 (6.5 g, 8.61 mmol, 1 eq) in toluene (90 mL) was added NH2OH.HC1 (5.98 g, 86.06 mmol, 10 eq) and Et3N (8.71 g, 86.06 mmol, 11.98 mL, 10 eq) and the reaction mixture was stirred at 100 °C for 12 h. Most of the reactant was consumed and a new spot with larger polarity was formed on TLC (PE / EtOAc = 10 / 1). After completion, the reaction mixture was cooled to 20 °C and poured into ACN (300 mL), then the precipitated solid was collected by filtration and slurry washed in methanol. The obtained solid was triturated with ACN (300 mL) and the solid was collected by filtration. After filtration, the obtained solid was dried under reduced pressure to give the title compound as a brown solid. The desired compound Label 3-2 (6.2 g, 8.05 mmol, 93.53% yield) was obtained as a brown solid. 1 H NMR (400 MHz, Chloroform-d) δ = 8.46 (s, 1H), 7.65 (d, J = 8.5 Hz, 1H), 6.51 - 6.41 (m, 2H), 4.03 - 3.91 (m, 4H), 1.87 - 1.71 (m, 4H), 1.50 - 1.42 (m, 4H), 1.27 (s, 72H), 0.89 (t, J = 6.8 Hz, 6H) ppm.

[1375] Step 3: Synthesis of [2,4-bis(dodecyloxy)phenyl]methanamine (Label 3-3)

[1376]

[1377] To a mixture of Label 3-2 (5 g, 6.49 mmol, 1 eq) in THF (50 mL) was added DIBALH (1 M, 19.47 mL, 3 eq) dropwise at 0 °C and the reaction mixture was stirred at 90 °C for 12 h. Most of the reactant was consumed and a new spot with small polarity was formed on TLC (PE / EtOAc = 10 / 1). The reaction mixture was poured into saturated sodium tartrate tetrahydrate (100 mL), then the precipitated solid was collected by filtration and slurry washed in methanol. The obtained solid was triturated with ACN (30 mL) and the solid was collected by filtration. After filtration, the obtained solid was dried under reduced pressure to give the title compound Label 3-3 (4.5 g, 5.95 mmol, 91.66% yield) as an off-white solid. 1H NMR (400 MHz, chloroform-d) δ = 6.61 - 6.29 (m, 3H), 4.14 - 3.65 (m, 6H), 2.84 (br s, 2H), 1.83 - 1.73 (m, 4H), 1.4 - 1.5 (m, 4H), 1.27 (br s, 72H), 0.89 (br t, J = 6.5 Hz, 6H) ppm.

[1378] Step 4: Synthesis of 9H-fluoren-9-ylmethyl N-[[4-[2-[[2,4-bis(dioleyloxy)phenyl]methylamino]-2-oxo-ethoxy]phenyl]-(2,4-dimethoxyphenyl)methyl]carbamate (Tag 3-4)

[1379]

[1380] To a solution of Tag 3-3 (2 g, 2.64 mmol, 1 eq) and 2-[4-[(2,4-dimethoxyphenyl)-(9H-fluoren-9-ylmethoxycarbonylamino)methyl]phenoxy]acetic acid Tag 3-3a (2.14 g, 3.97 mmol, 1.5 eq) in DCM (20 mL) were added DIC (500.58 mg, 3.97 mmol, 614.21 μL, 1.5 eq) and HOAt (539.89 mg, 3.97 mmol, 554.87 μL, 1.5 eq), and the resulting mixture was stirred at 20 °C for 2 h. Most of the reactants were consumed, and a new spot with large polarity was formed on TLC (PE / EtOAc = 3 / 1). To the reaction mixture was added ACN (100 mL), and the resulting suspension was filtered and washed with MeOH (30 mL x 2) to give off-white solid. The obtained solid was triturated with ACN (30 mL), and the solid was collected by filtration. After filtration, the obtained solid was dried under reduced pressure to give the title compound Tag 3-4 (2.3 g, 1.80 mmol, 68.06% yield) as off-white solid. 1H NMR (400 MHz, Chloroform-d) δ = 7.77 (br d, J = 7.1 Hz, 2H), 7.60 (br d, J = 6.8 Hz, 1H), 7.49 - 7.27 (m, 5H), 7.22 - 7.02 (m, 4H), 6.95 - 6.69 (m, 2H), 6.57 - 6.36 (m, 4H), 6.02 (br d, J = 8.6 Hz, 1H), 5.75 (br d, J = 7.6 Hz, 1H), 4.55 - 4.14 (m, 5H), 4.08 - 3.66 (m, 10H), 4.08 - 3.63 (m, 1H), 1.95 - 1.67 (m, 4H), 1.54 - 1.41 (m, 4H), 1.27 (s, 72H), 0.89 (t, J = 6.8 Hz, 6H) ppm.

[1381] Step 5: Synthesis of 2-[4-[amino-(2,4-dimethoxyphenyl)methyl]phenoxy]-N-[[2,4- bis(dodecauncoxy)phenyl]methyl]acetamide (Tag 3)

[1382]

[1383] To a solution of Tag 3-4 (2.2 g, 1.72 mmol, 1 eq) in THF (20 mL) was added piperidine (6.90 g, 81.01 mmol, 8 mL, 47.05 eq), and the resulting mixture was stirred at 20 °C for 2 h. Most of the reactant was consumed, and a new spot with large polarity was formed on TLC (PE / EtOAc = 9 / 1). LCMS showed that the desired mass was detected. To the reaction solution was added ACN (100 mL), and the resulting suspension was filtered and washed with MeOH (30 mL x 2) to give off-white solid. The obtained solid was triturated with ACN (20 mL), and the solid was collected by filtration. The obtained solid was dried under reduced pressure to give the title compound Tag 3 (1.8 g, 1.71 mmol, 99.04% yield) as off-white solid. LCMS (ESI): RT = 2.055 min for C 68 H 114 N2O6H, mass calcd 1055.9 [M+H] + , C 68 H 112 NO6 + 1038.8 [M-NH2] + , m / z found 1038.8 [M-NH2] + ; LCMS conditions: Method 12. 1H NMR (400 MHz, Chloroform-d) δ = 7.22 (br d, J = 8.5 Hz, 1H), 7.16 - 7.04 (m, 2H), 6.84 - 6.67 (m, 2H), 6.52 (d, J = 2.3 Hz, 1H), 6.49 - 6.29 (m, 4H), 5.36 (s, 1H), 4.43 - 4.24 (m, 1H), 4.05 - 3.85 (m, 5H), 3.82 - 3.70 (m, 6H), 3.19 (s, 2H), 1.83 - 1.75 (m, 4H), 1.26 (s, 76H), 0.90 - 0.87 (m, 6H) ppm.

[1384] 6.2 Synthesis of Tag 3-nAA

[1385] The following Figure 4 and Table 6 summarize the synthesis of M2 using Tag 3.

[1386] Table 6

[1387]

[1388] General procedure D for amide coupling to produce N-Fmoc-Tag 3-nAA

[1389] To a solution of Tag 3 or Tag 3-(n-l)AA (1 eq) and AAn (1.5 eq) in DCM (20 V) was added DIC (1.5 eq) and HOAt (1.5 eq), then the resulting mixture was stirred at 20 °C for 2 h. Most of the reactants were consumed, and a new spot with large polarity was formed on TLC (PE / EtOAc = 3 / 1). A small sample was taken and piperidine was added to the small sample to remove the Fmoc group. The sample was checked by LCMS, which showed that the desired mass was detected. ACN (80 V) was added to the reaction solution, and the resulting suspension was filtered and washed with MeOH (30 V x 2) to give off-white solid. The obtained solid was triturated with ACN (20 V), and the solid was collected by filtration. After filtration, the obtained solid was dried under reduced pressure to give the title compound N-Fmoc-Tag 3-nAA as off-white solid.

[1390] General procedure E for Fmoc deprotection to produce Tag 3-nAA:

[1391] A solution of N-Fmoc-tag 3-nAA (1 eq) in 20% piperidine in THF (10 V) was stirred at 20 °C for 1 h. Most of the reactant was consumed and a new spot was formed on TLC (PE / EtOAc = 9 / 1). LCMS showed the desired mass was detected. To the reaction solution was added ACN (100 V), and the resulting suspension was filtered and washed with MeOH (30 V x 2) to give the product. The obtained solid was triturated with ACN (20 V) and the solid was collected by filtration. The title compound tag 3-nAA was obtained as an off-white solid.

[1392] Step f: Synthesis of 9H-fluoren-9-ylmethyl N-[(1S)-1-[[[4-[2-[[2,4-bis(dioleyloxy)phenyl]methylamino]-2-oxo-ethoxy]phenyl]-(2,4-dimethoxyphenyl)methyl]aminocarbonyl]-4-(3,5-dimethylphenyl)butyl]carbamate (N-Fmoc-tag 3-1AA)

[1393]

[1394] The reaction was performed using General Procedure D for amide coupling using tag 3 (1 g, 947.29 pmol, 1 eq) and AA1 (630.23 mg, 1.42 mmol, 1.5 eq) in DCM (20 mL). N-Fmoc-tag 3-1AA was obtained as an off-white solid (1.1 g, 742.66 pmol, 78.40% yield). LCMS (ESI): RT = 2.252 min, for C 81 H 131 N3O7H, mass calcd 1259.9, m / z found 1260.0 [M-Fmoc+H] + ; LCMS conditions: Method 12.

[1395] 1 H NMR (400 MHz, Chloroform-d) δ = 7.83 - 7.50 (m, 4H), 7.45 - 7.26 (m, 5H), 7.12 - 6.61 (m, 9H), 6.55 - 6.17 (m, 4H), 5.55 - 5.27 (m, 1H), 4.54 - 4.09 (m, 6H), 4.06 - 3.70 (m, 8H), 3.69 - 3.55 (m, 3H), 3.49 (br s, 6H), 3.17 (br s, 2H), 2.50 (br s, 2H), 2.27 (br s, 6H), 2.01 (s, 2H), 1.27 (br s, 76H), 0.91 - 0.87 (m, 6H) ppm.

[1396] Step g: Synthesis of (2S)-2-amino-N-[[4-[2-[[2,4-bis(dodecyloxy)phenyl]methylamino]-2-oxo-ethoxy]phenyl]-(2,4-dimethoxyphenyl)methyl]-5-(3,5-dimethylphenyl)pentanamide (Tag 3-1AA)

[1397]

[1398] The reaction was performed using General Procedure E for Fmoc deprotection using N-Fmoc-Tag 3-1AA (1 g, 675.15 pmol, 1 eq) in THF (10 mL). Tag 3-1AA was obtained as off-white solid (0.81 g, 643.41 pmol, 95.30% yield). LCMS (ESI): RT = 6.848 min, for C 81 H 131 N3O7H, mass calcd 1258.9 [M+H] + , m / z found 1259.0 [M+H] + ; LCMS conditions: Method 12.

[1399] Step h: Synthesis of 9H-fluoren-9-ylmethyl N-[(1S)-1-[[4-[4-(4-azidobutoxy)-2- ethyl-phenyl]phenyl]methyl]-2-[[(1S)-1-[[[4-[2-[[2,4-bis(dodecyloxy)phenyl]methylamino]-2-oxo-ethoxy]phenyl]-(2,4-dimethoxyphenyl)methyl]aminocarbonyl]-4-(3,5-dimethylphenyl)butyl]amino]-2-oxo- ethyl]carbamate (N-Fmoc-Tag 3-2AA)

[1400]

[1401] The reaction was performed using General Procedure D for amide coupling using Tag 3-1AA (0.6 g, 476.60 pmol, 1 eq) and AA2 (432.30 mg, 714.90 pmol, 1.5 eq). N-Fmoc-Tag 3-2AA 5 was obtained as off-white solid (0.56 g, 303.43 pmol, 63.66% yield). LCMS (ESI): RT = 6.048 min, for C 49 H 54 N6O5Na, mass calcd 829.4, m / z found 829.4 [M-Rink amide-anchor + 2H] + ; Rink amide = C 17 H 18 O4, anchor = C 51 H97 NO2; LC-MS conditions: Method 7.

[1402] 1 H NMR (400 MHz, Chloroform-d) δ = 7.75 (br d, J = 6.3 Hz, 2H), 7.57 - 7.28 (m, 5H), 7.24 - 6.89 (m, 10H), 6.86 - 6.62 (m, 5H), 6.54 - 6.31 (m, 5H), 6.20 (br s, 1H), 5.27 (br s, 1H), 4.53 - 4.07 (m, 7H), 4.06 - 3.53 (m, 12H), 3.40 (br t, J = 6.3 Hz, 2H), 3.17 (br d, J = 7.3 Hz, 2H), 2.64 - 2.45 (m, 4H), 2.22 (br d, J = 3.3 Hz, 6H), 1.95 - 1.71 (m, 8H), 1.27 (s, 82H), 1.06 (br t, J = 7.2 Hz, 3H), 0.90 - 0.87 (m, 6H) ppm.

[1403] Step i: Synthesis of (2S)-2-((S)-2-amino-3-(4'-(4-azidobutoxy)-2'-ethyl-[l,l'-biphenyl]-4-yl) propanamido)-N-((4-(2-((2,4-bis(dodecaunyloxy)benzyl)amino)-2-oxoethoxy)phenyl)(2,4- dimethoxyphenyl)methyl)-5-(3,5-dimethylphenyl)pentanamide (Tag 3-2AA)

[1404]

[1405] The reaction was performed using the general procedure E for Fmoc deprotection using N-Fmoc-Tag 3-2AA (400 mg, 216.73 pmol, 1 equiv) to obtain Tag 3-2AA as a light yellow solid (380 mg, 210.67 pmol, 97.20% yield, 90% purity). LC-MS conditions: Method 7.

[1406] Step j: Synthesis of (3S)-tert-butyl 3-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-4- (((2S)-3-(4'-(4-azidobutoxy)-2'-ethyl-[l,l'-biphenyl]-4-yl)-l-(((2S)-l-(((4-(2-((2,4- bis(dodecaunyloxy)benzyl)amino)-2-oxoethoxy)phenyl)(2,4-dimethoxyphenyl)methyl)amino)- 5-(3,5-dimethylphenyl)-l-oxopentan-2-yl)amino)-l-oxopropan-2-yl)amino)-4- oxobutanoate (N-Fmoc-Tag 3-3AA)

[1407]

[1408] The reaction was performed using General Procedure D for amide coupling using Tag 3-2AA (380 mg, 210.67 pmol, 90% purity, 1 eq) and AA3 (130.02 mg, 316.01 pmol, 1.5 eq). N-Fmoc-Tag 3-3AA was obtained as a light yellow solid (350 mg, 156.19 pmol, 74.14% yield, 90% purity). LCMS (ESI): RT = 5.542 min, for C 53 H 60 N7O8, m / z calcd 922.44, [M-Rink Amide-Anchors-tBu+3H] + ; Rink Amide = C 17 H 18 O4, Anchors = C 51 H 97 NO2; m / z found 922.3; LC-MS conditions: Method 7.

[1409] Step k: Synthesis of (3S)-tert-butyl 3-amino-4-(((2S)-3-(4'-(4-azidobutoxy)-2'- ethyl-[l,l'-biphenyl]-4-yl)-l-(((2S)-l-(((4-(2-((2,4-bis(dodecaunoxyl)benzyl)amino)-2- oxoethoxy)phenyl)(2,4-dimethoxyphenyl)methyl)amino)-5-(3,5-dimethylphenyl)-l- oxopentan-2-yl)amino)-l-oxopropan-2-yl)amino)-4-oxoheptanoate (Tag 3-3AA)

[1410]

[1411] The reaction was performed using General Procedure E for Fmoc deprotection using N-Fmoc-Tag 3-3AA (350 mg, 156.19 pmol, 90% purity, 1 eq). Tag 3-3AA was obtained as a light yellow solid (280 mg, 140.43 pmol, 89.91% yield, 90% purity). LCMS (ESI): RT = 3.810 min, for C 38 H 50 N7O6, m / z calcd 700.37, [M-Rink Amide-Anchors-tBu+3H] + ; Rink Amide = C 17 H 18 O4, Anchors = C 51 H 97NO2; m / z found 700.3; LC-MS conditions: Method 7.

[1412] Step l: Synthesis of (3S)-tert-butyl 3-((S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)- 3-(tert-butoxy)propanamido)-4-(((2S)-3-(4'-(4-azidobutoxy)-2'-ethyl-[l,l'-biphenyl]-4-yl)- 1-(((2S)-l-(((4-(2-((2,4-bis(dodecaunyloxy)benzyl)amino)-2-oxoethoxy)phenyl)(2,4- dimethoxyphenyl)methyl)amino)-5-(3,5-dimethylphenyl)-l-oxopentan-2-yl)amino)-l- oxoprop-2-yl)amino)-4-oxobutanoate (N-Fmoc-tag 3-4AA)

[1413]

[1414] The reaction was performed using General Procedure D for amide coupling using Tag 3-3AA (280 mg, 140.35 pmol, 90% purity, 1 equiv) and AA4 (80.72 mg, 210.52 pmol, 1.5 equiv). N-Fmoc-Tag 3-4AA was obtained as a light yellow solid (300 mg, 124.94 pmol, 89.02% yield, 90% purity). LCMS (ESI): RT = 5.337 min, for C 56 H 65 N8O 10 , m / z calculated 1009.47, [M-Rink amide-anchor-2tBu + 4H] + ; Rink amide = C 17 H 18 O4, anchor = C 51 H 97 NO2; m / z found 1009.4; LC-MS conditions: Method 7.

[1415] Step m: Synthesis of (3S)-tert-butyl 3-((S)-2-amino-3-(tert-butoxy)propanamido)-4-(((2S)- 3-(4'-(4-azidobutoxy)-2'-ethyl-[l,l'-biphenyl]-4-yl)-l-(((2S)-l-(((4-(2-((2,4-bis(dodecaunyloxy)benzyl)amino)-2-oxoethoxy)phenyl)(2,4-dimethoxyphenyl)methyl)amino)-5-(3,5- dimethylphenyl)-l-oxopentan-2-yl)amino)-l-oxoprop-2-yl)amino)-4-oxobutanoate (Tag 3-4AA)

[1416]

[1417] The general procedure E for Fmoc deprotection was used to perform the reaction using N-Fmoc-Tag 3-4 AA (280 mg, 67 pmol, 90% purity, 1 equiv). Tag 3-4 AA was obtained as a light yellow solid (230 mg, 106.83 pmol, 91.56% yield, 90% purity). LC-MS conditions: Method 7.

[1418] Step n: Synthesis of (5S,8S,11S)-tert-butyl 11-(((2S)-3-(4'-(4-azidobutoxy)-2'- ethyl-[1,1'-biphenyl]-4-yl)-1-(((2S)-1-(((4-(2-((2,4-bis(dodecaunoxyl)benzyl)amino)-2- oxoethoxy)phenyl)(2,4-dimethoxyphenyl)methyl)amino)-5-(3,5-dimethoxyphenyl)-1- oxopentan-2-yl)amino)-1-oxopropan-2-yl)carbamoyl)-5-(CAN-1-(tert-butoxy)ethyl)-8- (tert-butoxymethyl)-1-(9H-fluoren-9-yl)-3,6,9-trioxo-2-oxa-4,7,10-triazatridecan-13-oate (N-Fmoc-Tag 3-5 AA)

[1419]

[1420] To a solution of Tag 3-4 AA (230 mg, 106.83 pmol, 90% purity, 1 equiv), AA5 (63.69 mg, 160.24 pmol, 1.5 equiv) and HOAt (21.81 mg, 160.24 pmol, 22.42 pL, 1.5 equiv) in DCM (4 mL) was added DIC (20.22 mg, 160.24 pmol, 24.81 pL, 1.5 equiv) in one portion at 20 °C under N2. The mixture was stirred at 20 °C for 2 h. To monitor the reaction, a small amount of residue was subjected to acid cleavage by using TFA mixture (0.1 mL, TFA:TIPS:H2O:anisole = 95:2.5:2.5:4.5) for 1 h. LCMS showed complete conversion of the reaction. The mixture was triturated with ACN (20 mL*3) to give the crude product N-Fmoc-Tag 3-4 AA as a light yellow solid (250 mg, 97.10 pmol, 90.90% yield, 90% purity).

[1421] LCMS (ESI): RT = 5.170 min for C 60 H 72 N9O 12, m / z calculated 1110.52, [M-Rink amide-anchor-3tBu+5H] + ; Rink amide = C 17 H 18 O4, anchor = C 51 H 97 NO2; m / z found 1110.4; LC-MS conditions: Method 7.

[1422] Example 7. Synthesis (LPPS) M2 on Tag 4

[1423] 7.1 Synthesis of Tag 4

[1424] The synthetic route for the preparation of Tag 4 is shown in Scheme 5 below.

[1425] Scheme 5

[1426]

[1427] Reagents and conditions a) C 22 H 45 Br (3.3 eq), K2CO3 (8 eq), DMF / THF, 90 °C, 12 h, 99.8%; b) LiAlH4 (3 eq), THF, 70 °C, 3 h, 92.3%; (c) PBr3 (3 eq), DCM, 20 °C, 3 h, 63%; d) 55A (1 eq), K2CO3 (6 eq), NMP, 90 °C, 16 h, 90.5%; e) NaBH4 (5 eq), EtOH (1 V), THF (10 V), 70 °C, 12 h, 89.9%; (f) I2 (1.8 eq), Li2CO3 (0.02 eq), TMSN3 (4.5 eq), DCM, 12 h, 37 °C, 80.9%; (g) Pme3 (3 eq), H2O, THF / DCM, 20 °C, 12 h, 66.8%. Overall yield: 25.5%

[1428] Trialkylation of 3,4,5-trihydroxybenzoyl ester with C22-alkyl bromide followed by LAH reduction of the ester tag 4-1 produced the alcohol tag 4-2 (Matsuda et al., “Solution-Phase Synthesis of Oligodeoxyribonucleotides Using the H-Phosphonate Method with N-Unprotected 5’-Phosphite Monomers,” RSC Adv. 11:38094-38107 (2021), which is hereby incorporated by reference in its entirety). Conversion of the alcohol tag 4-2 to the bromo analog tag 4-3 with triphenylphosphine and carbon tetrabromide followed by alkylation of tag 4-3a produced the hexaalkylated derivative tag 4-4. Conversion of the ketone tag 4-4 to the alcohol tag 4-5 with sodium borohydride and then conversion of the alcohol tag 4-5 to the azide tag 4-6 with trimethylsilyl azide as the nucleophile in the presence of lithium carbonate as the base, and finally reduction of the azide tag 4-6 to tag 4 with trimethyl phosphine produced tag 4.

