Universal solid supports and phosphoramidite building blocks for oligonucleotide synthesis

Abstract

Disclosed herein are the chemical preparation of oligonucleotides, chemical entities useful in such preparation, processes for such preparation, and uses related to the chemical preparation of oligonucleotides.Further disclosed herein are novel universal solid supports and phosphoramidite building blocks for synthesizing oligonucleotides on a solid phase, which achieve the removal of 3'-phosphate moieties during the process of oligonucleotide deprotection. JPEG2025528407000103.jpg2327

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Provisional Patent Application No. 63 / 373,619, filed August 26, 2022, which is incorporated herein by reference in its entirety.

[0002] References to a computer program reciting a "sequence listing," table, or attachment submitted on a compact disc This application contains a Sequence Listing that was submitted via EFS-Web and is incorporated herein by reference in its entirety. The XML copy, created on August 24, 2023, is named 09511l-1405629-000400PC.xml and is 6000 bytes in size.

[0003] Field The disclosure herein provides compounds, compositions and teachings related to the synthesis of oligonucleotides.For example, the disclosure provides non-nucleoside linker-based universal solid supports and phosphoramidite building blocks for synthesizing standard modified oligonucleotides, compositions comprising such non-nucleoside solid supports, phosphoramidite building blocks, and methods for using such supports and building blocks in the synthesis of modified oligonucleotides. [Background technology]

[0004] background Several innovations have been introduced in the field of oligonucleotide synthesis. Among these innovations are the development of superior orthogonal protecting groups, activators, reagents, and synthesis conditions. Numerous modifications and improvements have been made to the oligonucleotides themselves. Among these are chemistries that provide properties not present in naturally occurring oligonucleotides, such as low negative charge, hydrophobicity, the ability to emit fluorescence, and protein and receptor binding properties. These novel chemistries generally involve the modification of the non-nucleosidic building blocks that form the building blocks of oligonucleotides.

[0005] Oligonucleotides with a free 3'-hydroxy group, essential for enzymatic elongation, are among the most frequently used in the life sciences. Until the late 1990s, conventional synthesis of these oligonucleotides was almost exclusively performed on nucleoside-based solid supports, which contain a 3'-terminal nucleoside linked via a readily cleavable ester bond. An alternative approach uses universal solid supports, in which the 3'-terminal nucleoside residue is coupled to the support as a phosphoramidite building block in the first cycle of oligonucleotide synthesis. Assembly of the oligonucleotide chain is then continued until completion, and the support-bound material is deprotected. Importantly, the phosphate bridge formed between the universal linker and the 3'-terminal nucleoside can be cleaved in such a way that the phosphate bridge remains with the universal linker during the final deprotection process. The net result of deprotection is the formation of an oligonucleotide with a free, unprotected 3'-terminal hydroxy group identical to that prepared on the nucleoside-based solid support.

[0006] The deprotection process for synthetic oligonucleotides assembled on a universal solid support involves 1) removal of 2-cyanoethyl protecting groups from internucleoside phosphate or phosphorothioate residues; and 2) the removal of acetyl or benzoyl groups (C, respectively) for cytidine, as well as its 2'-deoxy, 2'-O-alkyl, and other analogs used in the art, in the standard, most robust protection scheme. ac and C bz ) and a benzoyl group (A ) for adenosine and its 2'-deoxy, 2'-O-alkyl, and other analogs used in the art. bz ) and an isobutyryl group (G) for guanosine, and its 2'-deoxy, 2'-O-alkyl, and other analogs used in the art. ib 1) deprotection of amino groups in nucleobases protected with 3) cleavage of nucleotides; 2) release of solid support-bound oligonucleotides in solution; and 3) 3'-dephosphorylation of 3'-hydroxy groups attached to universal linkers via phosphate or phosphorothioate linkages.

[0007] Among these processes, G ib The removal of the isobutyryl group from the residue and 3'-dephosphorylation are the most time-consuming and therefore control the length of deprotection time, and have been reported as the rate-limiting steps (Schwartz, ME, Breaker, RR, Asteriadis, GT, and Gough, GR 1995).

[0008] An analysis of the prior art reveals that all universal solid supports disclosed to date share one common drawback: 3'-dephosphorylation of oligonucleotides synthesized on said solid support remains the rate-limiting step in the process of deprotection of oligonucleotides. Summary of the Invention [Means for solving the problem]

[0009] Summary of the Invention One object of the present disclosure is to provide novel compounds that can serve as solid supports and phosphoramidite building blocks for preparing oligomeric compounds, natural oligonucleotide analogs, and chemically modified oligonucleotide analogs, wherein the non-nucleoside moiety, together with the phosphate moiety to which it is attached, is cleaved from the target oligonucleotide during the final deprotection process, thereby releasing a free 3'-hydroxy group in the oligonucleotide.

[0010] Disclosed herein are compounds of formula I [ka] (In the formula: R 1 and R 2 forms an orthoester functional group -C(CH3)(OCH3)- or R 1 and R 2 one of R is hydrogen, a trityl-type protecting group, or a xanthenyl-type protecting group; 1 and R 2 the other of which is acetyl, propionyl, n-butyryl, benzoyl, or L 1 and: L 1 is the linking moiety -C(=O)-Z-(C=O)-A 1 and: Z is selected from the group consisting of a covalent bond, a methylene group, —(CH)—, —(CH)—O—(CH)—, and —(CH)—O—CH—O—(CH)—; A 1 represents a hydroxy group, a salt of a hydroxy group with an inorganic cation or a tertiary amine, SP 1 Covalent bond to, or -NH(CH2) n -OR 7 and: SP 1is oxygen, amino, aminoalkyl, or hydroxyalkyl covalently attached to a solid phase material, including controlled pore glass, magnetic controlled pore glass, silica-containing particles, styrene-containing polymers or copolymers, divinylbenzene-containing polymers or copolymers, copolymers of styrene and divinylbenzene, controlled pore glass grafted with a styrene-containing polymer, controlled pore glass grafted with a copolymer of styrene and divinylbenzene, copolymers of styrene and divinylbenzene grafted with polyethylene glycol, or a flat glass surface; n is an integer from 2 to 10; R 7 is hydrogen or PA: [ka] R 8 is a methyl or 2-cyanoethyl group, and R 14 is alkyl, isoalkyl, sec-alkyl, or tert-alkyl; R 3 is selected from the group consisting of methyl, ethyl, propyl, isopropyl, and benzyl; R 4 is selected from the group consisting of a lone pair, hydrogen, methyl, ethyl, propyl, isopropyl, and benzyl; R 4 is other than a lone pair, N has a positive charge and forms a salt with a halide anion or with an intramolecular carboxyl functional group; R 5 and R 6 are independently hydrogen or methyl; Y is -(C=O)-, -CH(OR 9 )-, -CH(NR 10 R 11 )-, or -[C(OR 12 )(OR 13 )]-and: R 9is hydrogen, methyl, ethyl, benzyl, acetyl, propionyl, butyryl, isobutyryl, pivaloyl, benzoyl, t-butyldimethylsilyl, triisopropylsilyl, (t-butyl)diphenylsilyl, L 1 , PA, or -(CH2) m -O-PA: m is an integer ranging from 2 to 10; R 10 L 1 , -(C=O)-A 1 , or -(C=O)-W 1 -(CH2) p -W 2 and: W 1 is —(CH)—, —(NH)—, or —(NH)—(C═O)—; W 2 is hydroxy, amino, -O-PA, -(C=O)-A 1 , or -[NH(C=O)]-A 1 and; p is an integer from 2 to 10; R 11 is hydrogen, methyl, ethyl, or benzyl; R 12 and R 13 together form a ketal bridge -(CH2)2-, or -CH2-[C(CH3)2]-CH2-; R 1 , R 2 , and only one of Y is L 1 Or it may be PA, or L 1 or may contain PA).

[0011] Another object of the present disclosure is to provide a method for synthetically preparing said universal linker, a method for attaching it to a solid phase material, and a method for its use in oligonucleotide synthesis. Accordingly, a method for functionalizing a solid phase material with a first monomer subunit, comprising: (a) providing a solid phase material-binding compound of formula I [ka] (In the formula: R 1 and R 2 forms an orthoester functional group -C(CH3)(OCH3)- or R 1 and R 2 one of R is hydrogen, a trityl-type protecting group, or a xanthenyl-type protecting group; 1 and R 2 The other is acetyl, propionyl, n-butyryl, benzoyl, L 1 and: L 1 is the linking moiety -C(=O)-Z-(C=O)-A 1 and: Z is selected from the group consisting of a covalent bond, a methylene group, —(CH)—, —(CH)—O—(CH)—, and —(CH)—O—CH—O—(CH)—; A 1 SP 1 Covalent bond to -NH(CH2) n -OR 7 and: SP 1 is oxygen, amino, aminoalkyl, or hydroxyalkyl covalently attached to a solid phase material, including controlled pore glass, magnetic controlled pore glass, silica-containing particles, styrene-containing polymers or copolymers, divinylbenzene-containing polymers or copolymers, copolymers of styrene and divinylbenzene, controlled pore glass grafted with a styrene-containing polymer, controlled pore glass grafted with a copolymer of styrene and divinylbenzene, copolymers of styrene and divinylbenzene grafted with polyethylene glycol, and flat glass surfaces; n is an integer from 2 to 10; R 7 is PX: [ka] R 8 is a methyl or 2-cyanoethyl group, and X is oxygen or sulfur; R3 is selected from the group consisting of methyl, ethyl, propyl, isopropyl, and benzyl; R 4 is selected from the group consisting of a lone pair, hydrogen, methyl, ethyl, propyl, isopropyl, and benzyl; R 4 is other than a lone pair, N has a positive charge and forms a salt with a halide anion or with an intramolecular carboxyl functional group; R 5 and R 6 are independently hydrogen or methyl; Y is -(C=O)-, -CH(OR 9 )-, -CH(NR 10 R 11 )-, or -[C(OR 12 )(OR 13 )]-and: R 9 is hydrogen, methyl, ethyl, benzyl, acetyl, propionyl, butyryl, isobutyryl, pivaloyl, benzoyl, t-butyldimethylsilyl, triisopropylsilyl, or (t-butyl)diphenylsilyl group; L 1 , PX, or -(CH2) m -O-PX: m is an integer ranging from 2 to 10; R 10 L 1 , -(C=O)-A 1 , or -(C=O)-W 1 -(CH2) p -W 2 and: W 1 is -(CH2)-, -(NH)-, or -(NH)-(C=O)- W 2 is -O-PX or -[NH(C=O)]-A 1 and; p is an integer ranging from 2 to 10; R 11 is hydrogen, methyl, ethyl, or benzyl; R 12 and R 13together form a ketal bridge -(CH2)2-, or -CH2-[C(CH3)2]-CH2-; R 1 , R 2 , and only one of Y is L 1 Or it may be PX, or L 1 or may contain PX); (b) selectively removing one of the protecting groups of formula I to form a reactive hydroxyl group; (c) providing a first monomer subunit comprising an activated phosphorus group and a protected hydroxy group, and reacting the activated phosphorus group of the first monomer subunit with a reactive hydroxyl group of a compound of Formula I to form a monomer-functionalized solid support comprising a phosphite group; (d) treating the monomer-functionalized solid support with a capping agent and / or treating the monomer-functionalized solid support with an oxidizing solution or a sulfurizing agent to convert the phosphite triester groups to phosphotriesters or phosphothioate triesters, thereby forming an oxidized or sulfurized functionalized solid support; (e) optionally repeating steps (b), (c), and (d) one or more times on the oxidized or sulfurized functionalized solid support to form an oligomer-functionalized solid support, wherein the monomer subunits are the same or different for each repetition of steps (b), (c), and (d). Also disclosed herein are methods comprising: [Brief explanation of the drawings]

[0012] [Figure 1] FIG. 1 shows the synthetic scheme for preparing the starting material.

[0013] [Figure 2]FIG. 2 shows a synthetic scheme for preparing universal solid supports protected with 7-O-DMT-3-O-acyl or -3-O-silyl- and 7-O-TMT-3-O-acyl or -3-O-silyl, with a universal linker attached to the solid phase material via the 6-O-position.

[0014] [Figure 3] FIG. 3 shows a synthetic scheme for preparing 7-O-TMT-protected and 7-O-deprotected universal solid supports, in which the respective 3-O-alkylated universal linkers are attached to the solid phase material via the 6-O-position, followed by removal of the TMT group.

[0015] [Figure 4] Figure 4 shows a synthetic scheme for preparing a universal solid support in which a 7-O-TMT protected [3,2']spirodioxolane and a [3,2']spirodioxane universal linker are attached to the solid phase material via the 6-O-position.

[0016] [Figure 5] FIG. 5 shows a synthetic scheme for preparing a universal solid support in which a 7-O-DMT protected 6-O-acetylated universal linker and a 7-O-TMT protected 6-O-acetylated universal linker are attached to a solid phase material through the 3-N-position.

[0017] [Figure 6] FIG. 6 shows a synthetic scheme for preparing a universal solid support in which a 7-O-DMT protected 6-O-acetylated universal linker and a 7-O-TMT protected 6-O-acetylated universal linker are attached to a solid phase material through the 3-O-position.

[0018] [Figure 7]FIG. 7 shows synthetic schemes for preparing 7-O-DMT-protected universal phosphoramidites and 7-O-TMT-protected universal phosphoramidites in which the phosphoramidite moiety is attached to a universal linker via the 3-O- or 6-O-position of the linker.

[0019] [Figure 8] FIG. 8 shows a synthetic scheme for preparing 7-O-TMT protected universal phosphoramidites in which the phosphoramidite moiety is attached to a universal linker via the 3-N-position of the linker.

[0020] [Figure 9] FIG. 9 shows a synthetic scheme for preparing 6,7-orthoacetate protected universal phosphoramidites in which the phosphoramidite moiety is attached to a universal linker via the 3-O- or 3-N-position of the linker.

[0021] [Figure 10] FIG. 10 shows a DMT-protected solid support bearing tertiary amino groups.

[0022] [Figure 11] FIG. 11 shows a TMT-protected solid support bearing tertiary amino groups.

[0023] [Figure 12] FIG. 12 shows a DMT-protected universal solid support.

[0024] [Figure 13] FIG. 13 shows a TMT-protected universal solid support.

[0025] [Figure 14] FIG. 14 shows an unprotected universal solid support.

[0026] [Figure 15]FIG. 15 shows the reverse phase HPLC profile of oligonucleotide 40 synthesized on universal solid support 941c.

[0027] [Figure 16] FIG. 16 shows the time course of 3′-dephosphorylation of 40 by universal solid support 941 in 2.56 M MeNH 2 at 45° C.

[0028] [Figure 17] FIG. 17 shows a plot of the dependence of the pseudo-first order rate constant k for the dephosphorylation of compound 41 assembled on universal solid supports 941c and 943c on the concentration of aqueous methylamine at 45° C.

[0029] [Figure 18] FIG. 18 shows a plot of the time course of detransport of T2dGib 6T2 in 3.42 M aqueous methylamine at 40° C.

[0030] [Figure 19] FIG. 19 shows plots of the time required for dephosphorylation of oligonucleotide 40 synthesized on universal solid support 941c to 99% extent (t 99%) (solid line) and the time required for deprotection of N-isobutyrylguanosine bases in oligonucleotide 44 to 99% extent (t 99%) (dotted line) plotted against the concentration of aqueous methylamine at 45° C. DETAILED DESCRIPTION OF THE INVENTION

[0031] Detailed Description The present invention relates to 1) the synthesis of compounds for facilitating oligonucleotide synthesis, 2) the chemical preparation of oligonucleotides, 3) chemical entities useful in such preparation, 4) processes for such preparation, and 5) methods of use related to the chemical preparation of oligonucleotides. Specifically, the present invention provides novel non-nucleoside phosphoramidite building blocks and solid supports for incorporating a variety of useful ligands into natural oligonucleotides and their phosphorothioate analogs during solid-phase synthesis. More specifically, the present invention provides novel universal solid supports and phosphoramidite building blocks for synthesizing oligonucleotides on a solid phase, which achieve removal of the 3'-phosphate moiety during the oligonucleotide deprotection process. The phosphoramidite building blocks and solid supports of the present invention are highly efficient. These compounds are inexpensive to produce. They are stable in the solid state or in solution for extended periods of time. Their attachment to oligonucleotides does not create any novel chiral centers, thus not complicating the isolation of the ligand-modified oligonucleotides. The oligonucleotides do not undergo any undesired side reactions. All patents and publications cited in this specification are incorporated herein by reference in their entirety.

[0032] In order to improve the kinetics of 3'-dephosphorylation of oligonucleotides assembled on universal solid supports, new structural motifs of universal linkers that promote such 3'-dephosphorylation are needed.The present invention discloses a series of new universal linkers for oligonucleotide synthesis, solid supports and phosphoramidite building blocks derived from such linkers, and methods for their preparation.Compared with the kinetic data for the deprotection of N-isobutyryl-2'-deoxyguanosine residues in synthetic oligonucleotides, methods for synthesizing new structural motifs of universal linkers for 3'-dephosphorylation of oligonucleotides provided by the linkers are also disclosed, and deprotection protocols optimized for synthetic oligonucleotides are also disclosed.

[0033] Abbreviation Ac: acetyl; Bn: benzyl; Bz, bz: benzoyl; CDI: carbonyldiimidazole; DCM: dichloromethane; CPG: controlled pore glass; DIPEA: N-ethyl-N,N-diisopropylamine; DMF: N,N-dimethylformamide; DMT: bis(4-methoxyphenyl)phenylmethyl(4,4′-dimethoxytrityl); ES MS: mass spectrometry with electron spray ionization; Et: ethyl; Fmoc: (9-fluorenyl)methyloxycarbonyl; HPLC: high performance liquid chromatography; ib: isobutyryl; iPr: isopropyl; Me: methyl; MeCN: acetonitrile; MPPS: microporous polystyrene; NMI: N-methylimidazole; PTSA: p-toluenesulfonic acid; Py: pyridine; TBTU: [O-(benzotriazol-1-yl)-N,N,N',N'-tetramethyluronium tetrafluoroborate]; TEA: triethylamine; TCA: trichloroacetic acid; TFA: trifluoroacetic acid; TLC: thin layer chromatography; TMT: tris(4-methoxyphenyl)methyl (4,4′,4″-trimethoxytrityl); USS: Universal Solid Support.

[0034] Compounds of Formula I for Oligonucleotide Synthesis Disclosed herein are compounds of formula I [ka] (In the formula: R 1 and R 2 forms an orthoester functional group -C(CH3)(OCH3)- or R 1 and R 2 one of R is hydrogen, a trityl-type protecting group, or a xanthenyl-type protecting group; 1 and R 2 the other of which is acetyl, propionyl, n-butyryl, benzoyl, or L 1 and: L 1 is the linking moiety -C(=O)-Z-(C=O)-A 1 and: Z is selected from the group consisting of a covalent bond, a methylene group, —(CH)—, —(CH)—O—(CH)—, and —(CH)—O—CH—O—(CH)—; A 1 represents a hydroxy group, a salt of a hydroxy group with an inorganic cation or a tertiary amine, SP 1 Covalent bond to, or -NH(CH2) n -OR 7 and: SP 1is oxygen, amino, aminoalkyl, or hydroxyalkyl covalently attached to a solid phase material, including controlled pore glass, magnetic controlled pore glass, silica-containing particles, styrene-containing polymers or copolymers, divinylbenzene-containing polymers or copolymers, copolymers of styrene and divinylbenzene, controlled pore glass grafted with a styrene-containing polymer, controlled pore glass grafted with a copolymer of styrene and divinylbenzene, copolymers of styrene and divinylbenzene grafted with polyethylene glycol, or a flat glass surface; n is an integer from 2 to 10; R 7 is hydrogen or PA: [ka] R 8 is a methyl or 2-cyanoethyl group, and R 14 is alkyl, isoalkyl, sec-alkyl, or tert-alkyl; R 3 is selected from the group consisting of methyl, ethyl, propyl, isopropyl, and benzyl; R 4 is selected from the group consisting of a lone pair, hydrogen, methyl, ethyl, propyl, isopropyl, and benzyl; R 4 is other than a lone pair, N has a positive charge and forms a salt with a halide anion or with an intramolecular carboxyl functional group; R 5 and R 6 are independently hydrogen or methyl; Y is -(C=O)-, -CH(OR 9 )-, -CH(NR 10 R 11 )-, or -[C(OR 12 )(OR 13 )]-and: R 9is hydrogen, methyl, ethyl, benzyl, acetyl, propionyl, butyryl, isobutyryl, pivaloyl, benzoyl, t-butyldimethylsilyl, triisopropylsilyl, (t-butyl)diphenylsilyl, L 1 , PA, or -(CH2) m -O-PA: m is an integer ranging from 2 to 10; R 10 L 1 , -(C=O)-A 1 or -(C=O)-W 1 -(CH2) p -W 2 and: W 1 is —(CH)—, —(NH)—, or —(NH)—(C═O)—; W 2 is hydroxy, amino, -O-PA, -(C=O)-A 1 , or -[NH(C=O)]-A 1 and p is an integer from 2 to 10; R 11 is hydrogen, methyl, ethyl, or benzyl; R 12 and R 13 together form a ketal bridge -(CH2)2-, or -CH2-[C(CH3)2]-CH2-; R 1 , R 2 , and only one of Y is L 1 Or it may be PA, or L 1 or may contain PA).

[0035] In some examples of compounds of Formula I, R 1 and R 2 is hydroxy, tris-(4-methoxyphenyl)methyl, bis-(4-methoxyphenyl)phenylmethyl, 9-phenylxanthen-9-yl, or 9-(4-methoxyphenyl)xanthen-9-yl; and R 1 and R 2 The other one is L 1 R 1 and R2 One of them is L 1 In some cases, A 1 represents a hydroxy group, a salt of a hydroxy group with an inorganic cation or a tertiary amine, or SP 1 In some examples, Y is a covalent bond to -CH(OR 9 )- and R 9 is methyl, ethyl, benzyl, acetyl, propionyl, butyryl, isobutyryl, pivaloyl, benzoyl, t-butyldimethylsilyl, triisopropylsilyl, or phenyldimethylsilyl. In some examples, Y is -[C(OR 12 )(OR 13 )]-.

[0036] In some examples of compounds of Formula I, R 1 and R 2 is hydroxy, tris-(4-methoxyphenyl)methyl, or bis-(4-methoxyphenyl)phenylmethyl, and R 1 and R 2 and the other of Y is acetyl, propionyl, n-butyryl, or benzoyl. 9 )- and R 9 L 1 and A 1 is a hydroxy group optionally forming a salt with an inorganic cation or a tertiary amine, or SP 1 In another example, Y is —CH(NR 10 R 11 )- and R 11 is hydrogen, methyl, ethyl, or benzyl, and R 10 L 1 and A 1 represents a hydroxy group, a salt of a hydroxy group with an inorganic cation or a tertiary amine, or SP 1 In yet another example, Y is a covalent bond to —CH(NR 10 R 11 )- and R 11 is hydrogen, methyl, ethyl, or benzyl, and R 10-(C=O)-W 1 -(CH2) p -W 2 and W 2 is amino, -(C=O)-A 1 , or -[NH(C=O)]-A 1 and A 1 represents a hydroxy group, a salt of a hydroxy group with an inorganic cation or a tertiary amine, or SP 1 and optionally p is an integer from 3 to 10.

[0037] In some examples of compounds of Formula I, R 1 and R 2 one of R is tris-(4-methoxyphenyl)methyl or bis-(4-methoxyphenyl)phenylmethyl; 1 and R 2 The other one is L 1 In some cases, A 1 is -NH(CH2) n -OR 7 and Y is -CH(OR 9 ) In some cases, R 9 is methyl, ethyl, benzyl, acetyl, propionyl, butyryl, isobutyryl, pivaloyl, benzoyl, t-butyldimethylsilyl, triisopropylsilyl, or phenyldimethylsilyl.

[0038] In some examples of compounds of Formula I, R 1 and R 2 one of R is tris-(4-methoxyphenyl)methyl or bis-(4-methoxyphenyl)phenylmethyl; 1 and R 2 the other of which is acetyl, propionyl, n-butyryl, or benzoyl, and Y is -CH(OR 9 )-. In some cases, R 9 is PA. In some cases, R 9 Ha-(CH2) m -O-PA.

[0039] In some examples of compounds of Formula I, R 1 and R 2 one of R is tris-(4-methoxyphenyl)methyl or bis-(4-methoxyphenyl)phenylmethyl; 1 and R 2 the other is acetyl, propionyl, n-butyryl, or benzoyl, and Y is —CH(NR 10 R 11 )- and R 11 is hydrogen, methyl, ethyl, or benzyl. In some instances, R 10 -(C=O)-W 1 -(CH2) p -W 2 In some cases, W 1 is -(CH2)-, -(NH)-, or -(NH)-(C=O)-, and W 2 is hydroxy or O-PA.

[0040] In some examples of compounds of Formula I, R 1 and R 2 forms an orthoester functional group -C(CH3)(OCH3)-, and Y is -CH(OR 9 )- and R 9 is hydrogen, PA, or -(CH2) m -O-PA.

[0041] In some examples of compounds of Formula I, the trityl-type protecting group or the xanthenyl-type protecting group is selected from the group consisting of 4-methoxytrityl, 4,4′-dimethoxytrityl, 4,4′,4″-trimethoxytrityl, 9-phenylxanthen-9-yl, and 9-(4-methoxyphenyl)xanthen-9-yl.

[0042] In some examples, Z is selected from the group consisting of a covalent bond, a methylene group, —(CH)—, —(CH)—O—(CH)—, and —(CH)—O—C6H4—O—(CH)—, substituted or unsubstituted alkyl, substituted or unsubstituted alkene, substituted or unsubstituted alkenyl, substituted or unsubstituted cycloalkyl, or substituted or unsubstituted aryl.

[0043] In some examples of compounds of Formula I, R 1 and R 2 one of R is hydroxy, tris-(4-methoxyphenyl)methyl, bis-(4-methoxyphenyl)phenylmethyl, 9-phenylxanthen-9-yl, or 9-(4-methoxyphenyl)xanthen-9-yl; 1 and R 2 The other one is L 1 and;A 1 represents a hydroxy group, a salt of a hydroxy group with an inorganic cation or a tertiary amine, or SP 1 Y is a covalent bond to -CH(OR 9 )- and R 9 is methyl, ethyl, benzyl, acetyl, propionyl, butyryl, isobutyryl, pivaloyl, benzoyl, t-butyldimethylsilyl, triisopropylsilyl, or phenyldimethylsilyl. 1 and R 2 one of R is hydroxy, tris-(4-methoxyphenyl)methyl, bis-(4-methoxyphenyl)phenylmethyl, 9-phenylxanthen-9-yl, or 9-(4-methoxyphenyl)xanthen-9-yl; 1 and R 2 The other one is L 1 and;A 1 represents a hydroxy group, a salt of a hydroxy group with an inorganic cation or a tertiary amine, or SP 1 Y is a covalent bond to -[C(OR 12 )(OR 13 )]-.

[0044] In some examples of compounds of Formula I, R 1 and R 2 one of R is hydroxy, tris-(4-methoxyphenyl)methyl, bis-(4-methoxyphenyl)phenylmethyl, 9-phenylxanthen-9-yl, or 9-(4-methoxyphenyl)xanthen-9-yl; 1 and R 2The other one is L 1 and;A 1 represents a hydroxy group, a salt of a hydroxy group with an inorganic cation or a tertiary amine, or SP 1 Y is a covalent bond to -CH(NR 10 R 11 )- and R 10 -(C=O)-W 1 -(CH2) p -W 2 and W 1 is —(CH)—, —(NH)—, or —(NH)—(C═O)—; W 2 is hydroxy, amino, or -O-PA, and p is an integer from 3 to 10; R 11 is hydrogen, methyl, ethyl, or benzyl. 1 and R 2 one of R is hydroxy, tris-(4-methoxyphenyl)methyl, bis-(4-methoxyphenyl)phenylmethyl, 9-phenylxanthen-9-yl, or 9-(4-methoxyphenyl)xanthen-9-yl; 1 and R 2 The other one is L 1 and;A 1 represents a hydroxy group, a salt of a hydroxy group with an inorganic cation or a tertiary amine, or SP 1 and Y is —(C═O)—.

[0045] In some examples of compounds of Formula I, R 1 and R 2 one of R is hydroxy, tris-(4-methoxyphenyl)methyl, or bis-(4-methoxyphenyl)phenylmethyl; 1 and R 2 the other of which is acetyl, propionyl, n-butyryl, or benzoyl; Y is —CH(OR 9 )- and R 9 L 1 and A 1 is a hydroxy group optionally forming a salt with an inorganic cation or a tertiary amine, or SP 1In another example, R 1 and R 2 is hydroxy, tris-(4-methoxyphenyl)methyl, or bis-(4-methoxyphenyl)phenylmethyl, and R 1 and R 2 the other of which is acetyl, propionyl, n-butyryl, or benzoyl, and Y is —CH(NR 10 R 11 )- and R 11 is hydrogen, methyl, ethyl, or benzyl, and R 10 L 1 and A 1 represents a hydroxy group, a salt of a hydroxy group with an inorganic cation or a tertiary amine, or SP 1 In yet another example, R 1 and R 2 is hydroxy, tris-(4-methoxyphenyl)methyl, or bis-(4-methoxyphenyl)phenylmethyl, and R 1 and R 2 the other is acetyl, propionyl, n-butyryl, or benzoyl, and Y is —CH(NR 10 R 11 )- and R 10 -(C=O)-W 1 -(CH2) p -W 2 and W 1 is -(CH2)-, -(NH)-, or -(NH)-(C=O), and W 2 is hydroxy, amino, -O-PA, -(C=O)-A 1 , or -[NH(C=O)]-A 1 p is an integer from 3 to 10; R 11 is hydrogen, methyl, ethyl, or benzyl. 10 -(C=O)-W 1 -(CH2) p -W 2 and W 2 But -(C=O)-A 1 or -[NH(C=O)]-A 1In the example, A 1 SP 1 is a covalent bond to

[0046] In some embodiments, compounds of Formula I include compounds of Table 1: [Table 1-1] [Table 1-2] [Table 1-3] [Table 1-4] [Table 1-5] [Table 1-6] [Table 1-7] [Table 1-8] [Table 1-9] Methods for Oligonucleotide Synthesis

[0047] 1. A method for functionalizing a solid phase material with a first monomer subunit, comprising: (a) providing a solid phase material-binding compound of formula I [ka] (In the formula: R 1 and R 2 forms an orthoester functional group -C(CH3)(OCH3)- or R 1 and R 2one of R is hydrogen, a trityl-type protecting group, or a xanthenyl-type protecting group; 1 and R 2 The other is acetyl, propionyl, n-butyryl, benzoyl, L 1 and: L 1 is the linking moiety -C(=O)-Z-(C=O)-A 1 and: Z is selected from the group consisting of a covalent bond, a methylene group, —(CH)—, —(CH)—O—(CH)—, and —(CH)—O—CH—O—(CH)—; A 1 SP 1 Covalent bond to -NH(CH2) n -OR 7 and: SP 1 is oxygen, amino, aminoalkyl, or hydroxyalkyl covalently attached to a solid phase material, including controlled pore glass, magnetic controlled pore glass, silica-containing particles, styrene-containing polymers or copolymers, divinylbenzene-containing polymers or copolymers, copolymers of styrene and divinylbenzene, controlled pore glass grafted with a styrene-containing polymer, controlled pore glass grafted with a copolymer of styrene and divinylbenzene, copolymers of styrene and divinylbenzene grafted with polyethylene glycol, and flat glass surfaces; n is an integer from 2 to 10; R 7 is PX: [ka] R 8 is a methyl or 2-cyanoethyl group, X is oxygen or sulfur; R 3 is selected from the group consisting of methyl, ethyl, propyl, isopropyl, and benzyl; R 4 is selected from the group consisting of a lone pair, hydrogen, methyl, ethyl, propyl, isopropyl, and benzyl; R 4is other than a lone pair, N has a positive charge and forms a salt with a halide anion or with an intramolecular carboxyl functional group; R 5 and R 6 are independently hydrogen or methyl; Y is -(C=O)-, -CH(OR 9 )-, -CH(NR 10 R 11 )-, or -[C(OR 12 )(OR 13 )]-and: R 9 is hydrogen, methyl, ethyl, benzyl, acetyl, propionyl, butyryl, isobutyryl, pivaloyl, benzoyl, t-butyldimethylsilyl, triisopropylsilyl, or (t-butyl)diphenylsilyl group; L 1 , PX, or -(CH2) m -O-PX: m is an integer ranging from 2 to 10; R 10 L 1 , -(C=O)-A 1 or -(C=O)-W 1 -(CH2) p -W 2 and: W 1 is —(CH)—, —(NH)—, or —(NH)—(C═O)—; W 2 is -O-PX or -[NH(C=O)]-A 1 and; p is an integer ranging from 2 to 10; R 11 is hydrogen, methyl, ethyl, or benzyl; R 12 and R 13 together form a ketal bridge -(CH2)2-, or -CH2-[C(CH3)2]-CH2-; R 1 , R 2 , and only one of Y is L 1 Or it may be PX, or L 1or may contain PX); (b) selectively removing one of the protecting groups of formula I to form a reactive hydroxyl group; (c) providing a first monomer subunit comprising an activated phosphorus group and a protected hydroxy group, and reacting the activated phosphorus group of the first monomer subunit with a reactive hydroxyl group of a compound of Formula I to form a monomer-functionalized solid support comprising a phosphite group; (d) treating the monomer-functionalized solid support with a capping agent and / or treating the monomer-functionalized solid support with an oxidizing solution or a sulfurizing agent to convert the phosphite triester groups to phosphotriesters or phosphothioate triesters, thereby forming an oxidized or sulfurized functionalized solid support; (e) optionally repeating steps (b), (c), and (d) one or more times on the oxidized or sulfurized functionalized solid support to form an oligomer-functionalized solid support, wherein the monomer subunits are the same or different for each repetition of steps (b), (c), and (d). Also disclosed herein are methods comprising:

[0048] In some embodiments, the method further comprises deprotecting the oligomer-functionalized solid support of step (e) and cleaving the oligomer-functionalized solid support to form an oligomeric compound separated from the solid phase material, wherein cleavage forms a terminal hydroxy group on the oligomeric compound at the site of cleavage.

[0049] In some embodiments of the method, R 1 or R 2 One of them is L1.

[0050] In some embodiments of the method, R 7 is PX.

[0051] In some embodiments of the method, the activated phosphorus group comprises a phosphoramidite, an H-phosphonate, or a phosphate triester.

[0052] In some embodiments of the method, the oligomeric compound is an oligonucleotide, optionally comprising a non-natural sugar-modified nucleotide residue, a non-natural base-modified nucleotide residue, or a non-nucleotide monomer unit.

[0053] The solid phase binding compound of Formula I can be any of the compounds described herein that bind to a solid phase material. For example, the solid phase binding compound of Formula I can be any of the compounds described in Table 1 or throughout the Examples. For example, the solid phase binding compound of Formula I can be 701-707c / p, 711-713c / p, 721-728c / p, 731-738c / p, 741-748c / p, 751-757c / p, or 761-767c / p. For example, the solid phase material binding compound of Formula I can be 801-807c / p, 811-819c / p, 821-828c / p, 831-838c / p, 841-848c / p, 851-759c / p, 861-869c / p, 871c / p, 874c / p, or 875c / p. For example, the solid phase material binding compound of Formula I can be 901-905c / p, 911-919c / p, 925-928c / p, 931-936c / p, 941-948c / p, 958c / p, 961-969c / p, 971c / p, 974c / p, or 975c / p.

[0054] The step of deprotecting the hydroxyl group of Formula I by selectively removing one of the protecting groups can be achieved by any method known to those skilled in the art, depending on which protecting group is to be selectively removed. For example, a trityl-type protecting group can be removed by treatment with 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, or 95% trifluoroacetic acid. Alternatively, a trityl-type protecting group can be removed using a 5% anhydrous solution of trichloroacetic acid or trifluoroacetic acid in toluene to obtain an unprotected solid support. For example, xanthenyl-type protecting groups can be removed via treatment with 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, or 95% trifluoroacetic acid. Additional methods for selectively removing protecting groups are described in the examples of the present disclosure and are known to those of skill in the art.

