Selectively soluble triamide-derived supports for liquid-phase peptide and oligonucleotide synthesis

Abstract

The present invention provides a protecting reagent that can be removed in a high yield even under acidic conditions and can afford a resulting product at a high purity in an organic synthesis reaction such as peptide synthesis, oligonucleotide synthesis, and the like. The protecting reagent is a selectively soluble compound of Formula (I), which is derived from a triamide.

Description

SELECTIVELY SOLUBLE TRIAMIDE-DERIVED SUPPORTS FOR LIQUIDPHASE PEPTIDE AND OLIGONUCLEOTIDE SYNTHESISTECHNICAL FIELD

[0001] The field of the disclosure relates generally to amino acids, peptides, proteins, and nucleic acids for pharmaceutical industry and. more specifically, using selectively soluble supports for liquid phase organic synthesis of peptides and oligonucleotides.BACKGROUND OF THE INVENTION

[0002] The present invention relates to selectively soluble supports for liquid-phase peptide and oligonucleotide synthesis. The selectively soluble supports (also referred to herein as “tags”) are useful for precipitating an organic compound (e.g., a peptide, an oligonucleotide) in a solution wherein the organic compound uses the supports as a protecting group in peptide or oligonucleotide synthesis, particularly liquid phase synthesis, and methods of synthesis using such a compound.

[0003] Despite the widespread adoption of solid-phase synthesis of peptides and oligonucleotides due to the ease of use and automation, the method encounters significant challenges when scaling up. These challenges include inefficient diffusion of reactants to the solid support, the necessity for large excesses of reagents to drive reactions to completion, and the physical limitations of solid supports that lead to heterogeneous reaction conditions. Moreover, the solid phase method often results in a loss of yield and purity' with increased scale, necessitating complex and costly purification steps.

[0004] Conversely, liquid-phase synthesis on a soluble support offers a compelling alternative for scaling up peptide and oligonucleotide production. The soluble nature of the support in liquid-phase synthesis ensures homogenous reaction conditions, facilitating more efficient reactant interactions and reducing the requirement for excess reagents. This can lead to a more cost-effective process by minimizing waste and improving the overall yield and purity of the synthesized peptides and oligonucleotides.

[0005] Additionally, the liquid-phase approach can inherently accommodate larger reaction volumes without the physical constraints imposed by solid supports, thus enabling the synthesis of peptides and oligonucleotides at a larger scale. The ease of separating product from the soluble support through precipitation and washing steps circumvents the diffusion limitations of solid phase synthesis, offering a streamlined and potentially automatable purification process.

[0006] In an attempt to perform reactions in a homogeneous liquid phase while utilizing the advantages of the solid phase method in that isolation and purification after the reaction can be performed by filtration and washing alone, a method of isolating a particular component dissolved in a liquid as a solid has been used. This is because precipitation of a particular component alone facilitates isolation and purification after reaction.

[0007] A particular component dissolved in a solution can be precipitated only when predetermined conditions, such as chemical properties, property and relationship with solvents of the compound, are satisfied. However, determination of precipitation conditions requires trial and error and experimental searching in most cases. In liquid phase synthesis, moreover, some compounds to be synthesized are insoluble in organic solvents used for extraction or show low solubility therein, which necessitates confirmation of the property of each compound to search for isolation and purification methods therefor. Particularly, when sequential and multistep synthesis reactions are required as in peptide synthesis and the like, since isolation and purification conditions such as precipitation, extraction and the like need to be determined based on the properties unique to the compound synthesized in each step, a long time and high cost are required.

[0008] To solve such problems, a method using a carrier molecule wherein a dissolved state and an insolubilized state (precipitated state) irreversibly change according to the varying solvent composition has been developed. Using such a carrier, an isolation target compound can be selectively precipitated from a homogeneous solution state, in other words, a particular compound can be isolated after a liquid phase reaction when other soluble components still remain in a solution, thus obviating the need to consider extraction and precipitation conditions for each compound.

[0009] Soluble supports for peptide and oligonucleotide synthesis have been engineered to enhance the efficiency and scalability of the synthesis process. [3-Cyclodextrin supports, for instance, offer a streamlined chromatography and extraction process for short 5-mer DNA oligonucleotides, but may be limited by oligomer length. Pentaerythritol-based supports boast versatility' for both DNA and RNA syntheses up to 20 nucleotides, though the purification process is relatively complex. The adamantane-core support is notable for facilitating the synthesis of densely branched 8-mer oligonucleotides, yet the method may require extensive purification steps. PEG-based supports accommodate longer chains of up to 20-mers and are known for their efficient synthesis, though the need for multiple precipitation steps could be seen as a limitation in terms of process throughput and operational simplicity. ASS-based and Ajiphase supports have gained use in large scale production, providing a balance between efficiency and molecule stability.

[0010] Accordingly, there is a need in the art for new selectively soluble supports for liquid phase organic synthesis of peptides and oligonucleotides which are superior in broad utility' and stability7, which are useful as a protecting group (anchor) for peptide and oligonucleotide synthesis in the liquid phase.BRIEF SUMMARY OF THE INVENTION

[0011] In one aspect, disclosed herein is a selectively soluble support compound derived from an amino triamide the selectively soluble support compound represented by Formula (I):wherein Ri is H or a straight or branched C1-C40 alky l or aliphatic group; R2 is a straight or branched C6-C40 alkyl group; R3 is H or -O-alkyl; R4 is H or -O-alkyl; and Y is selectedfrom the group consisting of -OH, -COOH, -O-C(O)-X, wherein X is Cl, Br, I, F, imidazole or a leaving group, and an -NHR group, wherein R is H, a straight or branched C1-C30 alkyl group, or a C7-C30 aralkyl group, wherein the alkyl portion of the C7-C30 aralkyl group is a straight or branched C1-C30 alkyl.

[0012] In another aspect, disclosed herein is a liquid phase method of producing an oligonucleotide comprising a step of subjecting a 5'-hydroxyl group or 3'-hydroxyl group of a nucleoside or oligonucleotide having a selectively soluble support in at least one location selected from the group consisting of 2'-position. 3'-position, 5'-position and a nucleobase moiety and having a 5'-hydroxyl group or a 3'-hydroxyl group, to H- phosphonation to convert the 5'-hydroxyl group or the 3'-hydroxyl group into an H- phosphonated form, wherein the selectively soluble support is a compound represented by Formula (I):wh erein Ri is H or a straight or branched C1-C40 alkyl or aliphatic group; R2 is a straight or branched C6-C40 alkyl group; R3 is H or -O-alkyl; R4 is H or -O-alkyl; and Y is selected from the group consisting of -OH, -COOH, -O-C(O)-X, w herein X is Cl, Br, I, F, imidazole or a leaving group, and an -NHR group, wherein R is H, a straight or branched C1-C30 alkyl group, or a C7-C30 aralkyl group, wherein the alkyl portion of the C7-C30 aralkyl group is a straight or branched C1-C30 alkyl.

[0013] In yet another aspect, disclosed herein is a liquid phase method of producing a peptide comprising the following steps; obtaining C-protected amino acid or C-protected peptide, comprising condensing a compound of Formula (I):wherein Ri is H or a straight or branched C1-C40 alkyl or aliphatic group; R2 is a straight or branched C6-C40 alkyl group; Rs is H or -O-alkyl; R4 is H or -O-alkyl; and Y is selected from the group consisting of -OH, -COOH, -O-C(O)-X, wherein X is Cl, Br, I, F, imidazole or a leaving group, and an -NHR group, wherein R is H, a straight or branched Ci-Cso alkyl group, or a C7-C30 aralkyl group, wherein the alkyl portion of the C7-C30 aralkyl group is a straight or branched C1-C30 alkyl, with the C-terminal of N-protected amino acid or N- protected peptide; removing a temporary' protecting group of the N-terminal of the amino acid or peptide obtained in the above-mentioned step; condensing the N-terminal of the amino acid or peptide obtained in the above-mentioned step with N-protected amino acid or N-protected peptide; and precipitating the peptide obtained in the above-mentioned step.

[0014] Compounds of Formula (I) can be used as both a support for peptide and oligonucleotide synthesis and as a protecting agent for a functional group, such as a carboxy group, a hydroxy group, an amino group, and a mercapto group. The compound in which the carbonyl group is protected by a compound of Formula (I) is characterized by tending to be in a liquid state and its high solubility in a solvent. Accordingly, a compound in which a functional group is protected by a compound of Formula (I) and used as a protecting agent can be readily dissolved in an organic solvent and can be easily separated and purified by a procedure such as liquid-liquid phase separation. The protecting group used in the inventive compound can be readily eliminated by. for example, an acid, a base, or catalytic hydrogenation.

[0015] Compounds which can be protected by a compound of Formula (1) may be any compound having a functional group such as a carboxy group, a hydroxy group, a diol group, and an amino group, and examples thereof include amino acids, peptides, saccharides, proteins, nucleotides, various medicinal compounds and agrochemical compounds, and various polymers and dendrimers.

[0016] The method of synthesizing a peptide using a compound of Formula (I) as a protecting agent includes, for example, the following steps (1) to (4). In this peptide synthesizing method, the protected peptide obtained in each step and the target peptide can be separated by liquid-liquid separation.

[0017] (1) The compound of Formula (I) is condensed with the C-terminal carboxy group of an N-protected amino acid or N-protected peptide in a soluble solvent to obtain an N- protected C-protected amino acid or N-protected C-protected peptide in which the C- terminal is protected by the compound of Formula (I).

[0018] (2) The protecting group on the N-terminal of the resulting N-protected C- protected amino acid or N-protected C-protected peptide is removed to obtain a C- protected amino acid or C-protected peptide.

[0019] (3) An N-protected amino acid or N-protected peptide is condensed with the N- terminal of the resulting C-protected amino acid or C-protected peptide to obtain an N- protected C-protected peptide.

[0020] (4) The protecting groups on the N-terminal and the protecting group on the C- terminal of the resulting N-protected C-protected peptide are removed to obtain the target peptide.

[0021] This procedure using the selectively soluble support compounds of Formula (I) can also be used to synthesize an oligonucleotide in a liquid phase synthesis.

[0022] The tags of Formula (I) allow for easy in-process NMR analysis for confirmation.

[0023] The embodiments and features of the present invention can be used alone or in combinations with each other.BRIEF DESCRIPTION OF THE DRAWINGS

[0024] The foregoing will be apparent from the following more particular description of example embodiments of the invention, as illustrated in the accompanying drawings.

[0025] FIG. 1 is anXH NMR (400 MHz, CDCl3) of compound 9 of the Example 1 ;

[0026] FIG. 2 is an ’l l NMR (400 MHz, CDCl3) of compound 10 of the Example 1;

[0027] FIG. 3 is an1H NMR (400 MHz, D2O) spectrum of compound 24 of the Example 2;

[0028] FIG. 4 is a13C NMR (101 MHz, D2O) spectrum of compound 24 of the Example 2;

[0029] FIG. 5 is an LC / MS trace (EIC) chromatograph of purified compound 24 of the Example 2;

[0030] FIG. 6 is a LCMS (TIC, ESI) chromatograph of purified compound 24 of the Example 2;

[0031] FIG. 7 is anXH NMR (400 MHz. D2O) spectrum of compound 25 of the Example 2;

[0032] FIG. 8 is a13C NMR (101 MHz, D2O) spectrum of compound 25 of the Example 2;

[0033] FIG. 9 is an LC / MS trace (EIC) chromatograph of purified compound 25 of the Example 2;

[0034] FIG. 10 is an LCMS (TIC, ESI) chromatograph of purified compound 25 of the Example 2;

[0035] FIG. 11 is an1HNMR (400 MHz, CDCls) spectrum of compound 3 of the Example 3 after drying;

[0036] FIG. 12 is an the ’H NMR (400 MHz, DMSO-d6) spectrum of compound 4 of the Example 3 after drying;

[0037] FIG. 13 is an the H NMR (400 MHz, DMSO-d6) spectrum of compound 7 of the Example 3 after drying;

[0038] FIG. 14 is anXH NMR (400 MHz, CDCl3) spectrum of compound 12 of the Example 3 after drying;

[0039] FIG. 15 is anXH NMR (400 MHz, CDCl3) spectrum of compound 13 Tag 4B of the Example 3 after drying;

[0040] FIG. 16 is an LCMS (ES+) chromatograph of pentapeptide 25 of the Example 4;

[0041] FIG. 17 is an LCMS (TIC, ESI) chromatograph of purified pentapeptide 25 of the Example 4;

[0042] FIG. 18 is anXH NMR (400 MHz, D2O) spectrum of pentapeptide 25 of the Example 4;

[0043] FIG. 19 is a13C NMR (101 MHz, D2O) spectrum of pentapeptide 25 of the Example 4;

[0044] FIG. 20 is an LCMS (ES+) chromatograph of decapeptide 46 of the Example 4;

[0045] FIG. 21 is an LCMS (TIC, ESI) chromatograph of purified decapeptide 46 of the Example 4;

[0046] FIG. 22 is an1H NMR (400 MHz, D2O) spectrum of decapeptide 46 of the Example 4;

[0047] FIG. 23 is a13C NMR (101 MHz, D2O) spectrum of decapeptide 46 of the Example 4;

[0048] FIG. 24 is an LCMS (ES+) chromatograph of octreotide acetate of the Example 5;

[0049] FIG. 25 is an LCMS (TIC, ESI) chromatograph of purified octreotide acetate of the Example 5;

[0050] FIG. 26 is an ’H NMR (400 MHz, DMSO-tie) spectrum of octreotide acetate of the Example 5;

[0051] FIG. 27 is a13C NMR (400 MHz, DMSO-ife) spectrum of octreotide acetate of the Example 5; and

[0052] FIG. 28 is an 'H NMR (400 MHz, DMSO-d6) spectrum of octreotide acetate standard.DETAILED DESCRIPTION OF THE INVENTION

[0001] Unless otherwise specified in the sentences, any technique terms and scientific terms used in the present specification, have the same meaning as those generally understood by those of ordinary skill in the art in the art the present invention belongs to. Any methods and materials similar or equivalent to those described in the present specification can be used for practicing or testing the present invention, and preferable methods and materials are described in the following. All publications and patents referredto in the Specification are hereby incorporated by reference so as to describe and disclose constructed products and methodology described in, for example, publications usable in relation to the descnbed invention.Definitions

[0002] As used herein, the terms "a", "an", "the" and the like refer to both the singular and plural unless the context clearly indicates otherwise. "A bottle", for example, refers to a single bottle or more than one bottle.

[0003] As used herein, the terms “comprising’’ (and any form of comprising, such as “comprise”, “comprises”, and “comprised”), “having” (and any form of having, such as “have” and “has”), “including” (and any form of including, such as “includes” and “include”), or “containing” (and any form of containing, such as “contains” and “contain”), are inclusive and open-ended and include the options following the terms, and do not exclude additional, unrecited elements or method steps.

[0004] As used herein, the term “selectively soluble support” or “tag” is a compound that, when reacted with a peptide or a nucleotide, protects a reactive site on the peptide or nucleotide and supports the elongation of the peptide or nucleotide and can be solubilized or precipitated by changing the composition of the solvent in which the elongation reactions occur.

[0005] As used herein, the term “alkyl” means a saturated hydrocarbon group which is straight-chained or branched. In some embodiments, the alkyl group has from 1 to 20 carbon atoms, from 2 to 20 carbon atoms, from 1 to 10 carbon atoms, from 2 to 10 carbon atoms, from 1 to 8 carbon atoms, from 2 to 8 carbon atoms, from 1 to 6 carbon atoms, from 2 to 6 carbon atoms, from 1 to 4 carbon atoms, from 2 to 4 carbon atoms, from 1 to 3 carbon atoms, or 2 or 3 carbon atoms. Examples of alkyl groups include, but are not limited to, methyl (Me), ethyl (Et), propyl (e.g., n-propyl and isopropyl), butyl (e.g., n-butyl, t- butyl, isobutyl), pentyl (e.g., n-pentyl, isopentyd, neopentyl), hexyl, isohexyl, heptyl, octyl, nonyl, 4,4-dimethylpentyl, 2,2,4-trimethylpentyl, decyl, undecyl, dodecyl, 2-methyl-l- propyL 2-methyl-2 -propyl. 2-methyl-l -butyl, 3-methyl-l -butyl. 2-methyl-3-butyl, 2- methyl-1 -penty l, 2,2-dimethyl-l -propyl, 3-methyl-l-pentyl, 4-methyl-l -pentyl, 2-methyl-2- pentyl, 3-methyl-2-pentyl, 4-methyl-2-pentyl, 2,2-dimethyl-l -butyl, 3,3-dimethyl-l-butyl, 2-ethyl-l -butyl, and the like.

[0006] As used herein, the term “alkylamino” means an amino group substituted by at least one alkyd group. In some embodiments, the alkyl group is a lower alkyl group having from 1 to 6 carbon atoms. Alkylammo groups include, but are not limited to. -NHCH2CH3, -NH(CH2)2CH3, -NH(CH2)3CH3, -NH(CH2)4CH3, and -NH(CH2)5CH3, and the like.

[0007] As used herein, the term “amino” means -NH2.

[0008] As used herein, the term “aryl” means a monocyclic, bicyclic, or polycyclic (e.g.. having 2, 3 or 4 fused rings) aromatic hydrocarbon. In some embodiments, the aryl group has from 6 to 20 carbon atoms or from 6 to 10 carbon atoms. Examples of aryl groups include, but are not limited to, phenyl, naphthyl, anthracenyl, phenanthrenyl, indanyl, indenyl, and tetrahydronaphthyl, and the like.

[0009] As used herein, the term “aralkyl” or “arylalkyl” refers to a monovalent alkyl group substituted with one or more aryl groups. In certain embodiments, the aralkyl has from 7 to 30, from 7 to 20, or from 7 to 16 carbon atoms. Examples of aralkyl groups include, but are not limited to, benzy l, 1 -phenylethyl, 2 -phenylethyl, and 3 -phenylpropyl. In certain embodiments, the aralkyl is optionally substituted with one or more.

[0010] The term “halogen”, “halide” or “halo” refers to fluorine, chlorine, bromine, and / or iodine.