[1429] Step a: Synthesis of methyl 3,4,5-tris(dodecyloxy)benzoate (tag 4-1)

[1430]

[1431] To a mixture of methyl 3,4,5-trihydroxybenzoate (5 g, 27.15 mmol, 1 eq) and 1- bromododecane (34.90 g, 89.60 mmol, 3.3 eq) in DMF (100 mL) and THF (20 mL) was added K2CO3 (30.02 g, 217.22 mmol, 8 eq), and the reaction mixture was stirred at 90 °C for 12 h. Most of the reactants were consumed, and a new spot with less polarity was formed on TLC (PE / EtOAc = 3 / 1). After the reaction was completed, the reaction mixture was poured into purified water (1000 mL), the mixture was stirred for 1 h, and the precipitated solid was collected by filtration. The obtained solid was triturated with acetone (300 mL), and the solid was collected by filtration and slurry washed in methanol. After filtration, the obtained solid was dried under reduced pressure to give the title compound as an off-white solid. Compound tag 4-1 (30.1 g, 27.12 mmol, 99.88% yield) was obtained as a gray solid. 1H NMR (400 MHz, Chloroform-d) δ = 7.29 (s, 2H), 4.03 (dt, J = 2.5, 6.4 Hz, 6H), 3.91 (s, 2H), 3.93 - 3.87 (m, 1H), 1.85 - 1.71 (m, 6H), 1.54 - 1.45 (m, 6H), 1.28 (s, 108H), 0.90 (t, J = 6.8 Hz, 9H) ppm.

[1432] Step b: Synthesis of [3,4,5-tridodecyloxy)phenyl]methanol (Tag 4-2)

[1433]

[1434] To a solution of compound Tag 4-1 (1 g, 900.99 pmol, 1 eq) in THF (10 mL) was added LiAlH4(102.59 mg, 2.70 mmol, 2.70 mL, 3.0 eq) at 0 °C. Then the mixture was heated to 70 °C for 3 h. TLC (PE / EA = 20 / 1) showed most of the reactant was consumed completely and one major new spot was detected. The reaction mixture was quenched with 1 N HC1 and the mixture was stirred at 20 °C for 1 h. Then filtered and the filter cake was concentrated to give the product. Compound Tag 4-2 (0.9 g, 831.88 pmol, 92.33% yield) was obtained as a grey solid. 1 H NMR (400 MHz, Chloroform-d) δ ppm 6.58 (s, 2H) 4.62 (s, 2H) 3.93 - 4.04 (m, 6H) 1.72 - 1.87 (m, 6H) 1.43 - 1.55 (m, 6H) 1.28 (s, 108H) 0.90 (t, J = 6.79 Hz, 9H).

[1435] Step c: Synthesis of 5-(bromomethyl)-1,2,3-tridodecyloxy)benzene (Tag 4-3)

[1436]

[1437] To a solution of compound Tag 4-2 (0.9 g, 831.88 pmol, 1.0 eq) in DCM (10 mL) was added a solution of PBr3(675.54 mg, 2.50 mmol, 3.0 eq) in DCM (10 mL). The solution was then stirred at 20 °C for 3 h. TLC (PE / EA = 10 / 1) showed the reactant was consumed completely and one small polar spot was detected. The reaction mixture was quenched with H2O (50 mL) and ACN (50 mL), and the mixture was stirred at 20 °C for 1 h. Then filtered, and the filter cake was dried by evaporation of the solvent to give the product. Compound Tag 4-3 (0.6 g, 524.12 pmol, 63.00% yield) was obtained as a white solid. 1 H NMR (400 MHz, Chloroform-d) δ ppm 6.58 (s, 2H) 4.44 (s, 2H) 3.92 - 4.00 (m, 6H) 1.86 (dt, J = 6.53, 3.26 Hz, 7H) 1.42 - 1.52 (m, 8H) 1.26 (s, 125H) 0.86 - 0.93 (m, 12H).

[1438] Step d: Synthesis of Bis[4-[[3,4,5-tris(dodecyloxy)phenyl]methoxy]phenyl]methanone (Tag 4-4)

[1439]

[1440] A solution of compound Tag 4-3a (500 mg, 2.33 mmol, 1.0 eq) and K2CO3(1.94 g, 14.00 mmol, 6.0 eq) in NMP (150 mL) was degassed and purged with N2for 10 times for about 10 min. Then the mixture was heated to 90 °C, then a solution of Tag 4-3 (5.37 g, 4.69 mmol, 2.01 eq) in NMP (350 mL) was added under N2. Then the mixture was degassed and purged with N2for 10 times for about 10 min, stirred at 90 °C for 16 hours. TLC (PE / EA = 20 / 1) showed the starting material was consumed completely and the desired spot was detected. The reaction solution was slowly added to stirring ACN (1000 mL), then the reaction mixture was filtered; and the filter cake was washed with MeOH (250 mL*3) and ACN (250 mL*3). The filter cake was dried by evaporation of the residual solvent to give the product. Compound Tag 4-4 (5.5 g, 2.11 mmol, 90.55% yield, 90% purity) was obtained as a white solid. 1H NMR (400 MHz, Chloroform-d) δ ppm 7.80 (d, J = 8.75 Hz, 4 H) 6.99 - 7.09 (m, 4 H) 6.63 (s, 4 H) 5.03 (s, 4 H) 3.95 - 4.01 (m, 12 H) 1.80 (br dd, J = 14.07, 6.44 Hz, 12 H) 1.43 - 1.54 (m, 12 H) 1.26 (s, 226 H) 0.89 (t, J = 6.69 Hz, 18 H).

[1441] Step e: Synthesis of Bis[4-[[3,4,5-tris(docosyloxy)phenyl]methoxy]phenyl]methanol (Tag 4-5)

[1442]

[1443] To a solution of Tag 4-4 (5.5 g, 2.35 mmol, 1 eq) in THF (100 mL) and EtOH (25 mL), NaBH4(444.21 mg, 11.74 mmol, 5.0 eq) was added slowly and the mixture was heated to 70 °C and stirred at 77 °C for 5 h. TLC (PE / EA = 10 / 1) showed that the starting material was completely consumed and a new spot was detected. The reaction mixture was added slowly to saturated NH4Cl / ACN (550 mL / 750 mL). The slurry was filtered and the obtained crystals were washed with ACN (250 mL*3) and methanol (250 mL*3) to give the product. Compound Tag 4-5 (5.5 g, 2.11 mmol, 89.92% yield, 90% purity) was obtained as a white solid. 1 H NMR (400 MHz, Chloroform-d) δ ppm 7.30 (d, J = 8.63 Hz, 4 H) 6.96 (d, J = 8.63 Hz, 4 H) 6.62 (s, 4 H) 5.81 (br s, 1 H) 4.94 (s, 4 H) 3.95 - 4.01 (m, 12 H) 1.74 - 1.84 (m, 12 H) 1.48 (br s, 12 H) 1.28 (s, 226 H) 0.90 (t, J = 6.75 Hz, 18 H).

[1444] Step f: Synthesis of 5-[[4-[azido-[4-[[3,4,5-tris(docosyloxy)phenyl]methoxy]phenyl]methyl]phenoxy]methyl]-1,2,3-tris(docosyloxy)benzene (Tag 4-6)

[1445]

[1446] To a mixture of compound Label 4-5 (5.5 g, 2.35 mmol, 1 eq) and I2 (1.07 g, 4.22 mmol, 850.79 μL, 1.8 eq) in DCM (105 mL) was added Li2CO3 (3.47 mg, 46.93 μmol, 31.67 μL, 0.02 eq) and TMSN3 (1.22 g, 10.56 mmol, 1.39 mL, 4.5 eq), then the reaction mixture was stirred at 37 °C for 12 h. The reactants were consumed and the desired spot was formed on TLC (PE / EtOAc = 20 / 1). The reaction solution was slowly added to stirring ACN (750 ml) and saturated Na2SO3 (250 ml), then the reaction mixture was filtered; and the filter cake was washed with methanol (250 mL*3) and ACN (250 mL*3). The filter cake was dried by evaporating the residual solvent to give the product. Label 4-6 (5.0 g, 1.90 mmol, 80.95% yield, 90% purity) was obtained as a white solid. 1 H NMR (400 MHz, Chloroform-d) δ ppm 7.23 (br d, J=8.63 Hz, 4 H) 6.96 (br d, J=8.63 Hz, 4 H) 6.61 (s, 4 H) 5.62 (s, 1 H) 5.65 (s, 1 H) 4.93 (s, 4 H) 3.94 - 3.99 (m, 12 H) 1.76 - 1.84 (m, 12 H) 1.43 - 1.50 (m, 12 H) 1.27 (s, 226 H) 0.87 - 0.90 (m, 18 H).

[1447] Step g: Synthesis of Bis[4-[[3,4,5-tris(tetracosyloxy)phenyl]methoxy]phenyl]methanamine (Label 4)

[1448]

[1449] To a solution of Tag 4-6 (4.5 g, 1.90 mmol, 1 eq) in THF (50 mL), DCM (50 mL) and H2O (2.5 mL) was added PMe3 (1 M, 5.70 mL, 3.0 eq). Then, the reaction mixture was stirred at 20 °C for 12 h. TLC (PE / EA = 20 / 1) showed the reactant was consumed completely and TLC (PE / EA = 1 / 1) showed the desired spot was detected. The reaction solution was slowly added to stirring ACN (800 ml) and H2O (200 mL), then the reaction mixture was filtered; the filter cake was re-mulled with ACN (800 mL), and then the filter cake was washed with MeOH (250 mL*3) and ACN (250 mL*3). The filter cake was dried by evaporation of residual solvent to give the product Tag 4 (3.5 g, 1.27 mmol, 66.84% yield, 85% purity) as a white solid. 1 HNMR (400 MHz, Chloroform-d) δ ppm 7.28 - 7.33 (m, 4 H) 6.90 - 6.99 (m, 4 H) 6.60 - 6.66 (m, 4 H) 5.16 (s, 1 H) 4.93 (s, 4 H) 3.95 - 4.01 (m, 12 H) 1.76 - 1.82 (m, 12 H) 1.48 (br s, 12 H) 1.28 (s, 226 H) 0.88 - 0.92 (m, 18 H).

[1450] 7.2 Synthesis of Tag 4-M2

[1451] The following Figure 5 and Table 7 summarize the synthesis of M2 using Tag 4.

[1452] Table 7.

[1453]

[1454]

[1455] *: Low yield is due to insufficient cleavage of M2 from Tag 4.

[1456] General procedure F for amide coupling to make N-Fmoc-Tag 4-nAA

[1457] To a solution of Tag 4 or Tag 4-(n-1)AA (1 eq) and AAn (1.5 eq) and HOAt (1.5 eq) in DCM (20 V) was added DIC (1.5 eq) and HOAt (1.5 eq). The solution was stirred at 20 °C for 12 h. A small sample was taken and subjected to acidic cleavage by using TFA cocktail (TFA:TIPS:H2O = 95:2.5:2.5). The sample was filtered and analyzed by LCMS, which showed the reactant was consumed and the desired mass was detected. To the reaction solution was added ACN (80 V), and the resulting suspension was filtered and washed with ACN (20 V x 2) to give an off-white solid. The solvent was removed by filtration, and the filter cake was dried by evaporation of the residual solvent to give the product N-Fmoc-Tag 4-nAA as an off-white solid, which was used directly for the next step.

[1458] General procedure G for Fmoc deprotection to give Tag 4-nAA

[1459] A solution of compound N-Fmoc-Tag 4-nAA (1 eq) in 20% piperidine in THF and DCM (20 V) was stirred at 20 °C for 2 hours. TLC (PE / EA = 1 / 1) showed the reactant was completely consumed and the desired spot was detected. To the reaction solution was added ACN (200 V), and the resulting suspension was filtered and washed with ACN (40 V x 2) to give Tag 4-nAA as an off-white solid. The crude product was dried under reduced pressure and used directly for the next step without further purification.

[1460] Step 1: Synthesis of (2S)-2-amino-N-[bis[4-[[3,4,5-tris(tetracosyloxy)phenyl]methoxy]phenyl]methyl]-5- (3,5-dimethylphenyl)pentanamide (Tag 4-1AA)

[1461]

[1462] The reaction was performed according to general procedure F for amide coupling using Tag 4 (2.5 g, 1.07 mmol, 1 eq) and AA1 (709.88 mg, 1.60 mmol, 1.5 eq). N-Fmoc-Tag 4-1AA was obtained as an off-white solid (2.6 g, 751.31 pmol, 70.41% yield, 80% purity). LC-MS: RT = 4.061 min, for C 28 H 31 N2O3 + , mass calcd. 443.23, m / z found 443.20 [M-anchor+H] + ; (anchor = C 159H 286 MS = 2325.21). LCMS conditions: Method 12. (Anchors = Tag 4).

[1463] The reaction was performed according to the general procedure G for Fmoc deprotection using N-Fmoc-Tag 4-1 AA (2.0 g, 722.41 pmol, 1 equiv). Tag 4-1 AA was obtained as a white solid (1.6 g, crude) which was used directly in the next step. LC-MS: 443.2 (M+H) + .

[1464] Step 2: Synthesis of (2R)-2-[[(2S)-2-amino-3-[4-[4-(4-azidobutoxy)-2- ethyl-phenyl]phenyl]propanoyl]amino]-N-[bis[4-[[3,4,5-tris(dodecyloxy)phenyl]methoxy]phenyl]methyl]-5-(3,5-dimethylphenyl)pentanamide (Tag 4-2AA)

[1465]

[1466] The reaction was performed according to the general procedure F for amide coupling using Tag 4-1 AA (1.6 g, 628.37 pmol, 1 equiv) to obtain N-Fmoc-Tag 4-2AA as a white solid (1.9 g, 424.52 pmol, 67.56% yield, 70% purity) which was used directly in the next step. LC-MS: RT = 5.525 min, for C 49 H 55 N6O5 + , mass calcd. 807.42, m / z found 807.40 [M - Anchor + H] + ; (Anchor = C 159 H 286 MS = 2325.21). LCMS conditions: Method 12. (Anchors = Tag 4).

[1467] The reaction was performed according to the general procedure G for Fmoc deprotection using N-Fmoc-Tag 4-2AA (1.9 g, 606.46 pmol, 1 equiv). Tag 4-2AA was obtained as an off-white solid (1.7 g, 584.05 pmol, 96.31% yield). LC-MS: RT = 3.534 min, for C 47 H 55 N6O5 + , mass calcd. 783.42, m / z found 783.40 [M - Anchor + C 13 H 11 O2+H] + ; (Anchor = C 159 H286 O8, MS = 2325.21). LCMS conditions: Method 12. (Anchor = Tag 4) 34 H 45 N6O3 + , mass calcd. 585.35, m / z found 585.40 [M - Anchor + H] + ; (Anchor = C 159 H 286 O8, MS = 2325.21). LCMS conditions: Method 12. (Anchor = Tag 4)

[1468] Step 3: Synthesis of tert-butyl (3S)-3-amino-4-[[(1S)-1-[[4-[4-(4-azidobutoxy)-2- ethyl-phenyl]phenyl]methyl]-2-[[(1S)-1-[bis[4-[[3,4,5-tris(dodecyloxy)phenyl]methoxy]phenyl]methylamino carbonyl]-4-(3,5-dimethylphenyl)butyl]amino]-2-oxo- ethyl]amino]-4-oxo-butanoate (Tag 4-3AA)

[1469]

[1470] The reaction was performed following the general procedure F for amide coupling using Tag 4-2AA (1.7 g, 584.05 pmol, 1 equiv). N-Fmoc-Tag 4-3AA was obtained as off-white solid (1.9 g, 345.02 pmol, 59.07% yield, 60% purity). LC-MS: RT = 5.159 min, for C 53 H 60 N7O8 + , mass calcd. 922.41, m / z found 922.40 [M - Anchor + H] + ; (Anchor = C 159 H 286 O8, MS = 2325.21). LCMS conditions: Method 12. (Anchor = Tag 4)

[1471] The reaction was performed following the general procedure G for Fmoc deprotection using N-Fmc-Tag 4-3AA (1.9 g, 575.04 pmol, 1 equiv) to obtain Tag 4-3AA as off-white solid (1.50 g, 486.71 pmol, 84.64% yield). LC-MS: RT = 3.633 min, for C 51 H 60 N7O8 + , mass calcd. 898.44, m / z found 898.50 [M - tBu-Anchor + C 13 H 11 O2+H] +; (Anchor = C 159 H 286 O8, MS = 2325.21). LCMS conditions: Method 12. 38 H 50 N7O6 + Calcd 700.37, found 700.50 [M-tBu-Anchor + H] + ; (Anchor = C 159 H 286 O8, MS = 2325.21). LCMS conditions: Method 12.

[1472] (Anchor = Tag 4)

[1473] Step 4: Synthesis of tert-butyl (3S)-3-[[(2S)-2-amino-3-tert-butoxy- propanoyl]amino]-4-[[(1S)-1-[[4-[4-(4-azidobutoxy)-2-ethyl- phenyl]phenyl]methyl]-2-[[(1S)-1-[bis[4-[[3,4,5-tris(dodecyloxy)phenyl]methoxy]phenyl]methylamino carbonyl]-4-(3,5-dimethylphenyl)butyl]amino]-2-oxo- ethyl]amino]-4-oxo-butanoate (Tag 4-4AA)

[1474]

[1475] The reaction was performed following the general procedure F for amide coupling using Tag 4-3AA (1.5 g, 486.71 pmol, 1 equiv) in DCM (50 mL). N-Fmoc-Tag 4-4AA was obtained as an off-white solid (1.45 g, 420.62 pmol, 86.42% yield). LC-MS: RT = 5.019 min for C 56 H 65 N8O 10 + Calcd 1009.40, found 1009.40 [M-Anchor + H] + ; (Anchor = C 159 H 286 O8, MS = 2325.21). LCMS conditions: Method 12. (Anchor = Tag 4).

[1476] The reaction was performed following the general procedure G for Fmoc deprotection using N-Fmoc-Tag 4-4AA (1.4 g, 406.11 pmol, 1 equiv). Tag 4-4AA was obtained as an off-white solid (1.30 g, 403.09 pmol, 99.26% yield). LC-MS: RT = 3.527 min for C 54 H 65 N8O10 + , mass calculated 985.47, m / z found 985.50 [M-tBu-anchor+H] + ;(anchor=C 159 H 286 O8, MS = 2325.21); for C 41 H 55 N8O8 + , mass calculated 787.41, m / z found 787.50 [M-tBu-anchor+H] + ;(anchor=C 159 H 286 O8, MS = 2325.21); LCMS conditions: Method 12. (Anchor = Tag 4)

[1477] Step 5: Synthesis of (3S)-3-[[(2S)-2-[[(2S,3R)-2-amino-3-tert-butoxy-butyryl]amino]-3-tert-butoxy-propionyl]amino]-4-[[(1S)-1-[[4-[4-(4-azidobutyloxy)-2-ethyl-phenyl]phenyl]methyl]-2-[[(1S)-1-[bis[4-[[3,4,5-tris(docosanyloxy)phenyl]methoxy]phenyl]methylcarbamoyl]-4-(3,5-dimethylphenyl)butyl]amino]-2-oxo-ethyl]amino]-4-oxo-butyryl tert-butyl ester (label 4-5AA)

[1478]

[1479] The reaction was carried out according to General Procedure F for Amide Coupling using Tag 4-4AA (1.45 g, 420.62 μmol). N-Fmoc-Tag 4-5AA (1.3 g, 360.66 μmol, 85.74% yield) was obtained as an off-white solid. LC-MS: RT = 4.894 min, for C 60 H 72 N9O 12 + , mass calculated 1110.52, m / z found 1110.60 [M-tBu-anchor+H] + ;(anchor=C 159 H 286 O8, MS = 2325.21); for C 73 H 82 N9O 14 + , mass calculated 1308.57, m / z found 1308.60 [M-tBu-anchor+H] + ;(anchor=C 159 H 286O8, MS = 2325.21); LCMS conditions: Method 12. (Anchor = Tag 4)

[1480] The reaction was performed according to the general procedure G for Fmoc deprotection using N-Fmoc-Tag 4-5 AA (1.30 g, 360.66 pmol, 1 equiv). Tag 4-5 AA was obtained as off-white solid (1.0 g, 295.66 pmol, 81.98% yield) which was used directly for the next step. LC-MS: RT = 3.557 min, for C 58 H 72 N9O 12 + , mass calcd 1086.52, m / z found 1086.30 [M-3tBu-Anchor + 4H]+; (Anchor = C 13 H 11 O2+4H] + ; (Anchor = C 159 H 286 O8, MS = 2325.21); LCMS conditions: Method 12. (Anchor = Tag 4) 45 H 62 N9O 10 + , mass calcd 888.45, m / z found 888.50 [M-3tBu-Anchor + 4H]+; (Anchor = C 159 H 286 O8, MS = 2325.21); LCMS conditions: Method 12. (Anchor = Tag 4)