[0055] Each monomer subunit containing an activated phosphorus group and a protected hydroxy group may be reacted with a compound of Formula I by methods known in the art, including, but not limited to, those described in the specific examples disclosed herein. In some examples, each monomer subunit is a nucleotide or a derivative thereof. The reactive phosphorus group on the monomer subunit may include, but is not limited to, a phosphoramidite, an H-phosphonate, or a phosphate triester. The protected hydroxy group may include any protecting group known in the art. In some examples, the protected hydroxy group includes a trityl-type protecting group, such as, but not limited to, 4-methoxytrityl, 4,4'-dimethoxytrityl, or 4,4',4"-trimethoxytrityl. In other examples, the protected hydroxy group includes a xanthenyl-type protecting group, such as, but not limited to, 9-phenylxanthen-9-yl or 9-(4-methoxyphenyl)xanthen-9-yl. A monomer subunit containing an activated phosphorus group and a protected hydroxy group is reacted with a reactive hydroxyl group of a compound of Formula I to form a monomer-functionalized solid support containing a phosphite group.

[0056] Treating the monomer-functionalized solid support with a capping agent can be accomplished by methods known in the art, including, but not limited to, those described in the specific examples disclosed herein. In some examples, the capping agent is an organic ligand, organic amine, thiol, or pyridine-based capping agent. Treatment with the capping agent produces a 5'-capped oligonucleotide bound to the universal solid support. Treating the monomer-functionalized solid support with an oxidizing solution or sulfurizing agent can be accomplished by methods known in the art, including, but not limited to, those described in the specific examples disclosed herein. In some examples, the oxidizing solution consists of THF:pyridine:water. Treatment with the oxidizing solution converts the phosphite triester group to a phosphotriester. In some examples, the sulfurizing agent is DDTT (3-[[(dimethylamino)methylene]amino]-3H-1,2,4-dithiazole-5-thione). Treatment with the sulfurizing agent converts the phosphite triester group to a phosphothioate triester.

[0057] If desired, steps (b), (c), and (c) may be repeated to combine multiple monomer subunits to form an oligomer-functionalized solid support. When multiple monomer subunits react to form an oligomer, the monomer units may be the same or different, depending on the desired oligomer structure.

[0058] If the method further comprises deprotecting the oligomer-functionalized solid support in step (e) and cleaving the oligomer-functionalized solid support to form an oligomeric compound separated from the solid phase material, the cleavage forms a terminal hydroxy group on the oligomeric compound at the cleavage site. The step of deprotecting the oligomer-functionalized solid support can be accomplished by any method known to those of skill in the art, depending on which protecting groups are to be selectively removed. For example, certain methods for deprotecting the oligomer-functionalized solid support are described in the Examples disclosed herein. The step of cleaving to form an oligomeric compound separated from the solid phase material can also be accomplished by any method known to those of skill in the art, including, but not limited to, the methods described in the specific Examples disclosed herein.

[0059] The following examples disclose methods for synthesizing certain compounds of formula (I). Those skilled in the art will understand that these compounds, and others falling within the general scope of formula I, can be made by methods other than those specifically described herein, by adapting the methods described herein, and / or by adapting methods known in the art. In general, the compounds provided herein can be prepared in a multi-step synthesis as shown below. All amounts shown are approximate and are provided solely for illustrative purposes. [Example]

[0060] The following examples are intended to further illustrate certain preferred embodiments of the present invention and are not intended to be limiting in nature. Example 1 Preparation of Preferred Linkers for Universal Solid Supports Synthesis of starting materials (Figure 1)

[0061] Compounds 1-3 were prepared as described in Villacampa, M; Martinez, M.; Gonzalez-Trigo, G.; Sollhuber, M. M. Synthesis and stereochemistry of (3α)-6β,7β[3-dihydroxy- and 6β-hydroxy-8-alkyl-8-azabicyclo[3.2.1]octane-3-spiro-5′-imidazoline-2′,4′-diones. J. Heterocycl. Chem. 1992, 29(6), 1541-4. Compound 4 was prepared as described in Buchi, G.; Fliri, H.; Shapiro, R. Synthesis of betalains. J. Org. Chem. 1978, 43, 25, 4765-9. Compounds 7 and 8 were prepared as disclosed in Riche, F.; Masri, F.; Lopez, M. Diastereoselective synthesis of polyfunctionalized piperidines as precursors of dopamine transporter imaging agents. Tetrahedron Lett. 2007, 48, 9, 1609-1612, Paparin, J.-L.; Crevisy, C.; Toupet, L.; Gree, R. Synthesis and functionalization of new tropanes designed for use as scaffolds in combinatorial chemistry. Eur. J Org. Chem. 2000, (23), 3909-3918, and U.S. Pat. No. 4,001,249, respectively.

[0062] Conversion of 1-4, 7, and 8 to the orthoesters 11-14, 17, 18, and 26-28 was carried out as disclosed in Buchi, G.; Fliri, H.; Shapiro, R. Synthesis of betalains. J. Org. Chem. 1978, 43, 25, 4765-9.

[0063] Compounds 216, 218, and 221-224 were prepared as described in Affolter, O; Baro, A.; Laschat, S.; Fischer, P. Acylation of tropane alkaloids displaying reversed diastereoselectivities under enzymatic versus chemical conditions. Z. Naturforsch., B: Chemical Sciences 2007, 62(1), 82-92.

[0064] Compounds 25, 351, 353, and 451 were prepared from 11, 131, 133, and 231, respectively, by the action of hydroxylamine hydrochloride in pyridine followed by reduction with LiAlH4 as disclosed in Lewin, A.H.; Sun, G.; Fudala, L.; Navarro, H.; Zhou, L.-M.; Popick, P.; Faynsteyn, A.; Skolnick, P. Molecular Features Associated with Polyamine Modulation of NMDA Receptors. J. Medicinal Chem. 1998, 41(6), 988-995.

[0065] Compound 452 was prepared by reductive amination as disclosed in WO2009109477 or Baxter, Ellen W.; Reitz, Allen B. Reductive aminations of carbonyl compounds with borohydride and borane reducing agents. Organic Reactions (Hoboken, NJ, United States) 2002, p. 59. Synthesis of unprotected universal solid supports 911, 912, and 913

[0066] In a preferred embodiment, as shown in Figure 2, compounds 31, 33, and 34 were acylated at the free hydroxyl group with the corresponding carboxylic acid anhydride in pyridine under standard conditions. The resulting orthoesters 201, 203, and 205 were subjected to mild acidic hydrolysis using catalytic amounts of TFA and a threefold excess of water. Hydrolysis under the optimal conditions disclosed in Example 35 selectively formed the corresponding monoformates, which, upon removal of excess water by filtration through a drying agent, were treated with DMT or TMT chloride, followed by hydrolytic removal of the formate protecting group catalyzed by excess TEA, to give DMT-protected compounds 301, 303, and 305 and their TMT-protected counterparts 401, 403, and 405. The latter were succinylated with a threefold excess of succinic anhydride under standard conditions. The resulting hemisuccinates 501, 503, and 505, as well as 601, 603, and 605, were coupled to aminopropyl CPG and aminomethyl MPPS with various pore sizes. The loadings of these USSs and all other USSs in this application were consistent with the pore size and specific surface area of ​​the respective solid support materials (30-35, 40-50, 70-80, and 320-340 μmol / g for CPG1500, CPG1000, CPG500, and MPPS, respectively). Finally, the bridging tertiary amino groups were quaternized using excess methyl iodide and DIPEA to yield new DMT- and TMT-protected versions of universal solid supports 711, 712, and 713, as well as 811, 812, and 813, respectively.

[0067] Solid supports protected with trityl-type protecting groups are commonly used in oligonucleotide synthesis, and the protecting groups are removed under acidic conditions at the start of synthesis. However, detritylated universal solid supports can be attached to the instrument, allowing end users to skip the initial detritylation step, which is more convenient for the end user due to the savings in time, reagents, and solvents. Therefore, universal solid supports 811, 812, and 813 were detritylated with an anhydrous solution of 5% trichloro- or trifluoroacetic acid in toluene to obtain unprotected universal solid supports 911, 912, and 913. Synthesis of universal solid supports 817 and 819

[0068] In another preferred embodiment, 3-O-silyl-protected triols 216 and 218 were protected at one of the available hydroxy groups by reaction with TMT chloride (Figure 2). The steric bulk of the initially introduced TMT group substantially reduces the reactivity of the other hydroxy group. Thus, mono-TMT-protected species 416 and 418 were obtained in 69–72% yield and isolated by silica gel column chromatography. Subsequent succinylation afforded hemisuccinates 616 and 618, which were coupled to aminopropyl CPG and aminomethyl MPPS, followed by quaternization with methyl iodide to afford 3'-O-silyl-protected USSs 817 and 819. These were detritylated under standard conditions with an anhydrous solution of trichloro- or trifluoroacetic acid in toluene to afford universal solid supports 817 and 819. Synthesis of unprotected universal solid supports 971, 974, and 975

[0069] In another preferred embodiment, as shown in Figure 3, orthoacetate-protected compounds 21 and 24 were simultaneously alkylated at the amino and 3-hydroxy positions with methyl iodide or benzyl bromide to give quaternary salts 111, 114, and 115. Acidic hydrolysis of the orthoacetate gave the monoacetate, which was protected at the 7-hydroxy position with a TMT group without further purification to give 211, 214, and 215. In the next step, the acetyl group was removed with methanolic sodium methoxide, followed by purification by silica gel column chromatography to give 411, 414, and 415. Following the standard procedure described above, 411, 414, and 415 were reacted with succinic anhydride. The resulting hemisuccinates 671, 674, and 675 were coupled to aminopropyl CPG and aminomethyl MPPS to give TMT-protected USSs 871, 874, and 875. Finally, the TMT protection was removed by treatment with 5% trichloroacetic acid in toluene to give unprotected universal solid supports 971, 974, and 975. Synthesis of universal solid supports 921-924

[0070] In yet another preferred embodiment, readily available [3,2']spirodioxolane and [3,2']spirodioxane compounds 221–224, characterized by ketal protection of the carbonyl group in 6,7-dihydroxy-8-alkyltropan-3-one, were monoprotected at the 7-O-position with a TMT group (Figure 4). In the next step, the resulting 421–424 were succinylated at the 6-O-position to give compounds 621–624. Hemisuccinates 621–624 were coupled to aminopropyl CPG and aminomethyl MPPS to give compounds 821–824. The latter were quaternized to 821–824, which were then detritylated to give the [3,2']spirodioxolane and [3,2']spirodioxane universal solid supports 921–924. The spiroketal protection on all four supports was found to be stable to the acidic conditions of anhydrous detritylation. Use of 6-O-DMT- and 6-O-TMT-3-amino derivatives 351, 353, 451, and 452 in Figure 1

[0071] In one implementation of this preferred embodiment, compounds 351, 353, and 452 were selectively succinylated to form succinamic acid inner salts 551, 553, and 652 (Figure 5). These were coupled to aminopropyl- and hydroxypropyl-CPG, and aminomethyl- and hydroxymethyl-MPPS to give solid supports 751, 753, 852, and 859. The latter were converted to DMT-protected quaternary ammonium universal solid supports 761 and 763, and TMT-protected 862 and 869. Finally, 862 and 869 were detritylated to 962 and 969.

[0072] In another implementation of a preferred embodiment, the amino functional group of 451 was activated using carbonyldiimidazole, followed by reaction with 1,6-diaminohexane to attach a side chain to the amino functional group, forming compound 657. For column purification, 657 was activated with carbonyldiimidazole, and the activated species was loaded onto amino- and hydroxy-functionalized CPG and amino- and hydroxy-functionalized MPPS to form urea- and carbamate-immobilized universal solid supports 857 and 858, respectively. 8-N-Methylation was carried out as disclosed above to give 867 and 868, which, after detritylation, gave unprotected universal solid supports 967 and 968.

[0073] In yet another implementation of a preferred embodiment, TMT-protected compound 452 was acylated at the amino group with N-oxysuccinimidyl ester of 6-(trifluoroacetamido)hexanoic acid, followed by removal of the trifluoroacetyl protecting group with methanolic ammonia. Upon column purification, product 655 was activated with carbonyldiimidazole as previously disclosed, and the activated species was loaded onto amino- and hydroxy-functionalized CPG and amino- and hydroxy-functionalized MPPS to form urea- and carbamate-immobilized universal solid supports 855 and 856, respectively. These were 8-N-methylated to give universal solid supports 865 and 866, respectively, which were detritylated to give 965 and 966.

[0074] With the exception of 969, all linkers in this embodiment of the universal solid support are attached to the solid phase material via chemically stable spacers that are resistant to deprotection conditions using ammonia or methylamine. Therefore, only the 3'-dephosphorylated oligonucleotide product is expected to be released into solution during deprotection. Synthesis of universal solid supports 941-944, 947, and 948

[0075] In the most preferred embodiment, the universal linker is attached to the solid phase using the 3-O-position of the tropane system, as shown in Figure 6. Compounds 11-14, 17, and 18, protected at their vicinal hydroxy groups with orthoacetate moieties, are the most easily synthesized of all starting materials in this application. The hydroxy groups released upon mild acid-catalyzed hydrolysis of the orthoacetate protection with aqueous trifluoroacetic acid (TFA) were protected by reaction with DMT chloride to give compounds 131-134, 137, and 138, or by reaction with TMT chloride to give compounds 231-234, 237, and 238. The carbonyl functional groups in all test compounds were resistant to conventional reduction with NaBH4, but the addition of catalytic amounts of CeCl3 led to successful reduction, affording alcohols 331–334, 337, and 338, as well as 431–434, 437, and 438, without any loss of acetyl groups. In the next step, standard succinylation afforded 531–534, 537, and 538, as well as 631–634, 637, and 638. Similarly, treatment of 331 with diglycol anhydride afforded 535. Compound 636 was obtained from 431 and 1,4-hydroquinone diacetate activated with disuccinimidyl carbonate. All resulting linkers were coupled to aminopropyl CPG and aminomethyl MPPS according to standard protocols. The loading of all universal solid supports was commensurate with the pore size and specific surface area of ​​each solid support material. Subsequent methylation with iodomethane / DIPEA gave DMT-protected USSs 741–744, 747, and 748, and TMT-protected universal solid supports 841–844, 847, and 848. The TMT-protected universal solid supports were further detritylated to give 941–944, 947, and 948. Example 2 Providing universal phosphoramidites for oligonucleotide synthesis

[0076] Such phosphoramidites are coupled to underivatized solid supports bearing aminoalkyl or hydroxyalkyl groups, thereby converting the support into a universal solid support. This technique is advantageous for small-scale oligonucleotide synthesis, including but not limited to, the creation of oligonucleotide microarrays on glass slides or other supports. Synthesis of universal phosphoramidite 806a

[0077] In a preferred embodiment, hemisuccinate 406 was activated with N,N-disuccinimidyl carbonate to form the reactive N-oxysuccinimidyl ester, which was then treated with 6-aminohexanol to give compound 606a (Figure 7), which was methylated at the 8-amino position with iodomethane in the presence of DIPEA and finally converted to universal phosphoramidite 806a in the standard manner using 2-cyanoethylbis(N,N-diisopropylamino)phosphite activated with 1H-tetrazole. Synthesis of Universal Phosphoramidites 772-773 and 872-873

[0078] In another embodiment, compounds 331 and 431 disclosed above were methylated using iodomethane / DIPEA (Figure 7). The products were converted to DMT- and TMT-protected universal phosphoramidites 772 and 872.

[0079] Alternatively, 331 and 431 were methylated as disclosed above. In the next step, a side chain was attached to the 3-O-position by activating the 3-hydroxy function with 1,1-carbonyldiimidazole, followed by reaction with either 3-aminopropanol or 6-aminohexanol to form the carbamate derivative. After column purification, these gave the corresponding phosphoramidite 773 (R 1 =DMT, n=1) and converted to 873 (R 1 =DMT, n=4) was obtained. Synthesis of universal phosphoramidite 877

[0080] In yet another embodiment, compound 451 was first protected at its primary amino function with Fmoc-Cl in the presence of DIPEA (Figure 8). Without further purification, the product was acetylated at the 6-O-position with acetic anhydride in pyridine, and the Fmoc protecting group was selectively removed by treatment with diethylamine. The product was reacted with 1,1-carbonyldiimidazole, followed by the addition of 6-aminohexanol to form urea derivative 874. This was methylated with iodomethane in the presence of DIPEA, and the formed quaternary ammonium salt was converted to phosphoramidite 875 using standard protocols for this reaction. In a similar manner, compound 452 was acetylated at the 6-O-position by temporarily protecting the secondary amino function with Fmoc, followed by removal of the Fmoc protection to expose the amino function. In the next step, the secondary amino function was acylated with 9-hydroxynonanoic acid (pre-activated to its N-oxysuccinimidyl ester with N,N-disuccinimidyl carbonate) to give the extended alcohol 876. The remaining two steps of methylation and conversion to a phosphoramidite were carried out in the standard manner to give compound 877. Synthesis of universal phosphoramidites 881 and 883

[0081] In the most preferred embodiment, a universal phosphoramidite was generated in which the vicinal hydroxyl group was protected with an orthoacetate functional group (Figure 9). Therefore, the readily available compound 21 was methylated as described above and converted to phosphoramidite 278 in a very simple and rapid procedure. Those skilled in the art will appreciate that in certain cases, the distance between the surface of the solid support and the linker is required for specific applications. This requirement is met by the generation of phosphoramidites 881 and 883. Thus, compound 25 was acylated with N-oxysuccinimidyl 6-hydroxybutyrate to give the extended compound 880, which was then methylated and reacted with 2-cyanoethylbis(N,N-diisopropylamino)phosphite in the presence of 1H-tetrazole to give universal phosphoramidite 881. As shown in Figure 9, phosphoramidite 883 was synthesized in the same manner in three steps starting from orthoester-protected amine 25, except that the extension arm was introduced via a urea functionality. Example 3 Method for deprotecting oligonucleotides assembled on a universal solid support and simultaneously 3'-dephosphorylating said oligonucleotides

[0082] Here, several 2-deoxyoligonucleotides, their 2'-O-Me analogs, and their phosphorothioate analogs were synthesized on several universal solid supports of this disclosure, among which the properties of universal solid support 941c were studied in the most detail. Test activator

[0083] Three different activators have been tested and found to be equally effective with a typical coupling time of 20 seconds on the 1 μmol scale: 1) 1M 4,5-dicyanoimidazole + 0.1M N-methylimidazole in acetonitrile; 2) 0.1 M 5-[3,5-bis(trifluoromethyl)phenyl]-2H-tetrazole (Activator 42®) in acetonitrile, and 3) 0.45M 5-(benzylthio)-1H-tetrazole in acetonitrile. Oxidizing solutions with different compositions of I2, pyridine, and water were tested: 1) 0.02M I2 in 89:10:1 THF:water:pyridine; 2) 0.02M I2 in 88:10:2 THF:pyridine:water, and 3) 0.05M I2 in 88:10:2 THF:pyridine:water. An oxidizing solution with the composition 0.05M I2 in 88:10:2 THF:pyridine:water was determined to be the most efficient. sulfuration reaction

[0084] The sulfuration reaction was carried out with 0.1 M DDTT (3-[[(dimethylamino)methylene]amino]-3H-1,2,4-dithiazole-5-thione) in anhydrous pyridine for 10 min, followed by a capping step. Deprotection and cleavage of oligonucleotides

[0085] After synthesis, all oligonucleotides were treated with 30% aqueous diethylamine:acetonitrile (1:4) to remove the cyanoethyl protecting groups.

[0086] Cleavage of the oligonucleotides from the solid support and removal of the base-protecting groups was first carried out using concentrated aqueous ammonia at 60°C for 8 h and a 1:1 mixture of 40% aqueous methylamine and concentrated aqueous ammonia at 60°C for 2 h. Synthesis and 3'-dephosphorylation kinetics of oligonucleotide 40

[0087] The crude oligonucleotides were analyzed by RP and IE HPLC, revealing that the yield and purity of the oligonucleotides synthesized on universal solid support 941c were equal to or higher than those synthesized on universal solid support L. The RP HPLC profile of the crude reaction mixture of oligonucleotide 40, reproduced below, assembled on universal solid support 941c is shown in Figure 15 (and Table 2). 40:5'-DMT-d(TGT GAG TAC CAC TGA TTA)

[0088] Next, a model oligonucleotide 5'-T was assembled on a universal solid support 941. 10 The kinetics of the 3'-dephosphorylation of 5'-3'(41) was studied under pseudo-first-order conditions at 25.0, 35.0, and 45.0°C in aqueous methylamine solutions of various concentrations. Subsequently, the disappearance of the starting material and the release of the oligonucleotide from the solid phase with the linker still attached at the 3'-terminus were followed by RP HPLC for at least six half-lives. Product distribution studies indicated that 3'-dephosphorylation was the only reaction detected under all conditions tested. Therefore, the pseudo-first-order rate constant for the disappearance of the starting material was obtained by applying the integrated first-order rate equation to the time-dependent decrease in the concentration of the starting material. Similarly, the product 5'-T 10 The rate constant for the accumulation of -3' was obtained. Because data were collected at three different temperatures, the thermodynamic constants of the process were easily extracted.

[0089] The 3'-dephosphorylation of 41 was strictly first-order in both starting material and product accumulation. Without intending to be bound by theory, carrying out the process in 10 different concentrations of aqueous methylamine ranging from 11.55 M to 0.39 M (40% and 30-fold diluted methylamine, respectively) revealed a complex relationship between base concentration and rate constant (Figure 11). The reaction rate reached its maximum in the methylamine concentration range of 1.03 to 3.42 M (10-fold and 3-fold diluted, respectively). In the optimal range, the half-life for the disappearance of starting material at 45 °C ranged from 7 to 8 min, whereas at 40% aqueous methylamine the pseudo-first-order rate constant decreased by 8.7-fold, resulting in a half-life of 64.5 min.

[0090] The 3'-dephosphorylation of oligonucleotides containing a 2'-OMe-modified nucleotide residue at the 3'-terminus by universal solid support 941c was tested using 5'-T9(2'-OmeU)-3' as a model compound. Compared to 41, the rate of 3'-dephosphorylation was accelerated by 2.0 to 2.2-fold depending on the dilution, while the optimal concentration and optimal concentration window of methylamine remained within the same range.

[0091] When oligonucleotide 41 was attached to universal solid support 941c via a phosphorothioate linkage, as opposed to the phosphate linkage used in the experiments disclosed above, the rate of 3′-dephosphorylation decreased by 30 to 40% depending on the dilution, and the optimal concentration and optimal concentration window of methylamine remained within the same range.

[0092] In a separate experiment, universal phosphoramidite 878 was coupled to hydroxypropyl CPG1000 on a 1 μmol scale using the instrument's conventional coupling cycle. The resulting solid support was used to assemble oligonucleotide 40. After deprotection, the crude oligonucleotide was obtained, and its HPLC profile was indistinguishable from that obtained by synthesis on universal solid support 941c. Effect of Substituents at N-8 of the Tropane Ring

[0093] To assess the effect of substituents at N-8 of the tropane ring, oligonucleotide 41 was assembled on universal solid support 943c. The concentration-dependent pseudo-first-order rate constant for 3'-dephosphorylation of 41 was obtained as described for universal solid support 941c above. It was observed that 3'-dephosphorylation of 41 was reduced by approximately 30%, while the optimal concentration and optimal concentration window of methylamine remained within the same range (Figure 17). Without intending to be bound by theory, the delay is likely due to the steric bulkiness of the N-isopropyl group. However, universal solid support 943c remained highly efficient, with a half-life of 9.5 min at 45 °C.

[0094] Without intending to be bound by theory, the performance of Universal Solid Support 941 was very close to that of Universal Solid Support L in 40% methylamine, while in 3- to 10-fold dilutions of methylamine, 3'-dephosphorylation on Universal Solid Support 941 was approximately 9 times faster than that on Universal Solid Support L. Example 4 Development of optimal deprotection conditions for both nucleobases and universal solid support 941

[0095] To develop optimal deprotection conditions for both the nucleobase and the universal solid support 941, we investigated the deprotection of 1, 3, and 6 dG ib The deprotection kinetics of model oligonucleotides containing the α- and β-amino groups were investigated. For this purpose, the following four model oligonucleotides 42-45 were designed, taking into account the following considerations: a.dG ib The nucleotide residue was chosen because it exhibited the slowest deprotection kinetics among all other protected nucleotide residues; b. dG-rich oligonucleotides form complex inter- and intramolecular structures, such as dG quadruplets. Information about the behavior of partially protected oligonucleotides in solution is lacking. Without intending to be bound by theory, if such structures are formed by partially protected oligonucleotides, their presence may affect the rate of deprotection of the oligonucleotides. To determine whether such an effect exists, oligonucleotides 42 and 43 were prepared by combining dG-rich oligonucleotides separated by a thymidine nucleotide residue, such as 43. ib Nucleotide residues and dG grouped together like 44 ib Characterized by nucleotide residue. c. Long dG tracts can exhibit unexpected physicochemical properties. To determine whether their presence might slow the deprotection process, compound 45 was transformed into a 6-dG tract. ib Characterized by nucleotide residue. d. The presence of a negatively charged phosphate backbone in an oligonucleotide often retards the reaction of the oligonucleotide with nucleophiles. ib To exclude unnecessary fluctuations in the rate constants due to the potential for faster deprotection of nucleotide residues, all dGs at 42–45 were ib The residues were placed internally. Solid-supported synthesis of oligonucleotides 42-45 and their deprotection kinetics.

[0096] Compounds 42-45 were assembled on the conventional nucleoside-based solid support DMT-T CPG1000. The 5'-DMT group was removed at the end of synthesis, and all 2-cyanoethyl protecting groups were removed post-synthesis by treatment with 0.5 M piperidine in acetonitrile for 15 min. Vacuum-dried solid support-bound oligonucleotides 42-45 were stored at -80 °C as needed. 42:d(TTT TG ib T TTT T); 43:d(TG ib T.G. ib T.G. ibTTT T); 44:d(TTT G ib G ib G ib TTT T); 45:d(TTG ib G ib G ib G ib G ib G ib TT).

[0097] Deprotection of oligonucleotides 42–45 was carried out at 30.0, 40.0, and 50.0 °C using 1.37, 2.56, and 3.42 M aqueous methylamine (7.5-, 4.0-, and 3.0-fold dilutions, respectively). The progress of the fully base-protected starting material, all incompletely deprotected intermediates, and the final fully base-deprotected oligonucleotide released from the solid phase into solution was monitored by RP HPLC. Figure 18 shows the time-dependent product distribution in the reaction mixture of compound 45 in 3.42 M methylamine at 40 °C. The pseudo-first-order rate constant for the disappearance of the starting material was obtained by applying an integrated first-order rate equation to the time-dependent decrease in the concentration of the starting material. To extract the rate constants for the following steps in the reaction sequence, excluding the final step, differential rate equation (1) was applied and fitted to the experimental data by numerical integration. For the accumulation of fully deprotected 45, differential rate equation (2) was used and fitted to the experimental data by numerical integration.

number

[0098] Analysis of the kinetic data showed that each deprotection step for all tested oligonucleotides followed a first-order law with respect to the starting material, the partially deprotected intermediate, and the deprotecting agent methylamine. The rate constant for each deprotection step was normalized based on the number of residues available for deprotection at that step according to equation (3).

number

[0099] The normalized rate constants for all deprotection steps are ib It was also observed that the number of residues or their arrangement was virtually independent. This feature allowed the extraction of energy parameters for the removal of isobutyryl residues. [Table 2]

[0100] As seen in Table 2, the single dG in oligonucleotides 42-45 ib The determined activation energies for deprotection of residues are 1.4 to 1.5 times higher than those for 3'-dephosphorylation by USS 941 when using the universal solid support 941 under optimal conditions.

[0101] Based on the obtained kinetic and thermodynamic data, we optimized the deprotection conditions to determine the concentration of methylamine that would deprotect oligonucleotide 45 to such an extent that only 1% of one of the six dG residues would remain protected (0.167% per dG residue), while still allowing the oligonucleotide to be 3'-dephosphorylated to the same 99% extent. The plot shown in Figure 13 shows that as the concentration of methylamine increases, the time required for 99% 3'-dephosphorylation by universal solid support 941 increases, while the time for oligonucleotide 45 decreases. The intersection of the two curves represents an optimal concentration of methylamine of approximately 4.5 M at 45 °C and a deprotection time of 70-75 min (Figure 19).

[0102] While not intending to be bound by theory, the optimal deprotection time and concentration of methylamine were determined for oligonucleotides containing six dG residues, assuming that the residues are present in an average ratio of 25%, and such conditions are believed to be sufficient for the majority of synthetic oligonucleotides of 20-25 nt residues in length that are most commonly used in the art.

[0103] Thus, a novel universal solid support synthesis for oligonucleotide synthesis and the preparation of universal phosphoramidite building blocks, as well as their use in the assembly of oligonucleotides, and their deprotection under the optimal conditions disclosed herein are demonstrated. Beneficial to the overall deprotection time, under these conditions, the 3'-dephosphorylation of oligonucleotides proceeds more rapidly than the deprotection of the N-isobutyryl group in the guanidine residue. Example 5 [ka] rel-(1R,5S,6S,7R)-7-[(bis(4-methoxyphenyl)phenyl)methoxy]-8-methyl-3-oxo-8-azabicyclo[3.2.1]octan-6-yl acetate, 131

[0104] Compound 1 (4.9 g, 28.62 mmol), prepared as disclosed in J. Heterocyclic Chem., 29, 1541, 1992, (J. Am. Chem. Soc., 74, 3825-3828, 1952), was suspended in acetonitrile (40 mL) and trimethyl orthoacetate (5.2 g, 43 mmol) and placed in a 45° C. oil bath. Trifluoroacetic acid (0.6 mL, 15.2 mmol) was added, and the reaction mixture was stirred for 30 min. TLC analysis confirmed complete conversion of the starting material to the corresponding orthoester derivative. The reaction mixture was removed from the heating bath and cooled to room temperature. Water (0.772 g, 43 mmol) was added, and the reaction was stirred for 1 h. After 2 min, TLC analysis confirmed complete hydrolysis of orthoester 11 to the corresponding monoacetate. The reaction was quenched with 10% aqueous NaHCO3 (10 mL), and the volatiles were evaporated under vacuum. The resulting residue was dissolved in CHCl (50 mL) and washed with a 1:1 mixture of 10% aqueous NaHCO3 and brine (50 mL). The organic extract was dried (sodium sulfate) and evaporated to dryness. The oily solid was triturated with hexane and dried under high vacuum to give compound 131 (5.2 g, 85%) as a solid foam.

[0105] 1 H-NMR (500 MHz, CDCl3) δ 4.78 (1H, d); 4.1 (1H, d); 3.52 (1H, s); 3.51 (1H, s); 3.04 (1H, m) 2.53 (2H, m); 2.52 (3H, s); 2.3 (2H, m); 1.07 (6H, d). 13 C-NMR (100 MHz, CDCl3) δ 20.9, 34.38; 40.49; 40.83; 64.72; 67.89; 75.21; 170.65; 206.19. ES MS:[M+H] + 214.2 (measured value), 213.1 (calculated value).

[0106] The product of the previous step (2.2 g, 10.3 mmol) in CHCl (40 mL) and iPrNEt (4 g, 31 mmol) was treated with dimethoxytrityl chloride (3.85 g, 11.35 mmol). The reaction mixture was stirred for 2 h, diluted with CHCl (40 mL), and washed with 10% aqueous NaHCO. The organic extract was dried (sodium sulfate) and evaporated in vacuo. The residue was dissolved in methanol (100 mL) and cooled in an ice-water bath. After 30 min of magnetic stirring, the desired product precipitated from the solution. The product was filtered off and redissolved in CHCl (20 mL). The solution was evaporated, and the oily residue was dried in vacuo to give the product as a solid foam (4.4 g, 82%).

[0107] 1 7.25 (m, 2H); 7.23 (m, 5H); 6.9 (m, 4H); 4.64 (d, 1H); 3.79 (d, 1H); 3.78 (s, 6H); 3.32 (s, 2H); 2.62 (m, 2H); 2.5 (m, 2H); 2.44 (m, 1H); 2.09 (s, 3H); 1.09 (d, 1H); 1.50 (d, 1H). 13 C-NMR (100 MHz, DMSO-d6) δ 20.67, 21.01, 35.7, 41.47, 42.18, 55.05, 65.31, 65.61, 77.03, 77.66, 87.08, 113.25, 126.84, 127.79, 127.83, 129.71, 129.78, 135.91, 136.12, 145.27, 158.22, 158.25, 169.86, 206.73.ES MS:[M+H] + 516.1 (measured value), 515.2 (calculated value). Example 6 [ka] rel-(1R,5S,6S,7R)-7-(bis(4-methoxyphenyl)(phenyl)methoxy)-8-ethyl-3-oxo-8-azabicyclo[3.2.1]octan-6-yl acetate, 132.

[0108] Compound 132 was synthesized according to the procedure described in Example 1 above.

[0109] ES MS:[M+H] + 530.2 (measured value), 530.6 (calculated value). Example 7 [ka] rel-(1R,5S,6S,7R)-7-(bis(4-methoxyphenyl)(phenyl)methoxy)-8-isopropyl-3-oxo-8-azabicyclo[3.2.1]octan-6-yl acetate, 133.

[0110] Compound 133 was synthesized according to the procedure described in Example 5 above.

[0111] 1 7.26 (m, 2H); 7.23 (m, 5H); 6.9 (m, 4H); 4.72 (m, 1H); 3.74 (s, 1H); 3.72 (s, 6H); 3.57 (s, 1H); 3.32 (s, 1H); 2.96 (m, 1H); 2.74 (s, 1H); 2.24 (m, 1H); 2.13 (s, 3H); 1.99 (m, 1H); 1.47 (d, 1H); 1.02 (s, 3H); 0.9 (s, 3H). 13C-NMR (100 MHz, DMSO-d6) δ 21.39, 21.56, 41.25, 41.94, 44.47, 55.06, 60.91, 61.33, 76.59, 77.05, 87.0, 113.26, 126.80, 127.78, 127.83, 129.66, 129.77, 136.01, 136.17, 145.31, 158.20, 158.25, 170.01, 207.16.ES MS:[M+H] + 544.2 (measured value), 544.7 (calculated value). Example 8 [ka] rel-(1R,5S,6S,7R)-7-(bis(4-methoxyphenyl)(phenyl)methoxy)-8-benzyl-3-oxo-8-azabicyclo[3.2.1]octan-6-yl acetate, 134.

[0112] Compound 134 was synthesized according to the procedure described in Example 1 above.

[0113] ES MS:[M+H] + 592.3 (measured value), 592.7 (calculated value). Example 9 [ka] rel-(1R,5S,6S,7R)-7-(bis(4-methoxyphenyl)(phenyl)methoxy)-2,4,8-trimethyl-3-oxo-8-azabicyclo[3.2.1]octan-6-yl acetate, 137.

[0114] Compound 137 was synthesized according to the procedure described in Example 1 above.

[0115] ES MS:[M+H] + 544.1 (measured value), 544.7 (calculated value). Example 10 [ka] rel-(1R,5S,6S,7R)-7-(bis(4-methoxyphenyl)(phenyl)methoxy)-2,2,4,4,8-pentamethyl-3-oxo-8-azabicyclo[3.2.1]octan-6-yl acetate, 138.

[0116] Compound 138 was synthesized according to the procedure described in Example 1 above.

[0117] ES MS:[M+H] + 572.9 (measured value), 572.7 (calculated value). Example 11 [ka] rel-(1R,5S,6S,7R)-7-(tris(4-methoxyphenyl)methoxy)-8-ethyl-3-oxo-8-azabicyclo[3.2.1]octan-6-yl acetate, 231.

[0118] Compound 231 was synthesized according to the procedure described in Example 1 above.

[0119] ES MS:[M+H] + 548.0 (measured value), 547.7 (calculated value). Example 12 [ka] rel-(1R,5S,6S,7R)-7-(tris(4-methoxyphenyl)methoxy)-8-ethyl-3-oxo-8-azabicyclo[3.2.1]octan-6-yl acetate, 232.