[0011] As used herein, the term “nucleoside” is the constituent unit of an oligonucleotide means a compound wherein a nucleic acid base is bonded to the T-position of a sugar (e.g., ribose or deoxyribose wherein the carbon atoms at 2-position and carbon atom at 4-position of 2-deoxyribose, ribose, ribose ring or deoxyribose ring are bonded by a divalent organic group, and the like) by N-glycosidation. Examples of the ribose or deoxyribose wherein the carbon atom at 2-position and carbon atom at 4-position of the ribose ring or deoxyribose ring are bonded by a divalent organic group include the following compounds:

[0012] As used herein, the term "‘nucleotide” means a compound wherein a phosphoric acid group is bonded to a nucleoside.

[0013] As used herein, the term “oligonucleotide” means a compound wherein one or more of a plurality of nucleotides are bonded to a nucleoside. While the number of nucleosides in oligonucleotide in the present invention is not particularly limited, it is preferably 3 to 50, more preferably 5 to 30. In certain embodiments, one or more of the plurality of nucleosides is modified. In certain embodiments, an oligonucleotide comprises one or more ribonucleosides (RNA) and / or deoxyribonucleosides (DNA).

[0014] As used herein, the term “nucleoside” and “nucleotide” encompass morpholino nucleoside and morpholino nucleoside having a morpholine residue instead of a ribose residue or a deoxyribose residue.

[0015] Also as used herein, the description of one or more method steps does not preclude the presence of additional method steps before or after the combined recited steps. Additional steps may also be intervening steps to those described. In addition, it is understood that the lettering of process steps or ingredients is a convenient means foridentifying discrete activities or ingredients and the recited lettering can be arranged in any sequence.

[0016] Where a range of numbers is presented in the application, it is understood that the range includes all integers and fractions thereof between the stated range limits. A range of numbers expressly includes numbers less than the stated endpoints and those in-between the stated range. A range of from 1-3, for example, includes the integers one, two, and three as well as any fractions that reside between these integers.Compounds

[0017] The compounds of the present invention are selectively soluble support compounds derived from an amino triamide, the selectively soluble support compound represented by Formula (I):whereinRi is H or a straight or branched C1-C40 alkyl or aliphatic group;R2 is a C6-C40 straight or branched alkyl group;R3 and R are each independently selected from H and -O-alkyl; andY is selected from the group consisting of -OH, -COOH, -O-C(O)-X, wherein X is Cl, Br, I, F, imidazole or a leaving group, and an -NHR group, wherein R is H, a straight or branched C1-C30 alkyl group, or a C7-C30 aralkyl group, wherein the alkyl portion of the C7-C30 aralkyl group is a straight or branched C1-C30 alkyl.

[0018] A compound represented by Formula (I) of the present invention is bonded to a compound to be protected via group Y.

[0019] That is, the compound of the present invention wherein Y is a -OH, -COOH, O- C(O)-X, wherein X is Cl, Br, I, F, imidazole or a leaving group, or an -NHR group is bonded to the C-terminal of, for example, an amino acid or a peptide or an oligonucleotide for protection thereof.

[0020] Examples of the “alkyl group” for R include a C1-30 alkyl group, preferably a C 1-10 alkyl group, more preferably a C1-6 alkyl group. Preferable examples include methyl, ethyl, propyl, isopropyl, butyl, isobutyl, sec-butyL tert- butyl, and the like, and particularly preferred are methyl and ethyl.

[0021] In the present specification, examples of the “aralkyl group” for R include a C7-30 aralkyl group. Preferred is a C7-20 aralkyl group, more preferred is a C7-16 aralkyl group (a Ce-4o ary I -C 1-6 alkyl group). Preferable examples include benzyl, 1 -phenylethyl, 2- phenylethyl, 1 -phenylpropyl, naphthylmethyl, 1 -naphthylethyl, 1 -naphthylpropyl, and the like, and particularly preferred is benzyl.

[0022] As R, a hydrogen atom, a C1-6 alkyl group or a C7-16 aralkyl group is preferable, a hydrogen atom, methyl, ethyl or benzy l is more preferable, and a hydrogen atom is particularly preferable.

[0023] In some embodiments, Y is -OH. In other embodiments, Y is -OH and Ri is C18H37.

[0024] In other embodiments, Y is -NHR. In embodiments when Y is -NHR, R is H, a C1-C30 alkyl group, or a C7-C30 aralkyl group. In embodiments wherein R is C1-C30 alkyl, R is preferably methyl. In embodiments wherein R is C7-C30 aralkyl, R is preferably methyl.

[0025] In some embodiments, R is H.

[0026] Examples of the “aralky l group” for R include a C7-30 aralkyl group. Preferred is a C7-20 aralkyl group, more preferred is a C7-16 aralkyl group (a Cg-4o aryl-Ci-6 alkyl group). Preferable examples include benzy l, 1 -phenylethyl, 2-phenylethyl, 1 -phenylpropyl, naphthylmethyl, 1 -naphthylethyl, 1 -naphthylpropyl, and the like, and particularly preferred is benzy l.

[0027] The “C6-C40 aliphatic group” for R2 means a monovalent organic group having an aliphatic hydrocarbon group in the molecular structure. The R2 groups may be referred to herein “tails,” whose lengths can be selected to impact solubility of the compound.

[0028] The “aliphatic group” is an aliphatic hydrocarbon group consisting of straight chain or branched, saturated or unsaturated, and an aliphatic hydrocarbon group having a carbon number of not less than 6 is preferable, an aliphatic hydrocarbon group having acarbon number of 6 to 40 is particularly preferable, an aliphatic hydrocarbon group having a carbon number of 6 to 30 is more preferable, and an aliphatic hydrocarbon group having a carbon number of 10 to 30 is further preferable.

[0029] Examples of the "aliphatic group" include monovalent groups such as an alkyl group, a cycloalkyl group, an alkenyl group, a cycloalkenyl group, an alkynyl group and the like and divalent groups derived therefrom. Preferred are monovalent groups such as a methyl group, an ethyl group, a propyl group, an isopropyl group, a butyl group, an isobutyl group, a sec-butyl group, a tert-butyl group, a penty l group, a hexyl group, an octyl group, a decyl group, a dodecyl group, a lauryl group, a tridecyl group, a myristyl group, a cetyl group, a stearyl group, an aralkyl group, a behenyl group, an oleyl group, an isostearyl group and the like and divalent groups derived therefrom. In preferred embodiments, Ri is a stearyl group, C18H37.

[0030] The “C1-C40 aliphatic group” for Ri means a monovalent organic group having an aliphatic hydrocarbon group in the molecular structure. The Ri groups also may be referred to herein “tails,” whose lengths can be selected to impact solubility' of the compound.

[0031] In some embodiments, Ri is a Ci-6 aliphatic group and, preferably, is a methyl group. In other embodiments, Ri is H. In yet other embodiments, Ri is a Ce-40 aliphatic group. In yet other embodiments, Ri is a C16-30 aliphatic group and, in still other embodiments, each of Ri and R2 is a stearyl group, C18H37.

[0032] In some embodiments of Formula (I), R3 and R4 are not the same. In some embodiments, R3 is H and R4 is O-alkyl. In some embodiments, R3 is O-alkyl and R4 is H. In other embodiments, R3 is O-methyl and R4 is O-ethyl.

[0033] In some embodiments of Formula (I), wherein R3 is -OMe and R4 is H, an example of a selectively soluble support compound of Formula (I) is Formula (II):Another example of the selectively soluble support compound of Formula (I), wherein R3 is H and R4 is -OMe, is Formula (III):

[0034] Production Method of Compounds of Formula (I)

[0035] The compounds represented by Formula (I) of the present invention can be made by any method known to those skilled in the art. An exemplary method is detailed in Example 1 for the compound. which is a compound of Formula (I) wherein Y is -OH and each of Ri and R2 are C18H37. Below is a general schematic for the synthesis of this compound as well as that of, which is a compound of Formula (I) wherein Y is -OH, each Ri is methyl, and each R2 is C18H37. Notably, the starting compound, an amine triester derivative is readily available and is inexpensive.Synthesis of Tag 4A and Tag 4B:Use as a Selectively Soluble Support in Organic Synthesis for Peptides

[0036] The peptide synthesis method of the present invention can be suitably used in a peptide production method using a selectively soluble support (i.e., a carrier or Tag) capable of reversibly repeating the dissolved state and the insolubilized state.

[0037] As the peptide production method including the peptide synthesis method of the present invention, for example, steps as shown below are exemplified.

[0038] Compounds which can be protected by a compound of Formula (I) may be any compound having a functional group such as a carboxy group, a hydroxy group, a diol group, and an amino group, and examples thereof include amino acids, peptides, saccharides, proteins, nucleotides, various medicinal compounds and agrochemical compounds, and various polymers and dendrimers.

[0039] The compound of the present invention can be used as a reagent to protect a carboxyl group and the like, that is, a protecting reagent for amino acid or peptide, in organic synthesis reactions, preferably peptide synthesis and the like. Specifically, it is preferably introduced as a protecting group (anchor) of C-terminal functional groups such as a carboxyl group, a carboxamide group, a thiol group and the like, and side chain functional groups (hereinafter to be referred to as C-terminal etc.). When using as a protecting reagent, the compound may be activated to allow reaction with a substituent to be protected or may be converted to an equivalent to allow reaction.

[0040] The method of synthesizing a peptide using a compound of Formula (I) as a protecting agent includes, for example, the following steps (1) to (4). In this peptide synthesizing method, the protected peptide obtained in each step and the target peptide can be separated by liquid-liquid separation.

[0041] step (1) a step of dissolving the compound of the present invention in soluble solvent (dissolution step),

[0042] step (2) a step of bonding the compound of the present invention dissolved in soluble solvent as obtained in the above-mentioned step to a reaction substrate (bonding step),

[0043] step (3) a step of precipitating the bonded product obtained in the above- mentioned step (precipitation step), and

[0044] step (4) a step of redissolving the bonded product obtained in the above- mentioned step or a product resulting from the reaction in a soluble solvent and cleaving the tag from the bonded product or the resulting product (deprotection / cleavage step).

[0045] Step (1) (Dissolution Step)

[0046] In this step, the selectively soluble support compound of Formula (I) is dissolved in a soluble solvent.

[0047] As the solvent, a general organic solvent can be used. Since superior reactivity can be expected when the solubility in the solvent is higher, a solvent in which the compound of Formula (I) shows high solubility is preferably selected. Specifically, halogenated hydrocarbon such as chloroform, dichloromethane and the like; and a nonpolar organic solvent such as 1,4-dioxane, tetrahydrofuran and the like can be used. These solvents maybe used in a mixture of two or more kinds thereof at an appropriate ratio. In addition, the above-mentioned halogenated hydrocarbons and nonpolar organic solvent may be mixed with aromatic hydrocarbons such as benzene, toluene, xylene and the like; nitriles such as acetonitrile, propionitrile and the like; ketones such as acetone, 2-butanone and the like; amides such as N,N-dimethylformamide and the like; sulfoxides such as dimethyl sulfoxide and the like at an appropriate ratio and used as long as the compound of the present invention is dissolved.

[0048] Step (2) (Bonding Step)

[0049] In this step, the compound of Formula (I) dissolved in a soluble solvent, which is obtained in the above-mentioned step (1) is bonded to a reaction substrate.

[0050] Here, the reaction substrate has a — COOH group of protected amino acid and the like, and the amount of the reaction substrate to be used is 1-10 mol, preferably 1-5 mol, per 1 mol of the compound of the present invention.

[0051] When Y is a hydroxyl group, an ester bond can be formed by adding a condensing agent into a solvent that does not influence the reaction in the presence of a dimethylaminopyridine catalyst.

[0052] When Y is a group NHR, an amide bond can be formed by adding a condensing agent in the presence of a condensation additive (condensation promoter) such as 1- hydroxybenzotriazole (HOBt), ethyl l -hydroxy-lH-l,2,3-triazole-5-carboxylate (HOCt), 1- hydroxy-7-azabenzotriazole (HOAt) and the like.

[0053] The amount of the condensing additive to be used is preferably 0.05-1.5 mol per 1 mol of the compound of the present invention.

[0054] Examples of the condensing agent include dicyclohexylcarbodiimide (DCC), diisopropylcarbodiimide (DIC), N-ethyl-N'-3-dimethylaminopropylcarbodiimide and hydrochloride thereof (EDC HC1). hexafluorophosphoric acid (benzotriazol- 1- yloxy)tripyrrolizinophosphonium (PyBop), O-(benzotriazol-l-yl)-N,N,N',N'- tetramethyluronium tetrafluoroborate (TBTU), l-[bis(dimethylamino)methylene]-5-chloro- IH-benzotriazolium 3-oxide hexafluorophosphate (HCTU), O-benzotriazole-N,N,N'.N'- tetramethyluronium hexafluoroborate (HBTU), and the like.

[0055] The amount of the condensing agent to be used is 1-10 mol, preferably 1-5 mol. per 1 mol of the compound of the present invention.

[0056] Examples of the solvent include aromatic hydrocarbons such as toluene, xylene and the like; ethers such as diethyl ether, tetrahydrofuran, dioxane and the like; halogenated hydrocarbons such as chloroform, dichloromethane etc., and the like, and a mixture thereof. Of these, toluene, tetrahydrofuran and the like are preferable.

[0057] The reaction temperature is generally -10° C. to 30° C., preferably 0° C. to 20° C., and the reaction time is generally 1-30 hr.

[0058] For confirmation of the progress of the reaction, a method similar to general liquid phase organic synthesis reaction can be applied. That is, thin layer silica gel chromatography, high performance liquid chromatography and the like can be used to track the reaction.1HNMR may also be used to analyze / confirm the product, either in solution or as precipitated aliquot of the reaction mixture. The ability to use1HNMR is a benefit of using selectively soluble supports over solid-phase synthesis methods.

[0059] Step (3) (Precipitation Step)

[0060] In this step, the bonded product obtained in the above-mentioned step (2), or a product obtained by dissolving the bonded product in a soluble solvent and performing a desired organic synthesis reaction is isolated by changing the solvent in which the bonded product or the resulting product is dissolved (e.g., change of solvent composition, change of solvent kind) to allow precipitation. That is, the reaction is performed under the conditions where the bonded product is dissolved and, after the reaction, the solvent composition is changed to allow precipitation of the bonded product through dilution of the reaction mixture with a polar solvent in which the product is insoluble. As the substitution solvent, polar organic solvents such as methanol, acetonitrile, and the like are used. Thatis, the reaction is performed under conditions where the compound can be dissolved and, for solvent substitution after the reaction, halogenated solvents, THF and the like are used for dissolution and polar organic solvents such as methanol, acetonitrile and the like are used for precipitation. Isolation by fdtration and further washing of the isolated solid with additional portions of the polar solvent(s) should then afford the clean product.

[0061] Step (4) (Deprotection Step)

[0062] In this step, the selectively soluble support compound of Formula (I) is finally removed from the bonded product or resulting product isolated by precipitation in the above-mentioned step (3) to give the object compound.

[0063] When Y is a hydroxyl group, it reacts with — COOH of the initial reaction substrate to form an ester bond. After deprotection, the C-terminal of peptide becomes — COOH. On the other hand, when Y is an — NHR group, the — COOH group of the reaction substrate becomes an amide bond and, by removal of the support, the C-terminal is converted to a — CONHR group.

[0064] When Y is a hydroxyl group, the deprotection is preferably performed by an acid treatment. Examples of the acid to be used include trifluoroacetic acid (herein referred to as “TFA”), hydrochloric acid, sulfuric acid, methanesulfonic acid, p-toluenesulfonic acid and the like, with preference given to TFA. The deprotection is preferably performed under solution conditions using a solution such as chloroform, dichloromethane and THF, having an acid concentration of from about 0.1% to about 97%.

[0065] The amount of the acid to be used is appropriately determined according to the kind of the acid to be used, and an amount suitable for removing the support is used. The amount that can be used is 3-100 mol, preferably 5-50 mol, per 1 mol of the bonded product. When an acid is used, trifluoromethanesulfonic acid, trimethylsilyl trifluoromethanesulfonate, BF3, Et20, and the like can also be added as a further strong acid source.

[0066] The reaction temperature is generally 0°C to 80° C, preferably 0°C to 30° C. The reaction time is generally 0.5-24 hr.

[0067] By utilizing the above-mentioned steps, a peptide can be produced. The compound of Formula (I) of the present invention can be mainly used as, but is not limited to, a protecting reagent for C-terminal of amino acid or peptide and the like. In addition.since the compound of Formula (I) wherein Y is a hydroxyl group can be converted to a corresponding chloroformate form by a method conventionally used in the pertinent field, for example, reaction with phosgene, the chloroformate form can also be used as a protecting reagent of N-terminal and the like.

[0068] A method of producing a peptide utilizing the above-mentioned steps, comprising the following steps;

[0069] (1) a step of obtaining C-protected amino acid or C-protected peptide, comprising condensing a compound of Formula (I) with the C-terminal of N-protected amino acid or N-protected peptide (C-terminal protection step),

[0070] (2) a step of removing a temporary protecting group of the N-terminal of the amino acid or peptide obtained in the above-mentioned step (deprotection step of N- terminal),

[0071] (3) a step of condensing the N-terminal of the amino acid or peptide obtained in the above-mentioned step with N-protected amino acid or N-protected peptide (peptide chain elongation step), and

[0072] (4) a step of precipitating the peptide obtained in the above-mentioned step (precipitation step).

[0073] Step (1) (C -Terminal Protection Step)

[0074] In this step, the compound of Formula (I) is condensed with the C-terminal of N- protected amino acid or N-protected peptide to give C-protected amino acid or C-protected peptide. For example, the step can be performed according to the above-mentioned binding step.

[0075] In the present invention, the ‘"N-protected amino acid" and ‘"N-protected peptide" mean amino acid and peptide wherein amino group is protected and carboxyl group is unprotected, and may be referred to as “P-AA-OH” (P is amino-protecting group (or temporary protecting group on demand)).

[0076] The condensation reaction of the compound of Formula (I) and C-terminal of N- protected amino acid or N-protected peptide is preferably performed in a solvent that does not influence the reaction. For example, the reaction is performed in the presence of a condensing agent when Y is a hydroxyl group or — NHR group, and an ester bond isformed when Y is hydroxyl, and amide bond is formed when Y is an — NHR group. Examples of the condensing agent include di cyclohexylcarbodiimide. diisopropylcarbodiimide, N-ethyl-N'-3-dimethylaminopropylcarbodiimide and a hydrochloride thereof (EDC.HC1), and the like. An ester bond forming reaction is performed in the presence of dimethylaminopyridine, and an amide bond forming reaction is performed in the presence of a condensation additive such as HOBt, HOCt and the like.