[1481] Step 6: Synthesis of tert-butyl (3S)-3-[[(2S)-2-[[(2S,3R)-2-[[2-amino-3-(2- fluorophenyl)-2-methyl-propanoyl]amino]-3-tert-butoxy-butyryl]amino]-3-tert- butoxy-propionyl]amino]-4-[[(1S)-1-[[4-[4-(4-azidobutoxy)-2-ethylphenyl- phenyl]phenyl]methyl]-2-[[(1S)-1-[bis[4-[[3,4,5-tris(dodecyloxy)phenyl]methoxy]phenyl] methylcarbamoyl]-4-(3,5-dimethylphenyl)butyl]amino]-2-oxo-ethyl]amino]-4- oxo-butyrate (Tag 4-6 AA)

[1482]

[1483] The reaction was performed according to the general procedure F for amide coupling using Tag 4-5 AA (1.0 g, 295.66 pmol, 1 equiv). N-Fmoc-Tag 4-6 AA was obtained as off-white solid (0.8 g, 211.43 pmol, 71.51 % yield). LC-MS: RT = 5.208 min, for C 70 H 82 FN 10 O 13 + , mass calc. 1289.60, m / z found 1289.60 [M-3tBu-Anchors + 4H] + ; (Anchors = C 159 H 286 O8, MS = 2325.21 ); LCMS conditions: Method 12. (Anchors = Tag 4)

[1484] The reaction was performed according to the general procedure G for Fmoc deprotection using N-Fmoc-Tag 4-6 AA (0.8 g, 211.43 pmol, 1 equiv). Tag 4-6 AA was obtained as off-white solid (0.7 g, 196.55 pmol, 92.96% yield). LC-MS: RT = 3.697 min, for C 68 H 82 FN 10 O 13 + , mass calc. 1265.60, m / z found 1265.70 [M-3tBu-Anchors + 4H] 13 H 11 O2+ 4H] + ; (Anchors = C 159 H 286 O8, MS = 2325.21 ); RT = 3.630 min, for C 55 H 72 FN 10 O 11 + , mass calc. 1067.53, m / z found 1067.60 [M-3tBu-Anchors + 4H] + ; (Anchors = C 159 H 286 O8, MS = 2325.21 ); LCMS conditions: Method 12. (Anchors = Tag 4)

[1485] Step 7: Synthesis of tert-butyl (3S)-3-[[(2S)-2-[[(2S,3R)-2-[[2-[[(2S,3R)-2-amino-3-tert- butoxy-butyryl]amino]-3-(2-fluoro-phenyl)-2-methyl-propionyl]amino]-3-tert- butoxy-butyryl]amino]-3-tert-butoxy-propionyl]amino]-4-[[(1S)-1-[[4-[4-(4- azidobutoxy)-2-ethyl-phenyl]phenyl]methyl]-2-[[(1S)-1-[bis[4-[3,4,5-tris(dodecyloxy) phenyl]methoxy]phenyl]methylcarbamoyl]-4-(3,5-dimethylphenyl)butyl]amino]-2- oxo-ethyl]amino]-4-oxo-butyric acid (Tag 4-7AA)

[1486]

[1487] The reaction was performed according to the general procedure F for amide coupling using Tag 4-6AA (0.7 g, 196.55 pmol, 1 equiv). N-Fmoc-Tag 4-7AA was obtained as off-white solid (0.7 g, 177.62 pmol, 90.37% yield). LC-MS: RT = 5.224 min, for C 74 H 89 FN 11 O 15 + , mass calcd. 1390.64, m / z found 1390.60 [M-3tBu-anchor + 4H] + ; (anchor = C 159 H 286 O8, MS = 2325.21); LCMS conditions: Method 12. (anchor = Tag 4)

[1488] The reaction was performed according to the general procedure G for Fmoc deprotection using N-Fmoc-Tag 4-7AA (0.7 g, 177.62 pmol, 1 equiv). Tag 4-7AA was obtained as off-white solid (0.58 g, 155.97 pmol, 87.81% yield) which was used directly in the next step. LC-MS: RT = 3.869 min, for C 72 H 89 FN 11 O 15 + , mass calcd. 1366.65, m / z found 1366.70 [M-3tBu-anchor + C 13 H 11 O2+ 4H] + ; (anchor = C 159 H 286O8, MS = 2325.21); LCMS conditions: Method 12. (Anchors = Tag 4) 59 H 79 FN 11 O 13 + , mass calcd 1168.58, m / z found 1168.60 [M-3tBu-Anchors + 4H] + ; (Anchors = C 159 H 286 O8, MS = 2325.21); LCMS conditions: Method 12. (Anchors = Tag 4)

[1489] Step 8: Synthesis of tert-butyl (3S)-3-[[(2S)-2-[[(2S,3R)-2-[[2-[[(2S,3R)-2-[(2- aminoacetyl)amino-3-tert-butoxy-butyryl]amino]-3-(2-fluorophenyl)-2-methyl- propionyl]amino]-3-tert-butoxy-butyryl]amino]-3-tert-butoxy-propionyl]amino]- 4-[[(1S)-1-[[4-[4-(4-azidobutoxy)-2-ethyl-phenyl]phenyl]methyl]-2-[[(1S)-1- [bis[4-[3,4,5-tris(dodecyloxy)phenyl]methoxy]phenyl]methylcarbamoyl]-4-(3,5- dimethylphenyl)butyl]amino]-2-oxo-ethyl]amino]-4-oxo-butyric acid (Tag 4-8AA)

[1490]

[1491] The reaction was performed following the general procedure F for amide coupling using Tag 4-7AA (0.6 g, 161.35 pmol, 1 equiv). N-Fmoc-Tag 4-8AA was obtained as an off-white solid (0.56 g, 140.07 pmol, 86.81% yield). LC-MS: RT = 5.092 min, for C 76 H 92 FN 12 O 16 + , mass calcd 1447.67, [M-3tBu-Anchors + 4H] + ; (Anchors = C 159 H 286 O8, MS = 2325.21); LCMS conditions: Method 12. (Anchors = Tag 4)

[1492] The reaction was performed according to the general procedure G for Fmoc deprotection using N-Fmoc-Tag 4-8 AA (0.56 g, 140.07 pmol, 1 equiv). Tag 4-8 AA was obtained as off-white solid (0.5 g, 132.42 pmol) which was used directly for the next step. LC-MS: RT = 3.848 min, mass calculated 1225.60, m / z found 1225.70 [M-3tBu-Anchors + 4H] 61 H 82 FN 12 O 14 + , mass calculated 1225.60, m / z found 1225.70 [M-3tBu-Anchors + 4H] + , (Anchors = C 159 H 286 O8, MS = 2325.21); LCMS conditions: Method 12. (Anchors = Tag 4) 74 H 92 FN 12 O 16 + , mass calculated 1423.67, m / z found 1423.70 [M-3tBu-Anchors + 4H] 13 H 11 O2+ 4H] + ; (Anchors = C 159 H 286 O8, MS = 2325.21); LCMS conditions: Method 12. (Anchors = Tag 4)

[1493] Step 9: Synthesis of tert-butyl (3S)-3-[[(2S)-2-[[(2S,3R)-2-[[2-[[(2S,3R)-2-[[2-[[(2S)-2-amino-3-[1- [(2,4-dimethoxyphenyl)methyl]tetrazol-5-yl]propionyl]amino]acetyl]amino]-3-tert- butoxy-butyryl]amino]-3-(2-fluorophenyl)-2-methyl-propionyl]amino]-3-tert-butoxy- butyryl]amino]-3-tert-butoxy-propionyl]amino]-4-[[(1S)-1-[[4-[4-(4-azidobutoxy)-2- ethyl-phenyl]phenyl]methyl]-2-[[(1S)-1-[bis[4-[[3,4,5-tris(tetracosyloxy)phenyl]methoxy] phenyl]methylcarbamoyl]-4-(3,5-dimethylphenyl)butyl]amino]-2-oxo-ethyl]amino]-4- oxo-butyric acid (Tag 4-9 AA)

[1494]

[1495] The reaction was performed according to the general procedure F for amide coupling using Tag 4-8AA (105.19 mg, 198.64 pmol, 1.5 equiv), DMB-AA9 (105.19 mg, 198.64 pmol, 1.5 equiv), DIC (25.07 mg, 198.64 pmol, 30.76 pL, 1.5 equiv) and HOAt (27.04 mg, 198.64 pmol, 27.79 pL, 1.5 equiv). N-Fmoc-Tag 4-9AA was obtained as an off-white solid (0.5 g, 116.62 pmol, 88.07% yield). LC-MS: RT = 4.817 min, for C 80 H 98 FN 17 O 17 + , mass calc. 793.86, found 794.30 [M - 3tBu-PMB-anchor + 7H] 2+ ; (anchor = C 159 H 286 O8, MS = 2325.21); LCMS conditions: Method 12. (anchor = Tag 4) 95 H 106 FN 17 O 19 , mass calc. 892.89, found 893.50 [M - 3tBu-PMB-anchor + 7H] + ; (anchor = C 159 H 286 O8, MS = 2325.21); LCMS conditions: Method 12. (anchor = Tag 4)

[1496] The reaction was performed according to the general procedure G for Fmoc deprotection using N-Fmoc-Tag 4-9AA (0.5 g, 116.62 pmol, 1 equiv). Tag 4-9AA was obtained as an off-white solid (0.5 g, crude). LC-MS: RT = 3.838 min, for C 64 H 86 FN 17 O 16 + , mass calc. 683.80, m / z found 683.20 [M - 4tBu-DMB-anchor + 7H] 2+ ; (anchor = C 159 H 286 O8, MS = 2325.21); LCMS conditions: Method 12. (anchor = Tag 4) 77 H 96 FN 17 O 18 2+, mass calcd. 782.85, m / z found 782.30 [M-4tBu-DMB-Anchors + 7H] + ; (Anchors = C 159 H 286 O8, MS = 2325.21); LCMS conditions: Method 12. (Anchors = Tag 4)

[1497] Step 10: Synthesis of tert-butyl (3S)-4-[[(1S)-1-[[4-[4-(4-azidobutoxy)-2- ethyl-phenyl]phenyl]methyl]-2-[[(1S)-1-[bis[4-[[3,4,5-tris(tetracosyloxy)phenyl]methoxy]phenyl]methylamino carbonyl]-4-(3,5-dimethylphenyl)butyl]amino]-2-oxo-ethyl]amino]-3-[[(2S)-3-tert-butoxy-2-[[(2S,3R)-3-tert-butoxy-2-[[2-[[(2S,3R)-3-tert-butoxy-2-[[2-[[(2S)-3-[1-[(2,4-dimethoxyphenyl)methyl]tetrazol-5-yl]-2-[[2,2-dimethyl-3-oxo-3-[2-(3-tritylimidazol-4-yl)ethylamino]propionyl]amino]propionyl]amino]ethyl]amino]butyryl]amino]butyryl]amino]propionyl]amino]-4-oxo-butyric acid (Tag 4-10AA)

[1498]

[1499] The reaction was performed following the general procedure F for amide coupling using Tag 4-9AA (0.5 g, 123.00 µmol, 1 eq) in DCM (15 mL) and THF (15 mL), AA10 (86.26 mg, 184.50 µmol, 1.5 eq), DIC (23.28 mg, 184.50 µmol, 28.57 µL, 1.5 eq) and HOAt (25.11 mg, 184.50 µmol, 25.81 µL, 1.5 eq). Tag 4-10AA (0.5 g, 110.75 µmol, 90.04% yield) was obtained as an off-white solid which was used directly in the next step. LC-MS: RT = 3.874 min, for C 75 H 101 FN 20 O 172 + , mass calcd. 786.37, found 787.00 [M-4tBu-DMB-Trt-Anchors + 8H] 2+ ; (Anchors = C 159 H 286O8, MS = 2325.21); LCMS conditions: Method 12. Note: (Anchor = Tag 4) 88 H 111 FN 20 O 19 , mass calcd. 885.41, found 886.00 [M - 4tBu-DMB-Trt-Anchor + 8H] + ; (Anchor = C 159 H 286 O8, MS = 2325.21); LCMS conditions: Method 12. Note: (Anchor = Tag 4)

[1500] Step 11: Synthesis of Bis[4-[[3,4,5-tris(dodecyloxy)phenyl]methoxy]phenyl]methanamine (M2)

[1501]

[1502] To a mixture of Tag 4-10AA (432.77 mg, 95.86 pmol, 1 eq) in TFA (4.5 mL) and H20 (0.25 mL) was added triisopropylsilane (143.92 mg, 908.82 pmol, 186.67 pL, 9.48 eq) at 20 °C under N2in one portion. The mixture was stirred at 20 °C for 1.5 h. LCMS showed desired mass detected. LC-MS: RT = 3.822 min, for C 75 H 100 FN 20 O 17 , m / z calcd. 1570.75 [M - Boc-4tBu + 2H] 2+ , m / z found 786.20, LCMS conditions: Method 12. The mixture was filtered and the filtrate was diluted with t-BuOMe (1000 mL) to give a precipitate which was centrifuged for 10 min (5000 R) to give the crude product. The residue was purified by preparative HPLC (column: Welch Xtimate C18 100*40 mm*3 pm; mobile phase: [water (TFA)-ACN]; B%: 30%-60%, 15 min). Compound M2 (1 mg, 5.98e-1 pmol, 94% purity) was obtained as a white solid. LCMS (ESI): RT = 3.748 min, for C 75 H 100 FN 20 O 17 , m / z calcd. 1570.75 [M - Boc-4tBu + 2H] 2+ , m / z found 786.20, LCMS conditions: Method 12. HPLC RT = 9.411 min, 94% purity.

[1503] Example 8. Synthesis (LPPS) Ml and M3 on Tag 5

[1504] 8.1 Synthesis of Tag 5

[1505] Tag 5 was prepared via 10 steps as shown in Scheme 6 below.

[1506] Scheme 6

[1507]

[1508] Tag 5-3 was prepared from methyl 3,4,5-trihydroxybenzoate as described in Horenstein et al.,“Synthesis of Unprotected (±)-Tunichrome An-1, a Tunicate Blood Pigment,” J. Am. Chem. Soc. 111(16): 6242-6246 (1989), which is hereby incorporated by reference in its entirety. Reductive amination of 2 equivalents of Tag 5-3 gave Tag 5-4 in 70% yield. Removal of the N-allyl group of Tag 5-4 with 1,3-dimethylbarbituric acid in the presence of Pd(PPh3)4 catalyst provided Tag 5-5, which was coupled with Tag 5-5a to form Tag 5-6. Fmoc deprotection of Tag 5-6 in piperidine provided Tag 5-7. N-Alloc protection of Tag 5-7 and subsequent removal of the TBS in Tag 5-8 and alkylation with C22-alkyl bromide in one pot resulted in Tag 5-9. Removal of the N-alloc group from Tag 5-9 provided the final product, Tag 5. To further improve, an attempt was made to replace N-Fmoc of Tag 5-5a with N-alloc; however, Tag 5-5a without N-Fmoc was difficult to dissolve in organic solvents. The amounts and yields for each step are summarized in Table 8 below.

[1509] Table 8. Amounts and yields of intermediates and Tag 5

[1510]

[1511]

[1512]

[1513] Step a: Synthesis of methyl 3,4,5-tris((tert-butyldimethylsilyl)oxy)benzoate (Tag 5-1)

[1514]

[1515] To a clear solution of methyl 3,4,5-trihydroxybenzoate (3 kg, 16.29 mol, 1 eq) in DMF (24 L) was added imidazole (4.99 kg, 73.31 mol, 4.5 eq) in one portion to give a clear solution. TBDMSC1 (8.59 kg, 57.02 mol, 6.99 L, 3.5 eq) was added in four portions (2.148 kg x 4) under N2over a period of 2 h at a rate sufficient to maintain the internal temperature between 25 and 30 °C. After the addition, a yellow solution was obtained. The reaction was stirred at 25 °C under N2for 16 h until a thick suspension was formed. TLC results indicated that the starting material was completely consumed and one major new spot was detected (petroleum ether: EtOAc = 9:1, Rf= 0.58). The reaction mixture was pumped into a transfer drum (2*20 L). A saturated NaHC03(15 L) solution was charged into a 50 L jacketed flask. 20 L (1 / 2 volume) of the reaction suspension was added to the above saturated NaHC03(15 L) solution with vigorous stirring and extracted with PE (6 L x 2). Another portion (1 / 2 volume) of the reaction mixture was treated as above. All the organic layers were combined, washed with brine (10 L) and dried over Na2S04. The organic layer was filtered and concentrated under reduced pressure (~45 °C, -0.8 atm) to give ~10 kg of a light green oil. The light green oil was dissolved in MeOH (28 L) and stirred at 0-4 °C (internal temperature) for 12 h. A white solid was precipitated. The product label 5-1 (7.8 kg, 14.80 mol, 90.86% yield) was obtained as a white solid via filtration and dried under reduced pressure. 1 HNMR: (400 MHz, CDC13) δ = 7.23 (s, 2H), 3.85 (s, 3H), 1.00 (s, 9H), 0.99-0.86 (m, 18H), 0.24 (s, 12H), 0.15 (s, 6H); LC-MS: (analyzed by Method 1) Rt= 3.183 min, m / z = 527.3 [M+H] + .

[1516] Step b: Synthesis of [3,4,5-tris[[tert-butyl(dimethyl)silyl]oxy]phenyl]methanol (Label 5-2)

[1517]

[1518] A solution of Tag 5-1 (4 kg, 7.59 mol, 1 equiv) in THF (15 L) was added to a suspension of LAH (216.09 g, 5.69 mol, 0.75 equiv) in THF (16 L) at a rate sufficient to maintain gentle reflux (internal temperature: 66 °C). The reaction mixture was stirred at an internal temperature of 66 °C (oil bath temperature: 77 °C) for 3 h. TLC indicated that Tag 5-1 was completely consumed and a new spot was formed (petroleum ether: ethyl acetate = 10:1, Rf = 0.41). The reaction mixture was cooled (internal temperature: 0 °C) and diluted with 12 L of methyl tert-butyl ether (MTBE). The reaction mixture was stirred rapidly and 216 mL of water was added dropwise until the color changed from grey to white, followed by 216 mL of NaOH solution (wt%, 15%). The resulting mixture was stirred for 10 min. Water (620 mL) and silica gel (500 g) were added sequentially to the reaction mixture. The heterogeneous mixture was filtered through celite and the resulting filtrate was concentrated in vacuo to give a colorless oil. Further vacuum was pulled through an oil pump to remove solvent residue and give Tag 5-2 as a white solid (3.48 kg, 6.84 mol, 90.1% yield, 98% purity). 1 H NMR (400 MHz, CDC13) δ = 6.50 (s, 2H), 4.49 (s, 2H), 1.49 (br s, 1H), 1.00 (s, 9H), 0.95 (s, 18H), 0.21 (s, 12H), 0.13 (s, 6H); LC-MS: (analyzed by Method 1) Rt = 3.25 min, m / z = 481.3 [M+H-H20] + .

[1519] Step c: Synthesis of 3,4,5-tris[[tert-butyl(dimethyl)silyl]oxy]benzaldehyde (Tag 5-3)

[1520]

[1521] To a suspension of PCC (1.58 kg, 7.32 mol, 1.05 equiv) in DCM (12 L) was added a solution of Tag 5-2 (3.48 kg, 6.98 mol, 1 equiv) in DCM (16 L) at 0 °C. The reaction mixture was stirred at 20 °C for 20 h. TLC indicated that the reactant Tag 5-2 was consumed completely and one major new spot was detected (petroleum ether: ethyl acetate = 10:1, PMA, Rf = 0.61). MTBE (12 L) and silica gel (800 g) were added to the reaction mixture sequentially, which was then stirred for 10 min and filtered through celite. The filtrate was concentrated in vacuo; the resulting dark brown oil was diluted with petroleum ether (15 L) and passed through a short silica / celite pad (800 g / 400 g) and concentrated to give Tag 5-3 (3.13 kg, 6.30 mol, 90.34% yield) as a light yellow solid. 1 H NMR (400 MHz, CDC13) δ = 9.74 (s, 1H), 7.04 (s, 2H), 1.00 (s, 9H), 0.96 (s, 18H), 0.26 (s, 12H), 0.16 (s, 6H). LC-MS: (analyzed by Method 1) Rt = 3.550 min, m / z = 497.4 [M+H] + Tag 5-3 was used directly in the next step without purification.

[1522] Step d: Synthesis of N,N-bis[[3,4,5-tris[[tert-butyl(dimethyl)silyl]oxy]phenyl]methyl]pro-2-en-1-amine (Tag 5-4)

[1523]

[1524] To a solution of Tag 5-3a (240 g, 2.51 mol, 98% purity, 1 eq) in THF (8 L) and MeOH (12.5 L) was added NaOAc (721.82 g, 8.80 mol, 3.5 eq) and the mixture was stirred under N2for 30 min, then a solution of Tag 5-3 (1.40 kg, 2.77 mol, 98% purity, 1.1 eq) in THF (1.5 L) and HOAc (3.02 kg, 50.28 mol, 2.88 L, 20 eq) were added sequentially at 20 °C. The reaction mixture was stirred for 30 min. A solution of NaBH3CN (157.98 g, 2.51 mol, 1 eq) in THF (1.5 L) was added dropwise to the reaction mixture over a period of 30 min, then the reaction was stirred at 25 °C for 30 min. An additional solution of Tag 5-3 (1.66 kg, 3.27 mol, 98% purity, 1.3 eq) in THF (1.5 L) was added and the reaction was stirred for 30 min. An additional solution of NaBH3CN (236.98 g, 3.77 mol, 1.5 eq) in THF (1.5 L) was added dropwise to the reaction mixture over a period of 1 h, then the reaction was stirred at 25 °C for 12 h. TLC indicated that the reactant Tag 5-3 was consumed and one major spot appeared (petroleum ether / ethyl acetate = 20 / 1, staining reagent: PMA, Rf= 0.66). Two impurities, Tag 5-2 and Tag 5-4', were also detected. The reaction was quenched by the addition of saturated aqueous NaHC03solution (18 L, 3.6 kg NaHC03suspended in 16 L H20) and then the separated aqueous solution was extracted with MTBE (12 L*2). The combined organic layers were washed with brine (8 L), dried over Na2S04, filtered, and concentrated under reduced pressure to give the crude product as a colorless oil (~3.8 kg), which was identified by HNMR (ratio of 4:2:4' = 1.0:0.57:0.28). The crude material was mixed with silica gel (4.5 kg) and purified by column chromatography using petroleum ether / ethyl acetate = 1 / 0 to 100 / 10 to give Tag 5-4 (1.88 kg, 1.73 mol, 71.2% yield based on 3a). 1HNMR (400 MHz, CDC13) δ = 6.50 (s, 4H), 6.02 - 5.71 (m, 1H), 5.21 (d, J = 17.6 Hz, 1H), 5.12 (d, J = 10.0 Hz, 1H), 3.37 (br s, 4H), 2.98 (br d, J = 4.8 Hz, 2H), 0.99 (s, 18H), 0.93 (s, 36H), 0.20 (s, 24H), 0.11 (s, 12H). LC-MS: (analyzed by Method 1) Rt = 4.313 min, m / z = 1018.6 [M+H] + .