[0120] Compound 232 was synthesized according to the procedure described in Example 1 above.

[0121] ES MS:[M+H] + 561.2 (measured value), 561.7 (calculated value). Example 13 [ka] rel-(1R,5S,6S,7R)-7-(tris(4-methoxyphenyl)methoxy)-8-isopropyl-3-oxo-8-azabicyclo[3.2.1]octan-6-yl acetate, 233.

[0122] ES MS:[M+H] + 575.4 (measured value), 575.7 (calculated value). Example 14 [ka] rel-(1R,5S,6S,7R)-7-(tris(4-methoxyphenyl)methoxy)-8-benzyl-3-oxo-8-azabicyclo[3.2.1]octan-6-yl acetate, 234.

[0123] Compound 334 was synthesized according to the procedure described in Example 1 above.

[0124] ES MS:[M+H] + 624.1 (measured value), 623.7 (calculated value). Example 15 [ka] rel-(1R,5S,6S,7R)-7-(tris(4-methoxyphenyl)methoxy)-2,4,8-trimethyl-3-oxo-8-azabicyclo[3.2.1]octan-6-yl acetate, 237.

[0125] Compound 237 was synthesized according to the procedure described in Example 1 above.

[0126] ES MS:[M+H] + 575.1 (measured value), 575.7 (calculated value). Example 16 [ka] rel-(1R,5S,6S,7R)-7-(tris(4-methoxyphenyl)methoxy)-2,4,8-trimethyl-3-oxo-8-azabicyclo[3.2.1]octan-6-yl acetate, 238.

[0127] Compound 238 was synthesized according to the procedure described in Example 1 above.

[0128] ES MS:[M+H] + 602.9 (measured value), 603.8 (calculated value). Example 17 [ka] rel-(1R,5S,6S,7R)-7-(bis(4-methoxyphenyl)(phenyl)methoxy)-3-hydroxy-8-methyl-8-azabicyclo[3.2.1]octan-6-yl acetate, 331

[0129] Compound 131 (4.2 g, 8.15 mmol) in methanol (50 mL) was cooled in an ice bath. To this was added NaBH (12.22 mmol, 0.46 g) and CeCl (50 mg, 0.2 mmol) slowly over 15 minutes. The reaction mixture was stirred for 30 minutes and quenched with saturated NH Cl solution (10 mL). The volatiles were evaporated in vacuo, and the residue was redissolved in CHCl (50 mL) and washed with water (50 mL) and brine (50 mL). The organic extract was dried over sodium sulfate and evaporated. The crude mixture was purified by column chromatography (CHCl-MeOH) to give the desired compound 331 (3.5 g, 83%) as a white foam.

[0130] 16.87 (m, 4H); 4.6 (m, 1H); 4.49 (d, 1H); 3.97 (d, 1H); 3.74 (s, 6H); 3.13 (m, 1H); 2.94 (s, 1H); 2.53 (s, 1H); 2.35 (s, 3H); 1.99 (s, 3H); 1.5 (m, 1H); 1.33 (m, 1H); 1.16 (m, 2H). 13 C-NMR (100 MHz, DMSO-d6) δ 21.09, 33.5, 33.8, 37.02, 54.87, 55.04, 62.01, 65.55, 65.80, 76.92, 77.25, 87.04, 113.13, 126.75, 127.74, 127.98, 129.84, 129.91, 136.23, 136.38, 145.47, 158.14, 169.76.ES MS:[M+Na] + 517.25 (measured value), 518.4 (calculated value) [M+Na] + 540.2, [2M+Na] + 1057.2 (actual value). Example 18 [ka] rel-(1R,5S,6S,7R)-7-(bis(4-methoxyphenyl)(phenyl)methoxy)-3-hydroxy-8-ethyl-8-azabicyclo[3.2.1]octan-6-yl acetate, 332

[0131] Compound 332 was synthesized according to the procedure described in Example 17 above.

[0132] ES MS:[M+H] + 532.6 (measured value), 532.6 (calculated value) [M+Na] + 554.3 (actual value). Example 19 [ka] rel-(1R,5S,6S,7R)-7-(bis(4-methoxyphenyl)(phenyl)methoxy)-3-hydroxy-8-isopropyl-8-azabicyclo[3.2.1]octan-6-yl acetate, 333

[0133] Compound 333 was synthesized according to the procedure described in Example 17 above.

[0134] 1 6.9 (m, 4H); 4.69 (d, 1H); 4.45 (d, 1H); 3.88 (m, 1H); 3.73 (s, 6H); 3.23 (s, 1H); 3.14 (s, 1H); 2.98 (m, 1H); 2.66 (s, 1H); 2.04 (s, 3H); 1.49 (s, 1H); 1.26 (m, 1H); 1.19 (m, 2H), 0.93 (s, 3H); 0.84 (s, 3H). 13 C-NMR (100 MHz, DMSO-d6) δ 21.13, 2.42, 21.59, 32.89, 33.11, 44.34, 55.04, 60.59, 60.90, 62.11, 76.58, 76.63, 86.88, 113.14, 126.7, 127.74, 127.94, 129.76, 129.86, 136.37, 136.45, 145.56, 158.11, 158.14, 169.96.ES MS:[M+H] + 546.9 (measured value), 546.7 (calculated value) [M+Na] + 568.4 (actual value). Example 20 [ka] rel-(1R,5S,6S,7R)-7-(bis(4-methoxyphenyl)(phenyl)methoxy)-3-hydroxy-8-benzyl-8-azabicyclo[3.2.1]octan-6-yl acetate, 334

[0135] Compound 334 was synthesized according to the procedure described in Example 17 above.

[0136] ES MS:[M+H] + 594.3 (measured value), 594.7 (calculated value) [M+Na] + 616.2 (measured value). Example 21 [ka] rel-(1R,5S,6S,7R)-7-(bis(4-methoxyphenyl)(phenyl)methoxy)-2,4,8-trimethyl-3-hydroxy-8-azabicyclo[3.2.1]octan-6-yl acetate, 337.

[0137] Compound 337 was synthesized according to the procedure described in Example 17 above.

[0138] ES MS:[M+H] + 546.9 (measured value), 546.7 (calculated value). Example 22 [ka] rel-(1R,5S,6S,7R)-7-(bis(4-methoxyphenyl)(phenyl)methoxy)-2,2,4,4,8-pentamethyl-3-hydroxy-8-azabicyclo[3.2.1]octan-6-yl acetate, 338.

[0139] Compound 338 was synthesized according to the procedure described in Example 17 above.

[0140] ES MS:[M+H] + 574.0 (measured value), 574.7 (calculated value). Example 23 [ka]

[0141] rel-(1R,5S,6S,7R)-7-(tris(4-methoxyphenyl)methoxy)-3-hydroxy-8-methyl-8-azabicyclo[3.2.1]octan-6-yl acetate, 431

[0142] Compound 431 was synthesized according to the procedure described in Example 17 above.

[0143] ES MS:[M+H] + 550.2 (measured value), 549.7 (calculated value) [M+Na] + 582.1 (measured value). Example 24 [ka] rel-(1R,5S,6S,7R)-7-(tris(4-methoxyphenyl)methoxy)-3-hydroxy-8-ethyl-8-azabicyclo[3.2.1]octan-6-yl acetate, 432

[0144] Compound 432 was synthesized according to the procedure described in Example 17 above.

[0145] ES MS:[M+H] + 564.1 (measured value), 563.7 (calculated value) [M+Na] + 586.8 (actual value). Example 25 [ka] rel-(1R,5S,6S,7R)-7-(tris(4-methoxyphenyl)methoxy)-3-hydroxy-8-isopropyl-8-azabicyclo[3.2.1]octan-6-yl acetate, 433

[0146] Compound 433 was synthesized according to the procedure described in Example 17 above.

[0147] ES MS:[M+H] + 577.6 (measured value), 577.7 (calculated value) [M+Na] + 599.9 (actual value). Example 26 [ka] rel-(1R,5S,6S,7R)-7-(tris(4-methoxyphenyl)methoxy)-3-hydroxy-8-benzyl-8-azabicyclo[3.2.1]octan-6-yl acetate, 434

[0148] Compound 434 was synthesized according to the procedure described in Example 17 above.

[0149] ES MS:[M+H] + 626.6 (measured value), 625.7 (calculated value) [M+Na] + 647.1 (measured value). Example 27 [ka] rel-(1R,5S,6S,7R)-7-(tris(4-methoxyphenyl)methoxy)-2,4,8-trimethyl-3-hydroxy-8-azabicyclo[3.2.1]octan-6-yl acetate, 437.

[0150] Compound 437 was synthesized according to the procedure described in Example 17 above.

[0151] ES MS:[M+H] + 578.4 (measured value), 775.7 (calculated value). Example 28 [ka] rel-(1R,5S,6S,7R)-7-(tris(4-methoxyphenyl)methoxy)-2,2,4,4,8-pentamethyl-3-hydroxy-8-azabicyclo[3.2.1]octan-6-yl acetate, 438.

[0152] Compound 438 was synthesized according to the procedure described in Example 17 above.

[0153] ES MS:[M+H] + 606.5 (measured value), 605.8 (calculated value). Example 29 [ka] 9-Methyl-2-methoxy-hexahydro-6H-cyclohepta-1,3-dioxol-4,8-imin-6-ol, 31

[0154] Compound 31 was synthesized from compound 26 according to the procedure described in Example 17 above.

[0155] ES MS:[M+H] + 215.7 (measured value), 216.2 (calculated value). Example 30 [ka] 9-Isopropyl-2-methoxy-hexahydro-6H-cyclohepta-1,3-dioxol-4,8-imin-6-ol, 33

[0156] Compound 33 was synthesized from compound 27 according to the procedure described in Example 17 above.

[0157] ES MS:[M+H] + 244.9 (measured value), 244.3 (calculated value). Example 31 [ka] 9-Benzyl-2-methoxy-hexahydro-6H-cyclohepta-1,3-dioxol-4,8-imin-6-ol, 34

[0158] Compound 34 was synthesized from compound 28 according to the procedure described in Example 17 above.

[0159] ES MS:[M+H] + 291.5 (measured value), 292.3 (calculated value). Example 32 [ka] (9-methyl-2-methoxy-hexahydro-6H-cyclohepta-1,3-dioxol-4,8-imin-6-yl)acetate, 201

[0160] Acetic anhydride (g, 30 mmol) was added to a magnetically stirred solution of compound 31 (5.14 g, 20 mmol) in pyridine (30 mL) while cooling in an ice bath. The reaction mixture was stirred at room temperature overnight, and TLC showed complete disappearance of the starting material. The excess acylating agent was quenched by adding water (1.8 mL, 100 mmol) followed by TEA (4.04 g, 40 mmol). The mixture was stirred at room temperature for 2 hours and evaporated to an oil. The residue was dissolved in ethyl acetate (100 mL) and washed with 5% aqueous NaHCO3 (2 × 20 mL) and brine (20 mL). The organic phase was dried over Na2SO4, evaporated to an oil, and coevaporated with acetonitrile (2 × 25 mL). The residue was dissolved in ethyl acetate (10 mL) and added slowly to ice-cold hexane (100 mL), and the desired product precipitated as a colorless viscous oil. The solution was decanted and the residue was dried on an oil pump to give the desired product as a colorless viscous oil (6.71 g, 87%), which was a ca. 10:1 mixture of diastereomers.

[0161] 1 H-NMR (500 MHz, DMSO-d6) δ 5.97+5.70 (1H, s, 1:10), 5.28 (1H, m), 4.26 + 4.46 (2H, s, 1:10), 3.29 + 3.43 (3H, s, 10:1); 3H), 2.23 (2H, s), 2.12 (2H, m), 1.77 (3H, s), 1.47 (2H, m).ES MS:[M+H] + 257.4 (measured value), 258.3 (calculated value). Example 33 [ka] (9-Isopropyl-2-methoxy-hexahydro-6H-cyclohepta-1,3-dioxol-4,8-imin-6-yl)pivalate, 203

[0162] Compound 203 was synthesized from compound 33 according to the procedure described in Example 32 above.

[0163] ES MS:[M+H] + 239.1 (measured value), 238.4 (calculated value). Example 34 [ka] (9-benzyl-2-methoxy-hexahydro-6H-cyclohepta-1,3-dioxol-4,8-imin-6-yl)isobutyrate, 205

[0164] Compound 205 was synthesized from compound 34 according to the procedure described in Example 32 above.

[0165] ES MS:[M+H] + 361.8 (measured value), 362.4 (calculated value). Example 35 [ka] 9,9-Dimethyl-2,6-dimethoxy-hexahydro-6H-cyclohepta-1,3-dioxole-4,8-iminium iodide, 111

[0166] Compound 21 (5.73 g, 25 mmol) was dissolved in anhydrous DMF (20 mL) containing sodium hydride (60% suspension in oil, 1.05 g, 26.25 mmol). The mixture was magnetically stirred in a pressure flask at 60 °C for 30 min. The flask was brought to room temperature, and methyl iodide (14.2 g, 100 mmol) was added. The mixture was stirred at 45 °C overnight, after which TLC confirmed the reaction was complete. The mixture was evaporated in vacuo and coevaporated with toluene (5 × 50 mL). The residue was suspended in anhydrous acetone (50 mL), heated with stirring at 45 °C for 20 min, and cooled to 0 °C. The liquid phase was removed, and the process was repeated. The solid was filtered off, washed with cold acetone, and dried in vacuo to give the desired product as an off-white solid (g, 73.2%).

[0167] 1 H-NMR (500 MHz, DMSO-d6) δ 6.12 + 5.93 (total 1H, s, 1:10), 4.34 + 4.58 (total 2H, s), 3.60 + 3.58 (3H, s, 10:1); 3.41 (3H, s), 3.30 (3H, s), 3.25 (3H, s), 3.08 (1H, m), 2.31 (2H, m), 2.19 (2H, m).ES MS:[M+H] + 257.4 (measured value), 258.3 (calculated value). Example 36 [ka] 9-Methyl-9-benzyl-2-methoxy-6-benzyloxy-hexahydro-6H-cyclohepta-1,3-dioxol-4,8-iminium iodide, 114

[0168] Compound 114 (58.5%), an off-white solid, was synthesized from compound 21 according to the procedure described in Example 32. ES MS: [M] + 411.1 (measured value), 410.5 (calculated value). Example 37 [ka] 9-Methyl-9-benzyl-2-methoxy-6-benzyloxy-hexahydro-6H-cyclohepta-1,3-dioxol-4,8-iminium bromide, 114

[0169] Compound 114 (58.5%), an off-white solid, was synthesized from compound 21 according to the procedure described in Example 32. ES MS: [M] + 411.1 (measured value), 410.5 (calculated value). Example 38 [ka] 9-Methyl-9-benzyl-2,6-dimethoxy-hexahydro-6H-cyclohepta-1,3-dioxol-4,8-iminium bromide, 115

[0170] Compound 115 (63.6%), an off-white solid, was synthesized from compound 24 according to the procedure described in Example 32. ES MS: [M] + 333.6 (measured value), 334.4 (calculated value). Example 39 [ka] rel-(1R,5S,6S,7R)-7-[(tris(4-methoxyphenyl)methoxy]-8-methyl-3-acetoxy-8-azabicyclo[3.2.1]octan-6-ol, 401

[0171] Trifluoroacetic acid (0.5 M in anhydrous MeCN, 2.0 mL, 1 mmol) was added to a solution of compound 201 (5.15 g, 20 mmol) in MeCN (50 mL) containing water (0.72 g, 40 mmol). The mixture was maintained at room temperature for 6 hours, after which the hydrolysis of 201 to the corresponding formate ester was confirmed to be complete. A chromatography column packed with silica gel (30 mL) was washed with methanol (2 volumes, 60 mL) and anhydrous acetonitrile (2 volumes, 60 mL). The reaction mixture was passed through the column while the eluate was collected. The column was then washed with another 2 volumes of acetonitrile. The combined eluate was evaporated to dryness and coevaporated with acetonitrile (3 × 50 mL). The oily residue was dissolved in anhydrous acetonitrile (20 mL) and DIPEA (3.23 g, 25 mmol), followed by the addition of solid TMTCl (7.56 g, 20.5 mmol). The reaction mixture was stirred at room temperature for 3 hours, and water (1.8 g, 100 mmol) and TEA (2.02 g, 20 mmol) were added. After stirring for an additional 2 hours, the solvent was evaporated. The resulting oil was dissolved in ethyl acetate (200 mL) and washed with 5% aqueous NaHCO3 (2 x 50 mL) and brine (50 mL). The organic phase was dried over Na2SO4, evaporated, and co-evaporated with toluene (2 x 50 mL). The product was isolated by silica gel column purification using a linear gradient from ether to 5% MeOH in DCM. The collected fractions were evaporated and dried in vacuo to give the desired product as a solid foam (8.62 g, 78.7%). 1 H-NMR (500 MHz, DMSO-d6) δ 6.08 (m, 6H); 6.07 (m, 6H); 4.60 (m, 1H); 4.49 (d, 1H); 3.97 (d, 1H); 2.94 (s, 1H); 2.26 (s, 3H); (s, 3H); 1.70 - 1.5 (m, 4H); 1.32 (m, 2H).ES MS:[M+H] + 549.3 (measured value), 548.6 (calculated value). Example 40 [ka] rel-(1R,5S,6S,7R)-7-[(bis(4-methoxyphenyl)(phenyl)methoxy]-8-methyl-3-acetoxy-8-azabicyclo[3.2.1]octan-6-ol, 301

[0172] Compound 301 (79.5%), isolated as a solid foam, was synthesized from compound 201 according to the procedure described in Example 38. ES MS: [M] + 517.8 (measured value), 518.6 (calculated value). Example 41 [ka] rel-(1R,5S,6S,7R)-7-[(bis(4-methoxyphenyl)(phenyl)methoxy]-8-isopropyl-3-pivaloyloxy-8-azabicyclo[3.2.1]octan-6-ol, 303

[0173] Compound 301 (79.5%), isolated as a solid foam, was synthesized from compound 201 according to the procedure described in Example 38. ES MS: [M] + 588.7 (measured value), 588.8 (calculated value). Example 42 [ka] rel-(1R,5S,6S,7R)-7-[(bis(4-methoxyphenyl)(phenyl)methoxy]-8-benzyl-3-isobutyryloxy-8-azabicyclo[3.2.1]octan-6-ol, 305

[0174] Compound 305 (79.5%), isolated as a solid foam, was synthesized from compound 205 according to the procedure described in Example 38. ES MS: [M] + 622.0 (measured value), 622.8 (calculated value). Example 43 [ka] rel-(1R,5S,6S,7R)-7-[(tris(4-methoxyphenyl)methoxy]-8-isopropyl-3-pivaloyloxy-8-azabicyclo[3.2.1]octan-6-ol, 403

[0175] Compound 403 (81.2%), isolated as a solid foam, was synthesized from compound 203 according to the procedure described in Example 38. ES MS: [M] + 620.2 (measured value), 619.8 (calculated value). Example 44 [ka] rel-(1R,5S,6S,7R)-7-[(tris(4-methoxyphenyl)methoxy]-8-benzyl-3-isobutyryloxy-8-azabicyclo[3.2.1]octan-6-ol, 405

[0176] Compound 305 (73.1%), isolated as a solid foam, was synthesized from compound 205 according to the procedure described in Example 38. ES MS: [M] + 652.0 (measured value), 652.8 (calculated value). Example 45 [ka] rel-(1R,5S,6S,7R)-7-[(tris(4-methoxyphenyl)methoxy]-8-methyl-3-[(t-butyl)dimethylsilyloxy]-8-azabicyclo[3.2.1]octan-6-ol, 416

[0177] Compound 416 (72.6%), isolated as a solid foam, was synthesized from compound 216 according to the procedure described in Example 38. ES MS: [M] + 620.6, 621.6 (measured values), 620.3, 621.3 (calculated values). Example 46 [ka] rel-(1R,5S,6S,7R)-7-[(tris(4-methoxyphenyl)methoxy]-8-methyl-3-[(t-butyl)diphenylsilyloxy]-8-azabicyclo[3.2.1]octan-6-ol, 418

[0178] Compound 418 (69.6%), isolated as a solid foam, was synthesized from compound 218 according to the procedure described in Example 38. ES MS: [M] + 744.3, 745.6 (measured values), 744.4, 745.4 (calculated values). Example 47 [ka] rel-(1R,5S,6S,7R)-7-[(tris(4-methoxyphenyl)methoxy]-8,8-dimethyl-3-methoxy-8-azoniabicyclo[3.2.1]octan-6-ol acetate, 411

[0179] Trifluoroacetic acid (2.0 g, 1.5 mmol) was added to a solution of compound 211 (5.78 g, 15 mmol) in MeOH (50 mL) containing water (1.08 g, 60 mmol). The mixture was maintained at room temperature for 1 h, after which hydrolysis of 211 to the corresponding acetate ester was confirmed to be complete. The reaction mixture was evaporated to dryness and coevaporated with pyridine (3 × 50 mL). The oily residue was dissolved in anhydrous pyridine (50 mL) and DIPEA (3.23 g, 25 mmol), followed by the addition of solid TMTCl (5.90 g, 16 mmol). The reaction mixture was stirred at room temperature for 3 h. Sodium methoxide (30% w / w in MeOH, 8.1 g, 45 mmol) was added. After stirring for an additional 2 h, excess sodium methoxide was quenched with triethylammonium acetate buffer (1 M, 50 mL), and the solvent was evaporated. The resulting oil was dissolved in DCM-MeOH (19:1, 200 mL) and washed with brine (2 x 50 mL). The organic phase was dried over Na2SO4, evaporated, and dissolved in DCM-MeOH (19:1, 60 mL). The product was isolated by silica gel column purification using a linear gradient of 5% to 40% MeOH in DCM. The collected fractions were evaporated, and the residue was dissolved in DCM-MeOH (19:1, 200 mL) and washed with triethylammonium acetate buffer (0.5 M, 2 x 30 mL). The organic phase was dried over Na2SO4, evaporated, and dried in vacuo to give the desired product as a solid foam (5.40 g, 60.7%). ES MS: [M] + 533.6 (measured value), 534.7 (calculated value). Example 48 [ka] rel-(1R,5S,6S,7R)-7-[(tris(4-methoxyphenyl)methoxy]-8-methyl-8-benzyl-3-benzyloxy-8-azoniabicyclo[3.2.1]octan-6-ol acetate, 414

[0180] Compound 414 (73.1%), isolated as a solid foam, was synthesized from compound 214 according to the procedure described in Example 46. ES MS: [M]+ 686.9 (measured value), 686.4 (calculated value). Example 49 [ka] rel-(1R,5S,6S,7R)-7-[(tris(4-methoxyphenyl)methoxy]-8-methyl-8-benzyl-3-methoxy-8-azoniabicyclo[3.2.1]octan-6-ol acetate, 415

[0181] Compound 414 (65.3%), isolated as a solid foam, was synthesized from compound 215 according to the procedure described in Example 46. ES MS: [M] + 611.4 (measured value), 610.3 (calculated value). Example 50 [ka] rel-(1R,5S,6S,7R)-7-[(tris(4-methoxyphenyl)methoxy]-8-methyl-azaspiro[bicyclo[3.2.1]octan-3,2'-[1,3]dioxolan]-6-ol, 421

[0182] Solid TMTCl (5.90 g, 7.95 mmol) was added to compound 221 (1.61 g, 7.5 mmol) in anhydrous pyridine (40 mL) and DIPEA (1.25 g, 9.9 mmol). The reaction mixture was stirred at room temperature overnight, and the solvent was evaporated. The resulting oil was dissolved in ethyl acetate (200 mL) and washed with 5% aqueous NaHCO (2 × 50 mL) and brine (50 mL). The organic phase was dried over NaSO, evaporated, and coevaporated with toluene (2 × 50 mL). The product was isolated by silica gel column purification using a linear gradient from ether to 5% MeOH in DCM. The collected fractions were evaporated and dried in vacuo to give the desired product as a solid foam (3.10 g, 75.4%). ES MS: [M+H] + 548.1 (measured value), 547.3 (calculated value). Example 51 [ka] rel-(1R,5S,6S,7R)-7-[(tris(4-methoxyphenyl)methoxy]-8-isopropyl-azaspiro[bicyclo[3.2.1]octan-3,2'-[1,3]dioxolan]-6-ol, 423

[0183] Compound 423 (62.4%), isolated as a solid foam, was synthesized from compound 223 according to the procedure described in Example 50. ES MS: [M+H] + 577.5 (measured value), 576.7 (calculated value). Example 52 [ka] rel-(1R,5S,6S,7R)-7-[(tris(4-methoxyphenyl)methoxy]-5',5',8-trimethyl-azaspiro[bicyclo[3.2.1]octan-3,2'-[1,3]dioxolan]-6-ol, 422

[0184] Solid TMTCl (1.75 g, 4.75 mmol) was added to compound 222 (1.16 g, 4.5 mmol) in anhydrous pyridine (40 mL) and DIPEA (0.69 g, 5.5 mmol). The reaction mixture was stirred overnight at room temperature, and the solvent was evaporated. The resulting oil was dissolved in ethyl acetate (200 mL) and washed with 5% aqueous NaHCO (2 × 50 mL) and brine (50 mL). The organic phase was dried over NaSO, evaporated, and coevaporated with toluene (2 × 50 mL). The product was isolated by silica gel column purification using a linear gradient from ether to 5% MeOH in DCM. The collected fractions were evaporated and dried in vacuo to give the desired product as a solid foam (1.93 g, 72.6%). ES MS: [M+H] + 591.5 (measured value), 590.7 (calculated value). Example 53 [ka] rel-(1R,5S,6S,7R)-7-[(tris(4-methoxyphenyl)methoxy]-5',5'-dimethyl-8-benzyl-azaspiro[bicyclo[3.2.1]octan-3,2'-[1,3]dioxolan]-6-ol, 424

[0185] Compound 424 (72.9%), isolated as a solid foam, was synthesized from compound 224 according to the procedure described in Example 52. ES MS: [M+H] + 667.7 (measured value), 666.8 (calculated value). Example 54 [ka] rel-(1R,5S,6S,7R)-7-[(tris(4-methoxyphenyl)methoxy]-8-methyl-3-(methylamino)-aza[bicyclo[3.2.1]octan-6-ol, 452.

[0186] The reaction was carried out by modifying the method disclosed in Lewin, A.H.; Sun, G.; Fudala, L.; Navarro, H.; Zhou, L.-M.; Popick, P.; Faynsteyn, A.; Skolnick, P. Molecular Features Associated with Polyamine Modulation of NMDA Receptors. J. Medicinal Chem. 1998, 41(6), 988-995. A mixture of 40% aqueous methylamine (1.94 g, 25 mmol) and MeOH (40 mL) was neutralized to pH 6.5-7 with concentrated aqueous HCl. Compound 231 (3.27 g, 6.0 mmol) was added to this mixture, followed by NaBH3CN (1.2 g). The solution was stirred at room temperature for 3 days and evaporated. The residue was partitioned between HO (10 mL) and DCM (100 mL). The organic phase was washed with 1M NaH2PO4 (4 x 50 mL). The aqueous phase was basified with solid NaOH and extracted with DCM (3 x 50 mL). After drying over Na2SO4, the combined DCM solution was evaporated and dried under vacuum. The crude product (2.65 g, 85%) was used in the next step without further purification. Example 55 [ka] rel-(1R,5S,6S,7R)-7-[(tris(4-methoxyphenyl)methoxy]-8-methyl-3-[6-(trifluoroacetylamino)hexanmido]-aza[bicyclo[3.2.1]octane-6-ol), 655

[0187] N-Trifluoroacetyl-6-aminohexanoic acid was converted to its N-oxysuccinimidyl ester, 2,5-dioxo-1-pyrrolidinyl 6-[(2,2,2-trifluoroacetyl)amino]hexanoate, as disclosed in WO2008102606. This (1.78 g, 5.5 mmol) was dissolved in pyridine (25 mL), and the resulting solution was added to crude compound 452 (2.59 g, 5 mmol). The mixture was stirred overnight at room temperature and quenched by the addition of TEA (1.02 g, 10 mmol) and water (1 mL). After stirring for 2 hours, 10% methanolic ammonia (10 mL) was added, and stirring was continued for 3 hours. The solvent was evaporated, and the residue was dissolved in ethyl acetate (100 mL). The resulting solution was washed with 0.5 M aqueous NaOH (2 × 20 mL) and brine (2 × 50 mL). After drying over Na2SO4, the organic phase was evaporated. The resulting material was purified by silica gel column chromatography eluting with a linear gradient of DCM to DCM:MeOH:30% aqueous ammonia (80:15:5). Evaporation of the collected fractions afforded the desired product as a white solid foam (2.60 g, 82.4%). ES MS: [M+H] + 633.3 (measured value), 632.8 (calculated value). Example 56 [ka] rel-(1R,5S,6S,7R)-7-[(tris(4-methoxyphenyl)methoxy]-8-methyl-3-amino-aza[bicyclo[3.2.1]octan-6-ol, 451

[0188] Compound 451 was prepared by modifying the method disclosed in Lewin, AH; Sun, G.; Fudala, L.; Navarro, H.; Zhou, L.-M.; Popick, P.; Faynsteyn, A.; Skolnick, P. Molecular Features Associated with Polyamine Modulation of NMDA Receptors. J. Medicinal Chem. 1998, 41(6), 988-995.

[0189] A mixture of compound 231 (7.5 g, 5 mmol), HNOH-HCl (5.44 g, 78 mmol), and pyridine (10 mL) in EtOH (110 mL) was stirred at room temperature for 24 h. The mixture was concentrated, and the residue was partitioned between 2.5 M aqueous NaOH (40 mL) and ethyl acetate (100 mL). The aqueous phase was extracted twice with ethyl acetate (50 mL), and the combined organic phases were washed with brine (50 mL) and dried over KCO. The solvent was evaporated, and the residue was used in the next step without purification.

[0190] The product of the previous step was dissolved in anhydrous n-propanol (100 mL). To this was added sodium wiring (2.3 g, 100 mmol) over 20 minutes, and the mixture was refluxed for 1.5 hours. After cooling, water (5 mL) was added, and the solvent was evaporated. The residue was partitioned between water (50 mL) and ethyl acetate (100 mL). The aqueous phase was extracted twice with ethyl acetate (50 mL), and the combined organic phases were washed with brine (50 mL) and dried over K2CO3. The resulting crude material was purified by silica gel column chromatography eluting with a linear gradient of DCM to DCM:MeOH:30% aqueous ammonia (80:15:5). Evaporation of the collected fractions afforded the desired product as a white solid foam (1.48 g, 58.7%). ES MS: [M+H] + 506.5 (measured value), 505.6 (calculated value). Example 57 [ka] 2,9-Dimethyl-2-methoxy-6-amino-hexahydro-6H-cyclohepta-1,3-dioxol-4,8-imine, 25.

[0191] Compound 25 (46.7%), isolated as a hygroscopic solid, was synthesized from compound 11 according to the procedure described in Example 56. ES MS: [M+H] + 230.1 (measured value), 229.3 (calculated value). Example 58 [ka] rel-(1R,5S,6S,7R)-7-[(bis(4-methoxyphenyl)(phenyl)methoxy]-8-methyl-3-amino-aza[bicyclo[3.2.1]octan-6-ol, 351

[0192] Compound 351 (60.6%), isolated as a white solid foam, was synthesized from compound 131 according to the procedure described in Example 56. ES MS: [M+H] + 476.1 (measured value), 475.6 (calculated value). Example 59 [ka] rel-(1R,5S,6S,7R)-7-[(bis(4-methoxyphenyl)(phenyl)methoxy]-8-isopropyl-3-amino-aza[bicyclo[3.2.1]octan-6-ol, 353

[0193] Compound 353 (64.4%), isolated as a white solid foam, was synthesized from compound 133 according to the procedure described in Example 56. ES MS: [M+H] + 504.3 (measured value), 503.7 (calculated value). Example 60 [ka] N-(2,9-dimethyl-2-methoxy-6-amino-hexahydro-6H-cyclohepta-1,3-dioxol-4,8-imin-6-yl)-N'-(4-hydroxybutyl)urea, 882

[0194] Compound 25 (457 mg, 2.0 mmol) in pyridine (10 mL) was added dropwise to N,N'-carbonyldiimidazole (324 mg, 2.0 mmol) in pyridine (10 mL) while stirring and cooling in an ice bath. The mixture was stirred at room temperature overnight. Next, 4-aminobutanol (267 mg, 3.0 mmol) in acetonitrile (4 mL) was added, and the mixture was stirred for 24 h. The solvent was evaporated, and the residue was partitioned between dichloromethane (100 mL) and 1 M NaH2PO4 (20 mL). The organic phase was washed with 1 M NaH2PO4 (2 × 50 mL) and then with brine (30 mL). After drying over Na2SO4, the organic phase was evaporated and dried under vacuum. The product was isolated by silica gel column purification using a linear gradient from DCM to 15% MeOH in DCM. The collected fractions were evaporated and dried in vacuo to give the desired product as a solid foam (620 mg, 90.3%). ES MS: [M+H] + 345.0 (measured value), 344.4 (calculated value). Example 61 [ka] rel-(1R,5S,6S,7R)-N-[7-[(tris(4-methoxyphenyl)methoxy]-8-methyl-6-hydroxy-aza[bicyclo[3.2.1]octan-6-yl]-N'-(6-aminohexyl)urea, 657

[0195] Compound 657 (92.1%), isolated as a white solid foam, was synthesized from compound 451 according to the procedure described in Example 60. ES MS: [M+H] + 648.5 (measured value), 647.8 (calculated value). Example 62 [ka] rel-(1R,5S,6S,7R)-N-[7-[(tris(4-methoxyphenyl)methoxy]-8-methyl-6-acetoxy-aza[bicyclo[3.2.1]octan-6-yl]-N'-(9-hydroxynonyl)urea, 874

[0196] Fluorenylmethyloxycarbonyl chloride (543 mg, 2.1 mmol) in acetonitrile was added dropwise to compound 451 (1009 mg, 2.0 mmol) in acetonitrile (10 mL) while stirring in an ice bath. The mixture was stirred for 1 hour, after which pyridine (5 mL) and acetic anhydride (306 mg, 3 mmol) were added. The mixture was left overnight, and then 10% aqueous diethylamine solution (2.9 g, 4 mmol) was added. After stirring for 1 hour, the mixture was evaporated, and the residue was dissolved in ethyl acetate (50 mL) and washed with 5% aqueous NaHCO3 solution (2 x 50 mL) and brine (50 mL). The organic phase was dried over Na2SO4 and evaporated.

[0197] The residue was dissolved in pyridine (10 mL) and added dropwise to N,N'-carbonyldiimidazole (359 mg, 2.1 mmol) in pyridine (10 mL) with stirring and cooling in an ice bath. The mixture was stirred at room temperature overnight. Next, 9-aminononanol (477 mg, 3.0 mmol) in acetonitrile (4 mL) was added, and the mixture was stirred for 24 h. The solvent was evaporated, and the residue was partitioned between dichloromethane (100 mL) and 1 M NaH2PO4 (20 mL). The organic phase was washed with 1 M NaH2PO4 (2 × 50 mL) and then with brine (30 mL). After drying over Na2SO4, the organic phase was evaporated and dried under vacuum. The product was isolated by silica gel column purification using a linear gradient from DCM to 15% MeOH in DCM. The collected fractions were evaporated and dried in vacuo to give the desired product as a solid foam (1121 mg, 76.6%). ES MS:[M+H] + 733.4 (measured value), 732.9 (calculated value). Example 63 [ka] rel-(1R,5S,6S,7R)-N-methyl-N-[7-[(tris(4-methoxyphenyl)methoxy]-8-methyl-6-acetoxy-aza[bicyclo[3.2.1]octan-6-yl]-(9-hydroxy)nonanamide, 876

[0198] Fluorenylmethyloxycarbonyl chloride (310 mg, 1.2 mmol) in acetonitrile was added dropwise to compound 452 (561 mg, 1.0 mmol) and DIPEA (139 mg, 1.1 mmol) in acetonitrile (10 mL) while stirring in an ice bath. The mixture was stirred for 6 hours, after which pyridine (5 mL) and acetic anhydride (204 mg, 2 mmol) were added. The mixture was left overnight, and then 10% aqueous diethylamine solution (2.9 g, 4 mmol) was added. After stirring for 1 hour, the mixture was evaporated, and the residue was dissolved in ethyl acetate (50 mL) and washed with 5% aqueous NaHCO3 solution (2 x 50 mL) and brine (50 mL). The organic phase was dried over Na2SO4 and evaporated. The residue was dried on an oil pump.