[0077] Examples of the solvent to be used for the step include halogenated hydrocarbons such as chloroform, dichloromethane and the like; and nonpolar organic solvents such as 1,4-di oxane, tetrahydrofuran and the like. These solvents may be used in a mixture of two or more kinds thereof at an appropriate ratio, and preferred is dichloromethane. The amount of the solvent to be used is preferably 2- to 50-fold volume relative to the benzylic compound of the present invention.

[0078] The reaction temperature is generally -10 °C to 40 °C. preferably 0 °C to 30 °C. The reaction time is generally 1-70 hr.

[0079] Step (2) (N-Terminal Deprotection Step)

[0080] In this step, the temporary protecting group of the N-terminal of amino acid or peptide obtained in the above-mentioned step (1) is removed.

[0081] As the temporary protecting group of the N-terminal, the below-mentioned aminoprotecting groups generally used in the technical field of peptide chemistry and the like are usable. In the present invention, a tert-butoxycarbonyl group (hereinafter to be also referred to as Boc group), or a 9-fluorenylmethoxy carbonyl group (hereinafter to be also referred to as Fmoc group) are / is preferably used.

[0082] While the deprotection conditions are appropriately selected depending on the kind of the temporary' protecting group, a group that can be removed under conditions different from removal of the protecting reagent derived from the compound of the present invention is preferable. For example, Fmoc group can be removed by a treatment with a base, and Boc group can be removed by a treatment with an acid. The reaction is performed in a solvent which does not influence the reaction.

[0083] Examples of the base include dimethylamine, diethylamine, piperidine, and the like. Examples of the solvent include halogenated hydrocarbons such as chloroform, dichloromethane, and the like; aromatic hydrocarbons such as toluene, xylene, and the like;ethers such as diethyl ether, tetrahydrofuran, dioxane, and the like; nitriles such as acetonitrile and the like, and a mixture thereof.

[0084] Step (3) (Peptide Chain Elongation Step)

[0085] In this step, the N-terminal of amino acid or peptide deprotected in N-terminal in step (2) is condensed with N-protected amino acid or N-protected peptide.

[0086] This step is performed by using the condensing agent, condensation additive and the like described in the aforementioned step (1) and under peptide synthesis conditions generally used in the field of peptide chemistry.

[0087] Step (4) (Precipitation Step)

[0088] This step is performed in the same manner as in the precipitation step in the above-mentioned step (3).

[0089] In the production method of the peptide of the present invention, the N-protected amino acid or N-protected peptide obtained in step (4) can be subjected to steps (5)-(7) in a desired number of repeats:

[0090] (5) a step of removing the temporary protecting group of N-terminal of the peptide obtained in the precipitation step,

[0091] (6) a step of condensing N-protected amino acid or N-protected peptide with N- terminal of the peptide obtained in the above-mentioned step, and

[0092] (7) a step of precipitating the peptide obtained in the above-mentioned step.

[0093] Step (5)

[0094] The step is performed in the same manner as in the deprotection step of N- terminal in step (2).

[0095] Step (6)

[0096] This step is performed in the same manner as in the peptide chain elongation step of step (3).

[0097] Step (7)

[0098] This step is performed in the same manner as in the precipitation step of step (3).

[0099] In the production method of the peptide of the present invention, a step of deprotecting the C-terminal of the peptide, wherein the C-terminal is protected with a compound of Formula (I), may be further contained after the precipitation step of step (4) or step (7). For example, the step can be performed according to the above-mentioned step (4) for deprotection of the anchor of the present invention.

[0100] The resultant peptide as the final target can be isolated and purified according to methods normally used in peptide chemistry. For example, the reaction mixture can be subjected to extraction washing, crystallization, chromatography and the like, to isolate and purify a peptide as the final target.

[0101] When the organic synthesis reaction or peptide synthesis reaction of the present invention contains multi -steps, the aforementioned precipitation step may be appropriately omitted as long as the reaction in the next step is not influenced.

[0102] In each reaction, when the starting compound has a hydroxy group, an amino group, a carboxy group or a carbonyl group (particularly when amino acid or peptide has a functional group in the side chain), a protecting group generally used in the peptide chemistry and the like may be introduced into these groups, and the object compound can be obtained by removing the protecting group as necessary after the reaction.

[0103] In a final step (excluding any purification steps), the peptide is cleaved from the selectively soluble support of the present invention by addition of an acid or a base as is readily understood by a person of ordinary' skill in the art.

[0104] Specific examples of liquid phase peptide production with partially soluble supports can be found in, for example, U.S. Patent No. 11,420,997, U.S. Publication No. 2019 / 0023726. U.S. Publication No. 2011 / 0160433. U.S. Publication No. 2018 / 0215782, and U.S. Patent No. 9,206,230, the entireties of which are incorporated herein by reference.

[0105] The present invention allows for the attachment of up to six hydrophobic hydrocarbon tails via A / V-dialkyl amide linkages, which significantly enhances the solubility' profile of the peptide-tag conjugates during amino acid coupling reactions. This increased solubility' improves reaction efficiency, results in higher coupling yields, and enables the synthesis of longer peptide chains without premature precipitation or solubility issues. The improved amide-linked, selectively soluble supports have demonstrated thecapability to support the synthesis of peptides or peptide fragments comprising ten or more amino acids prior to cleavage.

[0106] The selectively soluble amide-derived supports of the present invention offer several improvements over the prior arts by introducing amide-connected hydrocarbon tails on the soluble support scaffold. These amide-linked hydrocarbon Tags enable the assembly of longer peptide sequences on the selectively soluble support.

[0107] (Composition Changing Means)

[0108] As the means for changing the composition of a solution which is preferably used in the method of the present invention, means capable of changing a composition of a solution dissolving a C-carrier protected peptide are used without particular restriction. The preferable means for changing a composition of a solution includes, for example, means in which a poor solvent is added to a solution dissolving a C-carner protected peptide as it is or a poor solvent is added after concentrating the solvent of the solution, to cause cry stallization. Here, concentrating means to distill off a part or all of the solvent. Thereafter, the precipitated crystal can be separated, for example, by filtration and centrifugal separation. Impurities separated together with the crystal can be removed completely from the crystallized C-carrier protected peptide, preferably by washing the separated cry stal with an organic solvent.

[0109] The poor solvent used in the present invention means a solvent in which a C- protected amino acid amide is poorly soluble, that it, a C-protected amino acid amide is not dissolved easily or is not dissolved. For C-protected amino acid amide is not dissolved easily or is not dissolved, the poor solvent may advantageously be a solvent which is liquid at normal temperature with which the solubility of a C-protected amino acid amide is less than 1% by mass at 25 °C, and preferable are acetonitrile, any proportioned aqueous acetonitrile, methanol, any proportioned aqueous methanol and water.

[0110] This procedure using the selectively soluble support compounds of Formula (I) can also be used to synthesize an oligonucleotide in a liquid phase synthesis.Use as a Selectively Soluble Support in Organic Synthesis of Oligonucleotides

[0111] Next, an oligonucleotide production method according to the present invention will be described. Specifically, there will be described a method in which a nucleoside or oligonucleotide (hereinafter, also written as the ”n-mer oligonucleotide7’) protected with aselectively soluble support of Formula (I) is elongated with a nucleoside or oligonucleotide (hereinafter, also written as the "p-mer oligonucleotide"’) to produce an oligonucleotide (hereinafter, also written as the “(n+p)-mer oligonucleotide”) protected with a selectively soluble support of Formula (I). For oligonucleotide synthesis, the Y portion of the compounds of Formula (I) is preferably selected from the group consisting of -OH, - COOH, O-C(O)-X, wherein X is Cl, Br, I, F, imidazole, and a leaving group, and an -NHR group, wherein R is H. a straight or branched C1-C30 alkyl group, or a C7-C30 aralkyl group, wherein the alkyl portion of the C7-C30 aralkyl group is a straight or branched C1-C30 alkyl. This is to accommodate an ester linkage to the 3’ OH group on a nucleoside via an O- C(O)- linkage. The n-mer oligonucleotide means an oligonucleotide in which n nucleoside units are linked together via a phosphorus-containing group. When n is 1, the n-mer oligonucleotide is understood as a nucleoside. The same applies to the p-mer oligonucleotide.

[0112] The oligonucleotide production method includes a step of subjecting a nucleoside or oligonucleotide having a selectively soluble support of Formula (I) in at least one location selected from the group consisting of 2'-position, 3'-position, 5'-position and a nucleobase moiety and having a 5'-hydroxyl group or a 3'-hydroxyl group, to H- phosphonation to convert the 5'-hydroxyl group or the 3'-hydroxyl group into an H- phosphonated form.

[0113] Further, the oligonucleotide production method includes at least one elongation reaction cycle including the above H-phosphonation step.

[0114] The oligonucleotide production method is characterized in that the elongation reaction cycle includes the step of subjecting a nucleoside or oligonucleotide having a selectively soluble support of Formula (I) to H-phosphonation to introduce an H- phosphonated form in place of the 5'-hydroxyl group or the 3'-hydroxyl group. The oligonucleotide production method preferably includes the following Step a to Step d in the elongation reaction cycle. Step (a) to Step (d) are performed in the order of Step (a), Step (b), Step (c) and Step (d), or in the order of Step (a). Step (d), Step (b) and Step (c), and are preferably performed in the order of Step (a), Step (b), Step (c) and Step (d).

[0115] Step (a)

[0116] Step (a) is a temporary7protecting group removal step including deprotecting a nucleoside or oligonucleotide which has a selectively soluble support of Formula (I) on at least one of a hydroxyl group that is not the elongation terminal, a nucleobase moiety and 2'-position and which has at the elongation terminal a hydroxyl group protected with a temporary7protecting group, to remove the temporary7protecting group to form a hydroxyl group.

[0117] Step (b)

[0118] Step (b) is a phosphonation step including converting the hydroxyl group resulting from the removal of the temporary protecting group into an H-phosphonated form using an H-phosphonate reagent.

[0119] Step (c)

[0120] Step (c) is a coupling step including adding a nucleoside or oligonucleotide having a hydroxyl group to the H-phosphonated nucleoside or oligonucleotide obtained in Step (b), and condensing the compounds by forming a phosphite diester bond via the hydroxyl group.

[0121] Step (d)

[0122] Step (d) is a conversion step including converting the phosphite diester bond into a phosphodi ester bond, a thiophosphodiester bond, an aminophosphodi ester bond, a boranophosphodiester bond, a phosphodiester bond protected with a basic protecting group (a phosphotriamide bond), a thiophosphodiester bond protected with a basic protecting group (a thiophosphate-O, O, S-triamide bond) or the like.

[0123] Here, the amino group in the aminophosphodiester bond is unsubstituted or is substituted with one or two Ci-6 alkyl groups.

[0124] The phosphodiester bond protected with a basic protecting group is a bond formed by substituting a phosphodi ester bond with, for example, a C1-40 alkyl group, a C3-6 cycloalkyl group, a Ce-io aryl group, a 5-10 membered heteroaryl group, an aralkyl group or a heteroaralkyl group in place of the hydrogen atom of the single hydroxyl group. Here, the C1-6 alkyl group is unsubstituted, or is substituted with a substituent such as a halogen atom or a cyano group. The C3-6 cycloalkyl group, the Ce-io aryl group, the 5-10 memberedheteroaryl group, the aralkyl group and the heteroaralkyl group are unsubstituted, or are substituted with a substituent such as a Ci-6 alkyl group, a halogen atom or a cyano group.

[0125] The thiophosphodiester bond protected with a basic protecting group is a bond formed by substituting a thiophosphodiester bond with, for example, a C1-40 alkyl group, a C3-6 cycloalkyl group, a Ce-io aryl group, a 5-10 membered heteroaryl group, an aralkyl group or a heteroaralkyl group in place of the hydrogen atom of the single thiol group. Here, the C1-40 alkyl group is unsubstituted, or is substituted with a substituent such as a halogen atom or a cyano group. The C3-6 cycloalkyl group, the Cs-io aryl group, the 5-10 membered heteroaryl group, the aralkyl group and the heteroaralkyl group are unsubstituted, or are substituted with a substituent such as a C1-6 alkyl group, a halogen atom or a cyano group.

[0126] In the nucleoside or oligonucleotide with a selectively soluble support of Formula (I) that is used in Step (a), the number n of the nucleoside units is not particularly limited as long as it is an integer of 1 or greater, but is preferably 1 to 50, more preferably 1 to 30, still more preferably 1 to 20, even more preferably 1 to 10, and particularly preferably 1 to 5.

[0127] In the nucleoside or oligonucleotide with a hydroxyl group that is used in Step (c), the number p of the nucleoside units is not particularly limited as long as it is an integer of 1 or greater, but is preferably 1 to 50, more preferably 1 to 30, still more preferably 1 to 20, even more preferably 1 to 5, further preferably 1 to 3, and particularly preferably 1. That is, it is particularly preferable to use a nucleoside.

[0128] The reaction mixture obtained in Step (d) may be directly used in Step (a). Treatment such as heating may be performed appropriately after the completion of Step (d), and thereby Step (a) may be performed concurrently.

[0129] The oligonucleotide production method may further include the following Step (e), in which case the nucleoside or oligonucleotide may be purified simply and effectively by removing the excess of raw materials and byproducts.

[0130] Step (e)

[0131] Step (e) is a separation step of adding a polar solvent to the reaction mixture obtained from any of Step (a) to Step (d) to precipitate the nucleoside or oligonucleotidehaving a pseudo solid phase-protecting group, and collecting the precipitate by solid liquid separation.

[0132] Step (e) is specific to the liquid phase synthesis method using a selectively soluble support of Formula (I), and cannot be present in a usual liquid phase synthesis method using no selectively soluble support of Formula (I) or in a solid phase synthesis method.

[0133] The number of Steps (e) present in the elongation reaction cycle is not particularly limited. Step (e) may be performed after any of Step (a) to Step (d).

[0134] For example. Step (e) may be performed 0 to 5 times, preferably 0 to 3 times, more preferably 0 to 2 times, and still more preferably 0 or 1 time independently after each of Step (a) to Step (d). Here, one or more Steps (e) are present after at least one of Steps(a) to (d).

[0135] The elongation reaction cycle preferably includes one to four Steps (e). To manage and control the generation of byproducts strictly and to lead the compounds to a high-purity oligonucleotide, it is preferable that Step (e) be performed one time after at least one of Step (a), Step (b) and Step (d) during the elongation reaction cycle. More preferably, Step (e) is performed one time after Step (b), one time after Step (d), or one time after each of Step (b) and Step (d) during the elongation reaction cycle.

[0136] In another embodiment, it is more preferable that Step (e) be performed one time after Step (a), one time after Step (b), or one time after each of Step (a) and Step (b), and it is still more preferable that Step (e) be performed one time after each of Step (a) and Step(b) during the elongation reaction cycle.

[0137] If the circumstances permit the amount of byproducts to be controlled by controlling the equivalents of the raw materials and by controlling the reaction, it is preferable that Step (e) be performed after Step (a) to Step (d) as a basic unit are repeated.

[0138] The oligonucleotide production method may further include Step (I), in which case the desired oligonucleotide can be isolated and produced.

[0139] Step (f)

[0140] Step (f) is a deprotection / cleavage step of removing all of the basic protecting group, the temporary protecting group and the selectively soluble support of Formula (I) in the oligonucleotide obtained by Step (a) through Step (e).

[0141] Specific examples of liquid phase oligonucleotide production with partially soluble supports can be found in, for example. U.S. Patent No. 10,464,966, U.S.Publication No. 2019 / 0169223, and U.S. Patent No. 9,029,528. the entireties of which are incorporated herein by reference.

[0142] One skilled in the art, using the preceding detailed description, can utilize the present invention to its fullest extent. The following example are provided to illustrate the invention but should not be construed as limiting the invention in any way except as indicated in the appended claims.EXAMPLESSynthesis of Tag 4A and Tag 4B:Synthesis ofl'ag 4ATag4ASynthesis ofTrimer Peptide with Tag 4ASynthesis of Pentamer Peptide with Tag 4ASynthesis of Primer Peptide with Tag 4BSynthesis of Pentamer Peptide with Tag 4BSynthesis of Decamer Peptide with Tag 4BExample 1: Synthesis of Tag 4AVanillin Triamide 9(intermediate 7 from Tag4B synthesis)Table 1. Summan' of Reagents

[0143] Compound 7 (8.50 g, 1.00 equiv.) was charged to a flask containing a stir bar. The material was dissolved in DCM (85 mL. 10.0 vol.). DIEA (21.18 g. 9.00 equiv.).EDCI HC1 (13.94 g, 4.00 equiv ), and DMAP (0.44 g, 0.20 equiv.) were added sequentially to the flask. A second aliquot of DCM (85 mL, 10.0 vol.) was added and the resulting mixture was agitated under ambient conditions until all solids dissolved (approximately 10 minutes). Amine 8 (16.50 g, 3.20 equiv.) was dissolved in DCM (85 mL, 10.0 vol.) and transferred to the reaction flask. The resulting mixture rapidly became an off-white slurry. After 30 minutes of aging, the flask contents remained as slurry. An additional allotment of DCM (85 mL, 10.0 vol.) was charged, but the slurry failed to transform into a homogenous mixture and was aged as is for 64 h.