[1525] Step e: Synthesis of 1-[3,4,5-tris[[tert-butyl(dimethyl)silyl]oxy]phenyl]-N-[[3,4,5- tris[[tert-butyl(dimethyl)silyl]oxy]phenyl]methyl]methane (Tag 5-5)

[1526]

[1527] To a solution of 1,3-dimethylbarbituric acid (CAS Number: 769-42-6, 885.13 g, 5.67 mol, 3.2 eq) in DCM (12.5 L) was added a solution of Tag 5-4 (1.9 kg, 1.77 mol, 95% purity, 1 eq) in DCM (4 L). The reaction was degassed and purged with N2 three times, and then Pd(PPh3)4 (61.41 g, 53.15 mmol, 0.03 eq) was added. The reaction mixture was degassed and purged with N2 three times, and stirred under N2 atmosphere for 4 hours at an internal temperature of 39 °C (oil bath temperature: 45 °C). TLC indicated that the reactant Tag 5-4 was completely consumed and one new spot was formed (petroleum ether: ethyl acetate = 10:1, NH3.H2O (0.5%, v / v), ninhydrin, Rf = 0.62). The reaction mixture was cooled to 10 °C and quenched by adding NH3.H2O (400 mL, wt%: 25%). White solid had precipitated. Mercaptosilica gel (350 g), silica gel (700 g) and Na2SO4 (1000 g) were added to the reaction, and the reaction mixture was stirred for 30 min. The reaction mixture was filtered through a plug of celite (500 g) to give Tag 5-5 (1.69 kg, 1.64 mol, 92.6% yield, 95% purity) as a yellow oil, which was dissolved in DCM (10 L) and used directly without purification. 1H NMR (400 MHz, CDC13) δ (ppm) = 6.46 (s, 4H), 3.59 (br s, 4H), 0.99 (s, 18H), 0.96 - 0.87 (m, 36H), 0.20 (s, 24H), 0.12 (s, 12H). LC-MS: (analyzed by Method 1) Rt = 4.21 min, m / z = 978.5 [M+H] + .

[1528] Step f: Synthesis of 9H-fluoren-9-ylmethyl N-[[4-[2-[bis[[3,4,5-tris[[tert- butyl(dimethyl)silyl]oxy]phenyl]methyl]amino]-2-oxo-ethoxy]phenyl]-(2,4- dimethoxyphenyl)methyl]carbamate (Tag 5-6)

[1529]

[1530] To a solution of Tag 5-5a (913.17 g, 1.69 mol, 1.13 eq) in DCM (6 L) was added HOAt (245.71 g, 1.81 mol, 252.53 mL, 1.2 eq) and DIC (227.82 g, 1.81 mol, 279.53 mL, 1.2 eq) and the reaction mixture was stirred at 25 °C for 30 min. Then a solution of Tag 5-5 (1.55 kg, 1.50 mol, 95% purity, 1 eq) in DCM (10 L) was added and the reaction mixture was stirred at 25 °C for 8 h. TLC indicated that the reactant Tag 5-5 was consumed and one major new spot was detected (petroleum ether: ethyl acetate = 5:1, 4% DCM to improve solubility, Rf = 0.39). The reaction mixture was quenched by adding saturated NaHC03(5 L*3) and then extracted with DCM (3 L*2). The combined organic layers were washed with brine (2.5 L), dried over Na2S04, filtered and concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (Si02, petroleum ether / ethyl acetate = 20 / 1 to 1 / 1) to provide Tag 5-6 (2.2 kg, 1.25 mol, 82.85% yield, 85% purity) as a white solid. 1H NMR (400 MHz, CDC13) δ (ppm) = 7.77 (br d, J = 7.3 Hz, 2H), 7.59 (br d, J = 6.4 Hz, 2H), 7.39 (br d, J = 6.6 Hz, 2H), 7.31 (br d, J = 6.4 Hz, 2H), 7.24 - 7.14 (m, 1H), 7.11 - 7.08 (m, 2H), 6.79 (br d, J = 8.5 Hz, 2H), 6.50 - 6.47 (m, 2H), 6.38 (m, 2H), 6.31 (s, 2H), 6.06 - 5.99 (m, 1H), 5.86 - 5.72 (m, 1H), 4.66 (s, 2H), 4.46 - 4.38 (m, 4H), 4.29 - 4.19 (m, 3H), 3.81 (s, 3H), 3.74 (s, 3H), 1.00 (s, 18H), 0.92 (s, 36H), 0.19 (s, 24H), 0.13 (s, 12H). LC-MS: (analyzed by Method 1) Rt = 5.39 min, m / z = 251.0 [M+6H] 6+ .

[1531] Step g: Synthesis of 2-[4-[amino-(2,4-dimethoxyphenyl)methyl]phenoxy]-N,N- bis[[3,4,5-tris[[tert-butyl(dimethyl)silyl]oxy]phenyl]methyl]acetamide (Tag 5-7)

[1532]

[1533] To a solution of Tag 5-6 (2.2 kg, 1.25 mol, 85% purity, 1 eq) in THF (16 L) was added piperidine (530.63 g, 6.23 mol, 615.44 mL, 5 eq). The reaction mixture was degassed and purged with N2 for 3 times, and then the mixture was stirred at 60 °C under N2 atmosphere for 2 hours. TLC showed one new major spot (petroleum ether: ethyl acetate = 1:1, Rf = 0.16) formed. The reaction mixture was concentrated to give the crude as a yellow oil (1.8 kg, 1.18 mol, 94.91% yield, 84% purity), which was dissolved in DCM (12 L), washed with saturated ammonium chloride (10 L*2), water (10 L), and brine (10 L). Then dried over Na2S04and filtered to give a solution, which was concentrated to give Tag 5-7 (1.8 kg, 94.9% yield, 84% purity). Tag 5-7 was used directly for the next step without purification. 1NMR (400 MHz, CDC13) δ (ppm) 7.27 (br d, J = 8.6 Hz, 2H), 7.12 (d, J = 8.0 Hz, 1H), 6.81 (d, J = 8.6 Hz, 2H), 6.44 (s, 2H), 6.39 (s, 2H), 6.31 (s, 2H), 5.38 (s, 1H), 4.66 (s, 2H), 4.43 (s, 2H), 4.29 (s, 2H), 3.79 - 3.76 (m, 6H), 1.00 (s, 18H), 0.92 (s, 36H), 0.25 - 0.16 (m, 24H), 0.13 (s, 12H). LC-MS: (analyzed by Method 1) Rt = 4.30 min, m / z = 1260.7 [M+H-NH3] + .

[1534] Step h: Synthesis of N-[[4-[2-[bis[[3,4,5-tris[[tert-butyl(dimethyl)silyl]oxy]phenyl]methyl]amino]-2-oxo- ethoxy]phenyl]-(2,4-dimethoxyphenyl)methyl]alanine allyl ester (Tag 5-8)

[1535]

[1536] To a solution of Tag 5-7 (1.8 kg, 1.18 mol, 84% purity, 1 eq) in DCM (20 L) was added DIEA (305.78 g, 2.37 mol, 412.10 mL, 2 eq) and allyl chloroformate (285.17 g, 2.37 mol, 250.15 mL, 2 eq). The reaction mixture was stirred at 25 °C for 12 h. TLC showed the starting material was completely consumed and one major new spot was detected (petroleum ether: ethyl acetate = 5:1, Rf = 0.45). The reaction mixture was quenched by the addition of saturated NH4Cl (5 L) and then extracted with DCM (5 L*2). The combined organic layers were washed with saturated NH4Cl (5 L*2), brine (2 L), dried over Na2S04, filtered and concentrated under reduced pressure to give the crude product as a yellow oil, which was purified by column chromatography (Si02, petroleum ether / ethyl acetate = 20:1 ~ 3:1, 2% DCM was added to the elution solvent to promote dissolution) to provide Tag 5-8, 1.59 kg in total from two portions (yield 97%, purity >98%). Fraction 1 (1.35 kg, 991.02 mmol, 99% purity) 83.78% yield. Fraction 2 (380 g, ~80% purity) was further purified to provide 236 g, 160.02 mmol, 98.1% purity as a light yellow oil. 1H NMR (400 MHz, CDC13) δ (ppm) = 7.10 (br d, J = 8.8 Hz, 3H, 26, 28, 37), 6.77 (d, J = 8.6 Hz, 2H), 6.52-6.42 (m, 2H), 6.37 (s, 2H), 6.30 (s, 2H), 6.01-5.78 (m, 3H), 5.30 (br dd, J = 1.6, 17.2 Hz, 1H), 5.21 (br dd, J = 1.3, 10.4 Hz, 1H), 4.64 (s, 2H), 4.61-4.58 (m, 2H), 4.42 (s, 2H), 4.26 (s, 2H), 3.80 (s, 3H), 3.71 (s, 3H), 0.99 (s, 18H), 0.95-0.88 (m, 36H), 0.18 (s, 24H), 0.12 (s, 12H). LC-MS: (Method 2) Rt = 2.22 min, m / z = 1379.74 [M+H30] + ; m / z = 2741.2 [2M+H30] +

[1537] Step i: Synthesis of N-[[4-[2-[bis[[3,4,5-tris(icosan-1,3,5,7,9,11,13,15,17,19,21- undecyloxy)phenyl]methyl]amino]-2-oxo-ethoxy]phenyl]-(2,4-dimethoxyphenyl)methyl] carbamic acid allyl ester (Tag 5-9)

[1538]

[1539] The reaction was performed in 50 g scale in parallel for 5 batches. To a mixture of Tag 5-8 (50.0 g, 33.80 mmol, 1 eq) and 1-bromodocosane (86.2 g, 206.19 mmol, 6.1 eq) was added K2CO3 (42.1 g, 304.2 mmol, 9 eq), 18-crown-6 (5.4 g, 20.3 mmol, 0.6 eq) and KF (17.67 g, 304.21 mmol, 9 eq). The reaction mixture was degassed and purged with N2 for 3 times, and then the mixture was stirred at 85 °C under N2 atmosphere for 24 hours. LCMS indicated Tag 5-8 was consumed. TLC showed one new major spot (THF / petroleum ether = 1:5, Rf = 0.35). The reaction mixture was suspended in THF (400 mL), filtered through a plug of silica gel to remove inorganic salts, and the filtrate was concentrated to give a residue. The residue was subjected to flash silica gel chromatography (petroleum ether / THF = 1:1) to give Tag 5-9 (50.0 g, 33.80 mmol, 99% yield) as a white solid. 330 g Silica Flash Column, 0% to 50% THF / petroleum ether gradient eluent: 120 mL / min) purification to give Tag 5-9 (76 g, 30.06 mmol, 88.94% yield).

[1540] Four additional reactions were performed and purified as described above. The five batches of purified Tag 5-9 were then combined and triturated with MeOH (200 mL, 70 °C, 2 hours), then cooled to 20 °C and filtered to give a total of 386 g of Tag 5-9 (purity: ~97.7%). 1 H NMR (400 MHz, CDC13) δ (ppm) = 7.12 (br d, J = 8.5 Hz, 3H), 6.81 (br d, J = 8.6 Hz, 2H), 6.50 - 6.43 (m, 2H), 6.40 (s, 2H), 6.30 (s, 2H), 6.00 (br d, J = 8.8 Hz, 1H), 5.97 - 5.87 (m, 1H), 5.86 - 5.72 (br d, J = 8.6 Hz, 1H), 5.38 - 5.26 (m, 1H), 5.25 - 5.08 (m, 1H), 4.71 (s, 2H), 4.59 (br d, J = 5.4 Hz, 2H), 4.50 (s, 2H), 4.40 (s, 2H), 3.93 (br t, J = 5.7 Hz, 4H), 3.90 - 3.81 (m, 8H), 3.80 (s, 3H), 3.72 (s, 3H), 1.82 - 1.68 (m, 12H), 1.53 - 1.20 (m, 228H), 0.89 (t, J = 6.8 Hz). LC-MS (Method 3): Rt = 11.33 min, m / z = 2544.7 / 2545.99 [M+H3O] + NMR data was matched to the target structure using HSQC. The chemical shift of C55 was 69.30 ppm and HMBC: H55 was correlated to C13.

[1541] Step j: Synthesis of 2-[4-[amino-(2,4-dimethoxyphenyl)methyl]phenoxy]-N,N- bis[[3,4,5-tridocosyloxy)phenyl]methyl]acetamide (Tag 5)

[1542]

[1543] To a solution of 1,3-dimethylhexahydropyrimidine-2,4,6-trione (15.3 g, 97.7 mmol, 3.2 eq) in DCM (560 mL) was added tag 5-9 (80 g, 30.54 mmol, 1 eq), the reaction was degassed and purged with N2 three times, and then Pd(PPh3)4 (1.41 g, 1.22 mmol, 0.04 eq) was added. The mixture was degassed and purged with N2 three times and stirred at 45°C under a N2 atmosphere for 6 hours. TLC showed that tag 5-9 was consumed, and a major new spot was detected (petroleum ether:THF=1:1, Rf=0.14, PMA). The reaction was concentrated, and the residue was triturated in MeOH (1 L) at 70°C for 1 h and then cooled to 20°C. The mixture was filtered to provide a crude product, which was further ground in ACN (1 L, 70° C., 1 hour), cooled to 20° C., filtered, and washed sequentially with ACN (400 mL), MeOH (400 mL), EtOAc (400 mL), and petroleum ether (400 mL), then dried under vacuum to give tag 5 (74.8 g, 29.32 mmol, 95.99% yield, 95.79% purity) as an off-white solid. Another four replicate reactions were performed as described above to produce total tag 5 (303.9 g, 95.5% purity). 1 H NMR (400MHz, CDCl3) δ (ppm) 7.14-7.25 (m, 2H), 7.03 (br d, J = 7.75Hz, 1H), 6.76 (brd, J = 8.25Hz, 2H), 6.34 (br d, J = 12.38Hz, 4H), 6.22 (br s, 2H), 5.30 (br s,1H),4.65(s,2H),4.23-4.50(m,4H),3.63-3.96(m,18H),1.67(br d,J=5.50Hz,12H),1.37(br s,12H),1.18(s,216H),0.81(br t,J=6.44Hz,18H)ppm. 13C NMR (101 MHz, CDC13) δ = 168.42, 159.83, 157.67, 156.74, 153.62, 153.27, 137.65 - 137.58, 137.58 - 137.50, 131.79, 131.09, 128.17, 128.07, 114.29, 107.04, 105.04, 104.00, 98.74, 77.25, 73.48, 73.43, 69.18, 69.14, 67.75, 55.37, 55.34, 52.88, 49.67, 48.50, 31.97, 30.42, 29.77, 29.71, 29.57, 29.51, 29.42, 26.23, 22.74, 14.16. LC-MS (Method 3): Rt = 11.20 min, m / z = 2444.02 [M+H] + .

[1544] 8.2 Characterization of Tag 5

[1545] Characterization of Label 5 is shown in Table 9.

[1546]

[1547] Table 9. Characterization of Label 5

[1548]

[1549] IR data

[1550] A powder sample of Label 5 was placed directly on the surface of the ATR, while the crystal (Znse) surface of the ATR was completely covered by the sample. A sample compression rod was used to compress the sample to evenly distribute the sample and bring it into full contact with the crystal surface of the ATR. The IR conditions used to collect the IR data are shown in Table 10.

[1551] Table 10. IR conditions

[1552]

[1553]

[1554] λ = 2915.54 cm -1 Indicative of C-H (CH3) bond; λ = 2848.94 cm -1 Indicative of C-H (CH2) bond; λ = 1647.99 cm -1 Indicative of C=0 (C=0NR2) bond; λ = 1505.99 cm -1 Indicative of C-N bond; λ = 1466.52 cm -1Indicates C=C(Ph) bond; l = 1379.34 cm -1 Indicates C-H(CH3) bond; and l = 1174.13 cm -1 Indicates C-O-C bond.

[1555] Determination of moisture content

[1556] The sample was weighed into a dry and clean vial. The vial was sealed. After the instrument reached the preset temperature (150 °C), the vial was placed in the oven. After the end of the Karl Fischer reaction, the water content of the sample was obtained (Table 11). The moisture content was determined to be 0.26%.

[1557] Table 11. Conditions used to determine the moisture content in the sample

[1558] Parameters Values Extraction time 30s Start drift 25 pg / min Stop criteria Drift relative to stop point 5 pg / min Anolyte solution HYDRANAL / Coulomat AK Catholyte solution HYDRANAL / Coulomat CG-K Agitation speed 45％ Atmosphere Dry air Gas flow 80 mL / min Vial volume 10L

[1559] Ion chromatography

[1560] The chromatography conditions used are shown in Tables 12-13. The following ion contents were determined: Cl - : 0.01%; Br - : 0.01%; F - : <0.01; CO3 2- : 0.02%.

[1561] Table 12. F ion chromatography conditions

[1562]

[1563] Table 13. Cl-, Br-, CO3 2- ion chromatography conditions

[1564] Instrument name Thermo ICS-6000 Mobile phase Isocratic 25 mM KOH Flow rate 1.0 mL / min Guard column Dionex IonPac AG11-HC, 4*50 mm Column Dionex IonPac AS11-HC, 4*250 mm Column temperature 30℃ Cell temperature 35℃ Detection mode Conductivity detection Suppressor current 62 mA Injection volume 25 pL Analysis time 10 min

[1565] Differential scanning calorimeter

[1566] The sample (1-8 mg) was placed in a high-pressure crucible and sealed with a matching tool. The prepared sample was placed in the corresponding position of the instrument, the parameters in the software were selected and the sequential run was started (Table 14).

[1567] The first peak (melting point, endotherm) was observed at 76.3 °C. The second peak (C22 should be volatile, endotherm) at ~ 371 °C.

[1568] Table 14. Differential scanning calorimeter (DSC) conditions

[1569]

[1570] Determination of residual solvent (RS)

[1571] Residual solvents (GC and HS conditions) are shown in Table 15. Instrument: SHIMADZU-GC2030 & HS-20. The column used for chromatography was a DB-624 (25 m x 0.2 mm, ID 1.12 μm). P / N: 128-1324. Split ratio: 30. Control mode: Linear velocity. Flow rate: 1.2 mL / min. Carrier gas: N2. Detector: Flame ionization detector (FID). All residual solvents were found to be below the limit of quantification (LOQ).

[1572] Table 15. Residual solvent conditions

[1573]

[1574]

[1575] Solubility test

[1576] Tag 5 (10 mg) was added to a solvent (100 μL) and shaken at different temperatures (Table 16). Tag 5 was found to be soluble in DCM and THF, PE / DCM, and PE / THF at all ratios, but insoluble in DMF and DMF / DCM, and insoluble in DMSO and DMSO / DCM (see Table 16).

[1577] Table 16. Solubility of Tag 5 in organic solvents

[1578]

[1579]

[1580] Stability testing

[1581] The stability test of Tag 5 was performed at pH = 2 for 2 hours using the following procedure. Tag 5 (1 g, 98.01% purity) was added to THF (20 mL) in a 50 mL flask with a magnetic stirrer, and the mixture was stirred for 5 min to dissolve the material. A solution of TFA in DCM (v / v=1:5, 2 mL) was added dropwise to the solution containing Tag 5 to reach pH 2, and the resulting solution was stirred continuously for 2 hours. The reaction was monitored by LCMS, which showed more than five peaks. The conclusion was that Tag 5 was not very stable in acidic solution (pH = 2).

[1582] 8.3 Synthesis of (LPPS) GLP1 peptoid on Tag 5

[1583] GLP1 peptidomimetic (M1) was synthesized on tag 5. Figure 6 and Figures 10A-10B and are shown in Table 17. The amino acids used to prepare the peptoids are shown in Table 18.

[1584] Table 17

[1585]

[1586]

[1587]

[1588]

[1589]

[1590] Table 18

[1591]

[1592]

[1593] a GB2551945 to Andrews et al., which is hereby incorporated by reference in its entirety

[1594] b WO2022 / 056494 to Han et al., which is hereby incorporated by reference in its entirety

[1595] c “Synthesis of Tetrazole Analogues of Amino Acids Using Fmoc Chemistry: Isolation of Amino Free Tetrazoles and Their Incorporation into Peptides,” Tetrahedron Lett. 48(39): 7038-7041 (2007) to Sureshbabu et al., which is hereby incorporated by reference in its entirety

[1596] d “Chemical Control Over Immune Recognition: A Class of Antibody-Recruiting Small Molecules That Target Prostate Cancer,” J. Am. Chem. Soc. 131(47): 17090-17092 (2009) to Murelli et al., which is hereby incorporated by reference in its entirety

[1597] General procedure H for amide coupling to give N-Fmoc-tagged 5-nAA

[1598] To a mixture of Tag 5 or Tag 5-(n-1)AA (1 eq) in DCM (50 V) was added AA n (1.5 eq), HOAt (1.5 eq) and DIC (1.5 eq). The resulting suspension was stirred at 20 °C for 2 h. A small sample of the reaction mixture was suspended with ACN and centrifuged 2 times (at 6000 rpm for 3 min). The formed precipitate was dissolved in 1 drop of DCM, cleaved using TFA / TIPS / H2O (95 / 2.5 / 2.5), diluted with MeOH / H2O (2 / 1) and filtered. The filtrate was checked by LCMS to confirm the desired mass. The reaction was concentrated under reduced pressure to give a residue which was added to ACN / MeOH (2 / 1, 60 V) and stirred for 1 h. The suspension was filtered and washed with ACN (10 V) twice. The precipitate was collected and used directly in the next step without any further purification. The desired compound N-Fmoc-Tag 5-nAA was obtained as a white solid.