[0199] Disuccinimidyl carbonate (384 mg, 1.5 mmol) was reacted with 9-hydroxynonanoic acid (261 mg, 1.5 mmol) in pyridine (5 mL) overnight. The resulting solution was added to the dried product from the previous step, and the mixture was allowed to react overnight. The solvent was evaporated, and the residue was partitioned between ethyl acetate (100 mL) and 5% aqueous NaHCO3 (20 mL). The organic phase was washed with 5% aqueous NaHCO3 (2 × 20 mL) and then with brine (30 mL). After drying over Na2SO4, the organic phase was evaporated and dried under vacuum. The product was isolated by silica gel column purification using a linear gradient from DCM to 15% MeOH in DCM. The collected fractions were evaporated and dried in vacuo to give the desired product as a solid foam (510 mg, 71.2%). ES MS: [M+H] + 718.7 (measured value), 717.9 (calculated value). Example 64 [ka] N-(2,9-dimethyl-2-methoxy-6-amino-hexahydro-6H-cyclohepta-1,3-dioxol-4,8-imin-6-yl)-(4-hydroxy)butanamide, 880

[0200] Disuccinimidyl carbonate (563 mg, 2.2 mmol) was reacted with 4-hydroxybutanoic acid (348 mg, 1.5 mmol) in pyridine (5 mL) overnight. The resulting solution was added to compound 25 (458 mg, 2.0 mmol), and the mixture was allowed to react overnight. The solvent was evaporated, and the residue was partitioned between ethyl acetate (100 mL) and 5% aqueous NaHCO3 (20 mL). The organic phase was washed with 5% aqueous NaHCO3 (2 × 20 mL) and then with brine (30 mL). After drying over Na2SO4, the organic phase was evaporated and dried under vacuum. The product was isolated by silica gel column purification using a linear gradient from DCM to 25% MeOH in DCM. The collected fractions were evaporated and dried in vacuo to give the desired product as a solid foam (560 mg, 89.1%). ES MS: [M+H] + 316.2 (measured value), 315.4 (calculated value). Example 65 [ka] (1S,5R,6R,7S)-[[6-acetoxy-7-(bis(4-methoxyphenyl)(phenyl)methoxy)-3-hydroxy-8-methyl-8-azabicyclo[3.2.1]octan-3-yl]oxy]-4-oxobutanoic acid, 531

[0201] To a solution of (1S,5R,6R,7S)-7-(bis(4-methoxyphenyl)(phenyl)methoxy)-3-hydroxy-8-methyl-8-azabicyclo[3.2.1]octan-6-yl acetate 331 (30.02 g, 58 mmol) in DCM (300 mL) was added iPrNEt (22.5 g, 174 mmol), dimethylaminopyridine (360 mg, 2.9 mmol), and succinic anhydride (11.7 g, 116.5 mmol). The mixture was stirred at room temperature for 3 h and quenched with water:triethylamine (9:1) (100 mL). The organic layer was diluted with DCM (300 mL) and extracted with water (500 mL). The organic extract was separated and evaporated. The crude residue was treated with a solution of water-triethylamine (19:1) (300 mL) and extracted with a solution of ethyl acetate:hexane (80:20) (500 mL). The organic extract was discarded, and the aqueous layer was acidified with 5% aqueous citric acid until a pH of 6.5-7.0 was reached. The product, which separated as an oil, was extracted with DCM (3 × 500 mL), and the extract was dried over sodium sulfate, evaporated, and dried on an oil pump. The product 531 was obtained as a white foam (34.03 g, 95.0%).

[0202] 1 H-NMR (500 MHz, CDCl3): δ 7.4 (m, 2H); 7.3 (m, 8H); 6.8 (m, 4H); 5.3 (m, 1H); 4.6 (m, 1H); 4.4 (d, 1H); 3.8 (s, 6H); 3.6 (s, 1H); 2.9 (s, 3H); 2.6 (m, 1H); 2.5 (m, 4H); 2.2 (s, 3H); 2.1 (m, 2H); 1.9 (m, 1H); 1.5 (m, 1H). 13 C-NMR (100 MHz, DMSO-d6): δ 14.05, 20.72, 20.96, 28.56, 28.7, 54.87, 55.08, 59.71, 113.33, 126.94, 127.89, 129.77, 129.86, 158.31, 169.65, 171.36, 173.19.ES MS:[M+H] + 618.3, [2M+H] +1235.5 (measured value); 617.3 (calculated value). Example 66 (rel-(1R,3-endo,5S,6S,7R)-7-(bis(4-methoxyphenyl)(phenyl)methoxy)-3-pivaloyloxy-8-(i-propyl)-8-azabicyclo[3.2.1]octan-6-yl)hemisuccinate, 503. (Figure 2)

[0203] A solution of compound 303 (2.17 mg, 3.69 mmol) and succinic anhydride (1.84 g, 18.44 mmol) in pyridine (67.1 mL) was stirred at 35° C. for 3 days. The mixture was quenched with water (0.66 mL) and triethylamine (5.0 mL) at room temperature for 2 hours and evaporated in vacuo. The residue was dissolved in DCM (100 mL) and washed with 0.5 M aqueous triethylammonium acetate. The organic phase was dried over Na2SO4, filtered, and evaporated in vacuo. The crude material was purified on a silica gel column using a linear gradient of MeOH (0 to 10% MeOH)-TEA (95:5) in DCM to give compound 503 (2.20 g, 86.7%) as a white solid foam. ES MS: [M-1] - 687.8. Example 67 (rel-(1R,3-endo,5S,6S,7R)-7-(bis(4-methoxyphenyl)(phenyl)methoxy)-3-isobutyryloxy-8-benzyl-8-azabicyclo[3.2.1]octan-6-yl) hemisuccinate, 505. (Figure 2)

[0204] A solution of compound 305 (3.07 mg, 4.94 mmol) and succinic anhydride (2.47 g, 24.68 mmol) in pyridine (89.8 mL) was stirred at 35° C. for 3 days. The mixture was quenched with water (0.89 mL) and triethylamine (5.0 mL) at room temperature for 2 hours and evaporated in vacuo. The residue was dissolved in DCM (100 mL) and washed with 0.5 M aqueous triethylammonium acetate. The organic phase was dried over Na2SO4, filtered, and evaporated in vacuo. The crude material was purified on a silica gel column using a linear gradient of MeOH (0 to 10% MeOH)-TEA (95:5) in DCM to give compound 505 (3.03 g, 85.0%) as a white solid foam. ES MS: [M-1] - 721.9. Example 68 (rel-(1R,3-endo,5S,6S,7R)-7-(tris(4-methoxyphenyl)methoxy)-3-acetoxy-8-methyl-8-azabicyclo[3.2.1]octan-6-yl) hemisuccinate, 601. (Figure 2)

[0205] A solution of compound 401 (2.82 mg, 5.15 mmol) and succinic anhydride (2.57 g, 25.73 mmol) in pyridine (93.6 mL) was stirred at 35 °C for 3 days. The mixture was quenched with water (0.93 mL) and triethylamine (5.0 mL) at room temperature for 2 hours and evaporated in vacuo. The residue was dissolved in DCM (100 mL) and washed with 0.5 M aqueous triethylammonium acetate. The organic phase was dried over Na2SO4, filtered, and evaporated in vacuo. The crude material was purified on a silica gel column using a linear gradient of MeOH (0 to 10% MeOH)-TEA (95:5) in DCM to give compound 601 (3.06 g, 91.7%) as a white solid foam. ES MS: [M-1] - 647.7. Example 69 (rel-(1R,3-endo,5S,6S,7R)-7-(tris(4-methoxyphenyl)methoxy)-3-pivaloyloxy-8-(i-propyl)-8-azabicyclo[3.2.1]octan-6-yl)hemisuccinate, 603. (Figure 2)

[0206] A solution of compound 403 (3.17 mg, 5.12 mmol) and succinic anhydride (2.56 g, 25.61 mmol) in pyridine (93.2 mL) was stirred at 35° C. for 3 days. The mixture was quenched with water (0.92 mL) and triethylamine (5.0 mL) at room temperature for 2 hours and evaporated in vacuo. The residue was dissolved in DCM (100 mL) and washed with 0.5 M aqueous triethylammonium acetate. The organic phase was dried over Na2SO4, filtered, and evaporated in vacuo. The crude material was purified on a silica gel column using a linear gradient of MeOH (0 to 10% MeOH)-TEA (95:5) in DCM to give compound 603 (3.04 g, 82.7%) as a white solid foam. ES MS: [M-1] - 717.9. Example 70 (rel-(1R,3-endo,5S,6S,7R)-7-(tris(4-methoxyphenyl)methoxy)-3-isobutyryloxy-8-methyl-8-azabicyclo[3.2.1]octan-6-yl) hemisuccinate, 605. (Figure 2)

[0207] A solution of compound 405 (3.07 mg, 4.70 mmol) and succinic anhydride (2.35 g, 23.51 mmol) in pyridine (85.6 mL) was stirred at 35° C. for 3 days. The mixture was quenched with water (0.85 mL) and triethylamine (5.0 mL) at room temperature for 2 hours and evaporated in vacuo. The residue was dissolved in DCM (100 mL) and washed with 0.5 M aqueous triethylammonium acetate. The organic phase was dried over Na2SO4, filtered, and evaporated in vacuo. The crude material was purified on a silica gel column using a linear gradient of MeOH (0 to 10% MeOH)-TEA (95:5) in DCM to give compound 605 (3.11 g, 88.1%) as a white solid foam. ES MS: [M-1] - 751.9. Example 71 (rel-(1R,3-endo,5S,6S,7R)-7-(tris(4-methoxyphenyl)methoxy)-3-[(t-butyl)dimethylsilyloxy]-8-methyl-8-azabicyclo[3.2.1]octan-6-yl)hemisuccinate, 616. (Figure 2)

[0208] A solution of compound 416 (2.67 mg, 4.30 mmol) and succinic anhydride (2.15 g, 21.51 mmol) in pyridine (78.3 mL) was stirred at 35° C. for 3 days. The mixture was quenched with water (0.77 mL) and triethylamine (5.0 mL) at room temperature for 2 hours and evaporated in vacuo. The residue was dissolved in DCM (100 mL) and washed with 0.5 M aqueous triethylammonium acetate. The organic phase was dried over Na2SO4, filtered, and evaporated in vacuo. The crude material was purified on a silica gel column using a linear gradient of MeOH (0 to 10% MeOH)-TEA (95:5) in DCM to give compound 616 (2.72 g, 87.9%) as a white solid foam. ES MS: [M-1] - 720.0. Example 72 (rel-(1R,3-endo,5S,6S,7R)-7-(tris(4-methoxyphenyl)methoxy)-3-[(t-butyl)diphenylsilyloxy]-8-methyl-8-azabicyclo[3.2.1]octan-6-yl) hemisuccinate, 618. (Figure 2)

[0209] A solution of compound 418 (3.13 mg, 4.21 mmol) and succinic anhydride (2.11 g, 21.05 mmol) in pyridine (76.6 mL) was stirred at 35° C. for 3 days. The mixture was quenched with water (0.76 mL) and triethylamine (5.0 mL) at room temperature for 2 hours and evaporated in vacuo. The residue was dissolved in DCM (100 mL) and washed with 0.5 M aqueous triethylammonium acetate. The organic phase was dried over Na2SO4, filtered, and evaporated in vacuo. The crude material was purified on a silica gel column using a linear gradient of MeOH (0 to 10% MeOH)-TEA (95:5) in DCM to give compound 618 (2.95 g, 82.9%) as a white solid foam. ES MS: [M-1] - 844.1. Example 73 (rel-(1R,3-endo,5S,6S,7R)-7-(tris(4-methoxyphenyl)methoxy)-8-methyl-8-azaspiro[bicyclo[3.2.1]octane-3,2'-[1,3]dioxolan]-6-yl) hemisuccinate, 621. (Figure 4)

[0210] A solution of compound 421 (2.19 mg, 4.01 mmol) and succinic anhydride (2.00 g, 20.03 mmol) in pyridine (72.9 mL) was stirred at 35° C. for 3 days. The mixture was quenched with water (0.72 mL) and triethylamine (5.0 mL) at room temperature for 2 hours and evaporated in vacuo. The residue was dissolved in DCM (100 mL) and washed with 0.5 M aqueous triethylammonium acetate. The organic phase was dried over Na2SO4, filtered, and evaporated in vacuo. The crude material was purified on a silica gel column using a linear gradient of MeOH (0 to 10% MeOH)-TEA (95:5) in DCM to give compound 621 (2.28 g, 87.8%) as a white solid foam. ES MS: [M-1] - 647.7. Example 74 (rel-(1R,3-endo,5S,6S,7R)-7-(tris(4-methoxyphenyl)methoxy)-8-methyl-8-azaspiro[bicyclo[3.2.1]octane-3,2'-[1,3]dioxan]-6-yl) hemisuccinate, 622. (Figure 4)

[0211] A solution of compound 422 (3.12 mg, 5.30 mmol) and succinic anhydride (2.65 g, 26.49 mmol) in pyridine (96.4 mL) was stirred at 35° C. for 3 days. The mixture was quenched with water (0.95 mL) and triethylamine (5.0 mL) at room temperature for 2 hours and evaporated in vacuo. The residue was dissolved in DCM (100 mL) and washed with 0.5 M aqueous triethylammonium acetate. The organic phase was dried over Na2SO4, filtered, and evaporated in vacuo. The crude material was purified on a silica gel column using a linear gradient of MeOH (0 to 10% MeOH)-TEA (95:5) in DCM to give compound 622 (3.20 g, 87.6%) as a white solid foam. ES MS: [M-1] - 689.8. Example 75 (rel-(1R,3-endo,5S,6S,7R)-7-(tris(4-methoxyphenyl)methoxy)-8-(i-propyl)-8-azaspiro[bicyclo[3.2.1]octane-3,2'-[1,3]dioxolan]-6-yl) hemisuccinate, 623. (Figure 4)

[0212] A solution of compound 423 (1.89 mg, 3.28 mmol) and succinic anhydride (1.64 g, 16.42 mmol) in pyridine (59.7 mL) was stirred at 35° C. for 3 days. The mixture was quenched with water (0.59 mL) and triethylamine (5.0 mL) at room temperature for 2 hours and evaporated in vacuo. The residue was dissolved in DCM (100 mL) and washed with 0.5 M aqueous triethylammonium acetate. The organic phase was dried over Na2SO4, filtered, and evaporated in vacuo. The crude material was purified on a silica gel column using a linear gradient of MeOH (0 to 10% MeOH)-TEA (95:5) in DCM to give compound 623 (1.99 g, 89.8%) as a white solid foam. ES MS: [M-1] - 675.8. Example 76 (rel-(1R,3-endo,5S,6S,7R)-7-(tris(4-methoxyphenyl)methoxy)-8-benzyl-8-azaspiro[bicyclo[3.2.1]octane-3,2'-[1,3]dioxan]-6-yl) hemisuccinate, 624. (Figure 4)

[0213] A solution of compound 424 (4.16 mg, 6.24 mmol) and succinic anhydride (3.12 g, 31.22 mmol) in pyridine (113.6 mL) was stirred at 35° C. for 3 days. The mixture was quenched with water (1.12 mL) and triethylamine (5.0 mL) at room temperature for 2 hours and evaporated in vacuo. The residue was dissolved in DCM (100 mL) and washed with 0.5 M aqueous triethylammonium acetate. The organic phase was dried over Na2SO4, filtered, and evaporated in vacuo. The crude material was purified on a silica gel column using a linear gradient of MeOH (0 to 10% MeOH)-TEA (95:5) in DCM to give compound 624 (4.16 g, 86.9%) as a white solid foam. ES MS: [M-1] - 765.9. Example 77 (rel-(1R,3-endo,5S,6S,7R)-7-(bis(4-methoxyphenyl)(phenyl)methoxy)-6-acetoxy-8-methyl-8-azabicyclo[3.2.1]octan-3-yl) hemisuccinate, 531. (Figure 6)

[0214] A solution of compound 331 (4.20 mg, 8.12 mmol) and succinic anhydride (4.06 g, 40.61 mmol) in pyridine (147.8 mL) was stirred at 35° C. for 3 days. The mixture was quenched with water (1.46 mL) and triethylamine (5.0 mL) at room temperature for 2 hours and evaporated in vacuo. The residue was dissolved in DCM (100 mL) and washed with 0.5 M aqueous triethylammonium acetate. The organic phase was dried over Na2SO4, filtered, and evaporated in vacuo. The crude material was purified on a silica gel column using a linear gradient of MeOH (0 to 10% MeOH)-TEA (95:5) in DCM to give compound 531 (4.38 g, 87.4%) as a white solid foam. ES MS: [M-1] - 617.7. Example 78 (rel-(1R,3-endo,5S,6S,7R)-7-(bis(4-methoxyphenyl)(phenyl)methoxy)-6-acetoxy-8-ethyl-8-azabicyclo[3.2.1]octan-3-yl) hemisuccinate, 532. (Figure 6)

[0215] A solution of compound 332 (2.39 mg, 4.49 mmol) and succinic anhydride (2.25 g, 22.46 mmol) in pyridine (81.7 mL) was stirred at 35° C. for 3 days. The mixture was quenched with water (0.81 mL) and triethylamine (5.0 mL) at room temperature for 2 hours and evaporated in vacuo. The residue was dissolved in DCM (100 mL) and washed with 0.5 M aqueous triethylammonium acetate. The organic phase was dried over Na2SO4, filtered, and evaporated in vacuo. The crude material was purified on a silica gel column using a linear gradient of MeOH (0 to 10% MeOH)-TEA (95:5) in DCM to give compound 532 (2.37 g, 83.4%) as a white solid foam. ES MS: [M-1] -631.7. Example 79 (rel-(1R,3-endo,5S,6S,7R)-7-(bis(4-methoxyphenyl)(phenyl)methoxy)-6-acetoxy-8-(i-propyl)-8-azabicyclo[3.2.1]octan-3-yl) hemisuccinate, 533. (Figure 6)

[0216] A solution of compound 333 (2.71 mg, 4.96 mmol) and succinic anhydride (2.48 g, 24.81 mmol) in pyridine (90.3 mL) was stirred at 35° C. for 3 days. The mixture was quenched with water (0.89 mL) and triethylamine (5.0 mL) at room temperature for 2 hours and evaporated in vacuo. The residue was dissolved in DCM (100 mL) and washed with 0.5 M aqueous triethylammonium acetate. The organic phase was dried over Na2SO4, filtered, and evaporated in vacuo. The crude material was purified on a silica gel column using a linear gradient of MeOH (0 to 10% MeOH)-TEA (95:5) in DCM to give compound 533 (2.80 g, 87.5%) as a white solid foam. ES MS: [M-1] - 645.8. (Example 80) (rel-(1R,3-endo,5S,6S,7R)-7-(bis(4-methoxyphenyl)(phenyl)methoxy)-6-acetoxy-8-benzyl-8-azabicyclo[3.2.1]octan-3-yl) hemisuccinate, 534. (Figure 6)

[0217] A solution of compound 334 (2.93 mg, 4.93 mmol) and succinic anhydride (2.47 g, 24.65 mmol) in pyridine (89.7 mL) was stirred at 35° C. for 3 days. The mixture was quenched with water (0.89 mL) and triethylamine (5.0 mL) at room temperature for 2 hours and evaporated in vacuo. The residue was dissolved in DCM (100 mL) and washed with 0.5 M aqueous triethylammonium acetate. The organic phase was dried over Na2SO4, filtered, and evaporated in vacuo. The crude material was purified on a silica gel column using a linear gradient of MeOH (0 to 10% MeOH)-TEA (95:5) in DCM to give compound 534 (2.95 g, 86.4%) as a white solid foam. ES MS: [M-1] - 693.8. Example 81 (rel-(1R,3-endo,5S,6S,7R)-7-(bis(4-methoxyphenyl)(phenyl)methoxy)-6-acetoxy-8-methyl-8-azabicyclo[3.2.1]octan-3-yl)diglycolate, 535. (Figure 6)

[0218] A solution of compound 331 (3.14 mg, 6.06 mmol) and succinic anhydride (3.03 g, 30.29 mmol) in pyridine (110.2 mL) was stirred at 35° C. for 3 days. The mixture was quenched with water (1.09 mL) and triethylamine (5.0 mL) at room temperature for 2 hours and evaporated in vacuo. The residue was dissolved in DCM (100 mL) and washed with 0.5 M aqueous triethylammonium acetate. The organic phase was dried over Na2SO4, filtered, and evaporated in vacuo. The crude material was purified on a silica gel column using a linear gradient of MeOH (0 to 10% MeOH)-TEA (95:5) in DCM to give compound 535 (3.19 g, 83.0%) as a white solid foam. ES MS: [M-1] - 633.7. Example 82 (rel-(1R,3-endo,5S,6S,7R)-7-(bis(4-methoxyphenyl)(phenyl)methoxy)-6-acetoxy-2,4,8-trimethyl-8-azabicyclo[3.2.1]octan-3-yl) hemisuccinate, 537. (Figure 6)

[0219] A solution of compound 337 (3.20 mg, 5.86 mmol) and succinic anhydride (2.93 g, 29.28 mmol) in pyridine (106.6 mL) was stirred at 35° C. for 3 days. The mixture was quenched with water (1.05 mL) and triethylamine (5.0 mL) at room temperature for 2 hours and evaporated in vacuo. The residue was dissolved in DCM (100 mL) and washed with 0.5 M aqueous triethylammonium acetate. The organic phase was dried over NaSO, filtered, and evaporated in vacuo. The crude material was purified on a silica gel column using a linear gradient of MeOH (0 to 10% MeOH)-TEA (95:5) in DCM to give compound 537 (3.48 g, 92.0%) as a white solid foam. ES MS: [M-1] - 645.8. Example 83 (rel-(1R,3-endo,5S,6S,7R)-7-(bis(4-methoxyphenyl)(phenyl)methoxy)-6-acetoxy-2,2,4,4,8-pentamethyl-8-azabicyclo[3.2.1]octan-3-yl) hemisuccinate, 538. (Figure 6)

[0220] A solution of compound 338 (4.00 mg, 6.97 mmol) and succinic anhydride (3.49 g, 34.83 mmol) in pyridine (126.8 mL) was stirred at 35° C. for 3 days. The mixture was quenched with water (1.25 mL) and triethylamine (5.0 mL) at room temperature for 2 hours and evaporated in vacuo. The residue was dissolved in DCM (100 mL) and washed with 0.5 M aqueous triethylammonium acetate. The organic phase was dried over NaSO, filtered, and evaporated in vacuo. The crude material was purified on a silica gel column using a linear gradient of MeOH (0 to 10% MeOH)-TEA (95:5) in DCM to give compound 538 (4.27 g, 91.0%) as a white solid foam. ES MS: [M-1] - 673.8. Example 84 (rel-(1R,3-endo,5S,6S,7R)-7-(tris(4-methoxyphenyl)methoxy)-6-acetoxy-8-methyl-8-azabicyclo[3.2.1]octan-3-yl) hemisuccinate, 631. (Figure 6)

[0221] A solution of compound 431 (3.49 mg, 6.36 mmol) and succinic anhydride (3.18 g, 31.78 mmol) in pyridine (115.7 mL) was stirred at 35° C. for 3 days. The mixture was quenched with water (1.14 mL) and triethylamine (5.0 mL) at room temperature for 2 hours and evaporated in vacuo. The residue was dissolved in DCM (100 mL) and washed with 0.5 M aqueous triethylammonium acetate. The organic phase was dried over Na2SO4, filtered, and evaporated in vacuo. The crude material was purified on a silica gel column using a linear gradient of MeOH (0 to 10% MeOH)-TEA (95:5) in DCM to give compound 631 (3.60 g, 87.5%) as a white solid foam. ES MS: [M-1] - 647.7. Example 85 (rel-(1R,3-endo,5S,6S,7R)-7-(tris(4-methoxyphenyl)methoxy)-6-acetoxy-8-ethyl-8-azabicyclo[3.2.1]octan-3-yl) hemisuccinate, 632. (Figure 6)

[0222] A solution of compound 432 (2.60 mg, 4.63 mmol) and succinic anhydride (2.32 g, 23.14 mmol) in pyridine (84.2 mL) was stirred at 35° C. for 3 days. The mixture was quenched with water (0.83 mL) and triethylamine (5.0 mL) at room temperature for 2 hours and evaporated in vacuo. The residue was dissolved in DCM (100 mL) and washed with 0.5 M aqueous triethylammonium acetate. The organic phase was dried over Na2SO4, filtered, and evaporated in vacuo. The crude material was purified on a silica gel column using a linear gradient of MeOH (0 to 10% MeOH)-TEA (95:5) in DCM to give compound 632 (2.69 g, 88.0%) as a white solid foam. ES MS: [M-1] - 661.8. Example 86 (rel-(1R,3-endo,5S,6S,7R)-7-(tris(4-methoxyphenyl)methoxy)-6-acetoxy-8-(i-propyl)-8-azabicyclo[3.2.1]octan-3-yl) hemisuccinate, 633. (Figure 6)

[0223] A solution of compound 433 (2.80 mg, 4.86 mmol) and succinic anhydride (2.43 g, 24.28 mmol) in pyridine (88.4 mL) was stirred at 35° C. for 3 days. The mixture was quenched with water (0.87 mL) and triethylamine (5.0 mL) at room temperature for 2 hours and evaporated in vacuo. The residue was dissolved in DCM (100 mL) and washed with 0.5 M aqueous triethylammonium acetate. The organic phase was dried over Na2SO4, filtered, and evaporated in vacuo. The crude material was purified on a silica gel column using a linear gradient of MeOH (0 to 10% MeOH)-TEA (95:5) in DCM to give compound 633 (2.94 g, 89.7%) as a white solid foam. ES MS: [M-1] - 675.8. Example 87 (rel-(1R,3-endo,5S,6S,7R)-7-(tris(4-methoxyphenyl)methoxy)-6-acetoxy-8-benzyl-8-azabicyclo[3.2.1]octan-3-yl) hemisuccinate, 634. (Figure 6)

[0224] A solution of compound 434 (2.50 mg, 4.00 mmol) and succinic anhydride (2.00 g, 19.98 mmol) in pyridine (72.7 mL) was stirred at 35° C. for 3 days. The mixture was quenched with water (0.72 mL) and triethylamine (5.0 mL) at room temperature for 2 hours and evaporated in vacuo. The residue was dissolved in DCM (100 mL) and washed with 0.5 M aqueous triethylammonium acetate. The organic phase was dried over Na2SO4, filtered, and evaporated in vacuo. The crude material was purified on a silica gel column using a linear gradient of MeOH (0 to 10% MeOH)-TEA (95:5) in DCM to give compound 634 (2.64 g, 91.2%) as a white solid foam. ES MS: [M-1] - 723.8. Example 88 (rel-(1R,3-endo,5S,6S,7R)-7-(tris(4-methoxyphenyl)methoxy)-6-acetoxy-8-methyl-8-azabicyclo[3.2.1]octan-3-yl)[2-[4-(carboxymethoxy)phenoxy]acetate], 636. (Figure 6)

[0225] A solution of compound 431 (2.83 mg, 5.17 mmol) and succinic anhydride (2.59 g, 25.83 mmol) in pyridine (94.0 mL) was stirred at 35° C. for 3 days. The mixture was quenched with water (0.93 mL) and triethylamine (5.0 mL) at room temperature for 2 hours and evaporated in vacuo. The residue was dissolved in DCM (100 mL) and washed with 0.5 M aqueous triethylammonium acetate. The organic phase was dried over Na2SO4, filtered, and evaporated in vacuo. The crude material was purified on a silica gel column using a linear gradient of MeOH (0 to 10% MeOH)-TEA (95:5) in DCM to give compound 636 (3.36 g, 86.0%) as a white solid foam. ES MS: [M-1]- 755.8. Example 89 (rel-(1R,3-endo,5S,6S,7R)-7-(tris(4-methoxyphenyl)methoxy)-6-acetoxy-2,4,8-trimethyl-8-azabicyclo[3.2.1]octan-3-yl) hemisuccinate, 637. (Figure 6)

[0226] A solution of compound 437 (3.00 mg, 5.20 mmol) and succinic anhydride (2.60 g, 25.98 mmol) in pyridine (94.6 mL) was stirred at 35° C. for 3 days. The mixture was quenched with water (0.94 mL) and triethylamine (5.0 mL) at room temperature for 2 hours and evaporated in vacuo. The residue was dissolved in DCM (100 mL) and washed with 0.5 M aqueous triethylammonium acetate. The organic phase was dried over NaSO, filtered, and evaporated in vacuo. The crude material was purified on a silica gel column using a linear gradient of MeOH (0 to 10% MeOH)-TEA (95:5) in DCM to give compound 637 (3.04 g, 86.7%) as a white solid foam. ES MS: [M-1] - 675.8. Example 90 (rel-(1R,3-endo,5S,6S,7R)-7-(tris(4-methoxyphenyl)methoxy)-6-acetoxy-2,2,4,4,8-pentamethyl-8-azabicyclo[3.2.1]octan-3-yl) hemisuccinate, 638. (Figure 6)

[0227] A solution of compound 438 (3.15 mg, 5.22 mmol) and succinic anhydride (2.61 g, 26.08 mmol) in pyridine (94.9 mL) was stirred at 35° C. for 3 days. The mixture was quenched with water (0.94 mL) and triethylamine (5.0 mL) at room temperature for 2 hours and evaporated in vacuo. The residue was dissolved in DCM (100 mL) and washed with 0.5 M aqueous triethylammonium acetate. The organic phase was dried over Na2SO4, filtered, and evaporated in vacuo. The crude material was purified on a silica gel column using a linear gradient of MeOH (0 to 10% MeOH)-TEA (95:5) in DCM to give compound 638 (3.19 g, 86.9%) as a white solid foam. ES MS: [M-1] - 703.8. Example 91 (rel-(1R,3-endo,5S,6S,7R)-7-(tris(4-methoxyphenyl)methoxy)-3-methoxy-8,8-dimethyl-8-azoniabicyclo[3.2.1]octan-6-yl) hemisuccinate, 671. (Figure 3)

[0228] A solution of compound 411 (2.83 mg, 4.77 mmol) and succinic anhydride (2.39 g, 23.86 mmol) in pyridine (86.8 mL) was stirred at 35 °C for 3 days. The mixture was quenched with water (0.86 mL) and triethylamine (5.0 mL) at room temperature for 2 hours and evaporated in vacuo. The residue was dissolved in DCM (100 mL) and washed with 0.5 M aqueous triethylammonium acetate. The organic phase was dried over Na2SO4, filtered, and evaporated in vacuo. The crude material was purified on a silica gel column using a linear gradient of MeOH (0 to 10% MeOH)-TEA (95:5) in DCM to give compound 671 (3.23 g, 85.2%) as a white solid foam. ES MS: [M-1] - 794.0. Example 92 (rel-(1R,3-endo,5S,6S,7R)-7-(tris(4-methoxyphenyl)methoxy)-3-benzyloxy-8-methyl-8-benzyl-8-azoniabicyclo[3.2.1]octan-6-yl) hemisuccinate, 674. (Figure 3)

[0229] A solution of compound 414 (3.55 mg, 4.77 mmol) and succinic anhydride (2.38 g, 23.83 mmol) in pyridine (86.7 mL) was stirred at 35° C. for 3 days. The mixture was quenched with water (0.86 mL) and triethylamine (5.0 mL) at room temperature for 2 hours and evaporated in vacuo. The residue was dissolved in DCM (100 mL) and washed with 0.5 M aqueous triethylammonium acetate. The organic phase was dried over Na2SO4, filtered, and evaporated in vacuo. The crude material was purified on a silica gel column using a linear gradient of MeOH (0 to 10% MeOH)-TEA (95:5) in DCM to give compound 674 (3.99 g, 88.4%) as a white solid foam. ES MS: [M-1] - 946.2. Example 93 (rel-(1R,3-endo,5S,6S,7R)-7-(tris(4-methoxyphenyl)methoxy)-3-methoxy-8-methyl-8-benzyl-8-azoniabicyclo[3.2.1]octan-6-yl) hemisuccinate, 675. (Figure 3)

[0230] A solution of compound 415 (3.39 mg, 5.06 mmol) and succinic anhydride (2.53 g, 25.31 mmol) in pyridine (92.1 mL) was stirred at 35° C. for 3 days. The mixture was quenched with water (0.91 mL) and triethylamine (5.0 mL) at room temperature for 2 hours and evaporated in vacuo. The residue was dissolved in DCM (100 mL) and washed with 0.5 M aqueous triethylammonium acetate. The organic phase was dried over Na2SO4, filtered, and evaporated in vacuo. The crude material was purified on a silica gel column using a linear gradient of MeOH (0 to 10% MeOH)-TEA (95:5) in DCM to give compound 675 (3.59 g, 81.4%) as a white solid foam. ES MS: [M-1]- 870.1. Example 94 N-[(rel-(1R,3-endo,5S,6S,7R)-7-(bis(4-methoxyphenyl)(phenyl)methoxy)-6-acetoxy-8-methyl-8-azabicyclo[3.2.1]octan-3-yl)]succinamic acid, 551. (Figure 5)

[0231] A solution of compound 351 (2.41 mg, 4.66 mmol) and succinic anhydride (2.33 g, 23.29 mmol) in pyridine (84.8 mL) was stirred at 35° C. for 3 days. The mixture was quenched with water (0.84 mL) and triethylamine (5.0 mL) at room temperature for 2 hours and evaporated in vacuo. The residue was dissolved in DCM (100 mL) and washed with 0.5 M aqueous triethylammonium acetate. The organic phase was dried over Na2SO4, filtered, and evaporated in vacuo. The crude material was purified on a silica gel column using a linear gradient of MeOH (0 to 10% MeOH)-TEA (95:5) in DCM to give compound 551 (2.45 g, 85.2%) as a white solid foam. ES MS: [M-1] - 616.7. Example 95 N-[(rel-(1R,3-endo,5S,6S,7R)-7-(bis(4-methoxyphenyl)(phenyl)methoxy)-6-acetoxy-8-(i-propyl)-8-azabicyclo[3.2.1]octan-3-yl)]succinamic acid, 553. (Figure 5)

[0232] A solution of compound 353 (2.00 mg, 3.67 mmol) and succinic anhydride (1.84 g, 18.35 mmol) in pyridine (66.8 mL) was stirred at 35° C. for 3 days. The mixture was quenched with water (0.66 mL) and triethylamine (5.0 mL) at room temperature for 2 hours and evaporated in vacuo. The residue was dissolved in DCM (100 mL) and washed with 0.5 M aqueous triethylammonium acetate. The organic phase was dried over NaSO, filtered, and evaporated in vacuo. The crude material was purified on a silica gel column using a linear gradient of MeOH (0 to 10% MeOH)-TEA (95:5) in DCM to give compound 553 (2.01 g, 84.9%) as a white solid foam. ES MS: [M-1] - 644.8. Example 96 N-[(rel-(1R,3-endo,5S,6S,7R)-7-(tris(4-methoxyphenyl)methoxy)-6-acetoxy-8-methyl-8-azabicyclo[3.2.1]octan-3-yl)]succinamic acid, 651. (Figure 5)

[0233] A solution of compound 451 (1.82 mg, 3.33 mmol) and succinic anhydride (1.66 g, 16.64 mmol) in pyridine (60.5 mL) was stirred at 35 °C for 3 days. The mixture was quenched with water (0.60 mL) and triethylamine (5.0 mL) at room temperature for 2 hours and evaporated in vacuo. The residue was dissolved in DCM (100 mL) and washed with 0.5 M aqueous triethylammonium acetate. The organic phase was dried over Na2SO4, filtered, and evaporated in vacuo. The crude material was purified on a silica gel column using a linear gradient of MeOH (0 to 10% MeOH)-TEA (95:5) in DCM to give compound 651 (1.80 g, 83.5%) as a white solid foam. ES MS: [M-1] - 646. Example 97 N-Methyl-N-[(rel-(1R,3-endo,5S,6S,7R)-7-(tris(4-methoxyphenyl)methoxy)-6-acetoxy-8-methyl-8-azabicyclo[3.2.1]octan-3-yl)]succinamic acid, 652.