[0144] A sample was prepared by partitioning a 1 mL aliquot of reaction mixture with brine. Reaction completion was subsequently assessed via a combination of1H NMR and TLC (10% MeOH / DCM). Both analyses showed approximately 70% conversion of starting material. The reaction was terminated at this point without further conversion. The crude mixture was partitioned with brine (85 mL, 10.0 vol.) and the product containing organic layer was further rinsed with 1 N HC1 (to a pH of 2.0). The combined aqueous layers were rinsed one time with DCM (85 mL, 10.0 vol.). The combined organic layers were rinsed 2 x with brine (2 x 85 mL, 2 x 10.0 vol ), dried over Na2SC>4, filtered, and concentrated under reduced pressure to afford ~10 mL of an auburn viscous oil. This oil w as directly loaded onto a 220 g normal phase column. A gradient eluent of 0 - 10% MeOH / DCM over 10 CV served as the mobile phase. The product eluted after 5 CV over 8 x 25 mL fractions.Combined fractions of compound 9 afforded 6.80 g (51% yield) of a yellow solid. FIG. 1 show s the 'H NMR (400 MHz, CDCls) spectrum of compound 9 after drying.1H NMR (400 MHz, CDC13) 5 9.82 (s. 1H), 8.03 - 7.81 (m, 1H), 7.43 - 7.35 (m, 2H), 6.97 (d, J= 8.1 Hz. 1H), 4.12 (t, J= 6.5 Hz, 2H), 3.89 (s, 3H). 3.25 (dt, J= 25.1, 7.4 Hz, 6H), 2.96 - 2.91 (m, 5H), 2.88 - 2.84 (m, 4H), 2.44 - 2.01 (m, 16H), 1.56 - 1.37 (m, 6H), 1.33 - 1.13 (m, 90H), 0.89 - 0.81 (m, 9H).Vanillin Triamide Alcohol (Tag 4A):,Table 2. Summary of Reagents

[0145] Compound 9 (14.41 g, 1.00 equiv.) was charged to a flask containing a stir bar and dissolved in DCM (144 mL, 10.0 vol.) alone. The solution was cooled to 0 °C and MeOH (144 mL, 10.0 vol.) was added. Sodium borohydride (4.32 g, 10.00 equiv.) was slowly added to the reaction mixture in portions to minimized hydrogen effervescence. Once complete addition was achieved, the flask contents were agitated at 0 °C for 1 h.

[0146] A 1 mL sample of the reaction mixture was removed and concentrated under reduced pressure. The residue was taken up into a 1: 1 DCM:brine and the organics were removed. The sample was concentrated under reduced pressure and taken up into CDCI3. JH NMR show ed the complete disappearance of the aldehyde proton, indicating reaction completion.

[0147] The crude reaction mixture was quenched with brine (144 mL, 10.0 vol.) followed by 1 V HCI (to a pH of 6.0). The resulting biphasic system was agitated for approximately 10 minutes at which point effervescence ceased. The aqueous layer was removed and rinsed with DCM (2 x 144 mL, 2 x 10 vol.). The combined organic layers were furtherrinsed 1 x with brine (144 mL, 10.0 vol.), dried over Na2SO4, fdtered, and concentrated under reduced pressure. The desired material 10 was isolated as a yellow solid with a yield of 6.54 g (97%). FIG. 2 shows the!H NMR (400 MHz. CDCl3) spectrum of compound 10 after drying. ‘H NMR (400 MHz, CDCl3) 8 7.61 - 7.45 (m, 1H), 6.91 (s, 1H), 6.83 - 6.79 (m, 2H), 4.57 (s, 2H), 4.02 (t, J= 6.3 Hz, 2H), 3.84 (s, 3H), 3.25 (dt, J= 25.5, 7.6 Hz, 6H), 2.95 - 2.91 (m, 5H), 2.88 - 2.84 (m, 4H), 2.37 - 2.25 (m, 8H), 2. 12 - 1.98 (m, 8H), 1.56 - 1.37 (m, 6H), 1.33 - 1.16 (m, 90H), 0.85 (t, J= 6.7 Hz, 9H).Example 2; Synthesis of Tag 4A PeptidesFmoc-Gly-Tag 4A (14)Table 3. Summary of Reagents

[0148] To a flask containing a stir bar was charged DIC (3.92 g, 3.20 equiv.), Oxyma (4.14 g, 3.00 equiv.), Fmoc-Gly-OH (5.78 g, 2.00 equiv.), and DCM (123 mL, 10.0 vol.). The flask contents were aged under ambient conditions for approximately 15 minutes until all solids dissolved, affording a neon yellow solution. Compound 10 (12.30 g, 1.00 equiv.)was dissolved in DCM (123 mL, 10.0 vol.) and slowly transferred to the flask containing the activated amino acid. The contents were then agitated under ambient conditions for 3 h. After the appropriate aging time had passed, the reaction mixture was sampled for completion. HPLC analysis demonstrated low crude purity, but the complete consumption of the starting material 10.

[0149] The crude reaction mixture was concentrated under reduced pressure to afford a yellow semi-solid. The concentrated crude product was treated with acetone (123 mL, 10.0 vol.) and again concentrated to dryness under reduced pressure. This process was repeated one additional time with acetone (123 mL, 10.0 vol.). Following the complete removal of DCM, acetone (243 mL, 20.0 vol.) was added to the flask containing the product residue. At ambient temperature, compound 5 displayed slight solubility' in acetone and afforded a yellow solution. Regardless, the product-containing mixture was cooled to 0 °C and aged for 30 minutes, during which time the homogenous solution slowly became a white slurry. The product was separated from the mother liquor via filtration and the product cake was gently rinsed with chilled acetone (2 x 123 mL, 2 x 10.0 vol.). The desired product was pushed through the filter into a clean flask using DCM and the solvent was removed under reduced pressure (bath temperature not exceeding 30 °C). The isolated solid was dried overnight at ambient temperature to afford 10.70 g (71%) of 14 as yellow solid.NH2-Gly-Tag 4A (15)Table 4. Summary of Reagents

[0150] Compound 14 (10.70 g, 1.00 equiv.) was charged to a flask containing a stir bar and dissolved in DCM (214 mL, 20.0 vol.) under ambient conditions. Piperidine (11.79 g, 20.00 equiv.) was slowly charged to the flask and the resulting yellow solution was aged for 3 h. After the allotted time had passed, a 0.1 mL sample of the crude reaction mixture was removed and diluted with MeOH. The diluted solution was analyzed via HPLC. The spectrum showed the disappearance of starting material 5, indicating reaction completion.

[0151] DCM solvent was removed under reduced pressure. The bath temperature did not exceed 30 °C due to potential stability concerns. MeCN (107 mL, 10.0 vol.) was added and the material was again concentrated to dry ness. This process was repeated one additional time with MeCN (107 mL. 10.0 vol.). To the resulting product residue was charged MeCN (428 mL. 40.0 vol.). The slurry’ was chilled to 0 °C and aged for approximately 30 minutes. Manual deagglomeration and sonication was periodically performed to ensure effective purification. Vacuum filtration separated the desired light-yellow precipitate from the mother liquor. The isolated product cake was rinsed with chilled MeCN (3 x 107 mL, 3 x 10.0 vol.) and dried under vacuum in the absence of heat. The desired product was pushed through the filter into a clean flask using DCM and the transfer solvent was removed under reduced pressure. Again, the bath temperature did not exceed 30 °C. The product was vacuum dried overnight at ambient temperature. A total of 8.31 g (91%) of yellow solid 15 was obtained.Fmoc-Gly-Gly-Tag 4A (16)Table 5. Summary of Reagents

[0152] To a flask containing a stir bar was charged DIC (2.54 g, 3.20 equiv.), Oxyma (2.68 g. 3.00 equiv ), Fmoc-Gly-OH (3.73 g, 2.00 equiv.), and DCM (83 mL, 10.0 vol.). The flask contents were aged under ambient conditions for approximately 15 minutes until all solids dissolved, affording a neon yellow solution. Compound 15 (8.31 g, 1.00 equiv.) was dissolved in DCM (83 mL, 10.0 vol.) and slowly transferred to the flask containing the activated amino acid. The contents were then agitated under ambient conditions for 3 h. After the appropriate aging time had passed, the reaction mixture was sampled for completion. HPLC analysis demonstrated low crude purity, but the complete consumption of the starting material 16.

[0153] The crude reaction mixture was concentrated under reduced pressure to afford a yellow semi-solid. The concentrated crude product was treated with acetone (83 mL, 10.0 vol.) and again concentrated to dry ness under reduced pressure. This process was repeated one additional time using acetone (83 mL, 10.0 vol.). Treatment with acetone eventually produced a yellow waxy solid. Acetone (166 mL, 20.0 vol.) was charged to the flask and the resulting mixture was agitated under ambient conditions for approximately 10 min. An off-white precipitate began to form even at ambient temperatures. The product-containing slurry was cooled to 0 °C and aged for 30 minutes, during which time further off-white precipitate formed. The desired product was separated from the mother liquor via filtration and the product cake was gently rinsed with chilled acetone (2 x 83 mL, 2 x 10.0 vol.). The product cake was transferred to a clean flask from the filter by treatment with DCM. Thesolution was concentrated under reduced pressure and dried overnight at ambient temperature. A total of 7. 10 g (71%) of 16 as yellow waxy solid was obtained.NH2-Glv-Glv-Tag 4A (17)Table 6. Summary of Reagents

[0154] Compound 16 (7. 10 g, 1.00 equiv.) was charged to a flask containing a stir bar and dissolved in DCM (142 mL, 20.0 vol.) under ambient conditions. Piperidine (7.55 g, 20.00 equiv.) was slowly charged to the flask and the resulting yellow solution was aged for 3 h. After the allotted time had passed, the reaction mixture was sampled for completion. HPLC analysis of the crude mixture demonstrated the complete consumption of starting material 7 and the reaction was terminated.

[0155] The DCM solvent was removed under reduced pressure with the temperature setting not exceeding 30 °C due to stability concerns. MeCN (71 mL, 10.0 vol.) was added and the material was again concentrated to dryness. This process was repeated one additional time using MeCN (71 mL, 10.0 vol). To the resulting product residue was charged MeCN (284 mL, 40.0 vol). The slurry was chilled to 0 °C and aged forapproximately 30 minutes. Manual deagglomeration and sonication was periodically performed to ensure effective purification. Vacuum filtration separated the desired lightyellow precipitate from the mother liquor. The isolated product cake was rinsed with chilled MeCN (3 x 71 mL, 3 x 10.0 vol.) and dried under vacuum in the absence of heat. The desired product was pushed through the filter into a clean flask using DCM and the transfer solvent was removed under reduced pressure at 30 °C. The product was vacuum dried overnight at ambient temperature. A total of 5.26 g (86%) of 17 as yellow solid was obtained from this reaction.Fmoc-Phe-Gly-Gly-Tag 4A (18)Table 7. Summary of Reagents

[0156] To a flask containing a stir bar was charged DIC (1.54 g, 3.20 equiv.), Oxyma (1.62 g, 3.00 equiv ), Fmoc-Phe-OH (2.37 g, 2.00 equiv.). and DCM (53 mL. 10.0 vol.). The flask contents were aged under ambient conditions for approximately 15 minutes until all solids dissolved, affording a neon yellow solution. Compound 17 (5.26 g, 1.00 equiv.)was dissolved in DCM (53 mL, 10.0 vol.) and slowly transferred to the flask containing the activated amino acid. The contents were then allowed to agitate under ambient conditions. After 3 hours, the reaction mixture was sampled for completion. HPLC analysis demonstrated low crude purity, but the complete consumption of the starting material 18

[0157] The crude reaction mixture was concentrated under reduced pressure at 30 °C to afford a yellow semi-solid. The concentrated crude product was treated with acetone (53 mL, 10.0 vol.) and again concentrated to dryness under reduced pressure. This process was repeated one additional time using acetone (53 mL, 10.0 vol.). Following complete removal of the reaction solvent, the flask was charged with acetone (105 mL, 20.0 vol.). At ambient temperature, crude compound 18 displayed slight solubility in acetone and afforded a yellow solution. Regardless, the product-containing mixture was cooled to 0 °C and aged for 30 minutes, during which time the homogenous solution slowly became a white slurry. The product was separated from the mother liquor via filtration and the product cake was gently rinsed with chilled acetone (2 x 10.0 vol.). The desired product was pushed through the filter into a clean flask using DCM and the solvent w as removed under reduced pressure (bath temperature not exceeding 30 °C). The isolated solid w as dried overnight at ambient temperature to afford 4.50 g (68%) of compound 18 as a yellow solid.NH2-Phe-Gly-Glv-Tag 4A (19)18 19Table 8. Summary of Reagents

[0158] Compound 18 (3.06 g, 1.00 equiv.) was charged to a flask containing a stir bar and dissolved in DCM (61 mL, 20.0 vol.) under ambient conditions. Piperidine (2.98 g, 20.00 equiv.) was charged to the flask and the resulting yellow solution was aged for 2 h. After the allotted time had passed, a 1 mL sample of the crude reaction mixture was removed and concentrated under reduced pressure. The resulting residue was taken up into CDCL and analyzed viaNMR spectroscopy. The spectrum showed the disappearance of the Fmoc methanetriyl proton signal, indicating reaction completion.

[0159] DCM solvent was removed under reduced pressure with a temperature setting of not exceeding 30 °C due to the low melting point of the desired material and stability concerns. MeCN (31 mL, 10.0 vol.) was added and the material was again concentrated to dryness. This process was repeated one additional time using MeCN (31 mL, 10.0 vol.). To the resulting product residue was charged MeCN (122 mL, 40.0 vol.). The slurry was chilled to 0 °C and aged for approximately 30 minutes. Manual deagglomeration and sonication was periodically performed to ensure effective solvent penetration. Vacuum filtration separated the desired light-yellow precipitate from the mother liquor. The isolated product cake was rinsed with chilled MeCN (3 x 31 mL, 3 x 10.0 vol.) and dried under vacuum in the absence of heat. The desired product was pushed through the filter into a clean flask using DCM and the transfer solvent was removed under reduced pressure (bath temperature of 30 °C). The product was vacuum dried overnight at ambient temperature. Compound 19 was obtained as a yellow solid with a yield of 2.40 g (99%).Fmoc-Ala-Phe-Gly-Glv-Tag 4A (20)19 20Table 9. Summary of Reagents

[0160] To a flask containing a stir bar was charged DIC (0.79 g, 3.20 equiv.), Oxyma (0.84 g, 3.00 equiv.), Fmoc-Ala-OH (1.22 g, 2.00 equiv.), and DCM (30 mL, 10.0 vol.). The flask contents were aged under ambient conditions for approximately 15 minutes until all solids dissolved, affording a neon yellow solution. Compound 19 (3.00 g, 1.00 equiv.) was dissolved in DCM (30 mL, 10.0 vol.) and slowly transferred to the flask containing the activated amino acid. The contents were then agitated under ambient conditions for 3 h. After the appropriate aging time had passed, the reaction mixture was sampled for completion. HPLC analysis demonstrated low crude purity, but the complete consumption of the starting material 19.

[0161] The crude reaction mixture was concentrated under reduced pressure at 30 °C to afford a yellow semi-solid. The concentrated crude product was treated with acetone (30 mL, 10.0 vol.) and similarly concentrated to dryness under reduced pressure. This process was repeated one additional time using acetone (30 mL, 10.0 vol.). Following complete removal of the reaction solvent, the flask was charged with acetone (60 mL, 20.0 vol ). At ambient temperature, the generated solid displayed slight solubility in acetone. Regardless, the product-containing mixture was cooled to 0 °C and aged for 30 minutes, during which time the flask contents became a white slurry7. The precipitated compound 20 was separated from the mother liquor via filtration and the product cake was gently rinsed with chilled acetone (2 x 30 mL, 2 x 10.0 vol.). The desired product was pushed through the filter into a clean flask using DCM and the solvent was removed under reduced pressure (bathtemperature not exceeding 30 °C). The isolated solid was dried overnight at ambient temperature to afford 2.58 g (72%) of compound 20 as a yellow solid.NH2-Ala-Phe-Glv-Glv-Tag 4A (21)20 21Table 10. Summary7of Reagents

[0162] Compound 20 (2.58 g, 1.00 equiv.) was charged to a flask containing a stir bar and dissolved in DCM (52 mL, 20.0 vol.) under ambient conditions. Piperidine (2.41 g, 20.00 equiv.) was slowly charged to the flask and the resulting yellow solution was aged for approximately 3 h. After the allotted time had passed, the reaction mixture was sampled for completion. HPLC analysis of the crude mixture demonstrated the complete consumption of the starting material 20 and the reaction was terminated.

[0163] The DCM solvent was removed under reduced pressure with the temperature setting not exceeding 30 °C due to stability concerns. MeCN (26 mL, 10.0 vol.) was added and the material was again concentrated to dryness. This process was repeated one additional time using MeCN (26 mL, 10.0 vol ). To the resulting product residue was charged MeCN (103 mL, 40.0 vol.). The slurry was chilled to 0 °C and aged for approximately 30 minutes. Manual deagglomeration and sonication was periodically performed to ensure effective solvent penetration. Vacuum filtration separated the desiredlight-yellow precipitate from the mother liquor. The isolated product cake was rinsed with chilled MeCN (3 x 26 mL, 3 x 10.0 vol.) and dried under vacuum in the absence of heat. The desired product was pushed through the filter into a clean flask using DCM and the transfer solvent was removed under reduced pressure (bath temperature of 30 °C). The product was vacuum dried overnight at ambient temperature. A total of 2.11 g (93%) of 21 as yellow solid was obtained.NH2-Val-Ala-Phe-Gly-Gly-Tag 4A (23)Table 11. Summary of Reagents

[0164] To a vial containing a stir bar was charged DIEA (0.34 g, 2.00 equiv.), Fmoc-Val- OH (0.47 g, 1.05 equiv.), and DCM (21 mL. 10.0 vol.). The resulting solution was allowed to age for approximately 5 minutes. HATU (0.53 g, 1.05 equiv.) was added to the vial and the solution was further aged for an additional 10 minutes to allow for activation of the amino acid. The contents of this vial were transferred to a separate flask containing compound 21 (2.11 g, 1.00 equiv.) dissolved in DCM (21 mL, 10.0 vol ). Once all reagents were combined, the orange solution was aged for 2 h under ambient conditions.

[0165] After the appropriate time had passed, the flask contents were transferred to a separatory funnel for extraction. The organic layer was partitioned with brine (42 mL, 20.0 vol.) and the remaining brine layer was rinsed a singular time with DCM (42 mL, 20.0 vol.) before being discarded. The combined organic layers were rinsed an additional time with brine (42 mL, 20.0 vol ), dried over sodium sulfate, and filtered to afford a light-yellow product solution. Solvent was removed under reduced pressure (bath temperature not exceeding 30 °C) and the yellow oily residue was directly carried forward to the deprotection step. Reagent / solvent charges were calculated assuming complete conversion to compound 22.