[1599] General procedure I for Fmoc deprotection to prepare Tag 5-nAA

[1600] A clear solution of N-Fmoc-Tag 5-nAA (1 eq) in 20% piperidine / THF (20 V) was stirred at 50 °C for 0.5 h. A small sample was suspended with CAN and centrifuged 2 times (at 6000 rpm for 3 min). The precipitate was collected, re-dissolved in 1 drop of DCM, cleaved using TFA / TIPS / H2O (95 / 2.5 / 2.5), diluted with MeOH / H2O (2 / 1) and filtered. The filtrate was checked by LCMS to confirm the reaction completion. The reaction was concentrated under reduced pressure to give a residue which was added to ACN / MeOH (2 / 1, 60 V) and stirred for 1 h. The suspension was filtered and washed with ACN (10 V) twice. The precipitate was collected and used directly in the next step without any further purification. The desired compound Tag 5-nAA was obtained as a white solid.

[1601] Step a: Synthesis of 9H-fluoren-9-ylmethyl N-[(1S)-1-[[[4-[2-[bis[[3,4,5-tris(icosan-1,3,5,7,9,11,13,15,17,19,21-undecyloxy)phenyl]methyl]amino]-2-oxo-ethoxy]phenyl]-(2,4-dimethoxyphenyl)methyl]aminocarbonyl]-4-(3,5-dimethylphenyl)butyl]carbamate (N-Fmoc-Tag 5-1AA)

[1602]

[1603] The reaction was performed using General Procedure H for amide coupling using Tag 5 (1 g, 409.15 pmol, 1 equiv), AA1 (272 mg, 0.62 mmol, 1.5 equiv), HOAt (84 mg, 0.62 mmol, 1.5 equiv) and DIC (77 mg, 0.62 mmol, 95.1 pL, 1.5 equiv). The reaction was monitored by LC-MS. N-Fmoc-Tag 5-1AA was obtained as a white solid (1.18 g, 344.59 pmol, 84.22% yield). LC-MS: RT = 5.17 min, for C 28 H 30 N2O3H, mass calcd 443.24, m / z found 443.2 [M-Tag + 2H] + .

[1604] Step b: Synthesis of (2S)-2-amino-N-[[4-[2-[bis[[3,4,5-tris(tetracosan-1,3,5,7,9,11,13,15,17,19,21-undecyloxy)phenyl]methyl]amino]-2-oxo-ethoxy]phenyl]-(2,4-dimethoxyphenyl)methyl]-5-(3,5-dimethylphenyl)pentanamide (Tag 5-1AA)

[1605]

[1606] The reaction was performed using General Procedure I for Fmoc deprotection. A clear solution of compound N-Fmoc-Tag 5-1AA (1.16 g, 404.24 pmol, 1 equiv) in 20% piperidine / THF (20 mL) was stirred at 50 °C for 0.5 h. The reaction was monitored by LC-MS. Tag 5-1AA was obtained as a white solid (1.06 g, 400.40 pmol, 99.05% yield). LC-MS: RT = 2.10 min, for C 13 H 20 N2OH, mass calcd 221.17, m / z found 221.1 [M-Tag + 2H] + .

[1607] Step c: Synthesis of 9H-fluoren-9-ylmethyl N-[(1 S)-1 -[[4-[4-(4-azidobutoxy)-2- ethyl-phenyl]phenyl]methyl]-2-[[(1 S)-1 -[[[4-[2-[bis[[3,4,5-tris(doxa-1,3,5,7,9,11,13,15,17,19,21 -undecyloxy)phenyl]methyl]amino]-2-oxo-ethoxy]phenyl]-(2,4- dimethoxyphenyl)methyl]aminocarbonyl]-4-(3,5-dimethylphenyl)butyl]amino]-2- oxo-ethyl]carbamate (N-Fmoc-tag 5-2AA)

[1608]

[1609] The reaction was performed using the general procedure H for amide coupling. To a mixture of compound tag 5-1 AA (1.06 g, 400.40 pmol, 1 equiv) in DCM (40 mL) was added AA2 (363 mg, 600.6 pmol, 1.5 equiv), HOAt (81.7 mg, 0.6 mmol, 84 pL, 1.5 equiv) and DIC (76 mg, 0.6 mmol, 93 pL, 1.5 equiv). The resulting suspension was stirred at 20 °C for 2 h. N-Fmoc-tag 5-2AA was obtained as a white solid (1.25 g, 386.51 pmol, 96.53% yield). LC-MS (ESI): RT = 6.72 min, for C 49 H 54 N6O5H, mass calcd. 807.43, m / z found 807.4 [M - tag + 2H] + .

[1610] Step d: Synthesis of (2S)-2-[[(2S)-2-amino-3-[4-[4-(4-azidobutoxy)-2-ethyl- phenyl]phenyl]propanoyl]amino]-N-[[4-[2-[bis[[3,4,5-tris(doxa-1,3,5,7,9,1 1,13,15,17,19,21 - undecyloxy)phenyl]methyl]amino]-2-oxo-ethoxy]phenyl]-(2,4-dimethoxyphenyl)methyl]- 5-(3,5-dimethylphenyl)pentanamide (tag 5-2AA)

[1611]

[1612] The reaction was performed using the general procedure I for Fmoc deprotection. A clear solution of compound N-Fmoc-Tag 5-2AA (1.25 g, 386.51 pmol, 1 equiv) in 20% piperidine / THF (25 mL) was stirred at 50 °C for 0.5 h. Tag 5-2AA was obtained as a white solid (1.15 g, crude). LC-MS: RT = 4.46 min, for C 34 H 44 N6O3H, mass calcd 585.36, m / z found 585.4 [M-Tag + 2H] + .

[1613] Step e: Synthesis of (3S)-4-[[(1S)-1-[[4-[4-(4-azidobutoxy)-2-ethyl- phenyl]phenyl]methyl]-2-[[(1S)-1-[[[4-[2-[bis[[3,4,5-tris(tetracosan-1,3,5,7,9,11,13,15,17,19,21-undecyloxy)phenyl]methyl]amino]-2-oxo- ethoxy]phenyl]-(2,4-dimethoxyphenyl)methyl]aminocarbonyl]-4-(3,5- dimethylphenyl)butyl]amino]-2-oxo-ethyl]amino]-3-(9H-fluoren-9- ylmethoxycarbonylamino)-4-oxo-butyric acid tert-butyl ester (N-Fmoc-Tag 5-3AA)

[1614]

[1615] The reaction was performed using the general procedure H for amide coupling. To a mixture of compound Tag 5-2AA (1.15 g, 381.83 pmol, 1 equiv) in DCM (60 mL) was added AA3 (235.5 mg, 0.57 mmol, 1.5 equiv), HOAt (78 mg, 0.57 mmol, 80.1 pL, 1.5 equiv) and DIC (72.3 mg, 0.57 mmol, 88.7 pL, 1.5 equiv). The resulting suspension was stirred at 20 °C for 2 h. N-Fmoc-Tag 5-3AA was obtained as a white solid (1.3 g, 381.76 pmol, 99.98% yield).

[1616] LCMS conditions (CR): Reverse phase LCMS was performed using a NanoChrom ChromCore 120 C18 3 pm, 3.0*30 mm column eluting with a gradient of 10% to 80% ACN containing 0.02% TFA (solvent B) and water containing 0.04% TFA (solvent A) at a flow rate of 1.5 mL / min.

[1617] LC-MS: RT = 6.25 min, for C53 H 59 N7O8H, mass calcd 922.45, m / z found 922.4 [M-tag-tBu+3H] + .

[1618] LCMS conditions (BF): Reverse phase LCMS was performed using an Xtimate C18 2.1*30mm 3pm column eluting with a gradient of 10% to 80% ACN containing 0.02% TFA (solvent B) and water containing 0.04% TFA (solvent A) at a flow rate of 1.5 mL / min.

[1619] Step f: Synthesis of tert-butyl (3S)-3-amino-4-[[(1S)-1-[[4-[4-(4-azidobutoxy)-2- ethyl-phenyl]phenyl]methyl]-2-[[(1S)-1-[[[4-[2-[bis[[3,4,5-tris(tetracosan-1,3,5,7,9,11,13,15,17,19,21-undecyloxy)phenyl]methyl]amino]-2-oxo-ethoxy]phenyl]-(2,4- dimethoxyphenyl)methyl]aminocarbonyl]-4-(3,5-dimethylphenyl)butyl]amino]-2- oxo-ethyl]amino]-4-oxo-butyrate (Tag 5-3AA)

[1620]

[1621] The reaction was performed using the general procedure I for Fmoc deprotection. A clear solution of compound N-Fmoc-Tag 5-3AA (1.30 g, 381.76 pmol, 1 equiv) in 20% piperidine / THF (26 mL) was stirred at 50 °C for 0.5 h. Compound Tag 5-3AA was obtained as a white solid (1.2 g, 377.00 pmol, 98.75% yield).

[1622] LC-MS: RT = 4.646 min, for C 38 H 49 N7O6H, mass calcd 700.38, m / z found 700.5 [M-tag-tBu+3H] + ; RT = 5.065 min, for C 42 H 57 N7O6Na, mass calcd 778.43, m / z found 778.5 [M-tag+2H+Na] + .

[1623] Step g: Synthesis of tert-butyl (3S)-4-[[(1 S)-1 -[[4-[4-(4-azidobutoxy)-2- ethyl-phenyl]phenyl]methyl]-2-[[(1 S)-1 -[[[4-[2-[bis[[3,4,5-tris(doxazolidin- 13-yl)phenyl]methyl]amino]-2-oxo-ethoxy]phenyl]-(2,4-dimethoxyphenyl) methyl]amino carbamoyl]-4-(3,5-dimethylphenyl)butyl]amino]-2-oxo- ethyl]amino]-3-[[(2S)-3-[tert-butyl(dimethyl)silyl]oxy-2-(9H-fluoren-9- ylmethoxycarbonylamino)propanoyl]amino]-4-oxo-butyric acid (N-Fmoc-Tag5-4AA)

[1624]

[1625] The reaction was performed using the general procedure H for amide coupling. To a mixture of compound Tag5-3AA (1.1 g, 345.59 pmol, 1 equiv) in DCM (50 mL) was added TBS-AA4 (229 mg, 0.52 mmol, 1.5 equiv), HOAt (70.5 mg, 0.52 mmol, 1.5 equiv) and DIC (65.4 mg, 0.52 mmol, 80.3 pL, 1.5 equiv). The resulting suspension was stirred at 20 °C for 2 h. The reaction was monitored by LC-MS. Compound N-Fmoc-Tag5-4AA (1.06 g, 293.91 pmol, 85.05% yield) was obtained as a white solid.

[1626] LC-MS: RT = 6.01 min for C 56 H 64 N8O 10 H, mass calcd. 1009.48, m / z found 1009.4 [M-Tag-tBu-TBS+4H] + LCMS conditions (BF): Reverse phase LCMS was performed using an Xtimate C18 2.1*30 mm 3 pm column eluting with a gradient of 10% to 80% ACN containing 0.02% TFA (solvent B) and water containing 0.04% TFA (solvent A) at a flow rate of 1.5 mL / min.

[1627] Step h: Synthesis of tert-butyl (3S)-3-[[(2S)-2-amino-3-[tert-butyl(dimethyl)silyl]oxy- propionyl]amino]-4-[[(1 S)-1 -[[4-[4-(4-azidobutoxy)-2-ethyl-phenyl]phenyl]methyl]-2- [[(1 S)-1 -[[[4-[2-[bis[[3,4,5-tris(doxazolidin-13-yl)phenyl]methyl]amino]-2-oxo- ethoxy]phenyl]-(2,4-dimethoxyphenyl)methyl]aminocarbamoyl]-4-(3,5- dimethylphenyl)butyl]amino]-2-oxo-ethyl]amino]-4-oxo-butanoate (Tag 5-4AA)

[1628]

[1629] A clear solution of compound N-Fmoc-Tag 5-4AA (1.05 g, 291.13 pmol, 1 eq) in 20% piperidine / THF (20 mL) was stirred at 50 °C for 0.5 h. Following the general precipitation procedure, the desired compound Tag 5-4AA was obtained as a white solid (985 mg, 291.05 pmol, 99.97% yield).

[1630] LC-MS: RT = 4.24 min for C 41 H 54 N8O8H, mass calcd 787.42, m / z found 787.4 [M - Tag - tBu-TBS + 4H] + ; RT = 5.10 min for C 47 H 68 N8O8SiH, mass calcd 901.50, m / z found 901.5 [M - Tag - tBu + 3H] + LCMS conditions (BF): Reverse phase LCMS was performed using an Xtimate C18 2.1*30 mm 3 pm column eluting with a gradient of 10% to 80% ACN containing 0.02% TFA (solvent B) and water containing 0.04% TFA (solvent A) at a flow rate of 1.5 mL / min.

[1631] Step i: Synthesis of tert-butyl (3S)-4-[[(1 S)-1 -[[4-[4-(4-azidobutoxy)-2- ethyl-phenyl]phenyl]methyl]-2-[[(1 S)-1 -[[[4-[2-[bis[[3,4,5-tris(tetracosan-1,3,5,7,9,1 1,13,15,17,19,21 -undecyloxy)phenyl]methyl]amino]-2-oxo-ethoxy]phenyl]-(2,4- dimethoxyphenyl)methyl]aminocarbonyl]-4-(3,5-dimethylphenyl)butyl]amino]- 2-oxo-ethyl]amino]-3-[[(2S)-3-[tert-butyl(dimethyl)silyl]oxy-2-[[(2S,3R)-3-[tert- butyl(dimethyl)silyl]oxy-2-(9H-fluoren-9-ylmethoxycarbonylamino)butanoyl]amino] propanoyl]amino]-4-oxo-butyric acid (N-Fmoc-tag5-5AA)

[1632]

[1633] To a mixture of compound tag5-4AA (985 mg, 291.05 pmol, 1 eq) in DCM (50 mL) was added TBS-AA5 (2S,3R)-3-[tert-butyl(dimethyl)silyl]oxy-2-(9H-fluoren-9- ylmethoxycarbonylamino)butanoic acid (199 mg, 0.44 mmol, 1.5 eq), HOAt (59.4 mg, 0.44 mmol, 1.5 eq) and DIC (55 mg, 0.44 mmol, 67.6 pL, 1.5 eq). The resulting suspension was stirred at 20 °C for 2 h. Following the general precipitation procedure, compound N-Fmoc-tag5-5AA was obtained as a white solid (1.05 g, 247.73 pmol, 94.39% yield).

[1634] LC-MS: RT = 5.91 min, for C 60 H 71 N9O 12 H, mass calcd. 1110.53, m / z found 1110.6 [M - tag - tBu-2TBS + 5H] + LCMS conditions (BF): Reverse phase LCMS was performed using an Xtimate C18 2.1*30 mm 3 pm column eluting with a gradient of 10% to 80% ACN containing 0.02% TFA (solvent B) and water containing 0.04% TFA (solvent A) at a flow rate of 1.5 mL / min.

[1635] Step j: Synthesis of tert-butyl (3S)-3-[[(2S)-2-amino-3-[tert-butyl(dimethyl)silyl]oxy- propionyl]amino]-4-[[(1S)-1-[[4-[4-(4-azidobutoxy)-2-ethyl-phenyl]phenyl]methyl]-2- [[(1S)-1-[[[4-[2-[bis[[3,4,5-tris(doxazolidin-13-yl)phenyl]methyl]amino]-2-oxo- ethoxy]phenyl]-(2,4-dimethoxyphenyl)methyl]aminocarbonyl]-4-(3,5- dimethylphenyl)butyl]amino]-2-oxo-ethyl]amino]-4-oxo-butanoate (Tag 5-5AA)

[1636]

[1637] A clear solution of compound N-Fmoc-Tag 5-5AA (1.05 g, 291.13 pmol, 1 equiv) in 20% piperidine / THF (20 mL) was stirred at 50 °C for 0.5 h. The desired compound Tag 5-5AA (0.83 g, 245.25 pmol, 84.24% yield) was obtained as a white solid following the general precipitation procedure. LC-MS: RT = 5.107 min, for C 51 H 75 N9O 10 SiH, mass calcd 1002.55, m / z found 1002.7 [M-Tag-tBu-TBS+4H] + LCMS conditions (CR): Reversed phase LCMS was performed using a NanoChrom ChromCore 120 C18 3 pm, 3.0*30 mm column eluting with a gradient of 10% to 80% ACN containing 0.02% TFA (solvent B) and water containing 0.04% TFA (solvent A) at a flow rate of 1.5 mL / min.

[1638] Step k: Synthesis of tert-butyl (3S)-4-[[(1 S)-1 -[[4-[4-(4-azidobutoxy)-2- ethyl-phenyl]phenyl]methyl]-2-[[(1 S)-1 -[[[4-[2-[bis[[3,4,5-tris(doxazolidin- 13,15,17,19,21 -undecyloxy)phenyl]methyl]amino]-2-oxo-ethoxy]phenyl]-(2,4- dimethoxyphenyl)methyl]aminocarbonyl]-4-(3,5-dimethylphenyl)butyl]amino]- 2-oxo-ethyl]amino]-3-[[(2S)-3-[tert-butyl(dimethyl)silyl]oxy-2-[[(2S,3R)-3-[tert- butyl(dimethyl)silyl]oxy-2-[[(2S)-2-(9H-fluoren-9-ylmethoxycarbonylamino)-3-(2- fluorophenyl)-2-methyl-propanoyl]amino]butanoyl]amino]propanoyl]amino]-4- oxo-butyric acid N-Fmoc-tag 5-6 AA

[1639]

[1640] To a mixture of compound Tag 5-5 AA (0.83 g, 245.25 pmol, 1 eq) in DCM (50 mL) was added AA6 (2S)-2-(9H-fluoren-9-ylmethoxycarbonylamino)-3-(2- fluorophenyl)-2-methyl-propanoic acid (154.3 mg, 367.9 pmol, 1.5 eq), HOAt (84 mg, 0.62 mmol, 1.5 eq) and DIC (78 mg, 0.62 mmol, 96 pL, 1.5 eq). The resulting suspension was stirred at 20 °C for 2 h. The desired compound N-Fmoc-Tag 5-6 AA (0.95 g, crude) was obtained as a white solid following the general precipitation procedure.

[1641] LC-MS: RT = 6.25 min for C 70 H 81 FN 10 O 13 H, mass calcd. 1289.61, m / z found 1290.8 [M - Tag - tBu-2TBS + 5H] + LCMS conditions (BV): Reverse phase LCMS was performed using a NanoChrom ChromCore 120 C18 3 pm, 3.0*30 mm column eluting with a gradient of 10% to 80% ACN containing 0.02% TFA (solvent B) and water containing 0.04% TFA (solvent A) at a flow rate of 1.5 mL / min.

[1642] LC-MS: RT = 6.25 min for C70 H 81 FN 10 O 13 H, mass calcd. 1289.61, m / z found 1289.5 [M-tag-tBu-2TBS+5H] + LCMS conditions (BF): Reverse phase LCMS was performed using an Xtimate C18 2.1*30mm 3pm column eluting with a gradient of 10% to 80% ACN with 0.02% TFA (solvent B) and water with 0.04% TFA (solvent A) at a flow rate of 1.5 mL / min.

[1643] Step 1 : Synthesis of tert-butyl (3S)-3-[[(2S)-2-[[(2S,3R)-2-[[(2S)-2-amino-3-(2- fluorophenyl)-2-methyl-propanoyl]amino]-3-[tert-butyl(dimethyl)silyl]oxy-butyryl]amino]- 3-[tert-butyl(dimethyl)silyl]oxy-propionyl]amino]-4-[[(1S)-1-[[4-[4-(4-azidobutoxy)-2- ethyl-phenyl]phenyl]methyl]-2-[[(1S)-1-[[[4-[2-[bis[[3,4,5-tris( docosa-1,3,5,7,9,11,13,15,17,19,21 -undecyloxy)phenyl]methyl]amino]-2-oxo- ethoxy]phenyl]-(2,4-dimethoxyphenyl)methyl]carbamoyl]-4-(3,5-dimethylphenyl)butyl]amino]- 2-oxo-ethyl]amino]-4-oxo-butyric acid tert-butyl ester (Tag 5-6AA)

[1644]

[1645] A clear solution of compound N-Fmoc-Tag 5-6AA (0.95 g, 237.43 pmol, 1 eq) in 20% piperidine / THF (20 mL) was stirred at 50 °C for 0.5 h. Following the general precipitation procedure, compound Tag 5-6AA was obtained as a white solid (0.85 g, 224.93 pmol, 94.74% yield).

[1646] LC-MS: RT = 4.64 min, for C 55 H 71 FN 10 O 11 H, mass calcd. 1067.54, m / z found 1067.7 [M-tag-tBu-2TBS+5H] +LCMS conditions (CR): Reverse phase LCMS was performed using a NanoChrom ChromCore 120 C18 3pm, 3.0*30 mm column eluting with a gradient of 10% to 80% ACN with 0.02% TFA (solvent B) and water with 0.04% TFA (solvent A) at a flow rate of 1.5 mL / min.