[0234] A solution of compound 452 (2.23 mg, 3.97 mmol) and succinic anhydride (1.99 g, 19.87 mmol) in pyridine (72.3 mL) was stirred at 35° C. for 3 days. The mixture was quenched with water (0.72 mL) and triethylamine (5.0 mL) at room temperature for 2 hours and evaporated in vacuo. The residue was dissolved in DCM (100 mL) and washed with 0.5 M aqueous triethylammonium acetate solution. The organic phase was dried over Na2SO4, filtered, and evaporated in vacuo. The crude material was purified on a silica gel column using a linear gradient of MeOH (0 to 10% MeOH)-TEA in DCM. Example 98 [ka] (rel-(1R,3-endo,5S,6S,7R)-7-[(bis(4-methoxyphenyl)(phenyl)methoxy]-3-acetoxy-8-methyl-aza[bicyclo[3.2.1]octan-6-yl]hemisuccinate, 701c) covalently bound to aminopropyl CPG500.

[0235] TBTU (0.26 g, 0.81 mmol) was added to a solution of compound 501 (0.48 g, 0.77 mmol) and N,N-diisopropylethylamine (0.239 g, 1.85 mmol) in a mixture of anhydrous pyridine (1.25 mL) and acetonitrile (8 mL). The mixture was stirred for 15 minutes, transferred to a suspension of aminopropyl CPG500 (10 g) in anhydrous acetonitrile (45 mL), and the resulting suspension was shaken for 4 hours. N-methylimidazole (2.0 mL) and acetic anhydride (2.0 mL) were then added to the suspension, which was then shaken again for 45 minutes. The solid support was filtered off, washed on the filter with acetonitrile (5 × 50 mL), and dried in vacuo. The loading of the finished solid support 701c (69.0 μmol / g), where applicable, was determined by standard di- or trimethoxytrityl assays as disclosed in Guzaev, AP and Pon, RT Curr. Protoc. Nucleic Acid Chem. 2013, 52, pp. 3.2.1-3.2.23. Example 99 (rel-(1R,3-endo,5S,6S,7R)-7-(bis(4-methoxyphenyl)(phenyl)methoxy)-3-pivaloyloxy-8-(i-propyl)-8-azabicyclo[3.2.1]octan-6-yl) hemisuccinate covalently bound to aminopropyl CPG1500, solid support 703c. (FIG. 10)

[0236] Following the procedure described in Example 98, compound 503 (0.36 g, 0.53 mmol) was activated with TBTU (0.178 g, 0.55 mmol) and N,N-diisopropylethylamine (0.164 g, 1.27 mmol) in a mixture of pyridine (0.85 mL) and acetonitrile (6 mL) and reacted with aminopropyl CPG (15.0 g) in anhydrous acetonitrile (68 mL). The solid support was capped with N-methylimidazole (1.4 mL) and acetic anhydride (1.4 mL), filtered, washed with acetonitrile (5 × 68 mL), and dried to give a loading of 32.0 μmol / g. Example 100 (rel-(1R,3-endo,5S,6S,7R)-7-(bis(4-methoxyphenyl)(phenyl)methoxy)-3-isobutyryloxy-8-benzyl-8-azabicyclo[3.2.1]octan-6-yl) hemisuccinate covalently bound to aminopropyl CPG1000, solid support 705c. (FIG. 10)

[0237] Following the procedure described in Example 98, compound 505 (0.71 g, 0.99 mmol) was activated with TBTU (0.334 g, 1.04 mmol) and N,N-diisopropylethylamine (0.307 g, 2.38 mmol) in a mixture of pyridine (1.60 mL) and acetonitrile (10 mL) and reacted with aminopropyl CPG (20.0 g) in anhydrous acetonitrile (90 mL). The solid support was capped with N-methylimidazole (2.6 mL) and acetic anhydride (2.6 mL), filtered, washed with acetonitrile (5 × 90 mL), and dried to give a loading of 43.7 μmol / g. Example 101 (rel-(1R,3-endo,5S,6S,7R)-7-(tris(4-methoxyphenyl)methoxy)-3-acetoxy-8-methyl-8-azabicyclo[3.2.1]octan-6-yl) hemisuccinate covalently bound to aminopropyl CPG1000, solid support 801c. (Figure 11)

[0238] Following the procedure described in Example 98, compound 601 (0.8 g, 1.24 mmol) was activated with TBTU (0.417 g, 1.30 mmol) and N,N-diisopropylethylamine (0.384 g, 2.97 mmol) in a mixture of pyridine (2.00 mL) and acetonitrile (13 mL) and reacted with aminopropyl CPG (25.0 g) in anhydrous acetonitrile (113 mL). The solid support was capped with N-methylimidazole (3.3 mL) and acetic anhydride (3.3 mL), filtered, washed with acetonitrile (5 × 113 mL), and dried to give a loading of 44.2 μmol / g. Example 102 (rel-(1R,3-endo,5S,6S,7R)-7-(tris(4-methoxyphenyl)methoxy)-3-pivaloyloxy-8-(i-propyl)-8-azabicyclo[3.2.1]octan-6-yl) hemisuccinate covalently bound to aminopropyl CPG1000, solid support 803c. (Figure 11)

[0239] Following the procedure described in Example 98, compound 603 (0.53 g, 0.74 mmol) was activated with TBTU (0.250 g, 0.78 mmol) and N,N-diisopropylethylamine (0.23 g, 1.78 mmol) in a mixture of pyridine (1.20 mL) and acetonitrile (8 mL) and reacted with aminopropyl CPG (15.0 g) in anhydrous acetonitrile (68 mL). The solid support was capped with N-methylimidazole (2.0 mL) and acetic anhydride (2 mL), filtered, washed with acetonitrile (5 × 68 mL), and dried to give a loading of 44.8 μmol / g. (Example 103) (rel-(1R,3-endo,5S,6S,7R)-7-(tris(4-methoxyphenyl)methoxy)-3-isobutyryloxy-8-methyl-8-azabicyclo[3.2.1]octan-6-yl) hemisuccinate covalently bound to aminopropyl CPG500, solid support 805c.

[0240] Following the procedure described in Example 98, compound 605 (1.05 g, 1.4 mmol) was activated with TBTU (0.473 g, 1.47 mmol) and N,N-diisopropylethylamine (0.435 g, 3.37 mmol) in a mixture of pyridine (2.27 mL) and acetonitrile (15 mL) and reacted with aminopropyl CPG (17.0 g) in anhydrous acetonitrile (77 mL). The solid support was capped with N-methylimidazole (3.7 mL) and acetic anhydride (3.7 mL), filtered, washed with acetonitrile (5 × 77 mL), and dried to give a loading of 73.1 μmol / g. Example 104 (rel-(1R,3-endo,5S,6S,7R)-7-(tris(4-methoxyphenyl)methoxy)-3-[(t-butyl)dimethylsilyloxy]-8-methyl-8-azabicyclo[3.2.1]octan-6-yl) hemisuccinate covalently bound to aminopropyl CPG1500, solid support 816c. (FIG. 11)

[0241] Following the procedure described in Example 98, compound 616 (0.3 g, 0.42 mmol) was activated with TBTU (0.142 g, 0.44 mmol) and N,N-diisopropylethylamine (0.131 g, 1.01 mmol) in a mixture of pyridine (0.68 mL) and acetonitrile (4 mL) and reacted with aminopropyl CPG (12.0 g) in anhydrous acetonitrile (54 mL). The solid support was capped with N-methylimidazole (1.1 mL) and acetic anhydride (1.1 mL), filtered, washed with acetonitrile (5 × 54 mL), and dried to give a loading of 32.4 μmol / g. Example 105 (rel-(1R,3-endo,5S,6S,7R)-7-(tris(4-methoxyphenyl)methoxy)-3-[(t-butyl)diphenylsilyloxy]-8-methyl-8-azabicyclo[3.2.1]octan-6-yl) hemisuccinate covalently bound to aminopropyl CPG1000, solid support 818c. (FIG. 11)

[0242] Following the procedure described in Example 98, compound 618 (0.29 g, 0.35 mmol) was activated with TBTU (0.117 g, 0.36 mmol) and N,N-diisopropylethylamine (0.107 g, 0.83 mmol) in a mixture of pyridine (0.56 mL) and acetonitrile (4 mL) and reacted with aminopropyl CPG (7.0 g) in anhydrous acetonitrile (32 mL). The solid support was capped with N-methylimidazole (0.9 mL) and acetic anhydride (0.9 mL), filtered, washed with acetonitrile (5 × 32 mL), and dried to give a loading of 43.8 μmol / g. Example 106 (rel-(1R,3-endo,5S,6S,7R)-7-(tris(4-methoxyphenyl)methoxy)-8-methyl-8-azaspiro[bicyclo[3.2.1]octane-3,2'-[1,3]dioxolan]-6-yl) hemisuccinate covalently bound to aminopropyl CPG500, solid support 821c. (Figure 11)

[0243] Following the procedure described in Example 98, compound 621 (0.85 g, 1.32 mmol) was activated with TBTU (0.445 g, 1.39 mmol) and N,N-diisopropylethylamine (0.409 g, 3.17 mmol) in a mixture of pyridine (2.14 mL) and acetonitrile (14 mL) and reacted with aminopropyl CPG (16.0 g) in anhydrous acetonitrile (72 mL). The solid support was capped with N-methylimidazole (3.5 mL) and acetic anhydride (3.5 mL), filtered, washed with acetonitrile (5 × 72 mL), and dried to give a loading of 74.4 μmol / g. (Example 107) (rel-(1R,3-endo,5S,6S,7R)-7-(tris(4-methoxyphenyl)methoxy)-8-methyl-8-azaspiro[bicyclo[3.2.1]octane-3,2'-[1,3]dioxan]-6-yl) hemisuccinate covalently bound to aminopropyl CPG1000, solid support 822c. (Figure 11)

[0244] Following the procedure described in Example 98, compound 622 (0.68 g, 0.99 mmol) was activated with TBTU (0.334 g, 1.04 mmol) and N,N-diisopropylethylamine (0.307 g, 2.38 mmol) in a mixture of pyridine (1.60 mL) and acetonitrile (10 mL) and reacted with aminopropyl CPG (20.0 g) in anhydrous acetonitrile (90 mL). The solid support was capped with N-methylimidazole (2.6 mL) and acetic anhydride (2.6 mL), filtered, washed with acetonitrile (5 × 90 mL), and dried to give a loading of 44.1 μmol / g. Example 108 (rel-(1R,3-endo,5S,6S,7R)-7-(tris(4-methoxyphenyl)methoxy)-8-(i-propyl)-8-azaspiro[bicyclo[3.2.1]octane-3,2'-[1,3]dioxolan]-6-yl) hemisuccinate covalently bound to aminopropyl CPG1500, solid support 823c. (Figure 11)

[0245] Following the procedure described in Example 98, compound 623 (0.55 g, 0.81 mmol) was activated with TBTU (0.273 g, 0.85 mmol) and N,N-diisopropylethylamine (0.251 g, 1.94 mmol) in a mixture of pyridine (1.31 mL) and acetonitrile (8 mL) and reacted with aminopropyl CPG (23.0 g) in anhydrous acetonitrile (104 mL). The solid support was capped with N-methylimidazole (2.1 mL) and acetic anhydride (2.1 mL), filtered, washed with acetonitrile (5 × 104 mL), and dried to give a loading of 31.6 μmol / g. Example 109 (rel-(1R,3-endo,5S,6S,7R)-7-(tris(4-methoxyphenyl)methoxy)-8-benzyl-8-azaspiro[bicyclo[3.2.1]octane-3,2'-[1,3]dioxan]-6-yl) hemisuccinate covalently bound to aminopropyl CPG500, solid support 824c. (Figure 11)

[0246] Following the procedure described in Example 98, compound 624 (1.26 g, 1.65 mmol) was activated with TBTU (0.556 g, 1.73 mmol) and N,N-diisopropylethylamine (0.512 g, 3.96 mmol) in a mixture of pyridine (2.67 mL) and acetonitrile (17 mL) and reacted with aminopropyl CPG (20.0 g) in anhydrous acetonitrile (90 mL). The solid support was capped with N-methylimidazole (4.3 mL) and acetic anhydride (4.4 mL), filtered, washed with acetonitrile (5 × 90 mL), and dried to give a loading of 72.9 μmol / g. Example 110 Solid support 731c covalently bound to aminopropyl CPG500 (rel-(1R,3-endo,5S,6S,7R)-7-(bis(4-methoxyphenyl)(phenyl)methoxy)-6-acetoxy-8-methyl-8-azabicyclo[3.2.1]octan-3-yl) hemisuccinate. (FIG. 10)

[0247] Following the procedure described in Example 98, compound 531 (2.55 g, 4.13 mmol) was activated with TBTU (1.391 g, 4.33 mmol) and N,N-diisopropylethylamine (1.28 g, 9.9 mmol) in a mixture of pyridine (6.67 mL) and acetonitrile (43 mL) and reacted with aminopropyl CPG (50.0 g) in anhydrous acetonitrile (225 mL). The solid support was capped with N-methylimidazole (10.9 mL) and acetic anhydride (10.9 mL), filtered, washed with acetonitrile (5 x 225 mL), and dried to give a loading of 73.9 μmol / g. Example 111 (rel-(1R,3-endo,5S,6S,7R)-7-(bis(4-methoxyphenyl)(phenyl)methoxy)-6-acetoxy-8-ethyl-8-azabicyclo[3.2.1]octan-3-yl) hemisuccinate covalently bound to aminopropyl CPG1000, solid support 732c. (FIG. 10)

[0248] Following the procedure described in Example 98, compound 532 (0.78 g, 1.24 mmol) was activated with TBTU (0.417 g, 1.30 mmol) and N,N-diisopropylethylamine (0.384 g, 2.97 mmol) in a mixture of pyridine (2.00 mL) and acetonitrile (13 mL) and reacted with aminopropyl CPG (25.0 g) in anhydrous acetonitrile (113 mL). The solid support was capped with N-methylimidazole (3.3 mL) and acetic anhydride (3.3 mL), filtered, washed with acetonitrile (5 × 113 mL), and dried to give a loading of 44.4 μmol / g. Example 112 (rel-(1R,3-endo,5S,6S,7R)-7-(bis(4-methoxyphenyl)(phenyl)methoxy)-6-acetoxy-8-(i-propyl)-8-azabicyclo[3.2.1]octan-3-yl) hemisuccinate covalently bound to aminopropyl CPG1500, solid support 733c. (Figure 10)

[0249] Following the procedure described in Example 98, compound 533 (0.5 g, 0.77 mmol) was activated with TBTU (0.261 g, 0.81 mmol) and N,N-diisopropylethylamine (0.24 g, 1.86 mmol) in a mixture of pyridine (1.25 mL) and acetonitrile (8 mL) and reacted with aminopropyl CPG (22.0 g) in anhydrous acetonitrile (99 mL). The solid support was capped with N-methylimidazole (2.0 mL) and acetic anhydride (2 mL), filtered, washed with acetonitrile (5 × 99 mL), and dried to give a loading of 31.8 μmol / g. Example 113 (rel-(1R,3-endo,5S,6S,7R)-7-(bis(4-methoxyphenyl)(phenyl)methoxy)-6-acetoxy-8-benzyl-8-azabicyclo[3.2.1]octan-3-yl) hemisuccinate covalently bound to aminopropyl CPG1000, solid support 734c. (FIG. 10)

[0250] Following the procedure described in Example 98, compound 534 (0.69 g, 0.99 mmol) was activated with TBTU (0.334 g, 1.04 mmol) and N,N-diisopropylethylamine (0.307 g, 2.38 mmol) in a mixture of pyridine (1.60 mL) and acetonitrile (10 mL) and reacted with aminopropyl CPG (20.0 g) in anhydrous acetonitrile (90 mL). The solid support was capped with N-methylimidazole (2.6 mL) and acetic anhydride (2.6 mL), filtered, washed with acetonitrile (5 × 90 mL), and dried to give a loading of 41.5 μmol / g. Example 114 (rel-(1R,3-endo,5S,6S,7R)-7-(bis(4-methoxyphenyl)(phenyl)methoxy)-6-acetoxy-8-methyl-8-azabicyclo[3.2.1]octan-3-yl)diglycolate covalently bound to aminopropyl CPG500, solid support 735c. (Figure 10)

[0251] Following the procedure described in Example 98, compound 535 (1.44 g, 2.27 mmol) was activated with TBTU (0.765 g, 2.38 mmol) and N,N-diisopropylethylamine (0.704 g, 5.45 mmol) in a mixture of pyridine (3.67 mL) and acetonitrile (24 mL) and reacted with aminopropyl CPG (27.5 g) in anhydrous acetonitrile (124 mL). The solid support was capped with N-methylimidazole (6.0 mL) and acetic anhydride (6 mL), filtered, washed with acetonitrile (5 × 124 mL), and dried to give a loading of 72.6 μmol / g. Example 115 (rel-(1R,3-endo,5S,6S,7R)-7-(bis(4-methoxyphenyl)(phenyl)methoxy)-6-acetoxy-2,4,8-trimethyl-8-azabicyclo[3.2.1]octan-3-yl) hemisuccinate covalently bound to aminopropyl CPG500, solid support 737c. (Figure 10)

[0252] Following the procedure described in Example 98, compound 537 (1.07 g, 1.65 mmol) was activated with TBTU (0.556 g, 1.73 mmol) and N,N-diisopropylethylamine (0.512 g, 3.96 mmol) in a mixture of pyridine (2.67 mL) and acetonitrile (17 mL) and reacted with aminopropyl CPG (20.0 g) in anhydrous acetonitrile (90 mL). The solid support was capped with N-methylimidazole (4.3 mL) and acetic anhydride (4.4 mL), filtered, washed with acetonitrile (5 × 90 mL), and dried to give a loading of 74.9 μmol / g. Example 116 (rel-(1R,3-endo,5S,6S,7R)-7-(bis(4-methoxyphenyl)(phenyl)methoxy)-6-acetoxy-2,2,4,4,8-pentamethyl-8-azabicyclo[3.2.1]octan-3-yl) hemisuccinate covalently bound to aminopropyl CPG500, solid support 738c. (Figure 10)

[0253] Following the procedure described in Example 98, compound 538 (1.39 g, 2.06 mmol) was activated with TBTU (0.695 g, 2.17 mmol) and N,N-diisopropylethylamine (0.64 g, 4.95 mmol) in a mixture of pyridine (3.34 mL) and acetonitrile (22 mL) and reacted with aminopropyl CPG (25.0 g) in anhydrous acetonitrile (113 mL). The solid support was capped with N-methylimidazole (5.4 mL) and acetic anhydride (5.4 mL), filtered, washed with acetonitrile (5 × 113 mL), and dried to give a loading of 73.5 μmol / g. Example 117 (rel-(1R,3-endo,5S,6S,7R)-7-(tris(4-methoxyphenyl)methoxy)-6-acetoxy-8-methyl-8-azabicyclo[3.2.1]octan-3-yl) hemisuccinate covalently bound to aminopropyl CPG500, solid support 831c. (Figure 11)

[0254] Following the procedure described in Example 98, compound 631 (1.07 g, 1.65 mmol) was activated with TBTU (0.556 g, 1.73 mmol) and N,N-diisopropylethylamine (0.512 g, 3.96 mmol) in a mixture of pyridine (2.67 mL) and acetonitrile (17 mL) and reacted with aminopropyl CPG (20.0 g) in anhydrous acetonitrile (90 mL). The solid support was capped with N-methylimidazole (4.3 mL) and acetic anhydride (4.4 mL), filtered, washed with acetonitrile (5 × 90 mL), and dried to give a loading of 72.7 μmol / g. Example 118 (rel-(1R,3-endo,5S,6S,7R)-7-(tris(4-methoxyphenyl)methoxy)-6-acetoxy-8-ethyl-8-azabicyclo[3.2.1]octan-3-yl) hemisuccinate covalently bound to aminopropyl CPG1000, solid support 832c. (Figure 11)

[0255] Following the procedure described in Example 98, compound 632 (0.66 g, 0.99 mmol) was activated with TBTU (0.334 g, 1.04 mmol) and N,N-diisopropylethylamine (0.307 g, 2.38 mmol) in a mixture of pyridine (1.60 mL) and acetonitrile (10 mL) and reacted with aminopropyl CPG (20.0 g) in anhydrous acetonitrile (90 mL). The solid support was capped with N-methylimidazole (2.6 mL) and acetic anhydride (2.6 mL), filtered, washed with acetonitrile (5 × 90 mL), and dried to give a loading of 42.2 μmol / g. Example 119 (rel-(1R,3-endo,5S,6S,7R)-7-(tris(4-methoxyphenyl)methoxy)-6-acetoxy-8-(i-propyl)-8-azabicyclo[3.2.1]octan-3-yl) hemisuccinate covalently bound to aminopropyl CPG1500, solid support 833c. (Figure 11)

[0256] Following the procedure described in Example 98, compound 633 (0.48 g, 0.7 mmol) was activated with TBTU (0.237 g, 0.74 mmol) and N,N-diisopropylethylamine (0.218 g, 1.69 mmol) in a mixture of pyridine (1.14 mL) and acetonitrile (7 mL) and reacted with aminopropyl CPG (20.0 g) in anhydrous acetonitrile (90 mL). The solid support was capped with N-methylimidazole (1.9 mL) and acetic anhydride (1.9 mL), filtered, washed with acetonitrile (5 × 90 mL), and dried to give a loading of 31.5 μmol / g. Example 120 (rel-(1R,3-endo,5S,6S,7R)-7-(tris(4-methoxyphenyl)methoxy)-6-acetoxy-8-benzyl-8-azabicyclo[3.2.1]octan-3-yl) hemisuccinate covalently bound to aminopropyl CPG1000, solid support 834c. (Figure 11)

[0257] Following the procedure described in Example 98, compound 634 (0.7 g, 0.97 mmol) was activated with TBTU (0.325 g, 1.01 mmol) and N,N-diisopropylethylamine (0.299 g, 2.32 mmol) in a mixture of pyridine (1.56 mL) and acetonitrile (10 mL) and reacted with aminopropyl CPG (19.5 g) in anhydrous acetonitrile (88 mL). The solid support was capped with N-methylimidazole (2.5 mL) and acetic anhydride (2.6 mL), filtered, washed with acetonitrile (5 × 88 mL), and dried to give a loading of 45.0 μmol / g. Example 121 (rel-(1R,3-endo,5S,6S,7R)-7-(tris(4-methoxyphenyl)methoxy)-6-acetoxy-8-methyl-8-azabicyclo[3.2.1]octan-3-yl)[2-[4-(carboxymethoxy)phenoxy]acetate] covalently bound to aminopropyl CPG500, solid support 836c. (Figure 11)

[0258] Following the procedure described in Example 98, compound 636 (0.53 g, 0.7 mmol) was activated with TBTU (0.237 g, 0.74 mmol) and N,N-diisopropylethylamine (0.218 g, 1.69 mmol) in a mixture of pyridine (1.14 mL) and acetonitrile (7 mL) and reacted with aminopropyl CPG (20.0 g) in anhydrous acetonitrile (90 mL). The solid support was capped with N-methylimidazole (1.9 mL) and acetic anhydride (1.9 mL), filtered, washed with acetonitrile (5 × 90 mL), and dried to give a loading of 33.6 μmol / g. Example 122 (rel-(1R,3-endo,5S,6S,7R)-7-(tris(4-methoxyphenyl)methoxy)-6-acetoxy-2,4,8-trimethyl-8-azabicyclo[3.2.1]octan-3-yl) hemisuccinate covalently bound to aminopropyl CPG500, solid support 837c. (Figure 11)

[0259] Following the procedure described in Example 98, compound 637 (1.12 g, 1.65 mmol) was activated with TBTU (0.556 g, 1.73 mmol) and N,N-diisopropylethylamine (0.512 g, 3.96 mmol) in a mixture of pyridine (2.67 mL) and acetonitrile (17 mL) and reacted with aminopropyl CPG (20.0 g) in anhydrous acetonitrile (90 mL). The solid support was capped with N-methylimidazole (4.3 mL) and acetic anhydride (4.4 mL), filtered, washed with acetonitrile (5 × 90 mL), and dried to give a loading of 75.0 μmol / g. Example 123 (rel-(1R,3-endo,5S,6S,7R)-7-(tris(4-methoxyphenyl)methoxy)-6-acetoxy-2,2,4,4,8-pentamethyl-8-azabicyclo[3.2.1]octan-3-yl) hemisuccinate covalently bound to aminopropyl CPG500, solid support 838c. (Figure 11)

[0260] Following the procedure described in Example 98, compound 638 (1.1 g, 1.56 mmol) was activated with TBTU (0.527 g, 1.64 mmol) and N,N-diisopropylethylamine (0.485 g, 3.75 mmol) in a mixture of pyridine (2.53 mL) and acetonitrile (16 mL) and reacted with aminopropyl CPG (19.0 g) in anhydrous acetonitrile (85 mL). The solid support was capped with N-methylimidazole (4.1 mL) and acetic anhydride (4.1 mL), filtered, washed with acetonitrile (5 × 85 mL), and dried to give a loading of 72.3 μmol / g. Example 124 (rel-(1R,3-endo,5S,6S,7R)-7-(tris(4-methoxyphenyl)methoxy)-3-methoxy-8,8-dimethyl-8-azoniabicyclo[3.2.1]octan-6-yl) hemisuccinate covalently bound to aminopropyl CPG1000, solid support 871c. (Figure 13)

[0261] Following the procedure described in Example 98, compound 671 (0.87 g, 1.09 mmol) was activated with TBTU (0.367 g, 1.14 mmol) and N,N-diisopropylethylamine (0.338 g, 2.61 mmol) in a mixture of pyridine (1.76 mL) and acetonitrile (11 mL) and reacted with aminopropyl CPG (22.0 g) in anhydrous acetonitrile (99 mL). The solid support was capped with N-methylimidazole (2.9 mL) and acetic anhydride (2.9 mL), filtered, washed with acetonitrile (5 × 99 mL), and dried to give a loading of 42.7 μmol / g. Example 125 (rel-(1R,3-endo,5S,6S,7R)-7-(tris(4-methoxyphenyl)methoxy)-3-benzyloxy-8-methyl-8-benzyl-8-azoniabicyclo[3.2.1]octan-6-yl) hemisuccinate covalently bound to aminopropyl CPG500, solid support 874c. (Figure 13)

[0262] Following the procedure described in Example 98, compound 674 (1.72 g, 1.82 mmol) was activated with TBTU (0.612 g, 1.91 mmol) and N,N-diisopropylethylamine (0.563 g, 4.36 mmol) in a mixture of pyridine (2.94 mL) and acetonitrile (19 mL) and reacted with aminopropyl CPG (22.0 g) in anhydrous acetonitrile (99 mL). The solid support was capped with N-methylimidazole (4.8 mL) and acetic anhydride (4.8 mL), filtered, washed with acetonitrile (5 × 99 mL), and dried to give a loading of 74.1 μmol / g. Example 126 (rel-(1R,3-endo,5S,6S,7R)-7-(tris(4-methoxyphenyl)methoxy)-3-methoxy-8-benzyl-8-azabicyclo[3.2.1]octan-6-yl) hemisuccinate covalently bound to aminopropyl CPG1000, solid support 875c. (Figure 13)

[0263] Following the procedure described in Example 98, compound 675 (0.99 g, 1.14 mmol) was activated with TBTU (0.384 g, 1.20 mmol) and N,N-diisopropylethylamine (0.353 g, 2.73 mmol) in a mixture of pyridine (1.84 mL) and acetonitrile (12 mL) and reacted with aminopropyl CPG (23.0 g) in anhydrous acetonitrile (104 mL). The solid support was capped with N-methylimidazole (3.0 mL) and acetic anhydride (3 mL), filtered, washed with acetonitrile (5 × 104 mL), and dried to give a loading of 43.3 μmol / g. Example 127 N-[(rel-(1R,3-endo,5S,6S,7R)-7-(bis(4-methoxyphenyl)(phenyl)methoxy)-6-acetoxy-8-methyl-8-azabicyclo[3.2.1]octan-3-yl)]succinamic acid covalently bound to aminopropyl CPG500, solid support 751c. (FIG. 10)

[0264] Following the procedure described in Example 98, compound 551 (0.81 g, 1.31 mmol) was activated with TBTU (0.442 g, 1.38 mmol) and N,N-diisopropylethylamine (0.407 g, 3.15 mmol) in a mixture of pyridine (2.12 mL) and acetonitrile (14 mL) and reacted with aminopropyl CPG (15.9 g) in anhydrous acetonitrile (72 mL). The solid support was capped with N-methylimidazole (3.5 mL) and acetic anhydride (3.5 mL), filtered, washed with acetonitrile (5 x 72 mL), and dried to give a loading of 74.8 μmol / g. Example 128 N-[(rel-(1R,3-endo,5S,6S,7R)-7-(bis(4-methoxyphenyl)(phenyl)methoxy)-6-acetoxy-8-(i-propyl)-8-azabicyclo[3.2.1]octan-3-yl)]succinamic acid covalently bound to aminopropyl CPG1500, solid support 753c. (FIG. 10)

[0265] Following the procedure described in Example 98, compound 553 (0.35 g, 0.54 mmol) was activated with TBTU (0.181 g, 0.56 mmol) and N,N-diisopropylethylamine (0.166 g, 1.29 mmol) in a mixture of pyridine (0.87 mL) and acetonitrile (6 mL) and reacted with aminopropyl CPG (15.2 g) in anhydrous acetonitrile (68 mL). The solid support was capped with N-methylimidazole (1.4 mL) and acetic anhydride (1.4 mL), filtered, washed with acetonitrile (5 × 68 mL), and dried to give a loading of 32.0 μmol / g. Example 129 N-[(rel-(1R,3-endo,5S,6S,7R)-7-(tris(4-methoxyphenyl)methoxy)-6-acetoxy-8-methyl-8-azabicyclo[3.2.1]octan-3-yl)]succinamic acid covalently bound to aminopropyl CPG1500, solid support 851c. (FIG. 11)

[0266] Following the procedure described in Example 98, compound 651 (0.28 g, 0.44 mmol) was activated with TBTU (0.148 g, 0.46 mmol) and N,N-diisopropylethylamine (0.137 g, 1.06 mmol) in a mixture of pyridine (0.71 mL) and acetonitrile (5 mL) and reacted with aminopropyl CPG (12.5 g) in anhydrous acetonitrile (56 mL). The solid support was capped with N-methylimidazole (1.2 mL) and acetic anhydride (1.2 mL), filtered, washed with acetonitrile (5 × 56 mL), and dried to give a loading of 32.9 μmol / g. Example 130 N-[(rel-(1R,3-endo,5S,6S,7R)-7-(tris(4-methoxyphenyl)methoxy)-6-acetoxy-8-methyl-8-azabicyclo[3.2.1]octan-3-yl)]succinamic acid covalently bound to hydroxypropyl CPG500, solid support 859c. (FIG. 11)

[0267] Following the procedure described in Example 98, compound 651 (0.67 g, 1.03 mmol) was activated with TBTU (0.348 g, 1.08 mmol) and N,N-diisopropylethylamine (0.32 g, 2.48 mmol) in a mixture of pyridine (1.67 mL) and acetonitrile (11 mL) and reacted with aminopropyl CPG (12.5 g) in anhydrous acetonitrile (56 mL). The solid support was capped with N-methylimidazole (2.7 mL) and acetic anhydride (2.7 mL), filtered, washed with acetonitrile (5 × 56 mL), and dried to give a loading of 74.6 μmol / g. Example 131 N-methyl-N-[(rel-(1R,3-endo,5S,6S,7R)-7-(tris(4-methoxyphenyl)methoxy)-6-acetoxy-8-methyl-8-azabicyclo[3.2.1]octan-3-yl)]succinamic acid covalently bound to aminopropyl CPG500, solid support 852c. (FIG. 11)

[0268] Following the procedure described in Example 98, compound 652 (0.66 g, 1 mmol) was activated with TBTU (0.338 g, 1.05 mmol) and N,N-diisopropylethylamine (0.311 g, 2.41 mmol) in a mixture of pyridine (1.62 mL) and acetonitrile (10 mL) and reacted with aminopropyl CPG (12.2 g) in anhydrous acetonitrile (55 mL). The solid support was capped with N-methylimidazole (2.6 mL) and acetic anhydride (2.7 mL), filtered, washed with acetonitrile (5 × 55 mL), and dried to give a loading of 73.6 μmol / g. Example 132 [ka] (rel-(1R,3-endo,5S,6S,7R)-7-[(bis(4-methoxyphenyl)(phenyl)methoxy]-3-acetoxy-8-methyl-aza[bicyclo[3.2.1]octan-6-yl]hemisuccinate, 701p) covalently bound to aminomethyl MPPS.