[0166] Crude compound 22 (2.51 g, 1.00 equiv.) was taken up into DCM (50 mL, 20.0 vol.) and piperidine (2.25 g, 20.00 equiv.) was slowly added to the flask. The reaction mixture was aged for 2 h under ambient conditions. After the allotted time had passed, the reaction mixture was sampled for completion. HPLC analysis of the crude mixture demonstrated the complete consumption of the starting material 22 and the reaction was terminated.

[0167] DCM was removed under reduced pressure with the temperature setting not exceeding 30 °C due to stability concerns. MeCN (25 mL, 10.0 vol.) was added and the material was again concentrated to dryness. This process was repeated one additional time using MeCN (25 mL, 10.0 vol.). To the resulting product residue was charged MeCN (100 mL, 40.0 vol.). The slurry was chilled to 0 °C and aged for approximately 30 minutes.Manual deagglomeration and sonication was periodically performed to ensure effective solvent penetration. Vacuum filtration separated the desired light-yellow precipitate from the mother liquor. The isolated product cake was rinsed with chilled MeCN (3 x 25 mL, 3 x 10.0 vol.) and dried under vacuum in the absence of heat. The desired product was pushed through the filter into a clean flask using DCM and the solvent was removed under reduced pressure at 30 °C. The product was vacuum dried overnight at ambient temperature. A total of 1.96 g (88% - two step yield) of compound 23 was obtained from this reaction as a yellow solid.NH2-Phe-Glv-Glv-OH (24)Table 12. Summary of Reagents

[0168] A solution of 92.5:2.5:2.5:2.5 TFA:TIPS:H2O:DODT (2 mL, 10.0 vol.) was charged to a flask containing a stir bar. The deprotection cocktail was chilled to 0 °C. Compound 19 (0.20 g, 1.00 equiv.) was charged to the reaction flask in portions. The solids rapidly dissolved to afford a yellow solution, which was allowed to agitate for 2 h. After the allotted time has passed, a sample (50 LIL diluted to 1 mL with water) of the crude reaction mixture was analyzed via HPLC. Results from the HPLC suggested reaction completion as compound 10 was not observed in the HPLC trace.

[0169] The crude solution was quenched with chilled MTBE (12 mL, 60.0 vol.) and aged for 30 minutes at 0 °C. No precipitate formed during MTBE addition and the flask contents were instead transferred to a separatory funnel. The product-containing aqueous layer was removed and the remaining organic layer was rinsed one with deionized water (12 mL, 60.0 vol.). The combined aqueous layers were subsequently rinsed one time with MTBE (12 mL, 60.0 vol.) to remove any residual organic impurities. Peptide 24 was subsequently isolated via HPLC. affording 0.010 g (25%) of white solid product. Synthesis of peptide 24 was confirmed via a combination of LC / MS and NMR.

[0170] FIG. 3 shows therH NMR (400 MHz, D2O) spectrum of compound 24. 'H NMR (400 MHz, D2O) 5 7.49 - 7.36 (m, 3H), 7.36 - 7.30 (m, 2H), 4.32 (t, J= 7.3 Hz, 1H), 4.08 - 3.88 (m, 4H), 3.25 (dd, J= 7.4, 2.6 Hz, 2H). Singlet around 2.2 ppm corresponds to residual acetone signal.

[0171] FIG. 4 shows the13C NMR (101 MHz, D2O) spectrum of compound 24.13C NMR (101 MHz, D2O) 5 173.65. 171.03, 169.72, 133.73, 129.37, 129.16. 128.01, 54.48, 42.14, 36.72, 30.21.

[0172] FIG. 5 shows the LC / MS trace (EIC) of purified compound 24. LC-MS (ESI): m / z | M+H | calculated for CnHisNsCL: 280. 1 ; found: 280.0.

[0173] FIG. 6 shows the mass extraction of purified compound 24 with a retention time of 7.416 minutes.NH2-Val-Ala-Phe-Glv-Gly-OH (25)Table 13. Summan' of Reagents

[0174] A solution of 92.5:2.5:2.5:2.5 TFA:TIPS:H2O:DODT (4 mL, 10.0 vol.) was charged to a flask containing a stir bar. The deprotection cocktail was chilled to 0 °C. Compound 23 (0.40 g, 1.00 equiv.) was charged to the reaction flask in portions. The solids rapidly dissolved to afford a yellow solution, which was allowed to agitate for 2 h. After the allotted time has passed, a sample (50 uL diluted to 1 mL with water) of the crude reaction mixture was analyzed via HPLC. Results from the HPLC suggested reaction completion as compound 20 was not observed in the HPLC trace.

[0175] The crude solution was quenched with chilled MTBE (24 mL. 60.0 vol.) and aged for 30 minutes at 0 °C. No precipitate formed during MTBE addition and the flask contents were instead transferred to a separatory funnel. The product-containing aqueous layer was removed and the remaining organic layer was rinsed one with deionized water (24 mL, 60.0 vol.). The combined aqueous layers were subsequently rinsed one time with MTBE (24 mL, 60.0 vol.) to remove any residual organic impurities. Peptide 25 was subsequently isolated via HPLC, affording 0.035 g (33%) of white solid product. Synthesis of peptide 25 was confirmed via a combination LC / MS and NMR.

[0176] FIG. 7 shows the1H NMR (400 MHz, D2O) spectrum of compound 25.1H NMR (400 MHz, D2O) 5 7.36 - 7.18 (m, 5H), 4.58 - 4.48 (m, 1H), 4.37 - 4.26 (m, 1H), 4.01 - 3.64 (m, 5H), 3.11 - 2.99 (m, 2H). 2.16 - 2.03 (m, 1H), 1.28 (d, J= 7.1 Hz, 3H), 0.91 (d, J = 6.9 Hz, 6H). Peaks J - K correspond to exchangeable amide proton signals.

[0177] FIG. 8 shows the13C NMR (101 MHz. D2O) spectrum of compound 25.13C NMR (101 MHz, D2O) 5 174.15, 173.45, 173.08, 171.52, 168.86, 136.08, 129.18, 128.78, 127.23, 58.29, 55.29, 49.43, 42.26, 40.98, 36.85, 29.92, 17.59, 16.78, 16.58.

[0178] FIG. 9 shows the LC / MS trace (ETC) of purified compound 25. LC-MS (ESI): m / z | M+H | calculated for C21H32N5O6: 450.2; found: 450.3.

[0179] FIG. 10 shows the mass extraction of purified compound 25 with a retention time of 5.547 minutes.Example 3: Synthesis of Tag 4BTable 14. Summary of reagents

[0180] Vanillin 2 (20.0 g, 131.4 mmol, 1.0 eq) and halide 1 (28.20 g, 144.6 mmol, 1.1 eq) were added to a flask followed by DMF (100.0 mL, 5.0 Vol) and K.2CO3 (19.98 g, 144.6 mmol, 1.1 eq). The flask was equipped with a condenser and the slurry was then stirred while heating to 65 °C. During the reaction, aliquots were removed and monitored by 1HNMR to test for reaction completion. After 4.5 hours, all vanillin was consumed so the reaction mixture was cooled to room temperature. Water (100 mL, 5.0 Vol) was added and the mixture was stirred for 5-10 minutes. A second portion of water (100 mL, 5.0 Vol) was then added, resulting in the precipitation of a white solid. The slurry was filtered and then dried in a vacuum oven overnight (35 °C for 16 hours). A total of 31.86 g of 3 as a white solid was isolated (91% yield). FIG. 11 shows the 'H NMR (400 MHz, CDCI3) spectrum of compound 3 after drying. 'H NMR (400 MHz, CDCl3) 8 9.78 (s, 1H), 7.41 - 7.31 (m, 2H). 6.92 (d, J= 8.1 Hz, 1H), 4.16 - 4.00 (m, 4H), 3.85 (s, 3H), 2.48 (t, J= 7.2 Hz, 2H), 2.18 - 2.06 (m, 2H), 1.19 (t, J = 7.1 Hz, 3H).Vanillin Acid (4):Table 15. Summary of reagents

[0181] Vanillin ester 3 (31.86 g, 119.6 mmol, 1.0 eq) was added in portions to a flask containing a solution ofNaOH in water / ethanol (NaOH: 7.1786 g, 179.5 mmol, 1.5 eq; H2O: 63.7 mL. 2.0 Vol; EtOH: 159.3 mL. 5.0 Vol), keeping the temperature below 30 °C during the addition. Aliquots were monitored by1HNMR for reaction completion. After 3 hours, all 3 was consumed and the reaction was cooled in an ice bath and quenched slowly with 1 JVHC1 (239.0 mL, 7.5 Vol). The resulting slurry was filtered and then dried in a vacuum oven overnight (35 °C for 16 hours). A total of 27. 10 g of 4 as a pale-yellow solid was isolated (95% yield). FIG. 12 shows the!H NMR (400 MHz, DMSO-r / e) spectrum of compound 4 after drying. *HNMR (400 MHz, DMSO-c76) 5 12.19 (s, 1H), 9.84 (s, 1H), 7.54 (dd, J= 8.3, 1.9 Hz, 1H), 7.40 (d, J= 1.9 Hz, 1H), 7.17 (d, J = 8.2 Hz, 1H), 4.10 (t, J = 6.5 Hz. 2H), 3.84 (s, 3H), 2.41 (t, J= 7.3 Hz, 2H), 1.98 (p, J= 6.9 Hz, 2H).Vanillin Triester (6):Table 16. Summary7of reagents

[0182] Vanillin acid 4 (5.00 g, 20.99 mmol, 1.0 eq) was added to a flask followed by aminotnester 5 (10.4660 g, 25.19 mmol, 1.2 eq), DCM (50 mL, 10.0 Vol), DIPEA (5.4336 g, 41.97 mmol, 2.0 eq), and HATU (11.97 g, 31.48 mmol, 1.5 eq). The mixture was stirred, forming a red-brown solution after 5-10 minutes. The solution was allowed to stir at room temperature overnight under a nitrogenous atmosphere. After 16 hours, the reactionsolution was diluted with water (100 mL), stirred for 5 minutes, the layers were separated, and the aqueous was then extracted with DCM (3 x 50 mL). Organics were combined, dried over sodium sulfate, filtered, and then reduced to yield 27.72 g of 6 as a dark amber oil (>100% yield). The crude 6 as an oil was used directly for the next step without further purification.Vanillin Triacid (7):Table 17. Summary of reagents

[0183] Vanillin triester 6 (4.00 g, 6.29 mmol, 1.0 eq) was added to a flask followed by a solution of TFA (14.4 mL, 30 eq) in DCM (40.0 mL, 10.0 Vol). After stirring for 16 hours, the reaction volume was reduced by half via rotary evaporation and the remainder of the solution was added dropwise to a flask containing stirring MTBE (~50 mL) to precipitate the product. Once all the crude was added, it was allowed to stir for 2 hours, forming sticky yellow agglomerates. The slurry was filtered, the flask was charged with fresh MTBE (50 mL), and the sticky crude was added back and stirred overnight. After stirring for 16 hours, the agglomerates broke up into a fine yellow powder, which was filtered and dried in a vacuum oven overnight (40 °C, 20 hours). A total of 0.3.6016 g (>100% yield) of 7 as a yellow powder was isolated after drying. FIG. 13 shows the H NMR (400 MHz, DMSO-de) spectrum of compound 7 after dry ing. 'H NMR (400 MHz, DMSO-c / e) 5 12. 11 (s, 4H), 9.84 (s. 1H), 7.54 (dd, .7= 8.3, 1.9 Hz, 1H), 7.40 (d, J= 1.8 Hz, 1H), 7.27 (s, 1H), 7.15 (d, J = 8.3 Hz. 1H), 4.06 (t, J = 6.5 Hz, 2H), 3.85 (s, 3H), 2.28 (dt. J= 17.8, 7.6 Hz. 4H), 2.20 - 2.05 (m, 7H), 1.94 (p, J= 7.0 Hz, 2H), 1.89 - 1.76 (m, 8H).Vanillin Triamide (12):Table 18. Summary of reagents

[0184] Vanillin triacid (7) (3.00 g, 6.42 mmol, 1.0 eq) was added to a flask followed by DCM (60 ml, 20.0 Vol), DIPEA (7.4767 g, 57.76 mmol, 9.0 eq), EDCI HC1 (4.9210 g, 25.67 mmol, 4.0 eq), and DMAP (0.1568 g, 1.28 mmol. 0.2 eq). After stirring for ~5 minutes, dioctadecylamine (11) (10.7198 g, 20.54 mmol, 3.2 eq) was added and the mixture was stirred overnight under nitrogen. After stirring for 16 hours, the reaction mixture was washed with water (60.0 mL), 1 N HC1 (60.0 mL), and then the aqueous washes were extracted with DCM (3 x 50 mL). The combined organics were dried over Na2SO4, filtered, and reduced. The resulting solid was stirred in 100 mL of acetone for 2hours and then filtered to remove acetone soluble by-products. The wet precipitate was dried in a vacuum oven overnight (30 °C, 20 hours). A total of 11.4436 g (90% yield) of 12 a pale-yellow powder was isolated after drying. FIG. 14 shows theNMR (400 MHz, CDCl3) spectrum of compound 12 after drying. 'H NMR (400 MHz, CDCl3) 5 9.77 (s, 1H), 7.99 (s, 1H), 7.39 - 7.29 (m, 2H), 6.92 (d, J= 8.1 Hz, 1H), 4.07 (t, J= 6.6 Hz, 2H), 3.84 (s, 3H), 3.22 - 3.04 (m, 12H), 2.89 - 2.77 (m, 1H), 2.27 (dt, J= 17.0, 7.3 Hz, 9H), 2.05 (dt, J = 18.1, 7.2 Hz. 9H), 1.42 (dp, J= 14.9, 7.3 Hz, 13H), 1.18 (d. J= 4. 1 Hz. 202H). 0.81 (t. J = 6.8 Hz, 21H).Vanillin Triamide Alcohol Tag 4B (13):13 Tag 4B

[0185] Vanillin triamide aldehyde 12 (0.6800 g, 0.344 mmol, 1.0 eq) was diluted in DCM / MeOH (1 :1. 14 mL total, 20.0 Vol) and cooled to 0 °C in an ice bath. Once the solution was cool, NaBH4 (0. 1301 g, 3.438 mmol. 10.0 eq) was added in 3-4 portions to the reaction vessel. After all the borohydride was added, the reaction was allowed to continue stir at 0 °C under nitrogen. After stirring for 2 hours, an aliquot was removed, reduced, diluted in DCM, washed with 1 1VHC1, reduced, and taken up in CDCl3for 'HNMR analysis. The spectrum showed that the aldehyde proton was consumed so the reaction was diluted with ~50 mL DCM then quenched with the slow addition of water followed by 1 N HC1 until pH <4. The biphasic mixture was added to a separatory funnel and the organic phase was drained. The aqueous phase was extracted with DCM (2 x 20 mL) and the combined organics were dried over Na2SO4. filtered, and reduced to yield milky oil that was then dried overnight in a vacuum oven. After the oil was dried (16 hours, 35 °C) 0.5914 g of 13 as a white solid was obtained (87% yield). FIG. 15 shows the 'H NMR (400 MHz, CDCl3) spectrum of compound 13 Tag 4B after drying. 'H NMR (400 MHz, CDCI3) 5 7.06 - 6.78 (m, 3H), 4.62 (s, 1H), 4.06 (q, J= 6.7 Hz, 2H), 3.88 (d, J= 3.1 Hz, 3H), 3.34- 3.21 (m, 12H), 2.50 - 2.35 (m, 8H), 2.11 (dq, J= 22.6, 6.6 Hz, 8H), 1.27 (d, J= 2.3 Hz, 189H), 0.92 - 0.88 (m. 18H).Example 4: Synthesis of Tag 4B PeptidesFmoc-Glv-Tag 4B (26)Table 19. Summary7of reagents

[0186] Dioctadecyl triamide Tag 4B (13) (5.00 g, 2.21 mmol, 1.0 eq) was added to a flask followed by EDCIHC1 (0.7269 g, 3.79 mmol, 1.5 eq). DMAP (0.0618 g, 0.506 mmol, 0.2 eq), DCM (100 mL, 20.0 Vol), and Fmoc-Gly-OH (1.1274 g, 3.79 mmol, 1.5 eq). The mixture was then stirred at room temperature. After stirring for 3 hours, the solvent was reduced, acetone (100 mL, 20.0 Vol) was added and reduced (two times total). Acetone (100 mL, 20.0 Vol) was added and the slurry was stirred at 0 °C for 30 minutes. The resulting off-white precipitate was filtered and then dried overnight. After drying for 16 hours at ambient temperature, 5.0333 g of 26 as an off-white solid was isolated (88% yield). This material was carried through to the next step.NH2-Gly-Tag 4B (27)Table 20. Summary of reagents

[0187] Tag 4B-Gly-Fmoc 26 (5.00 g, 2.21 mmol, 1.0 eq) was added to a solution of piperidine (3.77 g, 44.27 mmol, 20.0 eq) in DCM (100 mL, 20.0 Vol). The clear solution was allowed to stir at room temperature. After 3 hours, the solvent was reduced, acetonitrile (100 mL, 20.0 Vol) was added and reduced (two times total). Acetonitrile (100 mL, 20.0 Vol) was added and the slurry was stirred at room temperature for 15 minutes. The slum- was filtered and then briefly dried in a vacuum oven. After drying for 2 hours at ambient temperature. 4.01 g of 27 as a slightly wet off-white solid was isolated (89% yield). This wet solid was carried through to the next step.Fmoc-Glv-Gly-Tag 4B (28)Table 21. Summary of reagents

[0188] Tag 4B-Gly-NH227 (4.00 g, 1.96 mmol. 1.0 eq) was added to a flask followed by EDCI HC1 (0.5642 g, 2.94 mmol, 1.5 eq), DMAP (0.0618 g, 0.506 mmol, 0.2 eq), DCM (80 mL, 20.0 Vol), and Fmoc-Gly-OH (0.8751 g, 2.94 mmol, 1.5 eq). The mixture was then stirred at room temperature overnight. After 16 hours, the solvent was reduced, acetone (80 mL, 20.0 Vol) was added and reduced (two times total). Acetone (80 mL, 20.0 Vol) was added and the slurry was stirred at 0 °C for 30 minutes. The slurry was filtered and the precipitated solid 28 (5.2056g, >100% yield) was used directly as a wet solid for the next step.NH2-Gly-Glv-Tag 4B (29)Table 22. Summary' of reagents