[1647] Step m: Synthesis of tert-butyl (3S)-4-[[(1S)-1-[[4-[4-(4-azidobutoxy)-2- ethyl-phenyl]phenyl]methyl]-2-[[(1S)-1-[[[4-[2-[bis[[3,4,5-tris(tetracosan-1,3,5,7,9,11,13,15,17,19,21-undecyloxy)phenyl]methyl]amino]-2-oxo- ethoxy]phenyl]-(2,4-dimethoxyphenyl)methyl]aminocarbonyl]-4-(3,5- dimethylphenyl)butyl]amino]-2-oxo-ethyl]amino]-3-[[(2S)-3-[tert-butyl(dimethyl)silyl]oxy-2- [[(2S,3R)-3-[tert-butyl(dimethyl)silyl]oxy-2-(9H-fluoren-9-ylmethoxycarbonylamino)butyryl] amino]propanoyl]amino]-4-oxo-butyric acid (N-Fmoc-tag 5-7AA)

[1648]

[1649] To a mixture of compound tag 5-6AA (0.85 g, 224.93 pmol, 1 eq) in DCM (50 mL) was added TBS-AA7 (2S,3R)-3-[tert-butyl(dimethyl)silyl]oxy-2-(9H-fluoren-9- ylmethoxycarbonylamino)butyric acid (1.02 g, 2.25 mmol, 10 eq), HOAt (306.16 mg, 2.25 mmol, 314.65 pL, 10 eq) and DIC (283.87 mg, 2.25 mmol, 348.30 pL, 10 eq). The resulting suspension was stirred at 40 °C for 12 h. Following the general precipitation procedure, compound N-Fmoc-tag 5-7AA (0.91 g, crude) was obtained as a white solid.

[1650] LC-MS: RT = 6.24 min, for C 74 H 88 FN 11 O 15 H, mass calcd 1390.65, m / z found 1390.5 [M - tag - tBu-3TBS + 6H] +LCMS conditions (BF): Reverse phase LCMS was performed using an Xtimate C18 2.1*30mm 3pm column eluting with a gradient of 10% to 80% ACN with 0.02% TFA (solvent B) and water with 0.04% TFA (solvent A) at a flow rate of 1.5 mL / min.

[1651] Step n: tert-butyl (3S)-3-[[(2S)-2-[[(2S,3R)-2-amino-3-[tert-butyl(dimethyl)silyl]oxy- butanoyl]amino]-3-[tert-butyl(dimethyl)silyl]oxy-propionyl]amino]-4-[[(1S)-1-[[4-[4- (4-azidobutoxy)-2-ethyl-phenyl]phenyl]methyl]-2-[[(1S)-1-[[[4-[2-[bis[[3,4,5-tris( docosan-1,3,5,7,9,11,13,15,17,19,21 -undecyloxy)phenyl]methyl]amino]-2-oxo- ethoxy]phenyl]-(2,4-dimethoxyphenyl)methyl]aminocarbamoyl]-4-(3,5- dimethylphenyl)butyl]amino]-2-oxo-ethyl]amino]-4-oxo-butyric acid; synthesis of compound molecular hydrogen (Tag 5-7AA)

[1652]

[1653] A clear solution of compound N-Fmoc-Tag 5-7AA (0.91 g, 238.10 pmol, 1 eq) in 20% piperidine / THF (3 mL) was stirred at 50 °C for 0.5 h. Following the general precipitation procedure, compound Tag 5-7AA was prepared as a white solid (0.85 g, 236.13 pmol, 99.17% yield).

[1654] LC-MS: RT = 5.72 min, for C 65 H 92 FN 11 O 13 SiH, mass calcd 1282.67, m / z found 1282.7 [M-Tag-tBu-2TBS+5H] + LCMS conditions (CR): Reverse phase LCMS was performed using a NanoChrom ChromCore 120 C18 3pm, 3.0*30mm column eluting with a gradient of 10% to 80% ACN with 0.02% TFA (solvent B) and water with 0.04% TFA (solvent A) at a flow rate of 1.5 mL / min.

[1655] Step o: Synthesis of tert-butyl (3S)-4-[[(1S)-1-[[4-[4-(4-azidobutoxy)-2- ethyl-phenyl]phenyl]methyl]-2-[[(1S)-1-[[[4-[2-[bis[[3,4,5-tris(tetracosan-1,3,5,7,9,11,13,15,17,19,21-undecyloxy)phenyl]methyl]amino]-2-oxo- ethoxy]phenyl]-(2,4-dimethoxyphenyl)methyl]aminocarbonyl]-4-(3,5- dimethylphenyl)butyl]amino]-2-oxo-ethyl]amino]-3-[[(2S)-3-[tert-butyl(dimethyl)silyl]oxy-2- [[2-(9H-fluoren-9-ylmethoxycarbonylamino)acetyl]amino]butyryl]amino]propanoyl] amino]-4-oxo-butyric acid tert-butyl ester (N-Fmoc-tag 5-8AA)

[1656]

[1657] To a mixture of compound tag 5-7AA (0.85 g, 236.13 pmol, 1 eq) in DCM (50 mL) was added AA82-(9H-fluoren-9-ylmethoxycarbonylamino)acetic acid (105.3 mg, 354 pmol, 1.5 eq), HOAt (48.2 mg, 354 pmol, 1.5 eq) and DIC (44.7 mg, 354 pmol, 54.8 pL, 1.5 eq). The resulting suspension was stirred at 20 °C for 2 h. The desired compound N-Fmoc-tag 5-8AA (0.79 g, 184.86 pmol, 78.29% yield) was obtained as a white solid following the general precipitation procedure.

[1658] LC-MS: RT = 6.32 min, for C 76 H 91 FN 12 O 16 H, mass calcd. 1447.68, m / z found 1448.6 [M - tag - tBu-3TBS + 6H] + LCMS conditions (BV): Reverse phase LCMS was performed using a NanoChrom ChromCore 120 C18 3 pm, 3.0*30 mm column eluting with a gradient of 10% to 80% ACN containing 0.02% TFA (solvent B) and water containing 0.04% TFA (solvent A) at a flow rate of 1.5 mL / min.

[1659] LC-MS: RT = 6.11 min, for C 76 H 91 FN 12 O 16 H, mass calcd. 1447.68, m / z found 1447.6 [M-tag-tBu-3TBS+6H] + LCMS conditions (BF): Reverse phase LCMS was performed using an Xtimate C18 2.1*30mm 3pm column eluting with a gradient of 10% to 80% ACN with 0.02% TFA (solvent B) and water with 0.04% TFA (solvent A) at a flow rate of 1.5 mL / min.

[1660] Step p: Synthesis of tert-butyl (3S)-3-[[(2S)-2-[[(2S,3R)-2-[[2-[[(2S,3R)-2-[(2- aminoacetyl)amino]-3-[tert-butyl(dimethyl)silyl]oxy-butyryl]amino]-3-(2- fluorophenyl)-2-methyl-propionyl]amino]-3-[tert-butyl(dimethyl)silyl]oxy- butyryl]amino]-3-[tert-butyl(dimethyl)silyl]oxy-propionyl]amino]-4-[[(1S)-1- [[4-[4-(4-azidobutoxy)-2-ethyl-phenyl]phenyl]methyl]-2-[[(1S)-1-[[[4-[2-[bis[[3,4,5- tris(doxa-1,3,5,7,9,11,13,15,17,19,21-undeca- yloxy)phenyl]methyl]amino]-2-oxo-ethoxy]phenyl]-(2,4-dimethoxyphenyl)methyl] aminocarbonyl]-4-(3,5-dimethylphenyl)butyl]amino]-2-oxo-ethyl]amino]-4-oxo- butyric acid (Tag 5-8AA)

[1661]

[1662] A clear solution of compound N-Fmoc-Tag 5-8AA (0.79 g, 184.86 pmol, 1 eq) in 20% piperidine / THF (20 mL) was stirred at 50 °C for 0.5 h. Following the general precipitation procedure, compound Tag 5-8AA was obtained as a white solid (0.74 g, 182.66 pmol, 98.81% yield). LC-MS: RT = 4.47 min, for C 61 H 81 FN 12 O 14 H, mass calcd. 1225.61, m / z found 1225.6 [M-tag-tBu-3TBS+6H]+ .

[1663] Step q: Synthesis of tert-butyl (3S)-4-[[(1S)-1-[[4-[4-(4-azidobutoxy)-2- ethyl-phenyl]phenyl]methyl]-2-[[(1S)-1-[[[4-[2-[bis[[3,4,5-tris(tetracosan-1,3,5,7,9,11,13,15,17,19,21-undecyloxy)phenyl]methyl]amino]-2-oxo- ethoxy]phenyl]-(2,4-dimethoxyphenyl)methyl]aminocarbonyl]-4-(3,5- dimethylphenyl)butyl]amino]-2-oxo-ethyl]amino]-3-[[(2S)-3-[tert-butyl(dimethyl)silyl]oxy-2- [[2-[[(2S,3R)-3-[tert-butyl(dimethyl)silyl]oxy-2-[[2-[[(2S,3R)-3-[tert-butyl(dimethyl)silyl]oxy- 2-[[2-[[(2S)-2-(9H-fluoren-9-ylmethoxycarbonylamino)-3-(1H-triazol-5-yl)propionyl]amino] acetyl]amino]butyryl]amino]-3-(2-fluorophenyl)-2-methyl-propionyl]amino]butyryl]amino] propionyl]amino]-4-oxo-butyric acid tert-butyl ester (N-Fmoc-tag5-9AA)

[1664]

[1665] Protected AA9 was used. To a mixture of compound tag5-8AA (50 mg, 12.34 pmol, 1 eq) in DCM (5 mL) was added AA9 (7 mg, 18.51 pmol, 1.5 eq), HOAt (2.52 mg, 18.51 pmol, 1.5 eq) and DIC (2.34 mg, 18.51 pmol, 2.87 pL, 1.5 eq). The resulting suspension was stirred at 20 °C for 2 h. No desired mass was detected (Table 19).

[1666] Table 19. Reaction between tag5-8AA and AA9

[1667]

[1668] LCMS conditions (CR): Reversed phase LCMS was performed using a NanoChrom ChromCore 120 C18 3 pm, 3.0*30 mm column eluting with a gradient of 10% to 80% ACN containing 0.02% TFA (solvent B) and water containing 0.04% TFA (solvent A) at a flow rate of 1.5 mL / min.

[1669] LCMS conditions (BF): Reverse phase LCMS was performed using an Xtimate C18 2.1*30mm 3pm column eluting with a gradient of 10% to 80% ACN with 0.02% TFA (solvent B) and water with 0.04% TFA (solvent A) at a flow rate of 1.5 mL / min.

[1670] Protected DMB-AA9 was used. To a mixture of compound Tag 5-8AA (0.5 g, 123.42 pmol, 1 eq) in DCM (5 mL) was added DMB-AA9 (98 mg, 185 pmol, 1.5 eq), HOAt (25.2 mg, 185 pmol, 1.5 eq) and DIC (23.36 mg, 185 pmol, 28.6 pL, 1.5 eq). The resulting suspension was stirred at 20 °C for 2 h. The desired compound N-Fmoc-Tag 5-9AA was obtained as a white solid (510 mg, 111.77 pmol, 90.57% yield) following the general precipitation procedure (Table 19).

[1671] LC-MS: RT = 5.68 min, for C 80 H 96 FN 17 O 17 H, mass calc. 1586.74 [M-Tag-tBu-3TBS-DMB+7H] + , C 80 H 96 FN 17 O 17 H2, 793.87 [M-Tag-tBu-3TBS-DMB+8H] 2+ , m / z found 794.3 [M-Tag-tBu-3TBS-DMB+8H] 2+ LCMS conditions (BV): Reverse phase LCMS was performed using a NanoChrom ChromCore 120 C18 3pm, 3.0*30mm column eluting with a gradient of 10% to 80% ACN with 0.02% TFA (solvent B) and water with 0.04% TFA (solvent A) at a flow rate of 1.5 mL / min.

[1672] LC-MS: RT = 5.68 min, for C 80 H 96 FN 17 O 17 H, mass calc. 1586.74 [M-Tag-tBu-3TBS-DMB+7H] + , C 80 H 96 FN17 O 17 H2, 793.87 [M-Tag-tBu-3TBS-DMB+8H] 2+ , m / z found 794.2 [M-Tag-tBu-3TBS-DMB+8H] 2+ LCMS conditions (BF): Reverse phase LCMS was performed using an Xtimate C18 2.1*30 mm 3 pm column eluting with a gradient of 10% to 80% ACN with 0.02% TFA (solvent B) and water with 0.04% TFA (solvent A) at a flow rate of 1.5 mL / min.

[1673] Step r: Synthesis of tert-butyl (3S)-3-[[(2S)-2-[[(2S,3R)-2-[[2-[[(2S,3R)-2-[[2-[[(2S)-2-amino-3-[1- [(2,4-dimethoxyphenyl)methyl]tetrazol-5-yl]propanoyl]amino]acetyl]amino]-3-(2- fluorophenyl)-2-methyl-propanoyl]amino]-3-[tert-butyl(dimethyl)silyl]oxy-butanoyl]amino]- 3-[tert-butyl(dimethyl)silyl]oxy-propanoyl]amino]-4-[[(1S)-1-[[4-[4-(4-azidobutoxy)-2- ethyl-phenyl]phenyl]methyl]-2-[[(1S)-1-[[[4-[2-[bis[[3,4,5-tris(doxadecan-1,3,5,7,9,11,13,15,17,19,21-undecoxy)phenyl]methyl]amino]-2-oxo-ethoxy]phenyl]-(2,4- dimethoxyphenyl)methyl]carbamoyl]-4-(3,5-dimethylphenyl)butyl]amino]-2-oxo-ethyl]amino]- 4-oxo-butyric acid tert-butyl ester (Tag 5-9AA)

[1674]

[1675] A clear solution of compound N-Fmoc-Tag 5-9AA (500 mg, 109.58 pmol, 1 eq) in 20% piperidine / THF (20 mL) was stirred at 50 °C for 0.5 h. Following the general precipitation procedure, the desired compound Tag 5-9AA was obtained as a white solid (0.47 g, 108.28 pmol, 98.81% yield).

[1676] LC-MS: RT = 4.78 min, for C 65 H 86 FN 17 O 15H, mass calcd. 1364.66, m / z found 1364.7 [M-Tag-tBu-3TBS-DMB+7H] + LCMS conditions (CR): Reverse phase LCMS was performed using a NanoChrom ChromCore 120 C18 3μm, 3.0*30 mm column eluting with a gradient of 10% to 80% ACN with 0.02% TFA (solvent B) and water with 0.04% TFA (solvent A) at a flow rate of 1.5 mL / min.

[1677] LC-MS: RT = 4.47 min, for C 65 H 86 FN 17 O 15 H, mass calcd. 1364.66, m / z found 1364.7 [M-Tag-tBu-3TBS-DMB+7H] + LCMS conditions (BF): Reverse phase LCMS was performed using a Xtimate C18 2.1*30 mm 3μm column eluting with a gradient of 10% to 80% ACN with 0.02% TFA (solvent B) and water with 0.04% TFA (solvent A) at a flow rate of 1.5 mL / min.

[1678] Step s: Synthesis of tert-butyl (3S)-4-[[(1S)-1-[[4-[4-(4-azidobutoxy)-2- ethyl-phenyl]phenyl]methyl]-2-[[(1S)-1-[[[4-[2-[bis[[3,4,5-tris(doxazolidin- 13-yl)phenyl]methyl]amino]-2-oxo-ethoxy]phenyl]-(2,4-dimethoxyphenyl) methyl]amino carbonyl]-4-(3,5-dimethylphenyl)butyl]amino]-2-oxo- ethyl]amino]-3-[[(2S)-3-[tert-butyl(dimethyl)silyl]oxy-2-[[(2S,3R)-3-[tert- butyl(dimethyl)silyl]oxy-2-[[2-[[(2S,3R)-3-[tert-butyl(dimethyl)silyl]oxy-2-[[2- [[(2S)-3-[1-[(2,4-dimethoxyphenyl)methyl]tetrazol-5-yl]-2-[[2,2-dimethyl-3- oxo-3-[2-(3-tritylimidazol-4-yl)ethylamino]propanoyl]amino]propanoyl] amino]butanoyl]amino]butanoyl]amino]-3-(2-fluorophenyl)-2-methyl- propanoyl]amino]propanoyl]amino]-4-oxo-butyric acid (Tag 5-10AA)

[1679]

[1680] To a mixture of compound Tag 5-9AA (450 mg, 103.67 pmol, 1 eq) in DCM (20 mL) was added AA10 (72.73 mg, 155.5 pmol, 1.5 eq), HOAt (21.2 mg, 155.5 pmol, 1.5 eq) and DIC (19.6 mg, 155.5 pmol, 54.1 pL, 1.5 eq). The resulting suspension was stirred at 20 °C for 2 h. The desired compound Tag 5-10AA was obtained as a white solid (480 mg, 100.21 pmol, 96.66% yield) following the general precipitation procedure. LC-MS (Method 4): RT = 4.456 min for C 75 H 99 FN 20 O 17 H, mass calcd. 1571.76 [M-Tag-tBu-3TBS-DMB-Trt+8H] + , C 75 H 99 FN 20 O 17 H2, 786.38 [M-Tag-tBu-3TBS-DMB-Trt+9H] 2+ , m / z found 786.7 [M-Tag-tBu-3TBS-DMB-Trt+9H] 2+ ; LC-MS (Method 4): RT = 4.85 min for C 75 H 99 FN 20 O 17 H, mass calcd. 1571.76 [M-Tag-tBu-3TBS-DMB-Trt+8H] + , C 75 H 99 FN 20 O 17 H2, 786.38 [M-Tag-tBu-3TBS-DMB-Trt+9H] 2+ , m / z found 786.8 [M-Tag-tBu-3TBS-DMB-Trt+9H] 2+ .

[1681] Step t: Synthesis of tert-butyl (3S)-4-[[(1S)-2-[[(1S)-1-[[[4-[2-[bis[[3,4,5-tris(tetracosan-1,3,5,7,9,11,13,15,17,19,21-undecyloxy)phenyl]methyl]amino]-2-oxo-ethoxy]phenyl]-(2,4-dimethoxyphenyl)methyl]aminocarbonyl]-4-(3,5-dimethylphenyl)butyl]amino]-1-[[4-[4-[4-[4-[2-[2-[2-[2-[2-[2-[2-[2-(tert-butoxycarbonylamino)ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxymethyl]triazol-1-yl]butoxy]-2-ethyl-phenyl]phenyl]methyl]-2-oxo-ethyl]amino]-3-[[(2S)-3-[tert-butyl(dimethyl)silyl]oxo-2-[[(2S,3R)-3-[tert-butyl(dimethyl)silyl]oxy-2-[[2-[[(2S,3R)-3-[tert-butyl(dimethyl)silyl]oxy-2-[[2-[[(2S)-3-[1-[(2,4-dimethoxyphenyl)methyl]tetrazol-5-yl]-2-[[2,2-dimethyl-3-oxo-3-[2-(3-tritylimidazol-4-yl)ethylamino]propanoyl]amino]propanoyl]amino]butanoyl]amino]butanoyl]amino]butanoyl]amino]-4-oxo-butyric acid tert-butyl ester (Tag 5-M1)

[1682]

[1683] Compound Tag 5-10AA (0.3 g, 62.63 pmol, 1 eq) was dissolved in H2O (1 mL) and THF (4 mL), then a solution of CuSO4.5H2O (23.46 mg, 93.94 pmol, 1.5 eq) and sodium ascorbate (18.61 mg, 93.94 pmol, 1.5 eq) in H2O (1 mL), a solution of TBTA (16.62 mg, 31.31 pmol, 0.5 eq) in DMSO (0.5 mL) and tert-butyl PEG8-Linker (95.37 mg, 187.89 pmol, 3 eq) in THF (4 mL) were added. The mixture was stirred at 40 °C for 12 h. The desired compound Tag 5-M1 (330 mg, 62.29 pmol, 99.46% yield) was obtained as a white solid following the general precipitation procedure.

[1684] LC-MS: RT = 3.72 min for C 94 H 136 FN 21 O25 H2, mass calcd 990.01 [M-tag-tBu-3TBS-DMB-Trt-Boc + 10H] 2+ , m / z found 990.6 [M-tag-tBu-3TBS-DMB-Trt-Boc + 10H] 2+ . LCMS conditions (CR): Reverse phase LCMS was performed using a NanoChrom ChromCore 120 C18 3 pm, 3.0*30 mm column eluting with a gradient of 10% to 80% ACN with 0.02% TFA (solvent B) and water with 0.04% TFA (solvent A) at a flow rate of 1.5 mL / min.

[1685] LC-MS: RT = 3.39 min, for C 94 H 136 FN 21 O 25 H2, mass calcd 990.01, m / z found 990.4 [M-tag-tBu-3TBS-DMB-Trt-Boc + 10H] 2+ ; RT = 3.99 min, for C 113 H 150 FN 21 O 25 H2, mass calcd 1111.06, m / z found 1111.5 [M-tag-tBu-3TBS-DMB-Boc + 9H] 2+ . LCMS conditions (BF): Reverse phase LCMS was performed using a Xtimate C18 2.1*30 mm 3 pm column eluting with a gradient of 10% to 80% ACN with 0.02% TFA (solvent B) and water with 0.04% TFA (solvent A) at a flow rate of 1.5 mL / min.