[0269] TBTU (0.948 g, 2.95 mmol) was added to a solution of compound 401 (1.736 g, 2.81 mmol) and N,N-diisopropylethylamine (0.87 g, 2.95 mmol) in a mixture of anhydrous pyridine (4.55 mL) and acetonitrile (29 mL). The mixture was stirred for 15 minutes, transferred to a suspension of aminomethyl MPPS (7.3 g) in anhydrous acetonitrile (37 mL), and the resulting suspension was shaken for 4 hours. N-methylimidazole (7.4 mL) and acetic anhydride (7.4 mL) were then added to the suspension, which was then shaken again for 45 minutes. The solid support was filtered off, washed on the filter with acetonitrile (5 × 37 mL), and dried in vacuo. The loading of the finished solid support 701p (341 μmol / g), where applicable, was determined by standard di- or trimethoxytrityl assays as disclosed in Guzaev, AP and Pon, RT Curr. Protoc. Nucleic Acid Chem. 2013, 52, pp. 3.2.1-3.2.23. Example 133 (rel-(1R,3-endo,5S,6S,7R)-7-(bis(4-methoxyphenyl)(phenyl)methoxy)-3-isobutyryloxy-8-benzyl-8-azabicyclo[3.2.1]octan-6-yl) hemisuccinate covalently bound to aminomethyl MPPS, solid support 705p. (Figure 10)

[0270] Following the procedure described in Example 132, compound 505 (2.255 g, 3.12 mmol) was activated with TBTU (1.053 g, 3.28 mmol) and N,N-diisopropylethylamine (0.969 g, 7.50 mmol) in a mixture of pyridine (5.05 mL) and acetonitrile (33 mL) and reacted with aminomethyl MPPS (8.1 g) in anhydrous acetonitrile (41 mL). The solid support was capped with N-methylimidazole (8.2 mL) and acetic anhydride (8.3 mL), filtered, washed with acetonitrile (5 × 41 mL), and dried to give a loading of 336 μmol / g. Example 134 (rel-(1R,3-endo,5S,6S,7R)-7-(tris(4-methoxyphenyl)methoxy)-3-acetoxy-8-methyl-8-azabicyclo[3.2.1]octan-6-yl) hemisuccinate covalently bound to aminomethyl MPPS, solid support 801p. (Figure 11)

[0271] Following the procedure described in Example 132, compound 601 (1.976 g, 3.05 mmol) was activated with TBTU (1.029 g, 3.20 mmol) and N,N-diisopropylethylamine (0.946 g, 7.32 mmol) in a mixture of pyridine (4.94 mL) and acetonitrile (32 mL) and reacted with aminomethyl MPPS (7.9 g) in anhydrous acetonitrile (40 mL). The solid support was capped with N-methylimidazole (8.0 mL) and acetic anhydride (8.1 mL), filtered, washed with acetonitrile (5 × 40 mL), and dried to give a loading of 336 μmol / g. Example 135 (rel-(1R,3-endo,5S,6S,7R)-7-(tris(4-methoxyphenyl)methoxy)-3-pivaloyloxy-8-(i-propyl)-8-azabicyclo[3.2.1]octan-6-yl) hemisuccinate covalently bound to aminomethyl MPPS, solid support 803p. (Figure 11)

[0272] Following the procedure described in Example 132, compound 603 (2.531 g, 3.53 mmol) was activated with TBTU (1.189 g, 3.70 mmol) and N,N-diisopropylethylamine (1.094 g, 8.46 mmol) in a mixture of pyridine (5.70 mL) and acetonitrile (37 mL) and reacted with aminomethyl MPPS (9.2 g) in anhydrous acetonitrile (46 mL). The solid support was capped with N-methylimidazole (9.3 mL) and acetic anhydride (9.3 mL), filtered, washed with acetonitrile (5 × 46 mL), and dried to give a loading of 320 μmol / g. Example 136 (rel-(1R,3-endo,5S,6S,7R)-7-(tris(4-methoxyphenyl)methoxy)-3-isobutyryloxy-8-methyl-8-azabicyclo[3.2.1]octan-6-yl) hemisuccinate covalently bound to aminomethyl MPPS, solid support 805p

[0273] Following the procedure described in Example 132, compound 605 (1.892 g, 2.52 mmol) was activated with TBTU (0.848 g, 2.64 mmol) and N,N-diisopropylethylamine (0.781 g, 6.04 mmol) in a mixture of pyridine (4.07 mL) and acetonitrile (26 mL) and reacted with aminomethyl MPPS (6.5 g) in anhydrous acetonitrile (33 mL). The solid support was capped with N-methylimidazole (6.6 mL) and acetic anhydride (6.6 mL), filtered, washed with acetonitrile (5 × 33 mL), and dried to give a loading of 333 μmol / g. Example 137 (rel-(1R,3-endo,5S,6S,7R)-7-(tris(4-methoxyphenyl)methoxy)-3-[(t-butyl)dimethylsilyloxy]-8-methyl-8-azabicyclo[3.2.1]octan-6-yl) hemisuccinate covalently bound to aminomethyl MPPS, solid support 816p. (Figure 11)

[0274] Following the procedure described in Example 132, compound 616 (2.277 g, 3.16 mmol) was activated with TBTU (1.066 g, 3.32 mmol) and N,N-diisopropylethylamine (0.981 g, 7.59 mmol) in a mixture of pyridine (5.12 mL) and acetonitrile (33 mL) and reacted with aminomethyl MPPS (8.2 g) in anhydrous acetonitrile (41 mL). The solid support was capped with N-methylimidazole (8.3 mL) and acetic anhydride (8.4 mL), filtered, washed with acetonitrile (5 × 41 mL), and dried to give a loading of 326 μmol / g. Example 138 (rel-(1R,3-endo,5S,6S,7R)-7-(tris(4-methoxyphenyl)methoxy)-3-[(t-butyl)diphenylsilyloxy]-8-methyl-8-azabicyclo[3.2.1]octan-6-yl) hemisuccinate covalently bound to aminomethyl MPPS, solid support 818p. (Figure 11)

[0275] Following the procedure described in Example 132, compound 618 (2.670 g, 3.16 mmol) was activated with TBTU (1.066 g, 3.32 mmol) and N,N-diisopropylethylamine (0.981 g, 7.59 mmol) in a mixture of pyridine (5.12 mL) and acetonitrile (33 mL) and reacted with aminomethyl MPPS (8.2 g) in anhydrous acetonitrile (41 mL). The solid support was capped with N-methylimidazole (8.3 mL) and acetic anhydride (8.4 mL), filtered, washed with acetonitrile (5 × 41 mL), and dried to give a loading of 334 μmol / g. Example 139 (rel-(1R,3-endo,5S,6S,7R)-7-(tris(4-methoxyphenyl)methoxy)-8-methyl-8-azaspiro[bicyclo[3.2.1]octane-3,2'-[1,3]dioxolan]-6-yl) hemisuccinate covalently bound to aminomethyl MPPS, solid support 821p. (Figure 11)

[0276] Following the procedure described in Example 132, compound 621 (1.308 g, 2.02 mmol) was activated with TBTU (0.681 g, 2.12 mmol) and N,N-diisopropylethylamine (0.626 g, 4.85 mmol) in a mixture of pyridine (3.27 mL) and acetonitrile (21 mL) and reacted with aminomethyl MPPS (5.2 g) in anhydrous acetonitrile (26 mL). The solid support was capped with N-methylimidazole (5.3 mL) and acetic anhydride (5.3 mL), filtered, washed with acetonitrile (5 × 26 mL), and dried to give a loading of 332 μmol / g. Example 140 (rel-(1R,3-endo,5S,6S,7R)-7-(tris(4-methoxyphenyl)methoxy)-8-methyl-8-azaspiro[bicyclo[3.2.1]octane-3,2'-[1,3]dioxan]-6-yl) hemisuccinate covalently bound to aminomethyl MPPS, solid support 822p. (Figure 11)

[0277] Following the procedure described in Example 132, compound 622 (2.363 g, 3.43 mmol) was activated with TBTU (1.155 g, 3.60 mmol) and N,N-diisopropylethylamine (1.063 g, 8.22 mmol) in a mixture of pyridine (5.54 mL) and acetonitrile (36 mL) and reacted with aminomethyl MPPS (8.9 g) in anhydrous acetonitrile (45 mL). The solid support was capped with N-methylimidazole (9.0 mL) and acetic anhydride (9.1 mL), filtered, washed with acetonitrile (5 × 45 mL), and dried to give a loading of 324 μmol / g. Example 141

[0278] (rel-(1R,3-endo,5S,6S,7R)-7-(tris(4-methoxyphenyl)methoxy)-8-(i-propyl)-8-azaspiro[bicyclo[3.2.1]octane-3,2'-[1,3]dioxolan]-6-yl) hemisuccinate covalently bound to aminomethyl MPPS, solid support 823p. (Figure 11)

[0279] Following the procedure described in Example 132, compound 623 (1.302 g, 1.93 mmol) was activated with TBTU (0.649 g, 2.02 mmol) and N,N-diisopropylethylamine (0.598 g, 4.62 mmol) in a mixture of pyridine (3.12 mL) and acetonitrile (20 mL) and reacted with aminomethyl MPPS (5.0 g) in anhydrous acetonitrile (25 mL). The solid support was capped with N-methylimidazole (5.1 mL) and acetic anhydride (5.1 mL), filtered, washed with acetonitrile (5 × 25 mL), and dried to give a loading of 324 μmol / g. Example 142

[0280] (rel-(1R,3-endo,5S,6S,7R)-7-(tris(4-methoxyphenyl)methoxy)-8-benzyl-8-azaspiro[bicyclo[3.2.1]octane-3,2'-[1,3]dioxan]-6-yl) hemisuccinate covalently bound to aminomethyl MPPS, solid support 824p. (Figure 11)

[0281] Following the procedure described in Example 132, compound 624 (2.721 g, 3.55 mmol) was activated with TBTU (1.198 g, 3.73 mmol) and N,N-diisopropylethylamine (1.102 g, 8.53 mmol) in a mixture of pyridine (5.75 mL) and acetonitrile (37 mL) and reacted with aminomethyl MPPS (9.2 g) in anhydrous acetonitrile (46 mL). The solid support was capped with N-methylimidazole (9.3 mL) and acetic anhydride (9.4 mL), filtered, washed with acetonitrile (5 × 46 mL), and dried to give a loading of 324 μmol / g. (Example 143) (rel-(1R,3-endo,5S,6S,7R)-7-(bis(4-methoxyphenyl)(phenyl)methoxy)-6-acetoxy-8-methyl-8-azabicyclo[3.2.1]octan-3-yl) hemisuccinate covalently bound to aminomethyl MPPS, solid support 731p. (Figure 10)

[0282] Following the procedure described in Example 132, compound 531 (1.633 g, 2.64 mmol) was activated with TBTU (0.891 g, 2.78 mmol) and N,N-diisopropylethylamine (0.820 g, 6.34 mmol) in a mixture of pyridine (4.28 mL) and acetonitrile (28 mL) and reacted with aminomethyl MPPS (6.9 g) in anhydrous acetonitrile (35 mL). The solid support was capped with N-methylimidazole (7.0 mL) and acetic anhydride (7.0 mL), filtered, washed with acetonitrile (5 × 35 mL), and dried to give a loading of 343 μmol / g. Example 144 (rel-(1R,3-endo,5S,6S,7R)-7-(bis(4-methoxyphenyl)(phenyl)methoxy)-6-acetoxy-8-ethyl-8-azabicyclo[3.2.1]octan-3-yl) hemisuccinate covalently bound to aminomethyl MPPS, solid support 732p. (Figure 10)

[0283] Following the procedure described in Example 132, compound 532 (1.583 g, 2.51 mmol) was activated with TBTU (0.845 g, 2.63 mmol) and N,N-diisopropylethylamine (0.777 g, 6.01 mmol) in a mixture of pyridine (4.05 mL) and acetonitrile (26 mL) and reacted with aminomethyl MPPS (6.5 g) in anhydrous acetonitrile (33 mL). The solid support was capped with N-methylimidazole (6.6 mL) and acetic anhydride (6.6 mL), filtered, washed with acetonitrile (5 × 33 mL), and dried to give a loading of 327 μmol / g. Example 145 (rel-(1R,3-endo,5S,6S,7R)-7-(bis(4-methoxyphenyl)(phenyl)methoxy)-6-acetoxy-8-(i-propyl)-8-azabicyclo[3.2.1]octan-3-yl) hemisuccinate covalently bound to aminomethyl MPPS, solid support 733p. (Figure 10)

[0284] Following the procedure described in Example 132, compound 533 (2.170 g, 3.36 mmol) was activated with TBTU (1.133 g, 3.53 mmol) and N,N-diisopropylethylamine (1.042 g, 8.06 mmol) in a mixture of pyridine (5.43 mL) and acetonitrile (35 mL) and reacted with aminomethyl MPPS (8.7 g) in anhydrous acetonitrile (44 mL). The solid support was capped with N-methylimidazole (8.8 mL) and acetic anhydride (8.9 mL), filtered, washed with acetonitrile (5 × 44 mL), and dried to give a loading of 326 μmol / g. Example 146 (rel-(1R,3-endo,5S,6S,7R)-7-(bis(4-methoxyphenyl)(phenyl)methoxy)-6-acetoxy-8-benzyl-8-azabicyclo[3.2.1]octan-3-yl) hemisuccinate covalently bound to aminomethyl MPPS, solid support 734p. (Figure 10)

[0285] Following the procedure described in Example 132, compound 534 (2.163 g, 3.12 mmol) was activated with TBTU (1.051 g, 3.27 mmol) and N,N-diisopropylethylamine (0.967 g, 7.48 mmol) in a mixture of pyridine (5.04 mL) and acetonitrile (33 mL) and reacted with aminomethyl MPPS (8.1 g) in anhydrous acetonitrile (41 mL). The solid support was capped with N-methylimidazole (8.2 mL) and acetic anhydride (8.2 mL), filtered, washed with acetonitrile (5 × 41 mL), and dried to give a loading of 331 μmol / g. Example 147 (rel-(1R,3-endo,5S,6S,7R)-7-(bis(4-methoxyphenyl)(phenyl)methoxy)-6-acetoxy-8-methyl-8-azabicyclo[3.2.1]octan-3-yl)diglycolate covalently bound to aminomethyl MPPS, solid support 735p. (Figure 10)

[0286] Following the procedure described in Example 132, compound 535 (1.762 g, 2.78 mmol) was activated with TBTU (0.937 g, 2.92 mmol) and N,N-diisopropylethylamine (0.862 g, 6.67 mmol) in a mixture of pyridine (4.50 mL) and acetonitrile (29 mL) and reacted with aminomethyl MPPS (7.2 g) in anhydrous acetonitrile (36 mL). The solid support was capped with N-methylimidazole (7.3 mL) and acetic anhydride (7.3 mL), filtered, washed with acetonitrile (5 × 36 mL), and dried to give a loading of 335 μmol / g. Example 148 (rel-(1R,3-endo,5S,6S,7R)-7-(bis(4-methoxyphenyl)(phenyl)methoxy)-6-acetoxy-2,4,8-trimethyl-8-azabicyclo[3.2.1]octan-3-yl) hemisuccinate covalently bound to aminomethyl MPPS, solid support 737p. (Figure 10)

[0287] Following the procedure described in Example 132, compound 537 (2.086 g, 3.23 mmol) was activated with TBTU (1.089 g, 3.39 mmol) and N,N-diisopropylethylamine (1.002 g, 7.75 mmol) in a mixture of pyridine (5.22 mL) and acetonitrile (34 mL) and reacted with aminomethyl MPPS (8.4 g) in anhydrous acetonitrile (42 mL). The solid support was capped with N-methylimidazole (8.5 mL) and acetic anhydride (8.5 mL), filtered, washed with acetonitrile (5 × 42 mL), and dried to give a loading of 326 μmol / g. Example 149 (rel-(1R,3-endo,5S,6S,7R)-7-(bis(4-methoxyphenyl)(phenyl)methoxy)-6-acetoxy-2,2,4,4,8-pentamethyl-8-azabicyclo[3.2.1]octan-3-yl) hemisuccinate covalently bound to aminomethyl MPPS, solid support 738p. (Figure 10)

[0288] Following the procedure described in Example 132, compound 538 (2.522 g, 3.74 mmol) was activated with TBTU (1.262 g, 3.93 mmol) and N,N-diisopropylethylamine (1.161 g, 8.98 mmol) in a mixture of pyridine (6.05 mL) and acetonitrile (39 mL) and reacted with aminomethyl MPPS (9.7 g) in anhydrous acetonitrile (49 mL). The solid support was capped with N-methylimidazole (9.8 mL) and acetic anhydride (9.9 mL), filtered, washed with acetonitrile (5 × 49 mL), and dried to give a loading of 331 μmol / g. Example 150 (rel-(1R,3-endo,5S,6S,7R)-7-(tris(4-methoxyphenyl)methoxy)-6-acetoxy-8-methyl-8-azabicyclo[3.2.1]octan-3-yl) hemisuccinate covalently bound to aminomethyl MPPS, solid support 831p. (Figure 11)

[0289] Following the procedure described in Example 132, compound 631 (2.362 g, 3.65 mmol) was activated with TBTU (1.230 g, 3.83 mmol) and N,N-diisopropylethylamine (1.131 g, 8.75 mmol) in a mixture of pyridine (5.90 mL) and acetonitrile (38 mL) and reacted with aminomethyl MPPS (9.5 g) in anhydrous acetonitrile (48 mL). The solid support was capped with N-methylimidazole (9.6 mL) and acetic anhydride (9.6 mL), filtered, washed with acetonitrile (5 × 48 mL), and dried to give a loading of 337 μmol / g. Example 151 (rel-(1R,3-endo,5S,6S,7R)-7-(tris(4-methoxyphenyl)methoxy)-6-acetoxy-8-ethyl-8-azabicyclo[3.2.1]octan-3-yl) hemisuccinate covalently bound to aminomethyl MPPS, solid support 832p. (Figure 11)

[0290] Following the procedure described in Example 132, compound 632 (1.897 g, 2.87 mmol) was activated with TBTU (0.966 g, 3.01 mmol) and N,N-diisopropylethylamine (0.889 g, 6.88 mmol) in a mixture of pyridine (4.64 mL) and acetonitrile (30 mL) and reacted with aminomethyl MPPS (7.4 g) in anhydrous acetonitrile (37 mL). The solid support was capped with N-methylimidazole (7.5 mL) and acetic anhydride (7.6 mL), filtered, washed with acetonitrile (5 × 37 mL), and dried to give a loading of 341 μmol / g. Example 152 (rel-(1R,3-endo,5S,6S,7R)-7-(tris(4-methoxyphenyl)methoxy)-6-acetoxy-8-(i-propyl)-8-azabicyclo[3.2.1]octan-3-yl) hemisuccinate covalently bound to aminomethyl MPPS, solid support 833p. (Figure 11)

[0291] Following the procedure described in Example 132, compound 633 (2.259 g, 3.34 mmol) was activated with TBTU (1.127 g, 3.51 mmol) and N,N-diisopropylethylamine (1.037 g, 8.02 mmol) in a mixture of pyridine (5.41 mL) and acetonitrile (35 mL) and reacted with aminomethyl MPPS (8.7 g) in anhydrous acetonitrile (44 mL). The solid support was capped with N-methylimidazole (8.8 mL) and acetic anhydride (8.8 mL), filtered, washed with acetonitrile (5 × 44 mL), and dried to give a loading of 338 μmol / g. Example 153 (rel-(1R,3-endo,5S,6S,7R)-7-(tris(4-methoxyphenyl)methoxy)-6-acetoxy-8-benzyl-8-azabicyclo[3.2.1]octan-3-yl) hemisuccinate covalently bound to aminomethyl MPPS, solid support 834p. (Figure 11)

[0292] Following the procedure described in Example 132, compound 634 (1.712 g, 2.36 mmol) was activated with TBTU (0.797 g, 2.48 mmol) and N,N-diisopropylethylamine (0.734 g, 5.68 mmol) in a mixture of pyridine (3.83 mL) and acetonitrile (25 mL) and reacted with aminomethyl MPPS (6.1 g) in anhydrous acetonitrile (31 mL). The solid support was capped with N-methylimidazole (6.2 mL) and acetic anhydride (6.2 mL), filtered, washed with acetonitrile (5 × 31 mL), and dried to give a loading of 343 μmol / g. Example 154 (rel-(1R,3-endo,5S,6S,7R)-7-(tris(4-methoxyphenyl)methoxy)-6-acetoxy-8-methyl-8-azabicyclo[3.2.1]octan-3-yl)[2-[4-(carboxymethoxy)phenoxy]acetate] covalently bound to aminomethyl MPPS, solid support 836p. (Figure 11)

[0293] Following the procedure described in Example 132, compound 636 (2.722 g, 3.60 mmol) was activated with TBTU (1.214 g, 3.78 mmol) and N,N-diisopropylethylamine (1.117 g, 8.64 mmol) in a mixture of pyridine (5.83 mL) and acetonitrile (38 mL) and reacted with aminomethyl MPPS (9.4 g) in anhydrous acetonitrile (47 mL). The solid support was capped with N-methylimidazole (9.5 mL) and acetic anhydride (9.5 mL), filtered, washed with acetonitrile (5 × 47 mL), and dried to give a loading of 343 μmol / g. Example 155 (rel-(1R,3-endo,5S,6S,7R)-7-(tris(4-methoxyphenyl)methoxy)-6-acetoxy-2,4,8-trimethyl-8-azabicyclo[3.2.1]octan-3-yl) hemisuccinate covalently bound to aminomethyl MPPS, solid support 837p. (Figure 11)

[0294] Following the procedure described in Example 132, compound 637 (1.811 g, 2.68 mmol) was activated with TBTU (0.904 g, 2.81 mmol) and N,N-diisopropylethylamine (0.831 g, 6.43 mmol) in a mixture of pyridine (4.34 mL) and acetonitrile (28 mL) and reacted with aminomethyl MPPS (7.0 g) in anhydrous acetonitrile (35 mL). The solid support was capped with N-methylimidazole (7.1 mL) and acetic anhydride (7.1 mL), filtered, washed with acetonitrile (5 × 35 mL), and dried to give a loading of 322 μmol / g. Example 156 (rel-(1R,3-endo,5S,6S,7R)-7-(tris(4-methoxyphenyl)methoxy)-6-acetoxy-2,2,4,4,8-pentamethyl-8-azabicyclo[3.2.1]octan-3-yl) hemisuccinate covalently bound to aminomethyl MPPS, solid support 838p. (Figure 11)

[0295] Following the procedure described in Example 132, compound 638 (1.959 g, 2.78 mmol) was activated with TBTU (0.938 g, 2.92 mmol) and N,N-diisopropylethylamine (0.863 g, 6.68 mmol) in a mixture of pyridine (4.50 mL) and acetonitrile (29 mL) and reacted with aminomethyl MPPS (7.2 g) in anhydrous acetonitrile (36 mL). The solid support was capped with N-methylimidazole (7.3 mL) and acetic anhydride (7.4 mL), filtered, washed with acetonitrile (5 × 36 mL), and dried to give a loading of 341 μmol / g. Example 157 (rel-(1R,3-endo,5S,6S,7R)-7-(tris(4-methoxyphenyl)methoxy)-3-methoxy-8,8-dimethyl-8-azoniabicyclo[3.2.1]octan-6-yl) hemisuccinate covalently bound to aminomethyl MPPS, solid support 871p. (Figure 13)

[0296] Following the procedure described in Example 132, compound 671 (2.296 g, 2.89 mmol) was activated with TBTU (0.973 g, 3.03 mmol) and N,N-diisopropylethylamine (0.896 g, 6.93 mmol) in a mixture of pyridine (4.67 mL) and acetonitrile (30 mL) and reacted with aminomethyl MPPS (7.5 g) in anhydrous acetonitrile (38 mL). The solid support was capped with N-methylimidazole (7.6 mL) and acetic anhydride (7.6 mL), filtered, washed with acetonitrile (5 × 38 mL), and dried to give a loading of 322 μmol / g. Example 158 (rel-(1R,3-endo,5S,6S,7R)-7-(tris(4-methoxyphenyl)methoxy)-3-benzyloxy-8-methyl-8-benzyl-8-azoniabicyclo[3.2.1]octan-6-yl) hemisuccinate covalently bound to aminomethyl MPPS, solid support 874p. (Figure 13)

[0297] Following the procedure described in Example 132, compound 674 (2.042 g, 2.16 mmol) was activated with TBTU (0.727 g, 2.26 mmol) and N,N-diisopropylethylamine (0.669 g, 5.17 mmol) in a mixture of pyridine (3.49 mL) and acetonitrile (23 mL) and reacted with aminomethyl MPPS (5.6 g) in anhydrous acetonitrile (28 mL). The solid support was capped with N-methylimidazole (5.7 mL) and acetic anhydride (5.7 mL), filtered, washed with acetonitrile (5 × 28 mL), and dried to give a loading of 329 μmol / g. Example 159 (rel-(1R,3-endo,5S,6S,7R)-7-(tris(4-methoxyphenyl)methoxy)-3-methoxy-8-benzyl-8-azabicyclo[3.2.1]octan-6-yl) hemisuccinate covalently bound to aminomethyl MPPS, solid support 875p. (Figure 13)

[0298] Following the procedure described in Example 132, compound 675 (2.683 g, 3.08 mmol) was activated with TBTU (1.038 g, 3.23 mmol) and N,N-diisopropylethylamine (0.955 g, 7.39 mmol) in a mixture of pyridine (4.98 mL) and acetonitrile (32 mL) and reacted with aminomethyl MPPS (8.0 g) in anhydrous acetonitrile (40 mL). The solid support was capped with N-methylimidazole (8.1 mL) and acetic anhydride (8.1 mL), filtered, washed with acetonitrile (5 × 40 mL), and dried to give a loading of 337 μmol / g. Example 160 N-[(rel-(1R,3-endo,5S,6S,7R)-7-(bis(4-methoxyphenyl)(phenyl)methoxy)-6-acetoxy-8-methyl-8-azabicyclo[3.2.1]octan-3-yl)]succinamic acid covalently bound to aminomethyl MPPS, solid support 751p. (Figure 10)

[0299] Following the procedure described in Example 132, compound 551 (1.585 g, 2.57 mmol) was activated with TBTU (0.866 g, 2.70 mmol) and N,N-diisopropylethylamine (0.797 g, 6.17 mmol) in a mixture of pyridine (4.16 mL) and acetonitrile (27 mL) and reacted with aminomethyl MPPS (6.7 g) in anhydrous acetonitrile (34 mL). The solid support was capped with N-methylimidazole (6.8 mL) and acetic anhydride (6.8 mL), filtered, washed with acetonitrile (5 × 34 mL), and dried to give a loading of 346 μmol / g. Example 161 N-[(rel-(1R,3-endo,5S,6S,7R)-7-(bis(4-methoxyphenyl)(phenyl)methoxy)-6-acetoxy-8-(i-propyl)-8-azabicyclo[3.2.1]octan-3-yl)]succinamic acid covalently bound to aminomethyl MPPS, solid support 753p. (Figure 10)

[0300] Following the procedure described in Example 132, compound 553 (1.626 g, 2.52 mmol) was activated with TBTU (0.850 g, 2.65 mmol) and N,N-diisopropylethylamine (0.782 g, 6.05 mmol) in a mixture of pyridine (4.08 mL) and acetonitrile (26 mL) and reacted with aminomethyl MPPS (6.6 g) in anhydrous acetonitrile (33 mL). The solid support was capped with N-methylimidazole (6.6 mL) and acetic anhydride (6.7 mL), filtered, washed with acetonitrile (5 × 33 mL), and dried to give a loading of 347 μmol / g. Example 162 N-[(rel-(1R,3-endo,5S,6S,7R)-7-(tris(4-methoxyphenyl)methoxy)-6-acetoxy-8-methyl-8-azabicyclo[3.2.1]octan-3-yl)]succinamic acid covalently bound to aminomethyl MPPS, solid support 851p. (Figure 11)

[0301] Following the procedure described in Example 132, compound 651 (1.508 g, 2.33 mmol) was activated with TBTU (0.786 g, 2.45 mmol) and N,N-diisopropylethylamine (0.724 g, 5.60 mmol) in a mixture of pyridine (3.77 mL) and acetonitrile (24 mL) and reacted with aminomethyl MPPS (6.1 g) in anhydrous acetonitrile (30 mL). The solid support was capped with N-methylimidazole (6.1 mL) and acetic anhydride (6.2 mL), filtered, washed with acetonitrile (5 × 30 mL), and dried to give a loading of 352 μmol / g. Example 163 N-[(rel-(1R,3-endo,5S,6S,7R)-7-(tris(4-methoxyphenyl)methoxy)-6-acetoxy-8-methyl-8-azabicyclo[3.2.1]octan-3-yl)]succinamic acid covalently bound to hydroxymethyl MPPS, solid support 851p. (Figure 11)

[0302] Following the procedure described in Example 132, compound 651 (1.508 g, 2.33 mmol) was activated with TBTU (0.786 g, 2.45 mmol) and N,N-diisopropylethylamine (0.724 g, 5.60 mmol) in a mixture of pyridine (3.77 mL) and acetonitrile (24 mL) and reacted with aminomethyl MPPS (6.1 g) in anhydrous acetonitrile (30 mL). The solid support was capped with N-methylimidazole (6.1 mL) and acetic anhydride (6.2 mL), filtered, washed with acetonitrile (5 × 30 mL), and dried to give a loading of 352 μmol / g. Example 164 N-methyl-N-[(rel-(1R,3-endo,5S,6S,7R)-7-(tris(4-methoxyphenyl)methoxy)-6-acetoxy-8-methyl-8-azabicyclo[3.2.1]octan-3-yl)]succinamic acid covalently bound to aminomethyl MPPS, solid support 852p. (Figure 11)

[0303] Following the procedure described in Example 132, compound 652 (1.526 g, 2.31 mmol) was activated with TBTU (0.778 g, 2.42 mmol) and N,N-diisopropylethylamine (0.716 g, 5.54 mmol) in a mixture of pyridine (3.73 mL) and acetonitrile (24 mL) and reacted with aminomethyl MPPS (6.0 g) in anhydrous acetonitrile (30 mL). The solid support was capped with N-methylimidazole (6.1 mL) and acetic anhydride (6.1 mL), filtered, washed with acetonitrile (5 × 30 mL), and dried to give a loading of 353 μmol / g. Example 165 [ka] (rel-(1R,3-endo,5S,6S,7R)-7-[(bis(4-methoxyphenyl)(phenyl)methoxy]-3-acetoxy-8,8-dimethyl-azoniabicyclo[3.2.1]octan-6-yl) hemisuccinate, 711c covalently bound to aminopropyl CPG500

[0304] Methyl iodide (0.4 M in MeCN, 43 mL) and N,N-diisopropylethylamine (0.535 g) were added to solid support 701c (10 g). The resulting suspension was shaken for 12 hours. The solid support was filtered off, washed on the filter with 0.25 M DIPEA-HCl in acetonitrile (55 mL), then washed with acetonitrile (5 × 50 mL), and dried in vacuo. The loading of the finished solid support 701c (69.0 μmol / g), where applicable, was determined by a standard di- or trimethoxytrityl assay as disclosed in Guzaev, AP and Pon, RT Curr. Protoc. Nucleic Acid Chem. 2013, 52, pp. 3.2.1-3.2.23. Example 166 (rel-(1R,3-endo,5S,6S,7R)-7-(bis(4-methoxyphenyl)(phenyl)methoxy)-3-pivaloyloxy-8-methyl-8-(i-propyl)-8-azoniabicyclo[3.2.1]octan-6-yl) hemisuccinate covalently bound to aminopropyl CPG1500, solid support 712c. (Figure 12)

[0305] Solid support 703c (10.00 g) was alkylated with methyl iodide (0.35 M in acetonitrile, 20.013 mL) and N,N-diisopropylethylamine (0.248 g) according to the method described in Example 165. The solid support was filtered off, washed with 0.25 M DIPEA-HCl in acetonitrile (26 mL), washed with acetonitrile (5 × 50 mL), and dried to give a loading of 32.0 μmol / g. Example 167 (rel-(1R,3-endo,5S,6S,7R)-7-(bis(4-methoxyphenyl)(phenyl)methoxy)-3-isobutyryloxy-8-methyl-8-benzyl-8-azoniabicyclo[3.2.1]octan-6-yl) hemisuccinate covalently bound to aminopropyl CPG1000, solid support 713c. (FIG. 12)

[0306] Solid support 705c (10.00 g) was alkylated with methyl iodide (0.35 M in acetonitrile, 27.313 mL) and N,N-diisopropylethylamine (0.339 g) according to the method described in Example 165. The solid support was filtered off, washed with 0.25 M DIPEA-HCl in acetonitrile (35 mL), washed with acetonitrile (5 x 50 mL), and dried to give a loading of 43.7 μmol / g. Example 168 (rel-(1R,3-endo,5S,6S,7R)-7-(tris(4-methoxyphenyl)methoxy)-3-acetoxy-8,8-dimethyl-8-azabicyclo[3.2.1]octan-6-yl) hemisuccinate covalently bound to aminopropyl CPG1000, solid support 811c. (Figure 13)

[0307] Solid support 801c (10.00 g) was alkylated with methyl iodide (0.35 M in acetonitrile, 27.625 mL) and N,N-diisopropylethylamine (0.343 g) according to the method described in Example 165. The solid support was filtered off, washed with 0.25 M DIPEA-HCl in acetonitrile (35 mL), washed with acetonitrile (5 × 50 mL), and dried to give a loading of 44.2 μmol / g. Example 169 (rel-(1R,3-endo,5S,6S,7R)-7-(tris(4-methoxyphenyl)methoxy)-3-pivaloyloxy-8-methyl-8-(i-propyl)-8-azoniabicyclo[3.2.1]octan-6-yl) hemisuccinate covalently bound to aminopropyl CPG1000, solid support 812c

[0308] Solid support 803c (10.00 g) was alkylated with methyl iodide (0.35 M in acetonitrile, 28.000 mL) and N,N-diisopropylethylamine (0.347 g) according to the method described in Example 165. The solid support was filtered off, washed with 0.25 M DIPEA-HCl in acetonitrile (36 mL), washed with acetonitrile (5 × 50 mL), and dried to give a loading of 44.8 μmol / g. Example 170 (rel-(1R,3-endo,5S,6S,7R)-7-(tris(4-methoxyphenyl)methoxy)-3-isobutyryloxy-8-methyl-8-benzyl-8-azoniabicyclo[3.2.1]octan-6-yl) hemisuccinate covalently bound to aminopropyl CPG500, solid support 813c. (Figure 13)

[0309] Solid support 805c (10.00 g) was alkylated with methyl iodide (0.35 M in acetonitrile, 45.688 mL) and N,N-diisopropylethylamine (0.567 g) according to the method described in Example 165. The solid support was filtered off, washed with 0.25 M DIPEA-HCl in acetonitrile, washed with acetonitrile (5 x 50 mL), and dried to give a loading of 73.1 μmol / g (58 mL). Example 171 (rel-(1R,3-endo,5S,6S,7R)-7-(tris(4-methoxyphenyl)methoxy)-3-[(t-butyl)dimethylsilyloxy]-8,8-dimethyl-8-azoniabicyclo[3.2.1]octan-6-yl) hemisuccinate covalently bound to aminopropyl CPG1500, solid support 817c. (Figure 13)

[0310] Solid support 816c (10.00 g) was alkylated with methyl iodide (0.35 M in acetonitrile, 20.250 mL) and N,N-diisopropylethylamine (0.251 g) according to the method described in Example 165. The solid support was filtered off, washed with 0.25 M DIPEA-HCl in acetonitrile (26 mL), washed with acetonitrile (5 × 50 mL), and dried to give a loading of 32.4 μmol / g. Example 172 (rel-(1R,3-endo,5S,6S,7R)-7-(tris(4-methoxyphenyl)methoxy)-3-[(t-butyl)diphenylsilyloxy]-8,8-dimethyl-8-azoniabicyclo[3.2.1]octan-6-yl) hemisuccinate covalently bound to aminopropyl CPG1000, solid support 819c. (Figure 13)

[0311] Solid support 818c (10.00 g) was alkylated with methyl iodide (0.35 M in acetonitrile, 27.375 mL) and N,N-diisopropylethylamine (0.340 g) according to the method described in Example 165. The solid support was filtered off, washed with 0.25 M DIPEA-HCl in acetonitrile (35 mL), washed with acetonitrile (5 × 50 mL), and dried to give a loading of 43.8 μmol / g. Example 173 (rel-(1R,3-endo,5S,6S,7R)-7-(tris(4-methoxyphenyl)methoxy)-8,8-dimethyl-8-azoniaspiro[bicyclo[3.2.1]octane-3,2'-[1,3]dioxolan]-6-yl) hemisuccinate covalently bound to aminopropyl CPG500, solid support 825p. (Figure 13)

[0312] Solid support 821c (10.00 g) was alkylated with methyl iodide (0.35 M in acetonitrile, 46.500 mL) and N,N-diisopropylethylamine (0.577 g) according to the method described in Example 165. The solid support was filtered off, washed with 0.25 M DIPEA-HCl in acetonitrile (60 mL), washed with acetonitrile (5 × 50 mL), and dried to give a loading of 74.4 μmol / g. Example 174 (rel-(1R,3-endo,5S,6S,7R)-7-(tris(4-methoxyphenyl)methoxy)-5',5',8-trimethyl-8-azoniaspiro[bicyclo[3.2.1]octane-3,2'-[1,3]dioxan]-6-yl) hemisuccinate covalently bound to aminopropyl CPG1000, solid support 826p. (Figure 13)

[0313] Solid support 822c (10.00 g) was alkylated with methyl iodide (0.35 M in acetonitrile, 27.563 mL) and N,N-diisopropylethylamine (0.342 g) according to the method described in Example 165. The solid support was filtered off, washed with 0.25 M DIPEA-HCl in acetonitrile (35 mL), washed with acetonitrile (5 x 50 mL), and dried to give a loading of 44.1 μmol / g. Example 175 (rel-(1R,3-endo,5S,6S,7R)-7-(tris(4-methoxyphenyl)methoxy)-8-methyl-8-(i-propyl)-8-azoniaspiro[bicyclo[3.2.1]octane-3,2'-[1,3]dioxolan]-6-yl) hemisuccinate covalently bound to aminopropyl CPG1500, solid support 827p. (Figure 13)

[0314] Solid support 823c (10.00 g) was alkylated with methyl iodide (0.35 M in acetonitrile, 19.750 mL) and N,N-diisopropylethylamine (0.245 g) according to the method described in Example 165. The solid support was filtered off, washed with 0.25 M DIPEA-HCl in acetonitrile (25 mL), washed with acetonitrile (5 × 50 mL), and dried to give a loading of 31.6 μmol / g. Example 176 (rel-(1R,3-endo,5S,6S,7R)-7-(tris(4-methoxyphenyl)methoxy)-5',5',8-trimethyl-8-azoniaspiro[bicyclo[3.2.1]octane-3,2'-[1,3]dioxan]-6-yl) hemisuccinate covalently bound to aminopropyl CPG500, solid support 828p. (Figure 13)