[0189] Fmoc-Gly-Gly-Tag 4B (28) (4.500 g, 1.943 mmol, 1.0 eq) was added to a solution of piperidine (3.3089 g, 38.86 mmol, 20.0 eq) in DCM (90 mL, 20.0 Vol). The clear yellow solution was allowed to stir at room temperature. After 3 hours, the solvent was reduced, acetonitrile (90 mL, 20.0 Vol) was added and reduced (two times total). Acetonitrile (90 mL, 20.0 Vol) was added and the slurry was stirred at room temperature for ~45 minutes. The slurry' was filtered and then the white precipitate was resubjected to acetonitrile (90 mL, 20.0 Vol) and allowed to stir an additional hour to break up hard agglomerates that had formed. The slurry was filtered and then briefly dried on the filter. After drying for ~ 15 minutes, 5.0487 g of 29 as a slightly wet off-white solid was isolated (>100%, wet). This wet solid was carried through to the next step without further drying.Fmoc-Phe-Gly-Gly-Tag 4B (30)Table 23. Summary of reagents

[0190] NFb-Gly-Gly-Tah 4B 29 (4.00 g, 1.91 mmol, 1.0 eq) was added to a flask followed by Fmoc-Phe-OH (1.4791 g, 3.82 mmol. 2.0 eq), DCM (80 mL. 20.0 Vol), oxyma (0.8138 g, 5.73 mmol, 3.0 eq), and DIC (0.7708 g, 6.11 mmol, 3.0 eq). The mixture was then stirred at room temperature overnight. After 16 hours, the solvent was then reduced.acetone (80 mL, 20.0 Vol) was added and reduced (two times total). Acetone (80 rnL, 20.0 Vol) was added and the slurry was stirred at 0 °C for 30 minutes. The slurry was filtered and then dried in a vacuum oven at ambient temperature. After drying for 72 hours, 3.41 g of solid 30 was isolated (72% yield).NH2-Phe-Gly-Gly-Tag 4B (31)Table 24. Summary of reagents

[0191] Fmoc-Phe-Gly-Gly-Tag 4B 30 (3.40 g, 1.38 mmol, 1.0 eq) was added to a solution of piperidine (2.35 g, 28.58 mmol, 20.0 eq) in DCM (68 mL. 20.0 Vol). The clear yellow solution was allowed to stir at room temperature. After 3 hours, the solvent was then reduced, acetonitrile (68 mL, 20.0 Vol) was added and reduced (two times total). Acetonitrile (68 mL, 20.0 Vol) was added and the slurry' was stirred at room temperature for ~45 minutes. The slum' was filtered and then dried in a vacuum oven at ambient temperature. After drying for 16 hours, 2.6470 g of 31 as a solid was isolated (86% yield).Tag4B-Gly-Glv-Phe-Ala-Fmoc (32)Table 25. Summary of reagents

[0192] Fmoc-Ala-OH (0.6794 g, 2.18 mmol, 2.0 eq) was added to a flask followed by DCM (48.9 ml, 20.0 Vol), oxyma (0.4652 g, 3.27 mmol, 3.0 eq), and DIC (0.4406 g, 3.49 mmol, 3.0 eq). This mixture was stirred for 5-10 minutes then 31 (2.4470 g, 1.091 mmol, 1.0 eq) was added and the resulting yellow solution was then stirred at room temperature overnight. After 16 hours, the solvent was then reduced, acetone (50 mL, 20.0 Vol) was added and reduced (two times total). Acetone (50 mL, 20.0 Vol) was added and the slurry was stirred at 0 °C for 30 minutes. The slurry was filtered and then dried further on the filter. After drying for ~20 minutes, 2.8606 g of 32 as a solid was isolated (>100% yield, wet). This wet solid was carried through to the next step without further drying.NH2-Ala-Phe-Glv-Gly-Tag 4B (33)Table 26. Summary7of reagents

[0193] Fmoc-Ala-Phe-Gly-Gly-Tag 4B (32) (2.50 g, 0.98 mmol, 1.0 eq) was added to a solution of piperidine (1.68 g, 19.72 mmol, 20.0 eq) in DCM (50 mL. 20.0 Vol). The clear yellow solution was allowed to stir at room temperature. After 2 hours, the solvent was reduced, acetonitrile (50 mL, 20.0 Vol) was added and reduced (two times total).Acetonitrile (50 mL, 20.0 Vol) was added and the slurry7was stirred at room temperature for ~45 minutes. The slurry7was filtered and then dried in a vacuum oven at ambient temperature. After drying for 16 hours, 2. 1564 g of 33 as a solid was isolated (95% yield).Tag4B-Gly-Glv-Phe-Ala-Val-Fmoc (34)Table 27. Summary of reagents

[0194] Fmoc-Val-OH (0.6527 g, 1.92 mmol, 2.0 eq) was added to a flask followed by DCM (43.1 mL, 20.0 Vol), oxyma (0.4099 g, 2.89 mmol, 3.0 eq), and DIC (0.3883 g, 3.08 mmol, 3.0 eq). This mixture was stirred for 5-10 minutes then 33 (2.1564 g, 0.962 mmol, 1 .0 eq) was added and the resulting yellow solution was then stirred at room temperature overnight. After 16 hours, the solvent was then reduced, acetone (43 mL, 20.0 Vol) w as added and reduced (two times total). Acetone (43 mL, 20.0 Vol) was added and the slurry was stirred at 0 °C for 30 minutes. The slurry was filtered and then dried further on the filter. After drying for ~20 minutes, 3.3630 g of 34 as a solid was isolated (>100% yield, wet). This w et solid was carried through to the next step without further drying.NH2-Val-Ala-Phe-Gly-Gly-Tag 4B (35)Table 28. Summary of reagents

[0195] Fmoc-Val-Ala-Phe-Gly-Gly (34) (2.50 g, 0.95 mmol, 1.0 eq) was added to a solution of piperidine (1.62 g, 18.97 mmol, 20.0 eq) in DCM (50 mL, 20.0 Vol). The clear yellow solution w as allowed to stir at room temperature. After 2 hours the solvent was reduced, acetonitrile (50 mL, 20.0 Vol) was added and reduced (two times total). Acetonitrile (50 mL, 20.0 Vol) was added and the slurry’ was stirred at room temperature for ~45 minutes. The slurry was then poured onto a frit. After filtration, more precipitate formed in the filtrate, so the mother liquor w as cooled in an ice bath and for ~10 minutes and then re-filtered. The resulting off-white precipitate was dried in a vacuum oven atambient temperature. After drying for 16 hours, 1.9104 g of 35 as a solid was isolated (84% yield).H-Val-Ala-Phe-Glv-Glv-OH (25)Table 29. Summary of reagents

[0196] NH2-Val-Ala-Phe-Gly-Gly-Tag 4B (35) (0.400 g, 0.166 mmol, 1.0 eq) was added to a solution of 92.5:2.5:2.5:2.5 TFA / TIPS / H2O / DODT (8.0 mL total, 20.0 Vol) at -10 °C. The mixture was allowed to stir for 4 hours while warming to room temperature before it was added dropwise to a flask containing MTBE (48 mL. 120.0 Vol) at -20 °C. The resulting precipitate was a gummy solid which was put in the freezer overnight to settle. After sitting for 16 hours at -20 °C the mixture was then filtered to yield a wet gel -like semisolid. Not much gel material was recovered and the filtrate appeared clear so it was then reduced to half its original volume and placed in the freezer overnight. The suspension was again filtered and more gel-like semisolid was recovered and immediately scraped into a vial to prevent it passing through the filter. A total of 0.9100 g of wet semisolid gel was isolated and purified by HPLC to yield 0.0065 g of peptide 25 as a fluffy white solid (9% yield).

[0197] FIG. 16 shows the LCMS (ES+) chromatograph of pentapeptide 25. LCMS m / z found: 450.2 (M+H ).

[0198] FIG. 17 shows the mass extraction of purified pentapeptide 25 with a retention time of 5.258 minutes.

[0199] FIG. 18 shows the ’H NMR (400 MHz, D2O) spectrum of pentapeptide 25. ’H NMR (400 MHz. D2O) 5 7.27 - 7.13 (m, 5H). 4.46 (t. J = 7.7 Hz. 1H), 4.24 (q. J = 7.2 Hz, 1H), 3.84 (s, 2H), 3.81 - 3.68 (m, 2H), 3.63 (d, J = 6.0 Hz, 1H), 3.05 - 2.89 (m, 2H), 2.10 - 1.96 (m, 1H), 1.21 (d, J = 7.2 Hz, 3H), 0.84 (d, J = 6.9 Hz, 6H).

[0200] FIG. 19 shows the13C NMR (101 MHz, D2O) spectrum of pentapeptide 25.13C NMR (101 MHz, D2O) 5 174.16, 173.46, 173.07, 171.53, 168.86, 163.14, 162.79, 136.08, 129.17, 128.78, 127.23, 117.75, 114.85, 58.29, 55.28, 42.26, 40.97, 36.85, 29.92. 17.58, 16.77.Fmoc-Thr(t-Bu)-Val-Ala-Phe-Gly-Gly-Tag 4B (36)Table 30. Summary of reagents

[0201] Fmoc-Thr(tBu)-OH (0.4942 g, 1.24 mmol, 2.0 eq) was added to a flask followed by DCM (30.0 mL, 20.0 Vol), oxyma (0.2650 g, 1.87 mmol, 3.0 eq), and DIC (0.2510 g, 1.99 mmol. 3.0 eq). This mixture was stirred for 5-10 minutes then 35 (1.50 g, 0.622 mmol, 1 .0 eq) was added and the resulting yellow solution was then stirred at room temperature overnight. After 16 hours, the solvent was reduced, acetone (50 mL, 20.0 Vol) was addedand reduced (two times total). Acetone (50 mL, 20.0 Vol) was added and the slurry was stirred at 0 °C for 30 minutes. The resulting off-white precipitate was filtered and then dried further on the filter. After drying for ~20 minutes. 2.3555 g of 36 as a solid was isolated (>100% yield, wet). This wet solid was carried through to the next step without further drying.NH2-Thr(tBu)-Val-Ala-Phe-Glv-Gly-Tag 4B (37)Table 31. Summary of reagents

[0202] Fmoc-Thr(tBu)-Val-Ala-Phe-Gly-Gly-Tag 4B (36) (1.700 g. 0.609 mmol. 1.0 eq) was added to a solution of piperidine (1.0368 g, 19.72 mmol, 20.0 eq) in DCM (34 mL, 20.0 Vol). The clear yellow solution was allowed to stir at room temperature. After 2 hours the solvent was reduced, acetonitrile (34 mL, 20.0 Vol) was added and reduced (two times total). Acetonitrile (34 mL, 20.0 Vol) was added and the slurry was stirred at room temperature for 20 minutes then cooled to 0 °C and stirred for 20 minutes. The slurry was filtered, and the resulting off-white precipitate was then dried in a vacuum oven at ambient temperature. After drying for 16 hours, 1.5125 g of 37 as a solid was isolated (95% yield).Fmoc-Arg(Pbf)-Thr(tBu)-Val-Ala-Phe-Gly-Gly-Tag 4B (38)Table 32. Summary of reagents

[0203] Fmoc-Arg(Pbf)-OH (0.7573 g, 1.16 mmol, 2.0 eq) was added to a flask followed by DCM (30.0 mL, 20.0 Vol), DEPBT (0.2096 g, 0.70 mmol, 1.2 eq), and DIPEA (0.1056 g, 0.82 mmol, 1.4 eq). This mixture was stirred for 5-10 minutes then 37 (1.50 g, 0.58 mmol, 1.0 eq) was added and the resulting yellow solution was then stirred at room temperature. After stirring for 16 hours, the solvent was then reduced, acetone (30 mL, 20.0 Vol) was added and reduced (two times total). Acetone (30 mL, 20.0 Vol) was added and the slurry' was stirred at 0 °C for 30 minutes. The slurry was filtered, and the resulting off-white precipitate was then dried in a vacuum oven at ambient temperature. After drying for 16 hours, 1.6737 g of 38 was isolated (>100% yield).NH2-Arg(Pbf)-Thr(tBu)-Val-Ala-Phe-Gly-Glv-Tag 4B (39)Table 33. Summary of reagents

[0204] Tag 4B-Gly-Gly-Phe-Ala-Val-Thr-Arg-Fmoc 38 (1.60 g, 0.50 mmol, 1.0 eq) was added to a solution of piperidine (0.85 g, 10.0 mmol, 20.0 eq) in DCM (32 rnL, 20.0 Vol). The clear yellow solution was allowed to stir at room temperature. After 2 hours the solvent was reduced. ~30 mL of acetonitrile was added and reduced (two times total), then 30 mL of acetonitrile was added and the slurry was stirred at room temperature for ~5 minutes then cooled to 0 °C and stirred for ~30 minutes. The slurry was then poured onto a frit and the isolated yellow precipitate was dried in a vacuum oven at ambient temperature. After drying for 16 hours, 1.4351 g of 39 was isolated (96% yield) as a solid.Fmoc-Ile-Arg(PbD-Thr(tBu)-Val-Ala-Phe-Glv-Gly-Tag 4B (40)Table 34. Summary of reagents

[0205] Fmoc-Ile-OH (0.3322 g, 0.94 mmol, 2.0 eq) was added to a flask followed by DCM (30.0 ml, 20.0 Vol), oxyma (0.2004 g, 1.41 mmol, 3.0 eq), and DIC (0.1898 g, 1.50 mmol, 3.0 eq). This mixture was stirred for 5-10 minutes then 39 (1.40 g, 0.47 mmol, 1.0 eq) was added and the resulting yellow solution was then stirred at room temperature overnight. After stirring for 16 hours, the solvent was reduced, acetone (28 mL, 20.0 Vol) was added and reduced (two times total). Acetone (28 mL, 20.0 Vol) was added and the si urn was stirred at 0 °C for 30 minutes. The slurry was filtered and the isolated solid was then dried further on the filter. After drying for ~20 minutes, 1.9302 g of 40 as a wet solid was isolated (>100% yield, wet). This wet solid was carried through to the next step without further drying.NH2-Ile-Arg(Pbf)-Thr(tBu)-Val-Ala-Phe-Glv-Gly-Tag 4B (41)Table 35. Summary of reagents

[0206] Fmoc-Ile-Arg(Pbf)-Thr(tBu)-Val-Ala-Phe-Gly-Gly-Tag 4B (40) (1.500 g, 0.453 mmol, 1.0 eq) was added to a solution of piperidine (0.77 g, 9.05 mmol, 20.0 eq) in DCM (30 mL, 20.0 Vol). The clear yellow solution was allowed to stir at room temperature.After 2 hours the solvent was reduced. ~30 mL of acetonitrile was added and reduced (two times total). Acetonitrile (30 mL, 20.0 Vol) was added and the slurry was stirred at room temperature for ~20 minutes then cooled to 0 °C and stirred for ~20 minutes. The slurrywas filtered and then dried in a vacuum oven at ambient temperature. After drying for 16 hours, 1.5031 g of solid 41 was isolated (>100% yield).Fmoc-Asn(Trt)-Ile-Arg(Pbf)-Thr(tBu)-Val-Ala-Phe-Gly-Glv-Tag 4B (42)Table 36. Summary7of reagents

[0207] Fmoc-Asn(Trt)-OH (0.5558 g, 0.93 mmol, 2.0 eq) was added to a flask followed by DCM (30 mL, 20.0 Vol), oxyma (0.1986 g, 1.40 mmol, 3.0 eq), and DIC (0.1881 g, 1.49 mmol, 3.2 eq). This mixture was stirred for 5-10 minutes then 41 (1.44 g, 0.47 mmol, 1.0 eq) was added and the resulting yellow solution was then stirred at room temperature overnight. After stirring for 16 hours, the solvent was reduced, acetone (29 mL, 20.0 Vol) was added and reduced (two times total). Acetone (29 mL, 20.0 Vol) was added and the slurry7was stirred at 0 °C for 30 minutes. The slurry was filtered and then dried further on the filter. The wet solid was placed in a vacuum oven to dry7overnight at ambient temperature. After drying for 16 hours, 1.5383 g of 42 was isolated (90% yield) as a yellow powder.NH2- Asn(Trt)-Ile-Arg(Pbf)-Thr(tBu)-Val-Ala-Phe-Gly-Gly-Tag 4B (43)Table 37. Summary of reagents

[0208] Fmoc-Asn(Trt)-Ile-Arg(Pbf)-Thr(tBu)-Val-Ala-Phe-Gly-Gly-Tag 4B (42) (1.5500 g, 0.42 mmol, 1.0 eq) was added to a solution of piperidine (0.7192 g, 8.45 mmol, 20.0 eq) in DCM (31 mL, 20.0 Vol). The clear yellow solution was allowed to stir at room temperature. After 2 hours, the solvent was reduced, acetonitrile (31 mL. 20.0 Vol) was added and reduced (two times total). Acetonitrile (31 mL, 20.0 Vol) was added and the si urn was stirred at room temperature for 20 minutes then cooled to 0 °C and stirred for 20 minutes. The slurry' was filtered and then dried in a vacuum oven at ambient temperature. After drying for 16 hours, 1.3540 g of solid 43 was isolated (93% yield).Fmoc-His(Boc)-Asn(Trt)-Ile-Arg(Pbf)-Thr(tBu)-Val-Ala-Phe-Glv-Gly-Tag 4B (44)Table 38. Summary' of reagents

[0209] Fmoc-His(Boc)-OH (0.3877 g, 0.81 mmol, 2.0 eq) was added to a flask followed by DCM (28.0 mL, 20.0 Vol), HATU (0.3088 g, 2.0 mmol, 2.0 eq), and DIPEA (0.2099 g, 1.62 mmol, 4.0 eq). This mixture was stirred for 5-10 minutes then 43 (1.40 g, 0.41 mmol, 1.0 eq) was added and the resulting yellow solution was then stirred at room temperature overnight. After stirring for 16 hours, the solvent was reduced, acetone (28 mL, 20.0 Vol) was added and reduced (tw o times total). Acetone (28 mL, 20.0 Vol) was added and the si urn w as stirred at 0 °C for 30 minutes. The yellow slurry was filtered and the wet solid 44 was then used directly for the next deprotection reaction.NH2- His(Boc)-Asn(Trt)-Ile-Arg(Pbf)-Thr(tBu)-Val-Ala-Phe-Gly-Gly-Tag 4B (45)Table 39. Summary of reagents

[0210] Fmoc-His(Boc)-Asn(Trt)-Ile-Arg(Pbf)-Thr(tBu)-Val-Ala-Phe-Gly-Gly-Tag 4B (44) (1.50 g, 0.384 mmol, 1.0 eq) was added to a solution of piperidine (0.6537 g, 7.68 mmol, 20.0 eq) in DCM (30 mL, 20.0 Vol). The clear yellow solution was allowed to stir at room temperature. After 2 hours, the solvent w as reduced, acetonitrile (30 mL, 20.0 Vol)was added and reduced (two times total). Acetonitrile (30 mL, 20.0 Vol) was added and the slurry was then cooled to 0 °C and stirred for 20 minutes. The yellow slurry was filtered and then dried in a vacuum oven at ambient temperature. After drying for 16 hours, 1.2959 g of 45 was isolated as a solid (92% yield).NH2-His-Asn-Ile-Arg-Thr-Val-Ala-Phe-Gly-Gly-QH (46)Table 40. Summary of reagents

[0211] NH2- His(Boc)-Asn(Trt)-Ile-Arg(Pbf)-Thr(tBu)-Val-Ala-Phe-Gly-Gly-Tag 4B (45) (1.29 g, 0.35 mmol, 1.0 eq) was added to a solution of 92.5:2.5:2.5:2.5 TFA / TIPS / H2O / DODT (12.9 mL total, 10.0 Vol) at -10 °C. After stirring for 2 hours, the reaction turned from a tan mixture to a red-orange slurry. After stirring for a total of 4 hours, the reaction was stopped. Once stirring was ceased, the mixture separated into two layers- a thick tan layer on top (-10% of the volume) and a magenta translucent layer on the bottom (-90% of the volume). The top layer was removed, and the bottom layer was added dropwise to -20 °C MTBE (70.0 mL) in an effort to precipitate the peptide. The resulting mixture was then stored in the freezer overnight to settle. After sitting in the freezer for 16 hours, the mixture was filtered, but no material was isolated on the frit. Water (100.0 mL) was added to the cloudy filtrate and the resulting biphasic mixture was stirred for 1 hour. The mixture was then added to a separatory funnel and the aqueous layer was drained. The organic layer was washed with water (2 x 25 mL) and the combined water washes werelyophilized to yield 0.3501 g of crude 46 as a yellow solid. The dry crude 46 was then purified by HPLC to yield 0.0805 g of purified 46 as a white solid (21% yield).