[1686] Step u: Synthesis of (3S)-4-[[(1S)-1-[[4-[4-(4-azidobutoxy)-2-ethyl- phenyl]phenyl]methyl]-2-[[(1S)-1-aminocarbonyl-4-(3,5-dimethylphenyl)butyl] amino]-2-oxo-ethyl]amino]-3-[[(2S)-2-[[(2S,3R)-2-[[3-(2-fluorophenyl)-2- [[(2S,3R)-3-hydroxy-2-[[2-[[(2S)-2-[[3-[2-(1H-imidazol-5-yl)ethylamino]-2,2- dimethyl-3-oxo-propionyl]amino]-3-(1H-tetrazol-5-yl)propionyl]amino] acetyl]amino]butyryl]amino]-2-methyl-propionyl]amino]-3-hydroxy-butyryl] amino]-3-hydroxy-propionyl]amino]-4-oxo-butyric acid (M1)

[1687]

[1688] To a mixture of Tag 5-M1 with all side chain protecting groups (430 mg, 81.17 pmol, 1 eq) in DCM (10 mL) was added TFA mixture (TFA / TIPS / H20 = 95 / 2.5 / 2.5) (30 ml). The resulting suspension was stirred at 20 °C for 2 h. A sample was taken, suspended with PE / MTBE (3 / 1, 1 mL) and centrifuged to give a solid. The solid was diluted with MeOH and H20, filtered and the filtrate was checked by LCMS, which showed the desired mass of M1. The reaction was concentrated under reduced pressure to give a residue, which was added to PE / TBME (3 / 1, 20V) and stirred for 1 h. The suspension was filtered to give a white solid. The residue was purified by preparative HPLC (basic conditions): Column: Boston Prime C18 150*30mm*5pm; Mobile Phase: [Water (Ammonium Hydroxide v / v) - ACN]; Gradient: 25% - 45% B in 11 min. The desired compound M1 was obtained as a white solid (5.1 mg, 2.45 pmol, 3.02% yield, 95% purity). LC-MS: RT = 3.399 min, m / z found 1979.01 [M+H], m / z calc 1978.92 [M+H]. 94 H 136 N 21 O 25 FH, m / z found 990.5 [M+2H], m / z calc 990.01 [M+2H] + , C 94 H 136 N 21 O 25 FH2 990.01 [M+2H] 2+ , m / z found 990.5 [M+2H] 2+; LCMS conditions: Method 7.

[1689] Using 1 HNMR, 19 FNMR (non-TFA salt) and M1 (TFA salt), F NMR, 13 CNMR and UPLC characterized M1. UPLC of M1 showed an impurity at RT = 6.5 min which was a system impurity. LCMS of M1 showed the main peak at a retention time of 1.564 min corresponding to the desired product M1 with observed masses of [M+H]+= 1980.3, [M / 2+H]+= 990.3, [M / 3+H]+= 660.7, [M / 4+H]+= 495.8.

[1690] M1-HCl salt qualification according to IC showed a HC1 mass content of 2.08% and a molar content of 1.15 mol equivalents.

[1691] 8.4 Synthesis of (LPPS) M3 on Tag 5

[1692] Synthesis of M3 on Tag 5 is shown herein. Figure 7 Synthesis of Tag 5-M3 is shown, and Figure 12 Extended conditions and results for the preparation of M3 using Tag 5 are shown. Step a: Synthesis of 9H-fluoren-9-ylmethyl N-[rac-(1S)-1-[[4-[4-(4-azidobutoxy)-2- ethyl-phenyl]phenyl]methyl]-2-[[[4-[2-[bis[4-[[3,4,5-tris(dodecyloxy)phenyl]methoxy]phenyl]methylamino]-2-oxo-ethoxy]phenyl]-(2,4-dimethoxyphenyl)methyl]amino]-2-oxo- ethyl]carbamate (N-Fmoc-Tag 5-2AA)

[1693]

[1694] To a mixture of Tag 5 (2 g, 757 pmol, 1 eq) in DCM (50 mL) was added AA2 (687 mg, 1.14 mmol, 1.5 eq) in THF (40 mL) and HOAt (155 mg, 1.14 mmol, 1.5 eq) and DIC (143.28 mg, 1.14 mmol, 175.81 pL, 1.5 eq). The solution was then stirred at 50 °C for 12 h. Traces were monitored by LC-MS. The reaction was carried out using the general procedure H for amide coupling to give N-Fmoc-Tag 5-2AA as a white solid (2.3 g, 712.30 pmol, 94.11% yield). LC-MS: RT = 5.506 min, for C36 H 37 N5O4Na, mass calcd. 626.03, m / z found 626.70 [M-tag + H + Na] + .

[1695] Step b: Synthesis of rac-(2S)-2-amino-3-[4-[4-(4-azidobutoxy)-2- ethyl-phenyl]phenyl]-N,N-bis[[3,4,5-tris(dodecyloxy)phenyl]methyl]propanamide (Tag 5-2AA)

[1696]

[1697] A clear solution of compound N-Fmoc-Tag 5-2AA (1 g, 366.11 pmol, 1 equiv) in 20% piperidine / THF (20 mL) was stirred at 50 °C for 2 h without monitoring. The general precipitation procedure was followed to give Tag 5-2AA as a white solid (0.9 g, 358.68 pmol, 97.97% yield).

[1698] Step c: Synthesis of rac-(3S)-3-(9H-fluoren-9-ylmethoxycarbonylamino)-4-oxo-4-[[rac-(1S)-1-[[4-[4-(4-azidobutoxy)-2-ethyl-phenyl]phenyl]methyl]-2-[[[4-[2-[bis[[3,4,5-tris(dodecyloxy)phenyl]methyl]amino]-2-oxo-ethoxy]phenyl]-(2,4-dimethoxyphenyl)methyl]amino]-2-oxo- ethyl]amino]butanoic acid tert-butyl ester (N-Fmoc-Tag 5-3AA)

[1699]

[1700] To a mixture of compound Tag 5-2AA (0.9 g, 320.45 pmol, 1 equiv) in DCM (40 mL) was added AA3 (395.55 mg, 961.36 pmol, 3 equiv), HOAt (130.85 mg, 961.36 pmol, 134.48 pL, 3 equiv) and DIC (121.32 mg, 961.36 pmol, 148.86 pL, 3 equiv). The resulting suspension was stirred at 20 °C for 2 h. The general precipitation procedure was followed to give the desired compound N-Fmoc-Tag 5-3AA as a white solid (1.2 g, crude). LC-MS: RT = 5.038 min, for C 40 H 43 N6O7, mass calcd. 719.31, m / z found 719.30 [M-tag-tBu + 2H] +.

[1701] Step d: Synthesis of rac-(3S)-3-amino-4-oxo-4-[[rac-(lS)-l-[[4-[4-(4-azidobutoxy)-2- ethyl-phenyl]phenyl]methyl]-2-[bis[[3,4,5-tris(dodecyloxy)phenyl]methyl]amino]-2- oxo-ethyl]amino]butyric acid tert-butyl ester (Tag 5-3AA)

[1702]

[1703] A clear solution of compound N-Fmoc-Tag 5-3AA (1.2 g, crude) in 20% piperidine / THF (25 mL) was stirred at 50 °C for 2 hours without monitoring. The general precipitation procedure was followed to give the desired compound Tag 5-3AA (1.11 g, 414.12 pmol, 100.00% yield) as a white solid.

[1704] Step e: Synthesis of rac-(3S)-4-oxo-4-[[rac-(lS)-l-[[4-[4-(4-azidobutoxy)-2-ethyl- phenyl]phenyl]methyl]-2-[bis[[3,4,5-tris(dodecyloxy)phenyl]methyl]amino]-2-oxo- ethyl]amino]-3-[[rac-(2S)-3-[tert-butyl(dimethyl)silyl]oxy-2-(9H-fluoren-9- ylmethoxycarbonylamino)propanoyl]amino]butyric acid tert-butyl ester (N-Fmoc-Tag 5- 4AA)

[1705]

[1706] To a mi...

Claims

1. A compound having a structure of formula (I): in: m and n are independently integers from 1 to 5, and (m+n)≥3; R 1 It is C 1-5 alkyl; R 2 is independently at each occurrence an optionally substituted C 10-30 alkyl; and R 3 is independently at each occurrence an optionally substituted C 10-30 alkyl, or a salt thereof.

2. The compound according to claim 1, wherein R 1 It's methyl.

3. The compound according to claim 1, wherein R 2 is independently at each occurrence an optionally substituted C 18-26 Alkyl, and R 3 is independently at each occurrence an optionally substituted C 18-26 alkyl.

4. The compound according to claim 1, wherein each R 2 is independently optionally substituted C 22 alkyl, and each R 3 is independently optionally substituted C 22 alkyl.

5. The compound according to claim 1, wherein the compound has the structure of Formula (Ia): or a salt thereof.

6. A process for preparing a compound of formula (I) or a salt thereof: in: m and n are independently integers from 1 to 5, and (m+n)≥3; R 1 It is C 1-5 alkyl; R 2 is independently at each occurrence an optionally substituted C 10-30 alkyl; and R 3 is independently at each occurrence an optionally substituted C 10-30 alkyl, The process comprises: (a) providing a compound of formula (II) having the following structure: in PG is a suitable protecting group; and (b) forming the compound of formula (I) from the compound of formula (II).

7. The process of claim 6, wherein PG is selected from the group consisting of allyloxycarbonyl (Alloc), benzyloxycarbonyl (Cbz), tert-butoxycarbonyl (Boc) and 9-fluorenylmethoxycarbonyl (Fmoc).

8. The process according to claim 6, wherein the compound of formula (II) has the structure of formula (IIa):

9. The process of claim 6, wherein step (b) of forming the compound of formula (I) comprises: The compound of formula (II) is reacted with a protecting group removing agent to produce the compound of formula (I).

10. The process according to claim 9, wherein the protecting group removing agent is selected from the group consisting of Pd(PPh)3, PhSiH3, H2, piperidine and trifluoroacetic acid (TFA).

11. The process according to claim 6, further comprising the steps of: Provided are compounds of formula (III) having the following structure: in PG 1 is a suitable protecting group; PG 2 is a suitable protecting group; and The compound of formula (II) is formed from the compound of formula (III) prior to step (a).

12. The process according to claim 11, wherein PG 1 is independently at each occurrence selected from the group consisting of tert-butyldimethylsilyl (TBS), trimethylsilyl (TMS), triethylsilyl (TES), tert-butyldiphenylsilyl (TBDPS) and triisopropylsilyl (TIPS).

13. The process according to claim 11, wherein PG 2 is independently at each occurrence selected from the group consisting of tert-butyldimethylsilyl (TBS), trimethylsilyl (TMS), triethylsilyl (TES), tert-butyldiphenylsilyl (TBDPS) and triisopropylsilyl (TIPS).

14. The process of claim 11, wherein each PG 1 and each PG 2 same.

15. The process of claim 11, wherein PG 1 and PG 2 Different from each other.

16. The process of claim 11, wherein the compound of formula (III) has the structure of formula (IIIa):

17. The process of claim 11, wherein the step of forming the compound of formula (II) prior to step (a) comprises: reacting the compound of formula (III) with a protecting group removing agent to obtain a deprotected intermediate; Said intermediate is reacted with at least one compound of formula (IV): R-LG(IV), in R is optionally substituted C 10-30 alkyl; and LG is a suitable leaving group; to produce the compound of formula (II).

18. The process according to claim 17, wherein LG is selected from the group consisting of halogen, OTf, OMs and OTs.

19. The process of claim 17, wherein the protecting group removing agent is selected from the group consisting of tetrabutylammonium fluoride (TBAF), NH4F, SiF4, AcOH, HCl, LiAlH4 and K2CO3.

20. The process of claim 11, further comprising the steps of: Provided are compounds of formula (V) having the following structure: and The compound of formula (III) is formed from the compound of formula (V).

21. The process of claim 20, wherein the step of forming the compound of formula (III) comprises: The compound of formula (V) is reacted with a compound of formula (VIa) or formula (VIb): PG-X(VIa) or PG2O(VIb), in X is Cl or Br; to produce the compound of formula (III).

22. The process of claim 21, wherein the compound of formula (VIa) or formula (VIb) is selected from the group consisting of AllocCl, Alloc2O, Cbz2O, CbzCl, FmocCl, and Boc2O.

23. The process of claim 20, further comprising: Provided is a compound of formula (VII) having the following structure: Among them PG 3 is a suitable protecting group; and The compound of formula (V) is formed from the compound of formula (VII).

24. The process of claim 23, wherein PG 3 Selected from the group consisting of allyloxycarbonyl (Alloc), benzyloxycarbonyl (Cbz), tert-butoxycarbonyl (Boc) and 9-fluorenylmethoxycarbonyl (Fmoc).

25. The process of claim 23, wherein the compound of formula (VII) has the following structure:

26. The process of claim 23, wherein the step of forming the compound of formula (V) comprises: The compound of formula (VII) is reacted with a protecting group removing agent to produce the compound of formula (V).

27. The process of claim 26, wherein the protecting group removing agent is selected from the group consisting of Pd(PPh)3, PhSiH3, H2, piperidine and trifluoroacetic acid (TFA).

28. The process of claim 23, further comprising: Provided are compounds of formula (VIII) having the following structure: and The compound of formula (VII) is formed from the compound of formula (VIII).

29. The process of claim 28, wherein the step of forming the compound of formula (VII) comprises: The compound of formula (VIII) is reacted with a compound of formula (IX) having the following structure: to produce the compound of formula (VII).

30. The process of claim 28, wherein the compound of formula (VIII) has the following structure:

31. The process of claim 29, wherein the compound of formula (IX) has the following structure:

32. The process of claim 28, further comprising: Provided is a compound of formula (X) having the following structure: Among them PG 4 is a suitable protecting group; and The compound of formula (VIII) is formed from the compound of formula (X).

33. The process of claim 32, wherein PG 4 Selected from the group consisting of allyl, allyloxycarbonyl (Alloc), benzyloxycarbonyl (Cbz), tert-butoxycarbonyl (Boc) and 9-fluorenylmethoxycarbonyl (Fmoc).

34. The process of claim 32, wherein the compound of formula (X) has the structure:

35. The process of claim 32, wherein the step of forming the compound of formula (VIII) comprises: The compound of formula (X) is reacted with a protecting group removing agent.

36. The process of claim 35, wherein the protecting group removal agent is selected from the group consisting of Pd / K2CO3, 1,3-dimethylbarbituric acid / Pd(PPh3)4, Pd(PPh)3, PhSiH3, H2, piperidine and trifluoroacetic acid (TFA).

37. The process of claim 32, further comprising: Provided are compounds of formula (XI) having the following structure: and The compound of formula (X) is formed from the compound of formula (XI).

38. The process of claim 37, wherein the compound of formula (XI) has the structure:

39. The process of claim 37, wherein the step of forming the compound of formula (X) comprises: The compound of formula (XI) is reacted with a compound of formula (XII) or a salt thereof: to produce the compound of formula (X).

40. The process of claim 37, further comprising: Provided are compounds of formula (XIII) having the following structure: and The compound of formula (XI) is formed from the compound of formula (XIII).

41. The process of claim 40, wherein the compound of formula (XIII) has the following structure:

42. The process of claim 40, wherein the step of forming the compound of formula (XI) comprises: The compound of formula (XIII) is reacted with an oxidizing agent to produce the compound of formula (XI).

43. The process of claim 42, wherein the oxidizing agent is selected from the group consisting of pyridinium chlorochromate (PCC), pyridinium dichromate (PDC), silver carbonate (Ag2CO3), tetra-n-propylammonium perruthenate (TPAP), and Dess-Martin periodinane (DMP).

44. The process of claim 40, further comprising: Provided is a compound of formula (XIV) having the following structure: in Y is -OC 1-6 alkyl; and The compound of formula (XIII) is formed from the compound of formula (XIV).

45. The process of claim 44, wherein the compound of formula (XIV) has the structure:

46. ​​The process of claim 44, wherein the step of forming the compound of formula (XIII) comprises: The compound of formula (XIV) is reacted with a reducing agent.

47. The process of claim 46, wherein the reducing agent is selected from the group consisting of LiAlH4, DIBALH, and LiBH4.

48. The process of claim 44, further comprising: Provided are compounds of formula (XV) having the following structure: and The compound of formula (XIV) is formed from the compound of formula (XV).

49. The process of claim 48, wherein the compound of formula (XV) has the structure:

50. The process of claim 48, wherein the step of forming the compound of formula (XIV) comprises: The compound of formula (XV) is reacted with a protecting group-introducing agent to produce the compound of formula (XIV).

51. The process of claim 50, wherein the protecting group introducing agent is selected from the group consisting of TBSCl, TBSOTf, TMSCl, TMSOTf, TESCl, TESOTf, TBDPSCl, TBDPSOTf, TIPSCl and TIPSOTf.

52. A product prepared by the method of any one of claims 6 to 51.

53. The product of claim 52, wherein the product is a compound of formula (I): in: m and n are independently integers from 1 to 5, and (m+n)≥3; R 1 It is C 1-5 alkyl; R 2 is independently at each occurrence an optionally substituted C 10-30 alkyl; and R 3 is independently at each occurrence an optionally substituted C 10-30 alkyl, or a salt thereof.

54. The product of claim 53, wherein the product is a compound of formula (Ia): or a salt thereof.

55. A method for preparing a peptide or peptidomimetic using a compound of formula (I) or a salt thereof, in: m and n are independently integers from 1 to 5, and (m+n)≥3; R 1 It is C 1-5 alkyl; R 2 is independently at each occurrence an optionally substituted C 10-30 alkyl; and R 3 is independently at each occurrence an optionally substituted C 10-30 alkyl.

56. The method of claim 55, wherein the compound of formula (I) is used as a carrier for the preparation of a peptide or peptidomimetic.

57. The method of claim 55, wherein the peptidomimetic is a compound of the formula: or a salt thereof.

58. The method of claim 55, wherein the peptidomimetic is a compound of the formula: or a salt thereof.

59. A compound prepared using a compound of formula (I) or a salt thereof: in: m and n are independently integers from 1 to 5, and (m+n)≥3; R 1 It is C 1-5 alkyl; R 2 is independently selected at each occurrence from optionally substituted C 10-30 alkyl; and R 3 is independently selected at each occurrence from optionally substituted C 10-30 alkyl.

60. The compound of claim 59, wherein the compound has the formula: or a salt thereof.

61. The compound of claim 59, wherein the compound has the formula: or a salt thereof.

62. A pharmaceutical composition comprising the peptidomimetic of claim 57 or the compound of any one of claims 59-60.

63. A pharmaceutical dosage form comprising the peptidomimetic of claim 57 or the compound of any one of claims 59-60.

64. A method of selectively targeting GLP1R on the surface of a cell with the peptidomimetic of claim 57 or the compound of any one of claims 59-60.

65. The method of claim 64, wherein the cell is a mammalian cell.

66. The method of claim 64 or 65, wherein the cell is a human cell.

67. A method of enhancing GLP1R activity in an individual in need thereof, the method comprising administering to the individual an effective amount of the peptidomimetic of claim 57 or the compound of any one of claims 59-60, the composition of claim 62, or the dosage form of claim 63.

68. A method of lowering blood glucose levels in a subject in need thereof, comprising administering to the subject an effective amount of the peptidomimetic of claim 57 or the compound of any one of claims 59-60, the composition of claim 62, or the dosage form of claim 63.

69. A method of reducing body weight in a subject in need thereof, comprising administering to the subject an effective amount of the peptidomimetic of claim 57 or the compound of any one of claims 59-60, the composition of claim 62, or the dosage form of claim 63.

70. A method of treating a GLP1R-associated condition in an individual in need thereof, the method comprising administering to the individual an effective amount of the peptidomimetic of claim 57 or the compound of any one of claims 59-60, the composition of claim 62, or the dosage form of claim 63.

71. The method of claim 70, wherein the GLP1R-associated condition is type II diabetes, obesity, liver disease, coronary artery disease, or kidney disease.

72. The method of claim 70, wherein the GLP1R-associated condition is type II diabetes and / or obesity.

73. The method of any one of claims 67-72, wherein the peptidomimetic of claim 57 or the compound of any one of claims 59-60, the composition of claim 62, or the dosage form of claim 63 is administered subcutaneously, intravenously, intradermally, intraperitoneally, or intramuscularly.

74. A method for preparing a peptide or peptidomimetic, the method comprising the steps of: (a) providing a compound of formula (I): in: m and n are independently integers from 1 to 5, and (m+n)≥3; R 1 It is C 1-5 alkyl; R 2 is independently selected at each occurrence from optionally substituted C 10-30 alkyl; and R 3 is independently selected at each occurrence from optionally substituted C 10-30 alkyl, or its salts; (b) providing a first amino acid (AA1); and (c) coupling the first amino acid (AA1) to the compound of formula (I) to form a peptide bond between the first amino acid (AA1) and the compound of formula (I).

75. The method of claim 74, wherein the first amino acid (AA1) comprises a protected amino group.

76. The method of claim 74, wherein the first amino acid (AA1) is 77. The method of claim 74, further comprising the steps of: Prior to step (c), the chemical groups on the first amino acid (AA1) are activated to prepare the first amino acid (AA1) for coupling with the compound of formula (I).

78. The method of claim 74, further comprising the steps of: (d) removing the protecting group from the first amino acid (AA1); (e) providing a second amino acid (AA2); and (f) coupling the second amino acid (AA2) to the first amino acid (I-AA1) to form a peptide bond (I-AA1-AA2) between the second amino acid (AA2) and the first amino acid.

79. The method of claim 78, wherein the second amino acid (AA2) is 80. The method of claim 78, further comprising the steps of: (g) removing the protecting group from the second amino acid (AA2); (h) providing a third amino acid (AA3); and (i) coupling the third amino acid (AA3) to the second amino acid to form a peptide bond (I-AA1-AA2-AA3) between the third amino acid (AA3) and the second amino acid.

81. The method of claim 80, wherein the third amino acid (AA3) is 82. The method according to claim 80, further comprising the steps of: (j) removing the protecting group from the third amino acid (AA3); (k) providing a fourth amino acid (AA4); and (1) coupling the fourth amino acid (AA4) to the third amino acid to form a peptide bond (I-AA1-AA2-AA3-AA4) between the fourth amino acid (AA4) and the third amino acid.