[0315] Solid support 824c (10.00 g) was alkylated with methyl iodide (0.35 M in acetonitrile, 45.563 mL) and N,N-diisopropylethylamine (0.565 g) according to the method described in Example 165. The solid support was filtered off, washed with 0.25 M DIPEA-HCl in acetonitrile (58 mL), washed with acetonitrile (5 x 50 mL), and dried to give a loading of 72.9 μmol / g. Example 177 (rel-(1R,3-endo,5S,6S,7R)-7-(bis(4-methoxyphenyl)(phenyl)methoxy)-6-acetoxy-8,8-dimethyl-8-azoniabicyclo[3.2.1]octan-3-yl) hemisuccinate covalently bound to aminopropyl CPG500, solid support 741c. (Figure 12)

[0316] Solid support 731c (10.00 g) was alkylated with methyl iodide (0.35 M in acetonitrile, 46.188 mL) and N,N-diisopropylethylamine (0.573 g) according to the method described in Example 165. The solid support was filtered off, washed with 0.25 M DIPEA-HCl in acetonitrile (59 mL), washed with acetonitrile (5 x 50 mL), and dried to give a loading of 73.9 μmol / g. Example 178 (rel-(1R,3-endo,5S,6S,7R)-7-(bis(4-methoxyphenyl)(phenyl)methoxy)-6-acetoxy-8,8-diethyl-8-azoniabicyclo[3.2.1]octan-3-yl) hemisuccinate covalently bound to aminopropyl CPG1000, solid support 742c. (Figure 12)

[0317] Solid support 732c (10.00 g) was alkylated with ethyl iodide (0.35 M in acetonitrile, 27.750 mL) and N,N-diisopropylethylamine (0.344 g) according to the method described in Example 165. The solid support was filtered off, washed with 0.25 M DIPEA-HCl in acetonitrile (36 mL), washed with acetonitrile (5 x 50 mL), and dried to give a loading of 44.4 μmol / g. (Example 179) (rel-(1R,3-endo,5S,6S,7R)-7-(bis(4-methoxyphenyl)(phenyl)methoxy)-6-acetoxy-8-methyl-8-(i-propyl)-8-azoniabicyclo[3.2.1]octan-3-yl) hemisuccinate covalently bound to aminopropyl CPG1500, solid support 743c. (Figure 12)

[0318] Solid support 733c (10.00 g) was alkylated with methyl iodide (0.35 M in acetonitrile, 19.875 mL) and N,N-diisopropylethylamine (0.247 g) according to the method described in Example 165. The solid support was filtered off, washed with 0.25 M DIPEA-HCl in acetonitrile (25 mL), washed with acetonitrile (5 × 50 mL), and dried to give a loading of 31.8 μmol / g. Example 180 (rel-(1R,3-endo,5S,6S,7R)-7-(bis(4-methoxyphenyl)(phenyl)methoxy)-6-acetoxy-8-methyl-8-benzyl-8-azoniabicyclo[3.2.1]octan-3-yl) hemisuccinate covalently bound to aminopropyl CPG1000, solid support 744c. (Figure 12)

[0319] Solid support 734c (10.00 g) was alkylated with methyl iodide (0.35 M in acetonitrile, 25.938 mL) and N,N-diisopropylethylamine (0.322 g) according to the method described in Example 165. The solid support was filtered off, washed with 0.25 M DIPEA-HCl in acetonitrile (33 mL), washed with acetonitrile (5 × 50 mL), and dried to give a loading of 41.5 μmol / g. Example 181 (rel-(1R,3-endo,5S,6S,7R)-7-(bis(4-methoxyphenyl)(phenyl)methoxy)-6-acetoxy-8,8-dimethyl-8-azoniabicyclo[3.2.1]octan-3-yl)diglycolate covalently bound to aminopropyl CPG500, solid support 745c. (Figure 12)

[0320] Solid support 735c (10.00 g) was alkylated with methyl iodide (0.35 M in acetonitrile, 45.375 mL) and N,N-diisopropylethylamine (0.563 g) according to the method described in Example 165. The solid support was filtered off, washed with 0.25 M DIPEA-HCl in acetonitrile (58 mL), washed with acetonitrile (5 x 50 mL), and dried to give a loading of 72.6 μmol / g. Example 182 (rel-(1R,3-endo,5S,6S,7R)-7-(bis(4-methoxyphenyl)(phenyl)methoxy)-6-acetoxy-2,4,8,8-tetramethyl-8-azoniabicyclo[3.2.1]octan-3-yl) hemisuccinate covalently bound to aminopropyl CPG500, solid support 747c. (Figure 12)

[0321] Solid support 737c (10.00 g) was alkylated with methyl iodide (0.35 M in acetonitrile, 46.813 mL) and N,N-diisopropylethylamine (0.581 g) according to the method described in Example 165. The solid support was filtered off, washed with 0.25 M DIPEA-HCl in acetonitrile (60 mL), washed with acetonitrile (5 × 50 mL), and dried to give a loading of 74.9 μmol / g. (Example 183) (rel-(1R,3-endo,5S,6S,7R)-7-(bis(4-methoxyphenyl)(phenyl)methoxy)-6-acetoxy-2,2,4,4,8,8-hexamethyl-8-azoniabicyclo[3.2.1]octan-3-yl) hemisuccinate covalently bound to aminopropyl CPG500, solid support 748c. (Figure 12)

[0322] Solid support 738c (10.00 g) was alkylated with methyl iodide (0.35 M in acetonitrile, 45.938 mL) and N,N-diisopropylethylamine (0.570 g) according to the method described in Example 165. The solid support was filtered off, washed with 0.25 M DIPEA-HCl in acetonitrile (59 mL), washed with acetonitrile (5 x 50 mL), and dried to give a loading of 73.5 μmol / g. Example 184 (rel-(1R,3-endo,5S,6S,7R)-7-(tris(4-methoxyphenyl)methoxy)-6-acetoxy-8,8-dimethyl-8-azoniabicyclo[3.2.1]octan-3-yl) hemisuccinate covalently bound to aminopropyl CPG500, solid support 841c. (Figure 13)

[0323] Solid support 831c (10.00 g) was alkylated with methyl iodide (0.35 M in acetonitrile, 45.438 mL) and N,N-diisopropylethylamine (0.564 g) according to the method described in Example 165. The solid support was filtered off, washed with 0.25 M DIPEA-HCl in acetonitrile (58 mL), washed with acetonitrile (5 x 50 mL), and dried to give a loading of 72.7 μmol / g. Example 185 (rel-(1R,3-endo,5S,6S,7R)-7-(tris(4-methoxyphenyl)methoxy)-6-acetoxy-8,8-diethyl-8-azoniabicyclo[3.2.1]octan-3-yl) hemisuccinate covalently bound to aminopropyl CPG1000, solid support 842c. (Figure 13)

[0324] Solid support 832c (10.00 g) was alkylated with ethyl iodide (0.35 M in acetonitrile, 26.375 mL) and N,N-diisopropylethylamine (0.327 g) according to the method described in Example 165. The solid support was filtered off, washed with 0.25 M DIPEA-HCl in acetonitrile (34 mL), washed with acetonitrile (5 × 50 mL), and dried to give a loading of 42.2 μmol / g. Example 186 (rel-(1R,3-endo,5S,6S,7R)-7-(tris(4-methoxyphenyl)methoxy)-6-acetoxy-8-methyl-8-(i-propyl)-8-azoniabicyclo[3.2.1]octan-3-yl) hemisuccinate covalently bound to aminopropyl CPG1500, solid support 843c. (Figure 13)

[0325] Solid support 833c (10.00 g) was alkylated with methyl iodide (0.35 M in acetonitrile, 19.685 mL) and N,N-diisopropylethylamine (0.244 g) according to the method described in Example 165. The solid support was filtered off, washed with 0.25 M DIPEA-HCl in acetonitrile (25 mL), washed with acetonitrile (5 × 50 mL), and dried to give a loading of 31.5 μmol / g. Example 187 (rel-(1R,3-endo,5S,6S,7R)-7-(tris(4-methoxyphenyl)methoxy)-6-acetoxy-8-methyl-8-benzyl-8-azoniabicyclo[3.2.1]octan-3-yl) hemisuccinate covalently bound to aminopropyl CPG1000, solid support 844c. (Figure 13)

[0326] Solid support 834c (10.00 g) was alkylated with methyl iodide (0.35 M in acetonitrile, 28.125 mL) and N,N-diisopropylethylamine (0.349 g) according to the method described in Example 165. The solid support was filtered off, washed with 0.25 M DIPEA-HCl in acetonitrile (36 mL), washed with acetonitrile (5 × 50 mL), and dried to give a loading of 45.0 μmol / g. Example 188 (rel-(1R,3-endo,5S,6S,7R)-7-(tris(4-methoxyphenyl)methoxy)-6-acetoxy-8,8-dimethyl-8-azoniabicyclo[3.2.1]octan-3-yl)[2-[4-(carboxymethoxy)phenoxy]acetate] covalently bound to aminopropyl CPG500, solid support 846c. (Figure 13)

[0327] Solid support 836c (10.00 g) was alkylated with methyl iodide (0.35 M in acetonitrile, 20.982 mL) and N,N-diisopropylethylamine (0.260 g) according to the method described in Example 165. The solid support was filtered off, washed with 0.25 M DIPEA-HCl in acetonitrile (27 mL), washed with acetonitrile (5 × 50 mL), and dried to give a loading of 33.6 μmol / g. Example 189 (rel-(1R,3-endo,5S,6S,7R)-7-(tris(4-methoxyphenyl)methoxy)-6-acetoxy-2,4,8,8-tetramethyl-8-azoniabicyclo[3.2.1]octan-3-yl) hemisuccinate covalently bound to aminopropyl CPG500, solid support 847c. (Figure 13)

[0328] Solid support 837c (10.00 g) was alkylated with methyl iodide (0.35 M in acetonitrile, 46.875 mL) and N,N-diisopropylethylamine (0.582 g) according to the method described in Example 165. The solid support was filtered off, washed with 0.25 M DIPEA-HCl in acetonitrile (60 mL), washed with acetonitrile (5 × 50 mL), and dried to give a loading of 75.0 μmol / g. Example 190 (rel-(1R,3-endo,5S,6S,7R)-7-(tris(4-methoxyphenyl)methoxy)-6-acetoxy-2,2,4,4,8,8-hexamethyl-8-azoniabicyclo[3.2.1]octan-3-yl) hemisuccinate covalently bound to aminopropyl CPG500, solid support 848c. (Figure 13)

[0329] Solid support 838c (10.00 g) was alkylated with methyl iodide (0.35 M in acetonitrile, 45.188 mL) and N,N-diisopropylethylamine (0.561 g) according to the method described in Example 165. The solid support was filtered off, washed with 0.25 M DIPEA-HCl in acetonitrile (58 mL), washed with acetonitrile (5 x 50 mL), and dried to give a loading of 72.3 μmol / g. (Example 191) N-[(rel-(1R,3-endo,5S,6S,7R)-7-(bis(4-methoxyphenyl)(phenyl)methoxy)-6-acetoxy-8,8-dimethyl-8-azoniabicyclo[3.2.1]octan-3-yl)]succinamic acid covalently bound to aminopropyl CPG500, solid support 761c. (FIG. 12)

[0330] Solid support 751c (10.00 g) was alkylated with methyl iodide (0.35 M in acetonitrile, 46.750 mL) and N,N-diisopropylethylamine (0.580 g) according to the method described in Example 165. The solid support was filtered off, washed with 0.25 M DIPEA-HCl in acetonitrile (60 mL), washed with acetonitrile (5 × 50 mL), and dried to give a loading of 74.8 μmol / g. Example 192 N-[(rel-(1R,3-endo,5S,6S,7R)-7-(bis(4-methoxyphenyl)(phenyl)methoxy)-6-acetoxy-8-methyl-8-(i-propyl)-8-azoniabicyclo[3.2.1]octan-3-yl)]succinamic acid covalently bound to aminopropyl CPG1500, solid support 763c. (FIG. 12)

[0331] Solid support 753c (10.00 g) was alkylated with methyl iodide (0.35 M in acetonitrile, 20.000 mL) and N,N-diisopropylethylamine (0.248 g) according to the method described in Example 165. The solid support was filtered off, washed with 0.25 M DIPEA-HCl in acetonitrile (26 mL), washed with acetonitrile (5 × 50 mL), and dried to give a loading of 32.0 μmol / g. (Example 193) N-[(rel-(1R,3-endo,5S,6S,7R)-7-(tris(4-methoxyphenyl)methoxy)-6-acetoxy-8,8-dimethyl-8-azoniabicyclo[3.2.1]octan-3-yl)]succinamic acid covalently bound to aminopropyl CPG1500, solid support 861c. (Figure 13)

[0332] Solid support 851c (10.00 g) was alkylated with methyl iodide (0.35 M in acetonitrile, 20.563 mL) and N,N-diisopropylethylamine (0.255 g) according to the method described in Example 165. The solid support was filtered off, washed with 0.25 M DIPEA-HCl in acetonitrile (26 mL), washed with acetonitrile (5 × 50 mL), and dried to give a loading of 32.9 μmol / g. (Example 194) N-[(rel-(1R,3-endo,5S,6S,7R)-7-(tris(4-methoxyphenyl)methoxy)-6-acetoxy-8,8-dimethyl-8-azoniabicyclo[3.2.1]octan-3-yl)]succinamic acid covalently bound to hydroxypropyl CPG500, solid support 861c. (Figure 13)

[0333] Solid support 859c (10.00 g) was alkylated with methyl iodide (0.35 M in acetonitrile, 46.625 mL) and N,N-diisopropylethylamine (0.579 g) according to the method described in Example 165. The solid support was filtered off, washed with 0.25 M DIPEA-HCl in acetonitrile (60 mL), washed with acetonitrile (5 × 50 mL), and dried to give a loading of 74.6 μmol / g. Example 195 N-methyl-N-[(rel-(1R,3-endo,5S,6S,7R)-7-(tris(4-methoxyphenyl)methoxy)-6-acetoxy-8,8-dimethyl-8-azoniabicyclo[3.2.1]octan-3-yl)]succinamic acid covalently bound to aminopropyl CPG500, solid support 862c. (Figure 13)

[0334] Solid support 852c (10.00 g) was alkylated with methyl iodide (0.35 M in acetonitrile, 46.000 mL) and N,N-diisopropylethylamine (0.571 g) according to the method described in Example 165. The solid support was filtered off, washed with 0.25 M DIPEA-HCl in acetonitrile (59 mL), washed with acetonitrile (5 x 50 mL), and dried. (Example 196) [ka] (rel-(1R,3-endo,5S,6S,7R)-7-[(bis(4-methoxyphenyl)(phenyl)methoxy]-3-acetoxy-8,8-dimethyl-azoniabicyclo[3.2.1]octan-6-yl) hemisuccinate, 711p, covalently bound to aminomethyl MPPS. (Figure 12)

[0335] Methyl iodide (1 M in MeCN, 68 mL) and N,N-diisopropylethylamine (2.2 g) were added to solid support 701p (10 g). The resulting suspension was shaken for 12 hours. The solid support was filtered off, washed on the filter with 0.25 M DIPEA-HCl in acetonitrile (135 mL), then washed with acetonitrile (5 × 50 mL), and dried in vacuo. The loading of the finished solid support 701c (341.0 μmol / g), where applicable, was determined by a standard di- or trimethoxytrityl assay as disclosed in Guzaev, AP and Pon, RT Curr. Protoc. Nucleic Acid Chem. 2013, 52, pp. 3.2.1-3.2.23. Example 197 (rel-(1R,3-endo,5S,6S,7R)-7-(bis(4-methoxyphenyl)(phenyl)methoxy)-3-pivaloyloxy-8-(i-propyl)-8-azoniabicyclo[3.2.1]octan-6-yl) hemisuccinate covalently bound to aminomethyl MPPS, solid support 712p. (Figure 12)

[0336] Solid support 703p (10.0 g) was alkylated with methyl iodide (1 M in acetonitrile, 67.6 mL) and N,N-diisopropylethylamine (2.184 g) according to the method described in Example 196. The solid support was filtered off, washed with 0.25 M DIPEA-HCl in acetonitrile (135 mL), washed with acetonitrile (5 × 50 mL), and dried to give a loading of 338 μmol / g. Example 198 (rel-(1R,3-endo,5S,6S,7R)-7-(bis(4-methoxyphenyl)(phenyl)methoxy)-3-isobutyryloxy-8-benzyl-8-azoniabicyclo[3.2.1]octan-6-yl) hemisuccinate covalently bound to aminomethyl MPPS, solid support 713p. (Figure 12)

[0337] Solid support 705p (10.0 g) was alkylated with methyl iodide (1 M in acetonitrile, 67.29 mL) and N,N-diisopropylethylamine (2.174 g) according to the method described in Example 196. The solid support was filtered off, washed with 0.25 M DIPEA-HCl in acetonitrile (135 mL), washed with acetonitrile (5 × 50 mL), and dried to give a loading of 336 μmol / g. Example 199 (rel-(1R,3-endo,5S,6S,7R)-7-(tris(4-methoxyphenyl)methoxy)-3-acetoxy-8,8-dimethyl-8-azoniabicyclo[3.2.1]octan-6-yl) hemisuccinate covalently bound to aminomethyl MPPS, solid support 811p. (Figure 13)

[0338] Solid support 801p (10.0 g) was alkylated with methyl iodide (1 M in acetonitrile, 67.19 mL) and N,N-diisopropylethylamine (2.171 g) according to the method described in Example 196. The solid support was filtered off, washed with 0.25 M DIPEA-HCl in acetonitrile (134 mL), washed with acetonitrile (5 × 50 mL), and dried to give a loading of 336 μmol / g. Example 200 (rel-(1R,3-endo,5S,6S,7R)-7-(tris(4-methoxyphenyl)methoxy)-3-pivaloyloxy-8-methyl-8-(i-propyl)-8-azoniabicyclo[3.2.1]octan-6-yl) hemisuccinate covalently bound to aminomethyl MPPS, solid support 812p

[0339] Solid support 803p (10.0 g) was alkylated with methyl iodide (1 M in acetonitrile, 64.02 mL) and N,N-diisopropylethylamine (2.069 g) according to the method described in Example 196. The solid support was filtered off, washed with 0.25 M DIPEA-HCl in acetonitrile (128 mL), washed with acetonitrile (5 × 50 mL), and dried to give a loading of 320 μmol / g. (Example 201) (rel-(1R,3-endo,5S,6S,7R)-7-(tris(4-methoxyphenyl)methoxy)-3-isobutyryloxy-8-methyl-8-benzyl-8-azoniabicyclo[3.2.1]octan-6-yl) hemisuccinate covalently bound to aminomethyl MPPS, solid support 813p. (Figure 13)

[0340] Solid support 805p (10.0 g) was alkylated with methyl iodide (1 M in acetonitrile, 66.52 mL) and N,N-diisopropylethylamine (2.150 g) according to the method described in Example 196. The solid support was filtered off, washed with 0.25 M DIPEA-HCl in acetonitrile (133 mL), washed with acetonitrile (5 × 50 mL), and dried to give a loading of 333 μmol / g. (Example 202) (rel-(1R,3-endo,5S,6S,7R)-7-(tris(4-methoxyphenyl)methoxy)-3-[(t-butyl)dimethylsilyloxy]-8-methyl-8-azoniabicyclo[3.2.1]octan-6-yl) hemisuccinate covalently bound to aminomethyl MPPS, solid support 817p. (Figure 13)

[0341] Solid support 816p (10.0 g) was alkylated with methyl iodide (1 M in acetonitrile, 65.26 mL) and N,N-diisopropylethylamine (2.109 g) according to the method described in Example 196. The solid support was filtered off, washed with 0.25 M DIPEA-HCl in acetonitrile (131 mL), washed with acetonitrile (5 × 50 mL), and dried to give a loading of 326 μmol / g. (Example 203) (rel-(1R,3-endo,5S,6S,7R)-7-(tris(4-methoxyphenyl)methoxy)-3-[(t-butyl)diphenylsilyloxy]-8-methyl-8-azoniabicyclo[3.2.1]octan-6-yl) hemisuccinate covalently bound to aminomethyl MPPS, solid support 819p. (Figure 13)

[0342] Solid support 818p (10.0 g) was alkylated with methyl iodide (1 M in acetonitrile, 66.7 mL) and N,N-diisopropylethylamine (2.155 g) according to the method described in Example 196. The solid support was filtered off, washed with 0.25 M DIPEA-HCl in acetonitrile (133 mL), washed with acetonitrile (5 × 50 mL), and dried to give a loading of 334 μmol / g. (Example 204) (rel-(1R,3-endo,5S,6S,7R)-7-(tris(4-methoxyphenyl)methoxy)-8,8-dimethyl-8-azoniaspiro[bicyclo[3.2.1]octane-3,2'-[1,3]dioxolan]-6-yl) hemisuccinate covalently bound to aminomethyl MPPS, solid support 825p

[0343] Solid support 821p (10.0 g) was alkylated with methyl iodide (1 M in acetonitrile, 66.41 mL) and N,N-diisopropylethylamine (2.146 g) according to the method described in Example 196. The solid support was filtered off, washed with 0.25 M DIPEA-HCl in acetonitrile (133 mL), washed with acetonitrile (5 × 50 mL), and dried to give a loading of 324 μmol / g. (Example 205) (rel-(1R,3-endo,5S,6S,7R)-7-(tris(4-methoxyphenyl)methoxy)-5',5',8-trimethyl-8-azoniaspiro[bicyclo[3.2.1]octane-3,2'-[1,3]dioxan]-6-yl) hemisuccinate covalently bound to aminomethyl MPPS, solid support 826p. (Figure 13)

[0344] Solid support 822p (10.0 g) was alkylated with methyl iodide (1 M in acetonitrile, 64.88 mL) and N,N-diisopropylethylamine (2.096 g) according to the method described in Example 196. The solid support was filtered off, washed with 0.25 M DIPEA-HCl in acetonitrile (130 mL), washed with acetonitrile (5 × 50 mL), and dried to give a loading of 341 μmol / g. (Example 206) (rel-(1R,3-endo,5S,6S,7R)-7-(tris(4-methoxyphenyl)methoxy)-8-methyl-8-(i-propyl)-8-azoniaspiro[bicyclo[3.2.1]octane-3,2'-[1,3]dioxolan]-6-yl) hemisuccinate covalently bound to aminomethyl MPPS, solid support 827p. (Figure 13)

[0345] Solid support 823p (10.0 g) was alkylated with methyl iodide (1 M in acetonitrile, 64.78 mL) and N,N-diisopropylethylamine (2.093 g) according to the method described in Example 196. The solid support was filtered off, washed with 0.25 M DIPEA-HCl in acetonitrile (130 mL), washed with acetonitrile (5 × 50 mL), and dried to give a loading of 339 μmol / g. (Example 207) (rel-(1R,3-endo,5S,6S,7R)-7-(tris(4-methoxyphenyl)methoxy)-5',5',8-trimethyl-8-azoniaspiro[bicyclo[3.2.1]octane-3,2'-[1,3]dioxan]-6-yl) hemisuccinate covalently bound to aminomethyl MPPS, solid support 828p. (Figure 13)

[0346] Solid support 824p (10.0 g) was alkylated with methyl iodide (1 M in acetonitrile, 64.87 mL) and N,N-diisopropylethylamine (2.096 g) according to the method described in Example 196. The solid support was filtered off, washed with 0.25 M DIPEA-HCl in acetonitrile (130 mL), washed with acetonitrile (5 × 50 mL), and dried to give a loading of 328 μmol / g. (Example 208) (rel-(1R,3-endo,5S,6S,7R)-7-(bis(4-methoxyphenyl)(phenyl)methoxy)-6-acetoxy-8,8-dimethyl-8-azoniabicyclo[3.2.1]octan-3-yl) hemisuccinate covalently bound to aminomethyl MPPS, solid support 741p. (Figure 12)

[0347] Solid support 731p (10.0 g) was alkylated with methyl iodide (1 M in acetonitrile, 68.63 mL) and N,N-diisopropylethylamine (2.218 g) according to the method described in Example 196. The solid support was filtered off, washed with 0.25 M DIPEA-HCl in acetonitrile (137 mL), washed with acetonitrile (5 × 50 mL), and dried to give a loading of 328 μmol / g. (Example 209) (rel-(1R,3-endo,5S,6S,7R)-7-(bis(4-methoxyphenyl)(phenyl)methoxy)-6-acetoxy-8,8-diethyl-8-azoniabicyclo[3.2.1]octan-3-yl) hemisuccinate covalently bound to aminomethyl MPPS, solid support 742p. (Figure 12)

[0348] Solid support 732p (10.0 g) was alkylated with ethyl iodide (1 M in acetonitrile, 65.4 mL) and N,N-diisopropylethylamine (2.113 g) according to the method described in Example 196. The solid support was filtered off, washed with 0.25 M DIPEA-HCl in acetonitrile (131 mL), washed with acetonitrile (5 × 50 mL), and dried to give a loading of 334 μmol / g. (Example 210) (rel-(1R,3-endo,5S,6S,7R)-7-(bis(4-methoxyphenyl)(phenyl)methoxy)-6-acetoxy-8-methyl-8-(i-propyl)-8-azoniabicyclo[3.2.1]octan-3-yl) hemisuccinate covalently bound to aminomethyl MPPS, solid support 743p. (Figure 12)

[0349] Solid support 733p (10.0 g) was alkylated with methyl iodide (1 M in acetonitrile, 65.25 mL) and N,N-diisopropylethylamine (2.109 g) according to the method described in Example 196. The solid support was filtered off, washed with 0.25 M DIPEA-HCl in acetonitrile (131 mL), washed with acetonitrile (5 × 50 mL), and dried to give a loading of 331 μmol / g. Example 211 (rel-(1R,3-endo,5S,6S,7R)-7-(bis(4-methoxyphenyl)(phenyl)methoxy)-6-acetoxy-8-methyl-8-benzyl-8-azoniabicyclo[3.2.1]octan-3-yl) hemisuccinate covalently bound to aminomethyl MPPS, solid support 744p. (Figure 12)

[0350] Solid support 734p (10.0 g) was alkylated with methyl iodide (1 M in acetonitrile, 66.11 mL) and N,N-diisopropylethylamine (2.136 g) according to the method described in Example 196. The solid support was filtered off, washed with 0.25 M DIPEA-HCl in acetonitrile (132 mL), washed with acetonitrile (5 × 50 mL), and dried to give a loading of 334 μmol / g. Example 212 (rel-(1R,3-endo,5S,6S,7R)-7-(bis(4-methoxyphenyl)(phenyl)methoxy)-6-acetoxy-8,8-dimethyl-8-azoniabicyclo[3.2.1]octan-3-yl)diglycolate covalently bound to aminomethyl MPPS, solid support 745p. (Figure 12)

[0351] Solid support 735p (10.0 g) was alkylated with methyl iodide (1 M in acetonitrile, 67.09 mL) and N,N-diisopropylethylamine (2.168 g) according to the method described in Example 196. The solid support was filtered off, washed with 0.25 M DIPEA-HCl in acetonitrile (134 mL), washed with acetonitrile (5 × 50 mL), and dried to give a loading of 327 μmol / g. (Example 213) (rel-(1R,3-endo,5S,6S,7R)-7-(bis(4-methoxyphenyl)(phenyl)methoxy)-6-acetoxy-2,4,8,8-tetramethyl-8-azoniabicyclo[3.2.1]octan-3-yl) hemisuccinate covalently bound to aminomethyl MPPS, solid support 747p. (Figure 12)

[0352] Solid support 737p (10.0 g) was alkylated with methyl iodide (1 M in acetonitrile, 65.28 mL) and N,N-diisopropylethylamine (2.109 g) according to the method described in Example 196. The solid support was filtered off, washed with 0.25 M DIPEA-HCl in acetonitrile (131 mL), washed with acetonitrile (5 × 50 mL), and dried to give a loading of 345 μmol / g. (Example 214) (rel-(1R,3-endo,5S,6S,7R)-7-(bis(4-methoxyphenyl)(phenyl)methoxy)-6-acetoxy-2,2,4,4,8,8-hexamethyl-8-azoniabicyclo[3.2.1]octan-3-yl) hemisuccinate covalently bound to aminomethyl MPPS, solid support 748p. (Figure 12)

[0353] Solid support 738p (10.0 g) was alkylated with methyl iodide (1 M in acetonitrile, 66.26 mL) and N,N-diisopropylethylamine (2.141 g) according to the method described in Example 196. The solid support was filtered off, washed with 0.25 M DIPEA-HCl in acetonitrile (133 mL), washed with acetonitrile (5 × 50 mL), and dried to give a loading of 345 μmol / g. (Example 215) (rel-(1R,3-endo,5S,6S,7R)-7-(tris(4-methoxyphenyl)methoxy)-6-acetoxy-8,8-dimethyl-8-azoniabicyclo[3.2.1]octan-3-yl) hemisuccinate covalently bound to aminomethyl MPPS, solid support 841p. (Figure 13)

[0354] Solid support 831p (10.0 g) was alkylated with methyl iodide (1 M in acetonitrile, 67.34 mL) and N,N-diisopropylethylamine (2.176 g) according to the method described in Example 196. The solid support was filtered off, washed with 0.25 M DIPEA-HCl in acetonitrile (135 mL), washed with acetonitrile (5 × 50 mL), and dried to give a loading of 342 μmol / g. (Example 216) (rel-(1R,3-endo,5S,6S,7R)-7-(tris(4-methoxyphenyl)methoxy)-6-acetoxy-8,8-diethyl-8-azoniabicyclo[3.2.1]octan-3-yl) hemisuccinate covalently bound to aminomethyl MPPS, solid support 842p. (Figure 13)

[0355] Solid support 832p (10.0 g) was alkylated with ethyl iodide (1 M in acetonitrile, 68.11 mL) and N,N-diisopropylethylamine (2.201 g) according to the method described in Example 196. The solid support was filtered off, washed with 0.25 M DIPEA-HCl in acetonitrile (136 mL), washed with acetonitrile (5 × 50 mL), and dried to give a loading of 342 μmol / g. Example 217 (rel-(1R,3-endo,5S,6S,7R)-7-(tris(4-methoxyphenyl)methoxy)-6-acetoxy-8-methyl-8-(i-propyl)-8-azoniabicyclo[3.2.1]octan-3-yl) hemisuccinate covalently bound to aminomethyl MPPS, solid support 843p. (Figure 13)

[0356] Solid support 833p (10.0 g) was alkylated with methyl iodide (1 M in acetonitrile, 67.7 mL) and N,N-diisopropylethylamine (2.187 g) according to the method described in Example 196. The solid support was filtered off, washed with 0.25 M DIPEA-HCl in acetonitrile (135 mL), washed with acetonitrile (5 × 50 mL), and dried to give a loading of 343 μmol / g. Example 218 (rel-(1R,3-endo,5S,6S,7R)-7-(tris(4-methoxyphenyl)methoxy)-6-acetoxy-8-methyl-8-benzyl-8-azoniabicyclo[3.2.1]octan-3-yl) hemisuccinate covalently bound to aminomethyl MPPS, solid support 844p. (Figure 13)

[0357] Solid support 834p (10.0 g) was alkylated with methyl iodide (1 M in acetonitrile, 68.53 mL) and N,N-diisopropylethylamine (2.214 g) according to the method described in Example 196. The solid support was filtered off, washed with 0.25 M DIPEA-HCl in acetonitrile (137 mL), washed with acetonitrile (5 × 50 mL), and dried to give a loading of 343 μmol / g. (Example 219) (rel-(1R,3-endo,5S,6S,7R)-7-(tris(4-methoxyphenyl)methoxy)-6-acetoxy-8,8-dimethyl-8-azoniabicyclo[3.2.1]octan-3-yl)[2-[4-(carboxymethoxy)phenoxy]acetate] covalently bound to aminomethyl MPPS, solid support 846p. (Figure 13)

[0358] Solid support 836p (10.0 g) was alkylated with methyl iodide (1 M in acetonitrile, 68.57 mL) and N,N-diisopropylethylamine (2.216 g) according to the method described in Example 196. The solid support was filtered off, washed with 0.25 M DIPEA-HCl in acetonitrile (137 mL), washed with acetonitrile (5 × 50 mL), and dried to give a loading of 344 μmol / g. Example 220 (rel-(1R,3-endo,5S,6S,7R)-7-(tris(4-methoxyphenyl)methoxy)-6-acetoxy-2,4,8,8-tetramethyl-8-azoniabicyclo[3.2.1]octan-3-yl) hemisuccinate covalently bound to aminomethyl MPPS, solid support 847p. (Figure 13)

[0359] Solid support 837p (10.0 g) was alkylated with methyl iodide (1 M in acetonitrile, 64.42 mL) and N,N-diisopropylethylamine (2.082 g) according to the method described in Example 196. The solid support was filtered off, washed with 0.25 M DIPEA-HCl in acetonitrile (129 mL), washed with acetonitrile (5 × 50 mL), and dried to give a loading of 345 μmol / g. Example 221 (rel-(1R,3-endo,5S,6S,7R)-7-(tris(4-methoxyphenyl)methoxy)-6-acetoxy-2,2,4,4,8,8-hexamethyl-8-azoniabicyclo[3.2.1]octan-3-yl) hemisuccinate covalently bound to aminomethyl MPPS, solid support 848p. (Figure 13)

[0360] Solid support 838p (10.0 g) was alkylated with methyl iodide (1 M in acetonitrile, 68.2 mL) and N,N-diisopropylethylamine (2.204 g) according to the method described in Example 196. The solid support was filtered off, washed with 0.25 M DIPEA-HCl in acetonitrile (136 mL), washed with acetonitrile (5 × 50 mL), and dried to give a loading of 346 μmol / g. Example 222 N-[(rel-(1R,3-endo,5S,6S,7R)-7-(bis(4-methoxyphenyl)(phenyl)methoxy)-6-acetoxy-8,8-dimethyl-8-azoniabicyclo[3.2.1]octan-3-yl)]succinamic acid covalently bound to aminomethyl MPPS, solid support 751p. (Figure 10)

[0361] Solid support 751p (10.0 g) was alkylated with methyl iodide (1 M in acetonitrile, 69.24 mL) and N,N-diisopropylethylamine (2.237 g) according to the method described in Example 196. The solid support was filtered off, washed with 0.25 M DIPEA-HCl in acetonitrile (138 mL), washed with acetonitrile (5 × 50 mL), and dried to give a loading of 346 μmol / g. Example 223 N-[(rel-(1R,3-endo,5S,6S,7R)-7-(bis(4-methoxyphenyl)(phenyl)methoxy)-6-acetoxy-8-methyl-8-(i-propyl)-8-azoniabicyclo[3.2.1]octan-3-yl)]succinamic acid covalently bound to aminomethyl MPPS, solid support 753p. (Figure 10)

[0362] Solid support 753p (10.0 g) was alkylated with methyl iodide (1 M in acetonitrile, 69.47 mL) and N,N-diisopropylethylamine (2.245 g) according to the method described in Example 196. The solid support was filtered off, washed with 0.25 M DIPEA-HCl in acetonitrile (139 mL), washed with acetonitrile (5 × 50 mL), and dried to give a loading of 347 μmol / g. Example 224 N-[(rel-(1R,3-endo,5S,6S,7R)-7-(tris(4-methoxyphenyl)methoxy)-6-acetoxy-8,8-dimethyl-8-azoniabicyclo[3.2.1]octan-3-yl)]succinamic acid covalently bound to aminomethyl MPPS, solid support 851p. (Figure 11)

[0363] Solid support 851p (10.0 g) was alkylated with methyl iodide (1 M in acetonitrile, 70.39 mL) and N,N-diisopropylethylamine (2.275 g) according to the method described in Example 196. The solid support was filtered off, washed with 0.25 M DIPEA-HCl in acetonitrile (141 mL), washed with acetonitrile (5 × 50 mL), and dried to give a loading of 352 μmol / g. Example 225 N-[(rel-(1R,3-endo,5S,6S,7R)-7-(tris(4-methoxyphenyl)methoxy)-6-acetoxy-8,8-dimethyl-8-azoniabicyclo[3.2.1]octan-3-yl)]succinamic acid covalently bound to hydroxymethyl MPPS, solid support 859p. (Figure 11)