[0212] FIG. 20 shows the LCMS (ES+) chromatograph of decapeptide 46. LCMS m / z found: 1071.5 (M+H+).

[0213] FIG. 21 shows the mass extraction of purified decapeptide 46 with a retention time of 6.408 minutes.

[0214] FIG. 22 shows the 'H NMR (400 MHz, D2O) spectrum of decapeptide 46.1H NMR (400 MHz, D2O) 5 8.58 (s, 1H), 7.30 - 7.11 (m, 6H), 4.46 (t, J= 7.6 Hz, 1H), 4.32 - 3.96 (m, 7H), 3.94 - 3.64 (m, 5H), 3.27 (d, J= 6.7 Hz, 2H), 3.01 (dt, J= 32.1, 7.8 Hz, 4H), 2.79 - 2.54 (m, 2H), 1.90 (h, J= 6.8 Hz, 1H), 1.82 - 1.57 (m, 3H), 1.49 (tdd, J= 17.2, 8.7, 4.4 Hz, 2H), 1.39 - 1.24 (m, 1H), 1.17 (d, J= 7.1 Hz, 3H), 1.13 - 0.95 (m, 4H), 0.93 - 0.53 (m. 12H).

[0215] FIG. 23 shows the13C NMR (101 MHz, D2O) spectrum of decapeptide 46.13C NMR (101 MHz, D2O) 5 174.44, 173.85, 173.42, 173.38, 173.13, 173.09, 172.75, 171.99, 171.56, 167.65, 163.13, 162.77, 156.65, 136.12, 134.40, 129.18, 128.76, 127.21, 125.58, 118.47, 117.76, 114.86, 67.02, 59.25, 58.99, 58.52, 55.17, 53.23, 51.68, 50.43, 42.29, 41.00, 40.48, 36.84, 36.22. 30.22, 28.11, 25.97, 24.46, 18.83, 18.33, 17.54, 16.50, 14.78. 10.28.Example 5: Synthesis of Octreotide Acetate with Tag 4BFmoc-Thr(tBu)-Tag4B (50)Table 41. Summary of reagents

[0216] Dioctadecyl triamide 13 Tag 4B (10.0000 g, 5.047 mmol, 1.0 eq) was added to a flask followed by EDCEHC1 (1.4512 g, 7.570 mmol, 1.5 eq), DMAP (0.1233 g, 1.009 mmol, 0.2 eq), DCM (200 mL, 20.0 Vol), and Fmoc-Thr(tBu)-OH (3.0089 g. 7.570 mmol, 1.5 eq). The mixture was then stirred at ambient temperature under a nitrogenous atmosphere. After stirring for 22 hours, the solvent was reduced, acetone (100 mL) was added, and the resulting slurry was stirred at 0 °C for 30 minutes. The white slurry was filtered and the solid was dried overnight in a vacuum oven. After drying for 20 hours at 30 °C, 12.2702 g of 51 as a white solid was isolated (>100% yield).H-Thr(tBu)-Tag4B (52)Table 42. Summary7of reagents

[0217] Fmoc-Thr(tBu)-Tag4B 51 (11.9000 g, 5.040 mmol, 1.0 eq) was added to a solution of piperidine (8.5840 g, 100.800 mmol, 20.0 eq) in DCM (238 mL, 20.0 Vol). The clear yellow solution was allowed to stir at ambient temperature. After stirring for 2 hours, the solvent was reduced, acetonitrile (100 mL) was added and reduced. Acetonitrile (150 mL) was added and the resulting slurry' was stirred at 0 °C for 2 hours. The slurry was then filtered and dried in a vacuum oven. After drying for 20 hours at ambient temperature, 9.7420 g of 52 as a white solid was isolated (90% yield).Fmoc-Lys(Boc)-Thr(tBu)-Tag4B (53)Table 43. Summary of reagents

[0218] A flask was charged with Fmoc-Lys(Boc)-OH (3.1877 g, 6.803 mmol, 1.5 eq), DCM (194.0 mL, 20.0 Vol), oxyma (1.2891 g, 9.071 mmol. 2.0 eq), and DIC (1.2593 g, 9.978 mmol, 2.2 eq). The mixture was stirred at ambient temperature for 5-10 minutes. H-Thr(tBu)-Tag4B 52 (9.7000 g, 4.536 mmol, 1.0 eq) was then added to the flask and the mixture was allowed to stir at ambient temperature overnight. After stirring for 20 hours, the solvent was then reduced, acetone (100 mL) was added and reduced. Acetone (200 mL) was added and the slum- was stirred at ambient temperature for 30 minutes followed by 30 minutes at 0 °C. The slurry' was filtered and then dried in a vacuum oven at ambient temperature. After drying for 16 hours, 8.2977 g of 53 as an off-white solid was isolated (70% yield).H-Lys(Boc)-Thr(tBu)-Tag4B (54)Table 44. Summary of reagents

[0219] Fmoc-Lys(Boc)-Thr(tBu)-Tag4B 53 (8.2000 g, 3.170 mmol. 1.0 eq) was added to a solution of piperidine (5.3900 g, 63.300 mmol, 20.0 eq) in DCM (164.0 mL, 20.0 Vol). The clear yellow solution was allowed to stir at ambient temperature. After stirring for 2 hours, the solvent was reduced. Acetonitrile (200 mL) was added and the slurry' was stirred at ambient temperature for 30 minutes followed by 1 hour at 0 °C. The slurry was filtered and then briefly dried on the filter before it was transferred to a vacuum oven to dryovernight at ambient temperature. After drying for 20 hours, 7. 1747 g of 54 as a white solid was isolated (95% yield).Fmoc-D-Trp(Boc)-Lys(Boc)-Thr(tBu)-Tag4B (55)Table 45. Summary of reagents

[0220] A flask was charged with Fmoc-D-Trp(Boc)-OH (2.3900 g, 4.543 mmol, 1.5 eq), DCM (143.4 mL, 20.0 Vol), oxyma (0.7750 g, 5.453 mmol, 1.8 eq), and DIC (0.8410 g, 6.664 mmol, 2.2 eq). The mixture was stirred at ambient temperature for 5-10 minutes. H- Lys(Boc)-Thr(tBu)-Tag4B 54 (7.1700 g, 3.029 mmol. 1.0 eq) was then added to the flask and the mixture was allowed to stir at ambient temperature. After stirring for 3 hours, the solvent was reduced, then acetone (100 mL) was added and reduced. Acetone (200 mL) was added and the slurry' was stirred at ambient temperature for 30 minutes followed by 30 minutes at 0 °C. The slurry was filtered and then dried in a vacuum oven at ambient temperature. After drying for 17 hours, 7.8782 g of 55 (90% yield) was isolated as a pale yellowH-D-Trp(Boc)-Lys(Boc)-Thr(tBu)-Tag4B (56)

[0221] Fmoc-D-Trp(Boc)-Lys(Boc)-Thr(tBu)-Tag4B 55 (7.8700 g, 2.737 mmol, 1.0 eq) was added to a solution of piperidine (4.6610 g, 54.738 mmol, 20.0 eq) in DCM (157.4 mL, 20.0 Vol). The clear yellow solution was allowed to stir at ambient temperature. After 3 hours, the solvent was reduced, then acetonitrile (100 mL) was added and reduced.Acetonitrile (200 mL) was added and the slum- was stirred at ambient temperature for 30 minutes followed by 30 minutes at 0 °C. The slurry was filtered and then dried in a vacuum oven at ambient temperature. After drying for 16 hours, 6.6069 g 56 as an of off-white solid was isolated (91% yield).Fmoc-Phe-D-Trp(Boc)-Lys(Boc)-Thr(tBu)-Tag4B (57)Table 47. Summary of reagents

[0222] A flask was charged with Fmoc-Phe-OH (1.4471 g, 3.735 mmol, 1.5 eq), DCM (132.1 mL, 20.0 Vol), oxyma (0.6369 g. 4.482 mmol, 1.8 eq), and DIC (0.6914 g, 5.478 mmol, 2.2 eq). The mixture was stirred at ambient temperature for 5-10 minutes. H-D- Trp(Boc)-Lys(Boc)-Thr(tBu)-Tag4B 56 (6.6069 g, 2.490 mmol, 1.0 eq) was then added to the flask and the mixture was allowed to stir at ambient temperature. After stirring for 3 hours, the solvent was reduced, then acetone (100 mL) was added and reduced. Acetone (200 mL) was added and the slum' was stirred at ambient temperature for 30 minutes followed by 30 minutes at 0 °C. The slurry was filtered and then dried in a vacuum oven at ambient temperature. After drying for 18 hours, 6.9691 g of 57 as a pale yellow solid was isolated (92% yield).H-Phe-D-Trp(Boc)-Lys(Boc)-Thr(tBu)-Tag4B (58)Table 48. Summary of reagents

[0223] Fmoc-Phe-D-Trp(Boc)-Lys(Boc)-Thr(tBu)-Tag4B 57 (6.9691 g, 2.306 mmol, 1.0 eq) was added to a solution of piperidine (3.9260 g, 46. 112 mmol, 20.0 eq) in DCM (140 mL, 20.0 Vol). The clear yellow solution was allowed to stir at ambient temperature. After 3 hours, the solvent was reduced, then acetonitrile (100 mL) was added and reduced. Acetonitrile (200 mL) was added and the slurry was stirred at ambient temperature for 30 minutes followed by 30 minutes at 0 °C. The slurry was filtered and then dried in a vacuum oven at ambient temperature. After drying for 72 hours, 6.3701 g of 58 as an off-white solid was isolated (98% yield).Fmoc-Cvs(Trt)-Phe-D-Trp(Boc)-Lys(Boc)-Thr(tBu)-Tag4B (59)Table 49. Summary of reagents

[0224] A flask was charged with Fmoc-Cys(Trt)-OH (2.006 g, 3.416 mmol, 1.5 eq), DCM (127.5 mL, 20.0 Vol), oxyma (0.5825 g, 4.099 mmol. 1.8 eq), and DIC (0.6322 g, 5.010 mmol, 2.2 eq). The mixture was stirred at ambient temperature for 5-10 minutes. H- Phe-D-Trp(Boc)-Lys(Boc)-Thr(tBu)-Tag4B 58 (6.3770 g, 2.277 mmol, 1.0 eq) was then added to the flask and the mixture was allowed to stir at ambient temperature. After stirring for 3.5 hours, the solvent was reduced, then acetone (100 mL) was added and reduced.Acetone (200 mL) was added and the slurry was stirred at ambient temperature for 30 minutes followed by 30 minutes at 0 °C. The slurry was filtered and then dried in a vacuum oven at ambient temperature. After drying for 18 hours. 6.9925 g of 59 as a pale yellow solid was isolated (97% yield).H-Cys(Trt)-Phe-D-Trp(Boc)-Lys(Boc)-Thr(tBu)-Tag4B (60)Table 50. Summary of reagents

[0225] Fmoc-Cys(Trt)-Phe-D-Trp(Boc)-Lys(Boc)-Thr(tBu)-Tag4B 59 (3.8800 g, 1.152 mmol, 1.0 eq) was added to a solution of piperidine (1.9620 g, 23.039 mmol, 20.0 eq) in DCM (77.6 mL, 20.0 Vol). The clear yellow solution was allowed to stir at ambient temperature. After 3 hours, the solvent was reduced, then acetonitrile (100 mL) was added and reduced. Acetonitrile (120 mL) was added and the slurry was stirred at ambient temperature for 60 minutes followed by 45 minutes at 0 °C. The slurry was filtered and then dried in a vacuum oven at ambient temperature. After drying for 18 hours, 3.4283 g of 60 as an off-white solid was isolated (94% yield).Boc-D-Phe-Cys(Trt)-Phe-D-Trp(Boc)-Lys(Boc)-Thr(tBu)-Tag4B (61)Table 51. Summary of reagents

[0226] A flask was charged with Boc-D-Phe-OH (0.4326 g. 1.631 mmol. 1.5 eq). DCM (68.4 mL, 20.0 Vol), oxyma (0.2781 g, 1 .957 mmol, 1 .8 eq), and DIC (0.3018 g, 2.392 mmol, 2.2 eq). The mixture was stirred at ambient temperature for 5-10 minutes. H- Cys(Trt)-Phe-D-Trp(Boc)-Lys(Boc)-Thr(tBu)-Tag4B 60 (3.4200 g, 1.087 mmol, 1.0 eq) was then added to the flask and the mixture was allowed to stir at ambient temperature. After stirring for 4 hours, the solvent was reduced, then acetone (50 mL) was added and reduced. Acetone (100 mL) was added and the slurry was stirred at ambient temperature for 10 minutes followed by 30 minutes at 0 °C. The slurry was filtered and then dried in a vacuum oven at ambient temperature. After drying for 18 hours, 3.3203 g of 61 as a pale yellow solid was isolated (90% yield).Boc-D-Phe-Cvs(Trt)-Phe-D-Trp(Boc)-Lys(Boc)-Thr(tBu)-OH (62)Table 52. Summary of reagents

[0227] Boc-D-Phe-Cys(Trt)-Phe-D-Trp(Boc)-Lys(Boc)-Thr(tBu)-Tag4B 61 (3.3000 g, 0.972 mmol, 1.0 eq) was added to flask followed by 2-Me THF (16.5 mL, 5.0 Vol) and stirred. A solution of LiOH*H2O (0.8161 g, 19.451 mmol. 20.0 eq) in water (16.5 mL, 5.0 Vol) was added dropwise to the reaction mixture, which was then heated to 60 °C. After stirring for 16 hours, the reaction mixture was cooled to 0 °C, adjusted to pH 3-4 with a 10% solution of aqueous KHSO4. The two layers were separated and the organic layer was set aside while the aqueous layer was extracted with DCM (2 x 25 mL). The combined organics were dried over Na2SC>4, filtered, and reduced. The resulting residue was then stirred with MeCN (50 mL) at ambient temperature for 45 minutes followed by 0 °C for 45 minutes. The slurry was filtered and the filtrate was reduced before it was triturated with 5% MTBE in heptane. The slurry was stirred at ambient temperature for 45 minutes followed by 0 °C for 45 minutes before it was filtered. The wet solid was partially dried on the frit before it was transferred to a vacuum oven to dry overnight at ambient temperature. After drying for 20 hours, 0.7734 g of 62 as a solid was isolated (56% yield).Fmoc-Cys(Trt)-Thr(tBu)-ol (49)NHFmoc47 48Table 53. Summary of reagents

[0228] Fmoc-Cys(Trt)-OH 47 (1.0000 g, 1.707 mmol. 1.0 eq) was added to a vial followed by DMF (5.5 mL, 5.5 Vol). The mixture was stirred at ambient temperature then cooled to 0 °C. Threoninol 48 (0.3303 g, 2.049 mmol, 1.2 eq) and HOBt (0.3461 g, 2.561 mmol, 1.5 eq) were added sequentially to the cooled solution. Next, EDCHHC1 (0.5237 g, 2.732 mmol, 1.6 eq) was slurried in DMF (5.0 mL, 5.0 Vol) and added to the reaction vial which was then stirred for 2 hours at 10 °C. Water (100 mL) was added to quench the reaction and precipitate the product. The resulting slurry was allowed to stir at ambient temperature for 3 hours before it was filtered. The filter cake was washed with water (50 mL) followed by heptane (50 mL) then transferred to a vacuum oven to dry at ambient temperature overnight. After drying for 17 hours, 1.3318 g of 49 as a white solid was isolated (>100% yield), which was found to contain as small amount of residual DMF.H-Cys(Trt)-Thr(tBu)-ol (50)49 50Table 54. Summary of reagents