83. The method of claim 82, wherein the fourth amino acid (AA4) is 84. The method of claim 82, further comprising: (m) removing the protecting group from the fourth amino acid (AA4); (n) providing a fifth amino acid (AA5); and (o) coupling the fifth amino acid (AA5) to the fourth amino acid to form a peptide bond (I-AA1-AA2-AA3-AA4-AA5) between the fifth amino acid (AA5) and the fourth amino acid.

85. The method of claim 84, wherein the fifth amino acid (AA5) is 86. The method of claim 84, further comprising: (p) removing the protecting group from the fifth amino acid (AA5); (q) providing a sixth amino acid (AA6); and (r) coupling the sixth amino acid (AA6) to the fifth amino acid to form a peptide bond (I-AA1-AA2-AA3-AA4-AA5-AA6) between the sixth amino acid (AA6) and the fifth amino acid.

87. The method of claim 86, wherein the sixth amino acid (AA6) is 88. The method of claim 86, further comprising: (s) removing the protecting group from the sixth amino acid (AA6); (t) providing the seventh amino acid (AA7); and (u) coupling the seventh amino acid (AA7) to the sixth amino acid to form a peptide bond (I-AA1-AA2-AA3-AA4-AA5-AA6-AA7) between the seventh amino acid (AA7) and the sixth amino acid.

89. The method of claim 88, wherein the seventh amino acid (AA7) is 90. The method of claim 88, further comprising: (v) removing the protecting group from the seventh amino acid (AA7); (w) providing an eighth amino acid (AA8); and (x) coupling the eighth amino acid (AA8) to the seventh amino acid to form a peptide bond (I-AA1-AA2-AA3-AA4-AA5-AA6-AA7-AA8) between the eighth amino acid (AA8) and the seventh amino acid.

91. The method of claim 90, wherein the eighth amino acid (AA8) is 92. The method of claim 90, further comprising: (y) removing the protecting group from the eighth amino acid (AA8); (z) providing the ninth amino acid (AA9); and (aa) coupling the ninth amino acid (AA9) to the eighth amino acid to form a peptide bond (I-AA1-AA2-AA3-AA4-AA5-AA6-AA7-AA8-AA9) between the ninth amino acid (AA9) and the eighth amino acid.

93. The method of claim 92, wherein the ninth amino acid (AA9) is 94. The method of claim 92, further comprising: (bb) removing the protecting group from the ninth amino acid (AA9); (cc) providing the tenth amino acid (AA10); and (dd) coupling the tenth amino acid (AA10) to the ninth amino acid to form a peptide bond (I-AA1-AA2-AA3-AA4-AA5-AA6-AA7-AA8-AA9-AA10) between the tenth amino acid (AA10) and the ninth amino acid.

95. The method of claim 94, wherein the tenth amino acid (AA10) is 96. The method of claim 94, further comprising: Repeat the following steps: (i) from amino acid (AA n ) removing the protecting group; (ii) provide the subsequent amino acid (AA n+1 ); and (iii) making the subsequent amino acid (AA n+1 ) and the amino acid (AA n ) is coupled to the subsequent amino acid (AA n+1 ) and the amino acids (AA n ) form a peptide bond between wherein said repetition is performed 1 to 100 times.

97. The method of claim 94, further comprising: (ee) removing the protecting group from the tenth amino acid (AA10); (ff) providing a compound of formula (B): wherein x is an integer from 1 to 15; and (gg) coupling the compound of formula (B) to one of the amino acids in the peptide or peptidomimetic to form a triazole ring.

98. The method of claim 97, wherein the compound of formula (B) has the formula:

99. The method of any one of claims 74-98, further comprising: (hh) cleaving the peptide bond between the first amino acid (AA1) and the compound of formula (I) to obtain the peptide or the peptidomimetic.

100. The method of any one of claims 74-98, wherein the first amino acid (AA1), the second amino acid (AA2), the third amino acid (AA3), the fourth amino acid (AA4), the fifth amino acid (AA5), the sixth amino acid (AA6), the seventh amino acid (AA7), the eighth amino acid (AA8), the ninth amino acid (AA9), the tenth amino acid (AA10), and subsequent amino acids (AA n+1 ) are each independently selected from the group consisting of: Among them PG 5 is a suitable protecting group.

101. The method of claim 100, wherein PG 5 is independently at each occurrence selected from the group consisting of triphenylmethyl (Trt), allyloxycarbonyl (Alloc), benzyloxycarbonyl (Cbz), tert-butoxycarbonyl (Boc), and 9-fluorenylmethoxycarbonyl (Fmoc).

102. The method of any one of claims 74-98, wherein the first amino acid (AA1), the second amino acid (AA2), the third amino acid (AA3), the fourth amino acid (AA4), the fifth amino acid (AA5), the sixth amino acid (AA6), the seventh amino acid (AA7), the eighth amino acid (AA8), the ninth amino acid (AA9), the tenth amino acid (AA10), and subsequent amino acids (AA n+1 ) are each independently selected from the group consisting of:

103. The method of claim 99, wherein the peptidomimetic has the formula: or a salt thereof.

104. The method of claim 99, wherein the peptidomimetic has the formula: or a salt thereof.

105. A compound having a structure of formula (AI): in: m and n are independently integers from 0 to 5; R 1a It is C 1-5 alkyl; R 2a is independently at each occurrence an optionally substituted C 10-30 alkyl; and R 3a is independently at each occurrence an optionally substituted C 10-30 alkyl, or a salt thereof.

106. The compound according to claim 105, wherein R 1a It's methyl.

107. The compound according to claim 105, wherein R 2a is independently at each occurrence an optionally substituted C 18-26 Alkyl, and R 3a is independently at each occurrence an optionally substituted C 18-26 alkyl.

108. The compound of claim 105, wherein each R 2a is independently optionally substituted C 22 alkyl, and each R 3a is independently optionally substituted C 22 alkyl.

109. A compound having a structure of formula (BI): in: m is an integer from 1 to 5; R 1b It is C 1-5 alkyl; and R 2b is independently at each occurrence an optionally substituted C 10-30 alkyl; or a salt thereof.

110. The compound according to claim 109, wherein R 1b It's methyl.

111. The compound of claim 109, wherein each R 2b is independently optionally substituted C 18-26 alkyl.

112. The compound of claim 109, wherein each R 2b is independently optionally substituted C 22 alkyl.

113. A compound having a structure of formula (CI): in: m and n are independently integers from 1 to 5, and (m+n)≥3; R 2c is independently at each occurrence an optionally substituted C 10-30 alkyl; and R 3c is independently at each occurrence an optionally substituted C 10-30 alkyl, or a salt thereof.

114. The compound according to claim 113, wherein R 2c is independently at each occurrence an optionally substituted C 18-26 Alkyl, and R 3c is independently at each occurrence an optionally substituted C 18-26 alkyl.

115. The compound of claim 113, wherein each R 2c is independently optionally substituted C 22 alkyl, and each R 3c is independently optionally substituted C 22 alkyl.

116. A process for preparing a compound of formula (DI) or a salt thereof: in: k is an integer from 1 to 15; q is an integer from 0 to 10, R 1d It is C 1-6 alkyl; PG 6 is a suitable protecting group; and PG 7 is a suitable protecting group, The process comprises: (a) providing a compound of formula (DII) having the following structure: as well as (b) forming the compound of formula (DI) from the compound of formula (DII).

117. The process of claim 116, wherein PG 6 Selected from the group consisting of allyloxycarbonyl (Alloc), benzyloxycarbonyl (Cbz), tert-butoxycarbonyl (Boc) and 9-fluorenylmethoxycarbonyl (Fmoc).

118. The process of claim 116, wherein PG 7 Selected from the group consisting of allyloxycarbonyl (Alloc), benzyloxycarbonyl (Cbz), tert-butoxycarbonyl (Boc) and 9-fluorenylmethoxycarbonyl (Fmoc).

119. The process of claim 116, wherein the compound of formula (DI) has the structure of formula (DIa) or formula (DIb):

120. The process of claim 116, wherein the compound of formula (DII) has the structure of formula (DIIa) or formula (DIIb):

121. The process of claim 116, wherein step (b) of forming the compound of formula (DI) comprises: The compound of formula (DII) is reacted with a compound of formula (DIII): to produce the compound of formula (II).

122. The process of claim 121, wherein the compound of formula (DIII) has the structure of formula (DIIIa):

123. The process of claim 116, further comprising the steps of: Provided is a compound of formula (DIV) or a salt thereof having the following structure: and The compound of formula (DII) is formed from the compound of formula (DIV) prior to step (a).

124. The process of claim 123, wherein the compound of formula (DIV) has the structure of formula (DIVa) or formula (DIVb):

125. The process of claim 123, wherein the step of forming the compound of formula (DII) comprises: The compound of formula (DIV) is reacted with a compound of formula (DVa) or formula (DVb): PG 6 -X(DVa) or PG 6 2O(DVb), in X is OSu, OTf, Cl or Br; to produce the compound of formula (DII).

126. The process of claim 125, wherein the compound of formula (DVa) or formula (DVb) is selected from the group consisting of AllocCl, Alloc2O, Cbz2O, CbzCl, FmocCl, FmocOSu, and Boc2O.

127. The process of claim 123, further comprising the steps of: Provided are compounds of formula (DVI) having the following structure: in PG 8 is a suitable protecting group; and The compound of formula (DIV) is formed from the compound of formula (DVI).

128. The process of claim 127, wherein the step of forming the compound of formula (DIV) comprises: The compound of formula (DVI) is reacted with a protecting group removing agent to produce the compound of formula (DIV).

129. The process of claim 128, wherein the protecting group removing agent is selected from the group consisting of Pd(PPh)3, PhSiH3, H2, piperidine and trifluoroacetic acid (TFA).

130. The process of claim 127, wherein the compound of formula (DVI) has the structure of formula (DVIa) or formula (DVIb):

131. The process of claim 127, further comprising the steps of: Provided are compounds of formula (DVII) having the following structure: and The compound of formula (DVI) is formed from the compound of formula (DVII).

132. The process of claim 131, wherein the step of forming the compound of formula (DVI) comprises: The compound of formula (DVII) is reacted with a base to produce the compound of formula (DVI).

133. The process of claim 128, wherein the base is selected from the group consisting of LiOH, NaOH, and KOH.

134. The process of claim 131 , wherein the compound of formula (DVII) has the structure of formula (DVIIa) or formula (DVIIb):

135. The process of claim 131 , further comprising the steps of: Provided are compounds of formula (DVIII) having the following structure: in LG' is a suitable reactive group; and The compound of formula (DVII) is formed from the compound of formula (DVIII).

136. The process of claim 135, wherein the step of forming the compound of formula (DVII) comprises: The compound of formula (DVIII) is reacted with an azide group-introducing agent to produce the compound of formula (DVII).

137. The process of claim 136, wherein the azide group introducing agent is selected from the group consisting of: NaN3, TMSN3, (PhO)2P(O)N3, Zn(N3)2*2Py and n-Bu4NN3.

138. The process of claim 135, wherein LG' is selected from the group consisting of halogen, OTf, OMs, and OTs.

139. The process of claim 135, wherein the compound of formula (DVIII) has the structure of formula (DVIIIa) or formula (DVIIIb):

140. The process of claim 135, further comprising the steps of: Provided are compounds of formula (DIX) having the following structure: in LG" is a suitable reactive group; and The compound of formula (DVIII) is formed from the compound of formula (DIX).

141. The process of claim 140, wherein LG" is selected from the group consisting of halogens, OTf, OMs, and OTs.

142. The process of claim 140, wherein the step of forming the compound of formula (DVIII) comprises: The compound of formula (DIX) is reacted with a compound of formula (DX): to produce the compound of formula (DVII).

143. The process of claim 140, wherein the compound of formula (DIX) has the structure of formula (DIXa):

144. The process of claim 142, wherein the compound of formula (DX) has the structure of formula (Dxa):

145. The process of claim 140, further comprising the steps of: Provided are compounds of formula (DXI) having the following structure: in R 2d It is C 1-6 alkylene; and The compound of formula (DIX) is formed from the compound of formula (DXI).

146. The process of claim 145, wherein the step of forming the compound of formula (DIX) comprises: The compound of formula (DXI) is reacted with a reducing agent to produce the compound of formula (DIX).

147. The process of claim 145, wherein the reducing agent is selected from the group consisting of H2, NH4HCO2, NABH(Oac)3, and LiAlH4.

148. The process of claim 141, wherein the compound of formula (DXI) has the structure of formula (DXIa):

149. The process of claim 145, further comprising the steps of: Provided are compounds of formula (DXII) having the following structure: as well as The compound of formula (DXI) is formed from the compound of formula (DXII).

150. The process of claim 149, wherein the step of forming the compound of formula (DXI) comprises: The compound of formula (DXII) is reacted with a compound of formula (DXIII): PPh3*C 1-6 Alkyl-Hal(DXIII), in Hal is halogen, to produce the compound of formula (DXI).

151. The process of claim 149, wherein the compound of formula (DXII) has the structure of formula (DXIIa):

152. The process of claim 150, wherein the compound of formula (DXIII) has the structure of formula (DXIIIa): PPh3*MeBr(DXIIIa).

153. The process of claim 149, further comprising the steps of: Provided are compounds of formula (DXIV) having the following structure: and The compound of formula (DXII) is formed from the compound of formula (DXIV).

154. The process of claim 153, wherein the step of forming the compound of formula (DXII) comprises: The compound of formula (DXIV) is reacted with a compound of formula (DXV): in LG'' is a suitable active group, to produce the compound of formula (DXII).

155. The process of claim 154, wherein LG'' is selected from the group consisting of halogens, OTf, OMs, and OTs.

156. The process of claim 153, wherein the compound of formula (DXIV) has the structure of formula (DXIVa):

157. The process of claim 142, further comprising the steps of: Provided is a compound of formula (DXVI) having the following structure: as well as in LG* is a suitable active group, The compound of formula (DX) is formed from the compound of formula (DXVI).

158. The process of claim 157, wherein LG* is selected from the group consisting of halogens, OTf, OMs, and OTs.

159. The process of claim 157, wherein the step of forming the compound of formula (DX) comprises: The compound of formula (DXVI) is reacted with a compound of formula (DXVII): to produce the compound of formula (DX).

160. The process of claim 157, wherein the compound of formula (DXVI) has the structure of formula (DXVIc):

161. The process of claim 157, further comprising the steps of: Provided are compounds of formula (DXVIII) having the following structure: and The compound of formula (DXVI) is formed from the compound of formula (DXVIII).

162. The process of claim 161, wherein the step of forming the compound of formula (DXVI) comprises: The compound of formula (DXVIII) is reacted with a compound of formula (DXIXa) or formula (DXIXb): LG*-X(DXIXa) or LG*2O(DXIXb), in X is OSu, Cl or Br; to produce the compound of formula (DXVI).

163. The process of claim 162, wherein the compound of formula (DXIXa) or formula (DXIXb) is selected from the group consisting of TfCl, Tf2O, MsCl and Ms2O.

164. A process for preparing a compound of formula (DII) or a salt thereof: in: q is an integer from 0 to 10, R 1d It is C 1-6 alkyl; and PG 6 is a suitable protecting group, The process comprises: (a) providing a compound of formula (DXXI) or a salt thereof having the following structure: and (b) forming the compound of formula (DII) from the compound of formula (DXXI).

165. The process of claim 164, wherein PG 6 Selected from the group consisting of allyloxycarbonyl (Alloc), benzyloxycarbonyl (Cbz), tert-butoxycarbonyl (Boc) and 9-fluorenylmethoxycarbonyl (Fmoc).

166. The process of claim 164, wherein the compound of formula (DXXI) is treated with a compound deal with.

167. The process of claim 164, wherein the compound of formula (DXXI) is treated with a catalyst.

168. The process of claim 167, wherein the catalyst is Pd(dppf)Cl2-DCM.

169. The process of claim 164, wherein the compound of formula (DXXI) is treated with a base.

170. The process of claim 169, wherein the base is K2CO3.

171. The process of claim 164, wherein the preparation is carried out in a solvent mixture comprising H2O / iPrOH.

172. The process of claim 164, wherein the compound of formula (DII) has the structure of formula (DIIa) or formula (DIIb):

173. The process of claim 164, wherein the compound of formula (DXXI) is prepared by Exposure And formed.

174. The process of claim 173, wherein the compound of formula (DXXI) is formed in the presence of PPh3.

175. The process of claim 173, wherein the compound of formula (DXXI) is formed in the presence of DIAD.

176. The process of claim 173, wherein the compound of formula (DXXI) is in the presence of In the case of molecular sieves.

177. The process of claim 173, wherein the compound of formula (DXXI) is formed in an organic solvent.

178. The process of claim 177, wherein the organic solvent is THF.

179. The process of claim 173, wherein By Formed upon exposure to PBin2.

180. The process of claim 179, wherein Formed in the presence of Pd(OAc)2.

181. The process of claim 179, wherein Formed in the presence of PCy3.

182. The process of claim 179, wherein Formed in the presence of KOAc.

183. The process of claim 179, wherein Formed in the presence of dioxane.

184. The process of claim 179, wherein Formed at elevated temperatures over a period of time.

185. The process of claim 179, wherein By Exposure to Bu4N + Br3 - And formed.

186. The process of claim 185, wherein Formed in organic solvents.

187. The process of claim 186, wherein the organic solvent is a mixture of DCM and methanol.

188. A process for preparing a compound of formula (DII) or a salt thereof: in: q is an integer from 0 to 10, R 1d It is C 1-6 alkyl; and PG 6 is a suitable protecting group, The process comprises: (a) providing a compound of formula (DIV) or a salt thereof having the following structure: and (b) forming the compound of formula (DII) from the compound of formula (DIV).

189. The process of claim 188, wherein PG 6 Selected from the group consisting of allyloxycarbonyl (Alloc), benzyloxycarbonyl (Cbz), tert-butoxycarbonyl (Boc) and 9-fluorenylmethoxycarbonyl (Fmoc).

190. The process of claim 188, wherein the compound of formula (DII) has the structure of formula (DIIa) or formula (DIIb):

191. The process of claim 188, wherein the compound of formula (DIV) has the structure of formula (DIVa) or formula (DIVb):

192. The process of claim 188, wherein the step of forming the compound of formula (DII) comprises: The compound of formula (DIV) is reacted with a compound of formula (DVa) or formula (DVb): PG 6 -X(DVa) or PG 6 2O(DVb), in X is OSu, Otf, Cl or Br; to produce the compound of formula (DII).

193. The process of claim 192, wherein the compound of Formula (DVa) or Formula (DVb) is selected from the group consisting of AllocCl, Alloc2O, Cbz2O, CbzCl, FmocCl, FmocOSu, and Boc2O.

194. The process of claim 188, further comprising the steps of: Provided is a compound of formula (DXVI) having the following structure: in PG 8 is a suitable protecting group; and The compound of formula (DIV) is formed from the compound of formula (DXVI).

195. The process of claim 194, wherein the step of forming the compound of formula (DIV) comprises: The compound of formula (DXVI) is reacted with a protecting group removing agent to produce the compound of formula (DIV).

196. The process of claim 195, wherein the protecting group removing agent is selected from the group consisting of HCl, Pd(PPh)3, PhSiH3, H2, piperidine and trifluoroacetic acid (TFA).

197. The process of claim 194, wherein the compound of formula (DXVI) has the structure of formula (DXVIa) or formula (DXVIb):

198. The process of claim 194, further comprising the steps of: Provided are compounds of formula (DXVII) having the following structure: in LG' is a suitable reactive group; and The compound of formula (DXVI) is formed from the compound of formula (DXVII).

199. The process of claim 198, wherein the step of forming the compound of formula (DXVI) comprises: The compound of formula (DXVII) is reacted with an azide group-introducing agent to produce the compound of formula (DXVI).

200. The process of claim 199, wherein the azide group introducing agent is selected from the group consisting of: NaN3, TMSN3, (PhO)2P(O)N3, Zn(N3)2*2Py and n-Bu4NN3.

201. The process of claim 198, wherein LG' is selected from the group consisting of halogens, OTf, OMs, and OTs.

202. The process of claim 198, wherein the compound of formula (DXVII) has the structure of formula (DXVIIa) or formula (DXVIIb):

203. The process of claim 198, further comprising the steps of: Provided are compounds of formula (DIX) having the following structure: in LG" is a suitable reactive group; and The compound of formula (DXVII) is formed from the compound of formula (DIX).

204. The process of claim 203, wherein the LG" is selected from the group consisting of halogens, OTf, OMs, and OTs.

205. The process of claim 203, wherein the step of forming the compound of formula (DXVII) comprises: The compound of formula (DIX) is reacted with a compound of formula (DXVIII): to produce the compound of formula (DXVII).

206. The process of claim 203, wherein the compound of formula (DIX) has the structure of formula (DIXa):

207. The process of claim 205, wherein the compound of formula (DXVIII) has the structure of formula (DXVIIIa):

208. The process of claim 203, further comprising the steps of: Provided are compounds of formula (DXIX) having the following structure: and The compound of formula (DIX) is formed from the compound of formula (DXIX).

209. The process of claim 208, wherein the step of forming the compound of formula (DIX) comprises: The compound of formula (DXIX) is reacted with a compound of formula (DXV): in LG'' is a suitable active group, to produce the compound of formula (DXIX).

210. The process of claim 209, wherein the LG'' is selected from the group consisting of halogens, OTf, OMs, and OTs.

211. The process of claim 208, wherein the compound of formula (DXIX) has the structure of formula (DXIXa):

212. The process of claim 209, wherein the compound of formula (DXV) has the structure of formula (DXVa):

213. The process of claim 208, further comprising the steps of: Provided are compounds of formula (DXX) having the following structure: and The compound of formula (DXIX) is formed from the compound of formula (DXX).

214. The process of claim 213, wherein the step of forming the compound of formula (DXIX) comprises: The compound of formula (DXX) is reacted with a LG" introducing agent to produce the compound of formula (DXIX).

215. The process of claim 214, wherein the LG" introducing agent is NBu4Br3.

216. The process of claim 213, wherein the compound of formula (DXX) has the structure of formula (DXXa):

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