[0229] Fmoc-Cys(Trt)-Thr(tBu)-ol 49 (1.24 g, 1.701 mmol, 1.0 eq) and MeCN (18.6 mL, 15.0 Vol) were added to a flask and cooled to 0 °C. A solution of 1 M NaOH (3.6 mL, 2.9 Vol) was added slowly to the cooled stirnng solution. Heptane (6.2 mL, 5.0 Vol) was then added and the reaction mixture was allowed to stir while warming to ambient temperature. After stirring at ambient temperature for 3 hours, the reaction mixture was cooled to 0 °C and acidified to pH 4-5 with a 10% solution of aqueous H2SO4. The aqueous layer was then separated, washed with 30% MTBE in heptane, and neutralized with NaHCCL. The neutralized aqueous layer was extracted with MTBE (3 x 30 mL). The combined organics were washed with brine, dried over IkfeSCL, filtered, and reduced to a sticky solid, which was transferred to a vacuum oven to dry overnight. After drying for 18 hours at ambient temperature, 0.7418 g of 50 a white solid was isolated (85% yield).Boc-D-Phe-Cvs(Trt)-Phe-D-Trp(Boc)-Lvs(Boc)-Thr(tBu)-Cys(Trt)-Thr(tBu)-ol (63)Table 55. Summary of reagents

[0230] Boc-D-Phe-Cys(Trt)-Phe-D-Trp(Boc)-Lys(Boc)-Thr(tBu)-OH 62 (0.7500 g, 0.524 mmol, 1.0 eq) and DMF (5.3 mL, 7.0 Vol) were added to a vial and stirred for 5-10 minutes. The mixture was cooled to 0 °C then HOBt (0.0709 g, 0.524 mmol, 1.0 eq) and 50 (0.2658 g, 0.524 mmol, 1.0 eq) were added. A slum7ofEDCI’HCl (0.2011 g, 1.049 mmol, 2.0 eq) in DMF (2.3 mL, 3.0 Vol) was added and the mixture was allowed to stir while warming to ambient temperature. After stirring for 18 hours, the reaction was cooled to 0 °C and a 1 % solution of aqueous citric acid (10 mL) was added, forming a precipitate. The si urn was stirred for ~20 minutes before it was filtered. The filtered solid was transferred to a vial and stirred with heptane (20 mL) for 30 minutes. The slurry7was filtered, partially dried on the frit, then transferred to a vacuum oven to dry overnight at ambient temperature. After drying for 22 hours, 0.8054 g of 63 as a tan solid was isolated (80% yield).Linear OctreotideTable 56. Summary of reagents

[0231] Boc-D-Phe-Cys(Trt)-Phe-D-Trp(Boc)-Lys(Boc)-Thr(tBu)-Cys(Trt)-Thr(tBu)-ol 63 (0.2000 g, 0.104 mmol, 1.0 eq) was added to a solution of 92.5:2.5:2.5:2.5 TFA / TIPS / H2O / DODT (2.0 mL, 10.0 Vol) at 0 °C. After stirring at 0 °C for 1 hour and 45 minutes, the reaction mixture was reduced to a yellow oil hi ch was then added dropwise to a vial containing -20 °C MTBE (20 mL). The vial was stirred in an ice bath for 10 minutes then transferred to a -20 °C freezer to cool. After cooling in the freezer for 2 hours, the mixture was filtered, partially dried on the frit, then transferred to a vacuum oven to dry overnight at ambient temperature. After dry ing for 18 hours. 0.0969 g of Linear Octreotide an off- white solid was isolated (83% yield).Octreotide AcetateTable 57. Summary of reagents

[0232] Linear octreotide (0.0950 g, 0.085 mmol, 1.0 eq) was added to a 5% solution of aqueous NaHCCL (19.0 mL, 200.0 Vol) at 0 °C. The reaction mixture was stirred at 0 °C for 15 minutes then the ice bath was removed and the reaction w as allowed to stir exposed to air while warming to ambient temperature. After stirring for 46 hours, MeCN (20 mL)was added and the aqueous layer was removed then lyophilized. The resulting white powder was triturated with MeOH (20 mL) for 10 minutes, filtered, and then reduced to a white residue. The white residue was combined with the reduced MeCN wash, which was found to contain the product, and loaded onto a pre-equilibrated Cis reverse phase column and eluted with a gradient of 5-100% MeCN (+0.1% AcOH) in H2O (+0.1% AcOH). Fractions containing the desired product were combined, reduced, and then lyophilized to afford 0.0412 g of octreotide acetate (92% purity by AUC) as a white fluffy solid (42% yield).

[0233] FIG. 24 shows the LCMS (ES+) chromatograph of octreotide acetate. LCMS m / z found: 1019.3 (M+H+).

[0234] FIG. 25 shows the mass extraction of purified octreotide acetate with a retention time of 9.272 minutes.

[0235] FIG. 26 shows theXH NMR (400 MHz, DMSO-d6) spectrum of octreotide acetate. 'H NMR (400 MHz, DMSO-J6) 5 10.99 (s, 1H), 8.73 (d, J= 5.4 Hz, 1H), 8.47 (dd, J= 22.3. 7.6 Hz, 4H), 7.76 (d, J= 8.9 Hz. 1H), 7.65 (d, J= 8.9 Hz, 1H), 7.43 (dt, J= 7.9. 1.0 Hz, 1H), 7.37 - 7.30 (m. 1H), 7.28 - 7. 15 (m. 9H), 7. 10 - 7.03 (m, 3H), 7.00 - 6.91 (m, 2H), 5. 13 (s, 3H), 4.65 - 4.46 (m, 3H), 4.24 - 4.17 (m, 1H), 4.01 - 3.89 (m, 4H), 3.66 (td, J= 7.6, 2.6 Hz, 2H), 3.56 - 3.50 (m, 3H), 3.40 (dd, J= 10.5, 6.0 Hz, 3H), 3.09 - 2.81 (m, 10H), 2.74 - 2.64 (m, 1H). 2.60 - 2.52 (m, 1H), 1.84 (s. 6H), 1.75 - 1.60 (m, 1H), 1.37- 1.19 (m, 3H). 1.13 - 0.96 (m, 7H). 0.83 (d, J = 7.5 Hz, 2H).

[0236] FIG. 27 shows the13C NMR (400 MHz, DMSO-Je) spectrum of octreotide acetate.13C NMR (400 MHz, DMSO-d6) 6 175.15, 172.85. 171.53, 170.58, 170.35, 169.60, 168.73, 138.90, 136.86, 136.14, 129.41, 129.00, 128.09, 128.02, 126.98, 126.34, 126.07, 123.63, 120.82, 118.17, 111.31, 108.96, 67.10, 64.23, 60.46, 56.06, 55.93, 52.95, 52.15, 44.59, 41.30, 30.51, 29.67, 26.11, 22.32, 22.14, 20.06, 19.40, 2.69.

[0237] FIG. 28 shows the *HNMR (400 MHz, DMSO-c4>) spectrum of octreotide acetate standard. ’H NMR (400 MHz, DMSO-J6) 5 11.00 (s, 1H), 8.74 (d, J= 5.4 Hz, 1H), 8.48 (dd. J = 22.3, 8.4 Hz, 4H), 7.78 (d, J = 8.8 Hz, 1H), 7.65 (d, J = 8.9 Hz, 1H), 7.43 (d, J = 7.8 Hz, 1H), 7.34 (dt, J= 8.1, 1.0 Hz, 1H), 7.30 - 7.13 (m, 9H), 7.11 - 7.04 (m, 3H), 7.02- 6.95 (m, 2H), 5.12 (d, J = 18.2 Hz, 3H), 4.67 - 4.46 (m, 3H), 4.22 (q, J= 6.9 Hz, 1H), 4.04 - 3.86 (m, 4H), 3.67 (d, J= 6.4 Hz, 2H), 3.58 - 3.49 (m, 3H), 3.41 (dd, J= 10.5, 6.0Hz, 3H), 3.11 -2.80 (m, 10H), 2.74-2.66 (m, 1H), 2.62- 2.54 (m, 1H), 1.86 (d,J=2.9 Hz.6H), 1.69 (s, 1H), 1.29 (d, J= 37.2 Hz, 3H). 1.06 (dd.J=14.0, 6.4 Hz.7H), 0.83 (d, J = 8.2 Hz.2H).

Claims

[00238] CLAIMS1. A selectively soluble support compound derived from an amino triamide, the selectively soluble support compound represented by Formula (I):whereinRi is H or a straight or branched C1-C40 alkyl or aliphatic group;R2 is a straight or branched C6-C40 alkyl group;R3 and R4 are each independently selected from H and -O-alkyl; andY is selected from the group consisting of -OH, -COOH, -O-C(O)-X, wherein X is Cl, Br, I, F. imidazole or a leaving group, and an -NHR group, wherein R is H, a straight or branched C1-C30 alkyl group, or a C7-C30 aralkyl group, wherein the alkyl portion of the C7-C30 aralkyl group is a straight or branched C1-C30 alkyl.

2. The selectively soluble support compound of claim 1, wherein Y is -OH.

3. The selectively soluble support compound of claim 1, wherein Ri is a straight or branched C1-C40 alky l group and R2 is a straight or branched C1-C40 alkyd group.

4. The selectively soluble support compound of claim 1, wherein Ri is H and R2 is C18H37.

5. The selectively soluble support compound of claim 2, wherein Ri is methyl, R2 is CI8H37, and Y is -OH.

6. The selectively soluble support compound of claim 2, wherein Ri is C18H37, R2 is C18H 7, and Y is -OH.

7. The selectively soluble support compound of claim 1, wherein Y is -NHR.

8. The selectively soluble support compound of claim 7, wherein R is a C1-C30 alkyl.

9. The selectively soluble support compound of claim 7, wherein R is methyl.

10. The selectively soluble support compound of claim 7, wherein R is H.

11. The selectively soluble support compound of claim 7, wherein R is a C7-C30 aralky l group.

12. A liquid phase method of producing an oligonucleotide comprising a step of subjecting a 5'-hydroxyl group or 3'-hydroxyl group of a nucleoside or oligonucleotide having a selectively soluble support in at least one location selected from the group consisting of 2'-position, 3 '-position, 5'-position and a nucleobase moiety and having a 5'-hydroxyl group or a 3'-hydroxyl group, to H-phosphonation to convert the 5'-hydroxyl group or the 3'-hydroxyl group into an H-phosphonated form, wherein the selectively soluble support is a compound represented by Formula (I):whereinRi is H or a straight or branched C1-C40 alkyl or aliphatic group;R2 is a straight or branched C6-C40 alkyl group;R3 and R4 are each independently selected from H and -O-alkyk andY is selected from the group consisting of -OH, -COOH, -O-C(O)-X, wherein X is Cl, Br, I, F, imidazole or a leaving group, and an -NHR group, wherein R is H, a straight or branched C1-C30 alkyl group, or a C7-C30 aralkyl group, wherein the alkyl portion of the C7-C30 aralky l group is a straight or branched C1-C30 alkyl.

13. The liquid phase method of claim 12, further comprising at least one elongation reaction cycle comprising a step of subjecting a nucleoside or oligonucleotide having a selectively soluble support represented by Formula (I) in at least one location selected from the group consisting of 2'-position, 3'-position, 5'-position and a nucleobase moiety and having a 5'-hydroxyl group or a 3'-hydroxyl group, to H-phosphonation to convert the 5'-hydroxyl group or the 3'-hydroxyl group into an H-phosphonated form.

14. The liquid phase method of claim 13, wherein the elongation reaction cycle comprises: a first step of deprotecting a first nucleoside or first oligonucleotide having a selectively soluble support represented by Formula (I) in at least one location selected from the group consisting of 2'-position, 3'-position and a nucleobase moiety, having a 3'- hydroxyl group protected with a basic protecting group or a selectively soluble support represented by Formula (I), and having a 5'-hydroxyl group protected with a temporary’ protecting group, to remove the temporary protecting group to form a 5'-hydroxyl group. a second step of converting the resultant 5'-hydroxyl group into an H-phosphonated form using an H-phosphonate reagent, and a third step of forming an oligomer of the first nucleoside or first oligonucleotide with a second nucleoside or second oligonucleotide having a 3'-hydroxyl group and having a 5 '-hydroxyl group protected with a temporary protecting group, by forming a phosphite diester bond from the 5'-hydroxyl group, now converted to the H-phosphonated form, of the first nucleoside or first oligonucleotide and the 3 '-hydroxyl group of the second nucleoside or second oligonucleotide.

15. The liquid phase method according to claim 14, further comprising a fourth step of converting the phosphite diester bond of the oligomer into a phosphodiester bond, a thiophosphodiester bond, a boranophosphodiester bond, an aminophosphodiester bond, a phosphodiester bond protected with a basic protecting group, or a thiophosphodiester bond protected with a basic protecting group.

16. The liquid phase method according to claim 14, further comprising a fourth step of converting the phosphite diester bond of the oligomer into a phosphodiester bond, a thiophosphodiester bond, a boranophosphodiester bond or an aminophosphodiester bond.

17. The liquid phase method according to claim 15, further comprising a fifth step of adding a polar solvent to a reaction mixture obtained from at least one step selected from the group consisting of the first step to the fourth step to form a precipitate, and collecting the precipitate by solid liquid separation.

18. A liquid phase method of producing a peptide comprising the following steps;(1) obtaining a C-protected amino acid or C-protected peptide, comprising condensing a compound of Formula (I):whereinRi is H or a straight or branched C1-C40 alkyl or aliphatic group;R2 is a straight or branched C6-C40 alkyl group;R3 and R4 are each independently selected from H and -O-alkyl; andY is selected from the group consisting of -OH, -COOH, -O-C(O)-X, wherein X is Cl, Br, I, F. imidazole or a leaving group, and an -NHR group, wherein R is H, a straight or branched C1-C30 alkyl group, or a C7-C30 aralkyl group, wherein the alkyl portion of the C7-C30 aralkyl group is a straight or branched C1-C30 alkyl, with a C-terminal of N-protected amino acid or N-protected peptide;(2) removing a temporary protecting group of a N-terminal of the amino acid or peptide obtained in the above-mentioned step;(3) condensing the N-terminal of the amino acid or peptide obtained in the above-mentioned step with N-protected amino acid or N-protected peptide; and(4) precipitating the peptide obtained in the above-mentioned step.

19. The liquid phase method of claim 18 wherein Y is -OH.

20. The liquid phase method of claim 19 wherein Ri is C18H37.

21. The liquid phase method of claim 12, further comprising a step of cleaving the formed oligonucleotide from the compound of Formula (I).

22. The liquid phase method of claim 18, further comprising a step of cleaving the formed peptide from the compound of Formula (I).

23. The selectively soluble support compound of claim 1, wherein one of R3 and R4 is - O-Methyl.

24. The liquid phase method of claim 12, wherein one of R3 and R4 is -O-Methyl.

25. The liquid phase method of claim 18, wherein one of Rs and R4 is -O-Methyl.

26. The selectively soluble support compound of claim 1, wherein R3 and R4 are not the same.

27. The liquid phase method of claim 12, wherein R3 and R4 are not the same.

28. The liquid phase method of claim 18, wherein Rs and R4 are not the same.

29. The selectively soluble support compound of claim 1, wherein the selectively soluble support compound is represented by Formula (II):Ri is H or a straight or branched C1-C40 alkyl or aliphatic group;R2 is a straight or branched C6-C40 alkyl group; andY is selected from the group consisting of -OH, -COOH, -O-C(O)-X, wherein X is Cl, Br, I, F, imidazole or a leaving group, and an -NHR group, wherein R is H, a straight or branched C1-C30 alkyl group, or a C7-C30 aralkyl group, wherein the alkyl portion of the C7-C30 aralkyl group is a straight or branched C1-C30 alkyl.

30. The selectively soluble support compound of claim 1, wherein the selectively soluble support compound is represented by Formula (III):Ri is H or a straight or branched C1-C40 alkyl or aliphatic group;R2 is a straight or branched C6-C40 alkyl group; andY is selected from the group consisting of -OH, -COOH, -O-C(O)-X, wherein X is Cl, Br, I, F, imidazole or a leaving group, and an -NHR group, wherein R is H, a straight or branched C1-C30 alkyl group, or a C7-C30 aralkyl group, wherein the alkyl portion of the C7-C30 aralkyl group is a straight or branched C1-C30 alkyl.

31. The liquid phase method of claim 12, wherein the selectively soluble support is a compound represented by Formula (11):Ri is H or a straight or branched C1-C40 alkyl or aliphatic group:R2 is a straight or branched C6-C40 alkyl group; andY is selected from the group consisting of -OH, -COOH, -O-C(O)-X, wherein X is Cl, Br, I, F, imidazole or a leaving group, and an -NHR group, wherein R is H, a straight or branched C1-C30 alkyl group, or a C7-C30 aralkyl group, wherein the alkyl portion of the C7-C30 aralkyl group is a straight or branched C1-C30 alkyl.

32. The liquid phase method of claim 12, wherein the selectively soluble support is a compound represented by Formula (III):Ri is H or a straight or branched C1-C40 alkyl or aliphatic group;R2 is a straight or branched C6-C40 alkyl group; andY is selected from the group consisting of -OH, -COOH, -O-C(O)-X, wherein X is Cl, Br, I, F, imidazole or a leaving group, and an -NHR group, wherein R is H. a straight or branched C1-C30 alkyl group, or a C7-C30 aralkyl group, wherein the alkyl portion of the C7-C30 aralkyl group is a straight or branched C1-C30 alky l.

33. The liquid phase method of claim 18, wherein comprises condensing a compound of Formula (II):Ri is H or a straight or branched C1-C40 alkyl or aliphatic group;R2 is a straight or branched C6-C40 alkyl group; andY is selected from the group consisting of -OH, -COOH, -O-C(O)-X, wherein X is Cl, Br, I, F, imidazole or a leaving group, and an -NHR group, wherein R is H, a straight or branched C1-C30 alkyl group, or a C7-C30 aralkyl group, wherein the alkyl portion of the C7-C30 aralkyl group is a straight or branched C1-C30 alkyl.

34. The liquid phase method of claim 18, wherein comprises condensing a compound of Formula (III):Ri is H or a straight or branched C1-C40 alkyl or aliphatic group;R2 is a straight or branched C6-C40 alkyl group; andY is selected from the group consisting of -OH, -COOH, -O-C(O)-X, wherein X is Cl, Br, I, F. imidazole or a leaving group, and an -NHR group, wherein R is H, a straight or branched C1-C30 alkyl group, or a C7-C30 aralkyl group, wherein the alkyl portion of the C7-C30 aralkyl group is a straight or branched C1-C30 alkyl.

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