[0364] Solid support 851p (10.0 g) was alkylated with methyl iodide (1 M in acetonitrile, 70.39 mL) and N,N-diisopropylethylamine (2.275 g) according to the method described in Example 196. The solid support was filtered off, washed with 0.25 M DIPEA-HCl in acetonitrile (141 mL), washed with acetonitrile (5 × 50 mL), and dried to give a loading of 352 μmol / g. Example 226 N-methyl-N-[(rel-(1R,3-endo,5S,6S,7R)-7-(tris(4-methoxyphenyl)methoxy)-6-acetoxy-8,8-dimethyl-8-azoniabicyclo[3.2.1]octan-3-yl)]succinamic acid covalently bound to aminomethyl MPPS, solid support 852p. (Figure 11)

[0365] Solid support 852p (10.0 g) was alkylated with methyl iodide (1 M in acetonitrile, 70.51 mL) and N,N-diisopropylethylamine (2.278 g) according to the method described in Example 196. The solid support was filtered off, washed with 0.25 M DIPEA-HCl in acetonitrile (141 mL), washed with acetonitrile (5 × 50 mL), and dried to give a loading of 353 μmol / g. Example 227 (rel-(1R,3-endo,5S,6S,7R)-N-[7-[tris(4-methoxyphenyl)methoxy]-6-acetoxy-8,8-dimethyl-azoniabicyclo[3.2.1]octan-6-yl)]carbamic acid, 864c, covalently bound to aminopropyl CPG1000. (Figure 13)

[0366] Solid support 854c (10.0 g) was alkylated with methyl iodide (1 M in acetonitrile, 27.0 mL) and N,N-diisopropylethylamine (0.336 g) according to the method described in Example 196. The solid support was filtered off, washed with 0.25 M DIPEA-HCl in acetonitrile (40 mL), washed with acetonitrile (5 × 50 mL), and dried to give a loading of 43.3 μmol / g. Example 228 [6-[methyl[(rel-(1R,3-endo,5S,6S,7R)-N-[7-[tris(4-methoxyphenyl)methoxy]-6-acetoxy-8,8-dimethyl-azoniabicyclo[3.2.1]octan-6-yl)]amino]-6-oxohexyl]carbamate, 865c, covalently bound to aminopropyl CPG500. (Figure 13)

[0367] Solid support 855c (10.0 g) was alkylated with methyl iodide (1 M in acetonitrile, 45.7 mL) and N,N-diisopropylethylamine (0.567 g) according to the method described in Example 196. The solid support was filtered off, washed with 0.25 M DIPEA-HCl in acetonitrile (58 mL), washed with acetonitrile (5 x 50 mL), and dried to give a loading of 73.1 μmol / g. Example 229 [6-[methyl[(rel-(1R,3-endo,5S,6S,7R)-N-[7-[tris(4-methoxyphenyl)methoxy]-6-acetoxy-8,8-dimethyl-azoniabicyclo[3.2.1]octan-3-yl)amino]-6-oxohexyl]carbamate, 866c, covalently bound to hydroxypropyl CPG500. (Figure 13)

[0368] Solid support 856c (10.0 g) was alkylated with methyl iodide (1 M in acetonitrile, 43 mL) and N,N-diisopropylethylamine (0.534 g) according to the method described in Example 196. The solid support was filtered off, washed with 0.25 M DIPEA-HCl in acetonitrile (55 mL), washed with acetonitrile (5 x 50 mL), and dried to give a loading of 68.8 μmol / g. Example 230 [6-[3-[(rel-(1R,3-endo,5S,6S,7R)-N-[7-[tris(4-methoxyphenyl)methoxy]-6-acetoxy-8,8-dimethyl-azoniabicyclo[3.2.1]octan-3-yl)ureido]hexyl]carbamic acid, 868c, covalently bound to hydroxypropyl CPG500. (Figure 13)

[0369] Solid support 858c (10.0 g) was alkylated with methyl iodide (1 M in acetonitrile, 42.1 mL) and N,N-diisopropylethylamine (0.523 g) according to the method described in Example 196. The solid support was filtered off, washed with 0.25 M DIPEA-HCl in acetonitrile (54 mL), washed with acetonitrile (5 x 50 mL), and dried to give a loading of 67.4 μmol / g. Example 231 (rel-(1R,3-endo,5S,6S,7R)-N-[7-[tris(4-methoxyphenyl)methoxy]-6-acetoxy-8,8-dimethyl-azoniabicyclo[3.2.1]octan-6-yl)]carbamic acid, 864p, covalently bound to hydroxymethyl MPPS. (Figure 13)

[0370] Solid support 854p (5.0 g) was alkylated with methyl iodide (1 M in acetonitrile, 35.0 mL) and N,N-diisopropylethylamine (2.27 g) according to the method described in Example 196. The solid support was filtered off, washed with 0.25 M DIPEA-HCl in acetonitrile (35 mL), washed with acetonitrile (5 × 50 mL), and dried to give a loading of 354 μmol / g. Example 232 [6-[methyl[(rel-(1R,3-endo,5S,6S,7R)-N-[7-[tris(4-methoxyphenyl)methoxy]-6-acetoxy-8,8-dimethyl-azoniabicyclo[3.2.1]octan-6-yl)]amino]-6-oxohexyl]carbamic acid, 865p, covalently bound to hydroxymethyl MPPS. (Figure 13)

[0371] Solid support 855p (10.0 g) was alkylated with methyl iodide (1 M in acetonitrile, 45.7 mL) and N,N-diisopropylethylamine (0.567 g) according to the method described in Example 196. The solid support was filtered off, washed with 0.25 M DIPEA-HCl in acetonitrile (38 mL), washed with acetonitrile (5 × 50 mL), and dried to give a loading of 346 μmol / g. Example 233 [6-[methyl[(rel-(1R,3-endo,5S,6S,7R)-N-[7-[tris(4-methoxyphenyl)methoxy]-6-acetoxy-8,8-dimethyl-azoniabicyclo[3.2.1]octan-3-yl)amino]-6-oxohexyl]carbamate, 866p, covalently bound to hydroxymethyl MPPS. (Figure 13)

[0372] Solid support 856p (5.0 g) was alkylated with methyl iodide (1 M in acetonitrile, 35.4 mL) and N,N-diisopropylethylamine (1.15 g) according to the method described in Example 196. The solid support was filtered off, washed with 0.25 M DIPEA-HCl in acetonitrile (35 mL), washed with acetonitrile (5 × 50 mL), and dried to give a loading of 322 μmol / g. Example 234 [6-[3-[(rel-(1R,3-endo,5S,6S,7R)-N-[7-[tris(4-methoxyphenyl)methoxy]-6-acetoxy-8,8-dimethyl-azoniabicyclo[3.2.1]octan-3-yl)ureido]hexyl]carbamic acid, 868p, covalently bound to hydroxymethyl MPPS (Figure 13).

[0373] Solid support 858p (5.0 g) was alkylated with methyl iodide (1 M in acetonitrile, 35.5 mL) and N,N-diisopropylethylamine (1.14 g) according to the method described in Example 196. The solid support was filtered off, washed with 0.25 M DIPEA-HCl in acetonitrile (54 mL), washed with acetonitrile (5 x 50 mL), and dried to give a loading of 325 μmol / g. Example 235 [ka] (rel-(1R,3-endo,5S,6S,7R)-N-[7-[tris(4-methoxyphenyl)methoxy]-6-acetoxy-8-methyl-azabicyclo[3.2.1]octan-6-yl)]carbamic acid, 854c covalently bound to aminopropyl CPG1000

[0374] A solution of 1,1-carbonyldiimidazole (97 mg, 0.6 mmol) in DMF (2 mL) was added dropwise to compound 451 (290 mg, 0.57 mmol) in acetonitrile (4 mL) and DIPEA (72 g, 0.57 mmol) while stirring in an ice bath. The mixture was stirred overnight and transferred to a suspension of aminopropyl CPG1000 (11.5 g) in anhydrous acetonitrile (40 mL) and DIPEA (176 mg). The suspension was shaken overnight, and completion of the loading reaction was verified by a standard trimethoxytrityl assay as disclosed in Guzaev, AP and Pon, RT, Curr. Protoc. Nucleic Acid Chem. 2013, 52, pp. 3.2.1-3.2.23. N-methylimidazole (1.5 mL) was added, followed by acetic anhydride (1.5 mL). The mixture was shaken for an additional 6 h, which was required for complete acetylation of the hydroxy group at the 6-position of the tropane ring. The solid support was filtered off, washed on the filter with acetonitrile (5 × 50 mL), and dried in vacuo to give a loading of 43.3 μmol / g.

[0375] Universal solid supports 854p, 855c, 855p, 865c, 856p, 858c, and 858p (Figure 11) were prepared according to the same method to give loadings of 354, 73.1, 346, 68.8, 322, 67.4, and 325 μmol / g, respectively. Example 236 [ka] (rel-(1R,3-endo,5S,6S,7R)-7-hydroxy-3-acetoxy-8,8-dimethyl-azoniabicyclo[3.2.1]octan-6-yl) hemisuccinate, 911c, covalently bound to aminopropyl CPG1000

[0376] A detritylation agent solution containing an anhydrous solution of trichloroacetic acid in toluene (5%, 60 mL) was passed through a column containing solid support 811c (10 g) at a rate of 6 mL / min. The column was flushed with a stream of nitrogen and washed with a solution of pyridine in acetonitrile (5%, 40 mL) at a flow rate of 10 to 20 mL / min. If necessary, after the 5% pyridine in acetonitrile wash, the wash cycle could include an additional wash with 0.25 M DIPEA-HCl in acetonitrile (5 mL / g) to replace the trichloroacetic acid counterion with chloride. Finally, the support was washed with acetonitrile (5 × 50 mL), flushed with a stream of nitrogen, and dried in vacuo. The loading of the completed solid support 911c (69.0 μmol / g) was determined by coupling DMT-T phosphoramidite to a 1 μmol sample of the support, followed by a standard dimethoxytrityl assay as disclosed in Guzaev, AP and Pon, RT Curr. Protoc. Nucleic Acid Chem. 2013, 52, pp. 3.2.1-3.2.23.

[0377] Deprotected solid supports (Figure 14) 911c-913c, 917c, 919c, 925c, 927c, 929c, 942c-945c, 946c-948c, 961c-966c, 968c, 969c, 971c, 974c, and 975c were obtained according to the procedures disclosed above. Example 237 [ka] (rel-(1R,3-endo,5S,6S,7R)-7-hydroxy-3-acetoxy-8,8-dimethyl-azoniabicyclo[3.2.1]octan-6-yl) hemisuccinate, 911p, covalently bound to aminomethyl MPPS

[0378] A detritylation agent solution containing an anhydrous solution of trichloroacetic acid in toluene (5%, 60 mL) was passed through a column containing solid support 811p (10 g) at a rate of 6 mL / min. The column was flushed with a stream of nitrogen and washed with a solution of pyridine in acetonitrile (5%, 60 mL) at a flow rate of 10 to 20 mL / min. If necessary, after the 5% pyridine in acetonitrile wash, the wash cycle could include an additional wash with 0.25 M DIPEA-HCl in acetonitrile (5 mL / g) to replace the trichloroacetic acid counterion with chloride. Finally, the support was washed with acetonitrile (5 × 50 mL), flushed with a stream of nitrogen, and dried in vacuo. The loading of the finished solid support 911p (341 μmol / g) was determined by coupling DMT-T phosphoramidite to a 1 μmol sample of the support, followed by a standard dimethoxytrityl assay as disclosed in Guzaev, AP and Pon, RT Curr. Protoc. Nucleic Acid Chem. 2013, 52, pp. 3.2.1-3.2.23.

[0379] Deprotected solid supports (Figure 14) 911p-913p, 917p, 919p, 925p, 927p, 929p, 942p-945p, 946p-948p, 961p-966p, 968p, 969p, 971p, 974p, and 975p were obtained according to the procedures disclosed above. Example 238 [ka] Universal Phosphoramidite 806a

[0380] A mixture of disuccinimidyl carbonate (705 mg, 2.75 mmol) and compound 406 (2.41 g, 2.5 mmol) in anhydrous pyridine (15 mL) was stirred overnight, and 6-aminohexanol (366 mg, 3.12 mmol) was added. The mixture was stirred for 6 hours, the solvent was evaporated, and the residue was partitioned between dichloromethane (100 mL) and 1 M NaH2PO4 (20 mL). The organic phase was washed with 1 M NaH2PO4 (2 × 50 mL), 5% aqueous NaHCO3 (2 × 50 mL), and brine (30 mL). After drying over Na2SO4, the organic phase was evaporated and dried under vacuum to give crude compound 606a.

[0381] Crude 606a was dissolved in acetonitrile, DIPEA (1.26 g, 10 mmol) and methyl iodide (1.06 g, 7.5 mmol) were added, and the mixture was stirred at 45 °C for 6 h. The solvent was evaporated, the residue was dissolved in DCM (100 mL), and the solution was washed with 5% aqueous NaHCO (2 × 50 mL) and brine (30 mL). The solution was applied to a silica gel column, and the product was isolated by column purification using a linear gradient of DCM to TEA:MeOH:DCM (5:20:75). The collected fractions were evaporated and coevaporated with acetonitrile (4 × 25 mL), after which the material was dried on an oil pump.

[0382] A solution of 1H-tetrazole in acetonitrile (0.45 M, 3.9 mL, 1.75 mmol) was added to a mixture of the product of the previous step, 2-cyanoethylbis(N,N-diisopropylamino)phosphite (829 mg, 2.75 mmol), and acetonitrile (5 mL). The mixture was stirred overnight. Triethylamine (202 mg, 2 mmol) was added, and the mixture was diluted with DCM (100 mL), washed with 5% aqueous NaHCO3 (2 × 20 mL), and washed with brine (30 mL). The organic phase was dried over Na2SO4 and evaporated. The residue was applied to a silica gel column, and the product was isolated by column purification using a linear gradient of DCM to TEA:MeOH:DCM (5:5:90). After evaporation of the collected fractions and coevaporation with acetonitrile (4 × 25 mL), the material was dried on an oil pump to give the desired product as a solid foam (1840 mg, 70.8%). 31 P NMR(CD3CN):δ149.45, 149.6.ES MS:[M+H] + 1004.7 (measured value), 1005.2 (calculated value). Example 239 [ka] 2-Cyanoethyl rel-(1R,5S,6S,7R)-3-[[[[7-(tris(4-methoxyphenyl)methoxy)-6-acetoxy-8,8-dimethyl-8-azoniabicyclo[3.2.1]octan-3-yl]oxy]carbonyl]amino]propyl(N,N-diisopropylamino)phosphite 773

[0383] Compound 431 (1095 mg, 2.0 mmol) was dissolved in acetonitrile, DIPEA (1.26 g, 10 mmol) and methyl iodide (1.06 g, 7.5 mmol) were added, and the mixture was stirred at 45 °C for 6 h. The solvent was evaporated, and acetonitrile (10 mL), N,N-carbonyldiimidazole (405 mg, 2.5 mmol), and DIPEA (378 mg, 3 mmol) were added to the residue. The mixture was stirred at 45 °C overnight, cooled in an ice-water bath, and 3-aminopropanol (225 mg, 3.0 mmol) in acetonitrile (3 mL) was added to the residue. The reaction was stirred for 3 h, diluted with DCM (100 mL), and washed with 1 M NaH2PO4 (2 × 50 mL) and brine (30 mL). After drying over Na2SO4, the organic phase was applied to a silica gel column. The product was isolated by column purification using a linear gradient of DCM to TEA:MeOH:DCM (5:25:70). Collected fractions were evaporated and coevaporated with acetonitrile (4 × 25 mL), after which the material was dried in an oil pump.

[0384] A mixture of the product from the previous step and 2-cyanoethylbis(N,N-diisopropylamino)phosphite (663 mg, 2.2 mmol) was charged with a solution of 1H-tetrazole in acetonitrile (0.45 M, 3.1 mL, 1.4 mmol), and the mixture was stirred overnight. Triethylamine (202 mg, 2 mmol) was added, and the mixture was diluted with DCM (100 mL), washed with 5% aqueous NaHCO3 (2 × 20 mL), and then washed with brine (30 mL). The organic phase was dried over Na2SO4 and evaporated. The residue was applied to a silica gel column, and the product was isolated by column purification using a linear gradient of DCM to TEA:MeOH:DCM (5:10:85). After evaporation of the collected fractions and coevaporation with acetonitrile (4 × 25 mL), the material was dried on an oil pump to give the desired product as a solid foam (1480 mg, 82.3%). 31 P NMR (CD3CN): δ 149.12, 149.55.ES MS:[M+H] + 865.5 (measured value), 865.0 (calculated value). (Example 240) [ka] 2-Cyanoethyl rel-(1R,5S,6S,7R)-6-[[[[7-(bis(4-methoxyphenyl)(phenyl)methoxy)-6-acetoxy-8,8-dimethyl-8-azoniabicyclo[3.2.1]octan-3-yl]oxy]carbonyl]amino]hexyl(N,N-diisopropylamino)phosphite 773

[0385] Compound 773 (83.1%), isolated as a white solid foam, was synthesized from compound 331 according to the procedure described in Example 227. 31 P NMR (CD3CN): δ 149.22, 149.59.ES MS:[M+H] + 877.7 (measured value), 877.1 (calculated value). (Example 241) [ka] 2-Cyanoethyl rel-(1R,5S,6S,7R)-7-(tris(4-methoxyphenyl)methoxy)-6-acetoxy-8,8-dimethyl-8-azoniabicyclo[3.2.1]octan-3-yl(N,N-diisopropylamino)phosphite, 872

[0386] Compound 431 (1369 mg, 2.5 mmol) was dissolved in acetonitrile, DIPEA (1.26 g, 10 mmol) and methyl iodide (1.06 g, 7.5 mmol) were added, and the mixture was stirred at 45 °C for 6 h. The solvent was evaporated, the residue was dissolved in DCM (100 mL), and the solution was washed with 5% aqueous NaHCO (2 × 50 mL) and brine (30 mL). The solution was applied to a silica gel column, and the product was isolated by column purification using a linear gradient of DCM to TEA:MeOH:DCM (5:20:75). The collected fractions were evaporated and coevaporated with acetonitrile (4 × 25 mL), after which the material was dried on an oil pump.

[0387] A solution of 1H-tetrazole in acetonitrile (0.45 M, 3.9 mL, 1.75 mmol) was added to a mixture of the product of the previous step, 2-cyanoethylbis(N,N-diisopropylamino)phosphite (829 mg, 2.75 mmol), and acetonitrile (5 mL). The mixture was stirred overnight. Triethylamine (202 mg, 2 mmol) was added, and the mixture was diluted with DCM (100 mL), washed with 5% aqueous NaHCO3 (2 × 20 mL), and then washed with brine (30 mL). The organic phase was dried over Na2SO4 and evaporated. The residue was applied to a silica gel column, and the product was isolated by column purification using a linear gradient of DCM to TEA:MeOH:DCM (5:5:90). After evaporation of the collected fractions and coevaporation with acetonitrile (4 × 25 mL), the material was dried on an oil pump to give the desired product as a solid foam (1714 mg, 85.9%). 31 P NMR (CD3CN): δ 148.53, 149.22.ES MS:[M+H] + 763.3 (measured value), 763.9 (calculated value). (Example 242) [ka] 2-Cyanoethyl 2,9-dimethyl-2-methoxy-hexahydro-6H-cyclohepta-1,3-dioxol-4,8-imin-6-yl(N,N-diisopropylamino)phosphite, 878

[0388] Compound 878 (83.1%), isolated as a white solid foam, was synthesized from compound 21 according to the procedure described in Example 229. 31 P NMR (CD3CN): δ 149.11, 149.42.ES MS:[M+H] + 446.3 (measured value), 445.5 (calculated value). (Example 243) [ka] 2-Cyanoethyl [4-[[2,9-dimethyl-2-methoxy-hexahydro-6H-cyclohepta-1,3-dioxol-4,8-imin-6-yl]amino]-4-oxobutyl(N,N-diisopropylamino)phosphite, 881

[0389] Compound 881 (79.2%), isolated as a white solid foam, was synthesized from compound 880 according to the procedure described in Example 229. 31 P NMR (CD3CN): δ 149.34, 149.66.ES MS:[M+H] + 531.1 (measured value), 530.6 (calculated value). (Example 244) [ka] Universal 2-cyanoethyl (N,N-diisopropylamino) phosphite, 883

[0390] Compound 883 (80.4%), isolated as a white solid foam, was synthesized from compound 882 according to the procedure described in Example 229. 31 P NMR (CD3CN): δ 149.48, 149.82.ES MS:[M+H] + 560.5 (measured value), 559.7 (calculated value). (Example 245) [ka] Universal 2-cyanoethyl (N,N-diisopropylamino) phosphite, 875

[0391] Compound 875 (82.7%), isolated as a white solid foam, was synthesized from compound 874 according to the procedure described in Example 229. 31 P NMR (CD3CN): δ 149.14, 149.54.ES MS:[M+H] + 948.9 (measured value), 948.2 (calculated value). (Example 246) [ka] Universal 2-cyanoethyl (N,N-diisopropylamino) phosphite, 877

[0392] Compound 877 (83.9%), isolated as a white solid foam, was synthesized from compound 876 according to the procedure described in Example 229. 31 P NMR (CD3CN): δ 149.26, 149.63.ES MS:[M+H] + 9934.1 (measured value), 933.2 (calculated value). (Example 247) Oligonucleotide synthesis, cleavage, and analysis

[0393] Oligonucleotides were synthesized using standard solid-phase oligonucleotide synthesis on an ABI 394 synthesizer using commercially available 5'-O-(4,4'-dimethoxytrityl)-2'-deoxy and 5'-O-(4,4'-dimethoxytrityl)-2'-O-methyl-3'-O-(2-cyanoethyl N,N-diisopropyl) phosphoramidites of thymidine, uridine, 4-N-acetylcytidine, 6-N-benzoyladenosine, and 2-N-isobutyrylguanosine. Removal of the dimethoxytrityl group in 16 (or 17) requires longer treatment than the recommended 3% TCA-CHCl cycle. Therefore, the support was treated with 5% TCA-toluene to ensure complete removal of the dimethoxytrityl group before oligonucleotide synthesis. Three different activators were tested (1 M 4,5-dicyanoimidazole + 0.1 M N-methylimidazole in acetonitrile, 0.1 M 5-[3,5-bis(trifluoromethyl)phenyl]-2H-tetrazole (Activator 42®) in acetonitrile, and 0.45 M 5-(benzylthio)-1H-tetrazole in acetonitrile) and all were found to be equally effective. Oxidizing solutions with different compositions of I2, pyridine, and water were tested (0.02 M I2 in 89:10:1 THF:water:pyridine; 0.02 M I2 in 88:10:2 THF:pyridine:water; and 0.05 M I2 in 88:10:2 THF:pyridine:water). The oxidizing solution with a composition of 0.05 M I2 in 88:10:2 THF:pyridine:water was found to be the most efficient. The sulfuration reaction was carried out with 0.1 M DDTT (3-[[(dimethylamino)methylene]amino]-3H-1,2,4-dithiazole-5-thione) in anhydrous pyridine for 3 min, followed by a capping step. After synthesis, all oligonucleotides were treated with 30% aqueous diethylamine:acetonitrile (1:4) to remove the cyanoethyl protecting groups.

[0394] Cleavage of the oligonucleotides from the solid support and removal of the base-protecting groups were carried out using various concentrations of aqueous ammonia and methylamine. Time-controlled deprotection samples were evaporated in vacuo or, if used for kinetic studies, neutralized with 1.66 M aqueous citric acid.

[0395] Oligonucleotide analysis by RP-HPLC was performed on a Phenomenex Kinetex 5 μm EVO C18 100 Å column or a Luna 5 μm C18(2) 100 Å column using 50 mM Tris-HCl, pH 7.6 as buffer A, 50 mM Tris-HCl, pH 7.6:CH3CN (20:80) as buffer B, a column temperature of 60 °C, and UV detection at 260 nm. Ion-exchange HPLC analysis was performed on a Thermo Scientific DNAPac PA 200 column using 20 mM Na2PO4:CH3CN (90:10), pH 11.5 as buffer A, and 20 mM Na2PO4, 1 M NaBr:CH3CN (90:10), pH 11.5 as buffer B, a column temperature of 60 °C, and UV detection at 260 nm. Example 248 Reaction kinetic studies

[0396] T assembled on solid support 941 10 Hydrolytic 3'-dephosphorylation of oligonucleotides and the protected oligonucleotide T4dG ib T5, T3dG ib 3T4, (TdG ib )3T4, and T2dG ibA kinetic study of the deprotection of N-isobutyryl-2'-deoxyguanosine residues in 6T2 was carried out in 1.37 M, 2.56 M, and 3.42 M MeNH2 aqueous solutions (7.5x, 4x, and 3x dilutions of a 10.25 M aqueous solution, commercially available 40% methylamine solution) at temperatures of 20.0 °C, 30.0 °C, 40.0 °C, and 50.0 °C. For each run, approximately 4 mg of solid support-bound oligonucleotide was placed in an 8 mL glass vial with a valved screw cap. This vial and the vial containing the methylamine solution were maintained in a thermostat set at the desired temperature for at least 30 minutes. Approximately 4 mL of the methylamine solution was then transferred to the vial containing the oligo to initiate the deprotection reaction. Time-controlled samples were withdrawn according to a schedule appropriate for the concentration and temperature of the methylamine solution used and immediately neutralized to pH 7 using 1.66 M aqueous citric acid. The neutralized samples were analyzed by reverse-phase HPLC. HPLC analysis was performed on an Agilent 1260 HPLC system using a Waters XBridge C18, 2.5 μm, 4.6 × 75 mm column, 100 mM HFIP, 5 mM TEA as buffer A, and acetonitrile as buffer B, with a linear gradient of 1 to 14% B at 0.45 mL × min. -1 or 1 to 12% B at a flow rate of 0.675 mL x min over 15 min. -1 The analysis was carried out at a flow rate of 0.05% over 72 minutes using a column temperature of 50°C. HPLC traces were recorded at 252 nm. To extract the rate constants, the collected data were fitted to the equation shown in Figure 1 using the solver module in Microsoft Excel.

[0397] Although the foregoing has been described in some detail by way of illustration and example for purposes of clarity and understanding, it will be understood by those skilled in the art that certain changes and modifications may be made within the scope of the appended claims. All publications, patents, patent applications, and sequence accession numbers cited herein are hereby incorporated by reference in their entirety for all purposes.

[0398] Short sequence listing SEQ ID NO: 1 (synthetic construct) TTTTGTTTTT SEQ ID NO: 2 (synthetic construct) TTTGGGTTTT SEQ ID NO: 3 (synthetic construct) TGTGTGTTTT SEQ ID NO: 4 (synthetic construct) TTGGGGGGTT SEQ ID NO: 5 (synthetic construct) TTTTTTTTTT

Claims

1. Compound of formula I 【Chemistry 91】 [In the formula: R １ and R ２ form an orthoester functional group -C(CH ３ )(OCH ３ )-, or one of R １ and R ２ is hydrogen, a trityl protecting group or a derivative thereof, or a xanthenyl protecting group or a derivative thereof, and the other of R １ and R ２ is acetyl, propionyl, n-butyryl, benzoyl, or L １ : L １ The connecting part is -C(=O)-Z-(C=O)-A １ And: Z is a covalent bond, methylene group, -(CH ２ ) ２ -, - (CH ２ )-O-(CH ２ ) - and - (CH ２ )-O-C ６ H ４ -O-(CH ２ Selected from the group consisting of: A １ This refers to a hydroxyl group, a salt of a hydroxyl group with an inorganic cation or tertiary amine, and SP １ Covalent bond to -NH(CH ２ ) ｎ -OR ７ And: SP １ This includes oxygen, amino, aminoalkyl, or hydroxyalkyl atoms covalently bonded to a solid phase material including controlled porous glass, magnetic controlled porous glass, silica-containing particles, styrene-containing polymer or copolymer, divinylbenzene-containing polymer or copolymer, styrene-divinylbenzene copolymer, controlled porous glass grafted with styrene-containing polymer, controlled porous glass grafted with styrene-divinylbenzene copolymer, styrene-divinylbenzene copolymer grafted with polyethylene glycol, or a flat glass surface; n is an integer between 2 and 10; R ７ is hydrogen or PA: 【Chemistry 87】 R ８ is a methyl or 2-cyanoethyl group, R １４ is alkyl, isoalkyl, sec-alkyl, or tert-alkyl; R ３ It is selected from the group consisting of methyl, ethyl, propyl, isopropyl, and benzyl; R ４ R is selected from the group consisting of lone pairs of electrons, hydrogen, methyl, ethyl, propyl, isopropyl, and benzyl. ４ If it is anything other than a lone pair of electrons, then N has a positive charge and forms a salt with a halide anion or an intramolecular carboxyl functional group; R ５ and R ６ These are independently hydrogen or methyl; Y is -(C=O)-, -CH(OR ９ )-,-CH(NR １０ R １１ ) - or - [C ( OR １２ ) ( OR １３ )] - and: R ９ This includes hydrogen, methyl, ethyl, benzyl, acetyl, propionyl, butyryl, isobutyryl, pivaloyl, benzoyl, t-butyldimethylsilyl, triisopropylsilyl, (t-butyl)diphenylsilyl, -(C=O)A1, L １ , or PA: m is an integer in the range of 2 to 10; R １０ is, L １ -(C=O)-A １ , or -(C=O)-W １ - (CH ２ ) ｐ -W ２ And: W １ is, -(CH ２ )-, -(NH)-, or -(NH)-(C=O)-; W ２ hydroxy, amino, -O-PA, -(C=O)-A １ , or -[NH(C=O)]-A １ And; p is an integer between 2 and 10; R １１ is hydrogen, methyl, ethyl, or benzyl; R １２ and R １３ They came together, Ketalbridge-(CH ２ ) ２ -, or -CH ２ - [C(CH ３ ) ２ ]-CH ２ - forms; However, R １ , R ２ , and only one of Y is L １ Or it is PA, or L １ [or including PA].

2. R １ and R ２ One of them is hydrogen, tris-(4-methoxyphenyl)methyl, bis-(4-methoxyphenyl)phenylmethyl, 9-phenylxanthene-9-yl, or 9-(4-methoxyphenyl)xanthene-9-yl, R １ and R ２ The other of them is L １ And: A １ However, a hydroxyl group, a salt of a hydroxyl group with an inorganic cation or tertiary amine, or SP １ It is a covalent bond to; (i) Y is -CH(OR ９ ) - and R ９ is methyl, ethyl, benzyl, acetyl, propionyl, butyryl, isobutyryl, pivaloyl, benzoyl, t-butyldimethylsilyl, triisopropylsilyl, or phenyldimethylsilyl; or (ii) Y is -[C(OR １２ ) ( OR １３ The compound according to claim 1, wherein the compound is:

3. R １ and R ２ One of them is hydrogen, tris-(4-methoxyphenyl)methyl, or bis-(4-methoxyphenyl)phenylmethyl, R １ and R ２ The compound according to claim 1, wherein the other of the two is acetyl, propionyl, n-butyryl, or benzoyl.

4. (i) Y is -CH(OR ９ ) - and R ９ is L １ A １ However, it is a hydroxyl group that forms a salt with an inorganic cation or a tertiary amine as needed, or SP １ Whether covalent bonds are formed to as needed; or (ii) Y is -CH(NR １０ R １１ ) - and R １１ However, it is hydrogen, methyl, ethyl, or benzyl, (a) R １０ is L １ and A １ is a hydroxy group, a salt of a hydroxy group with an inorganic cation or a tertiary amine, or a covalent bond to SP １ or; or (b) R １０ is -(C=O)-W １ -(CH ２ ) ｐ -W ２ and: W ２ However, amino, -(C=O)-A １ , or -[NH(C=O)]-A １ A １ However, a hydroxyl group, a salt of a hydroxyl group with an inorganic cation or tertiary amine, or SP １ It is a covalent bond to; The compound according to claim 3, wherein p is an integer from 3 to 10.

5. R １ and R ２ One of them is tris-(4-methoxyphenyl)methyl or bis-(4-methoxyphenyl)phenylmethyl, R １ and R ２ The other of them is L １ And: (i) A １ ga-NH(CH ２ ) ｎ -OR ７ And; (ii) Y is -CH(OR ９ ) and R ９ The compound according to claim 1, wherein the compound is methyl, ethyl, benzyl, acetyl, propionyl, butyryl, isobutyryl, pivaloyl, benzoyl, t-butyldimethylsilyl, triisopropylsilyl, or phenyldimethylsilyl.

6. R １ and R ２ One of them is tris-(4-methoxyphenyl)methyl or bis-(4-methoxyphenyl)phenylmethyl, R １ and R ２ The other of these is acetyl, propionyl, n-butyryl, or benzoyl, and Y is -CH(OR ９ ) - and: (i) R ９ Is it a PA, or (ii) R ９ ga- (CH ２ ) ｍ The compound according to claim 1, wherein it is -O-PA.

7. R １ and R ２ One of them is tris-(4-methoxyphenyl)methyl or bis-(4-methoxyphenyl)phenylmethyl, R １ and R ２ The other of these is acetyl, propionyl, n-butyryl, or benzoyl, and Y is -CH(NR １０ R １１ ) - and R １１ However, it is hydrogen, methyl, ethyl, or benzyl: R １０ ga - (C=O) - W １ - (CH ２ ) ｐ -W ２ And: W １ However, - (CH ２ )-, -(NH)-, or -(NH)-(C=O)-, W ２ The compound according to claim 1, wherein the compound is hydroxyl or O-PA.

8. R １ and R ２ is an orthoester functional group -C(CH ３ ) (OCH ３ ) - forms (i) Y is -CH(OR ９ ) - and; (ii) R ９ However, hydrogen, PA, or -(CH ２ ) ｍ The compound according to claim 1, wherein it is -O-PA.

9. The compound according to claim 1, wherein the trityl protecting group or its derivative or the xanthenyl protecting group or its derivative is selected from the group consisting of 4-methoxytrityl, 4,4'-dimethoxytrityl, 4,4',4''-trimethoxytrityl, 9-phenylxanthene-9-yl, and 9-(4-methoxyphenyl)xanthene-9-yl.

10. A method for functionalizing a solid-phase material using a first monomer subunit, (a) Step of providing a solid-phase material bonded compound of formula I. 【Chemistry 92】 [In the formula: R １ and R ２ This is an orthoester functional group -C(CH ３ ) (OCH ３ ) - forming or R １ and R ２ One of them is hydrogen, a trityl protecting group or its derivative, or a xanthenyl protecting group or its derivative, R １ and R ２ The other of these is acetyl, propionyl, n-butyryl, benzoyl, L １ And: L １ The connecting part is -C(=O)-Z-(C=O)-A １ And: Z is a covalent bond, methylene group, -(CH ２ ) ２ -, - (CH ２ )-O-(CH ２ ) - and - (CH ２ )-O-C ６ H ４ -O-(CH ２ Selected from the group consisting of: A １ SP １ It is a covalent bond to -NH(CH ２ ) ｎ -OR ７ And: SP １ These are oxygen, amino, aminoalkyl or hydroxyalkyl atoms covalently bonded to a solid phase material including controlled porous glass, magnetic controlled porous glass, silica-containing particles, styrene-containing polymer or copolymer, divinylbenzene-containing polymer or copolymer, styrene-divinylbenzene copolymer, controlled porous glass grafted with styrene-containing polymer, controlled porous glass grafted with styrene-divinylbenzene copolymer, styrene-divinylbenzene copolymer grafted with polyethylene glycol, and a flat glass surface; n is an integer between 2 and 10; R ７ It is a PX: 【Chemistry 89】 R ８ is a methyl or 2-cyanoethyl group, and X is oxygen or sulfur; R ３ It is selected from the group consisting of methyl, ethyl, propyl, isopropyl, and benzyl; R ４ R is selected from the group consisting of lone pairs of electrons, hydrogen, methyl, ethyl, propyl, isopropyl, and benzyl. ４ If it is anything other than a lone pair of electrons, then N has a positive charge and forms a salt with a halide anion or an intramolecular carboxyl functional group; R ５ and R ６ These are independently hydrogen or methyl; Y is -(C=O)-, -CH(OR ９ )-,-CH(NR １０ R １１ ) - or - [C ( OR １２ ) ( OR １３ )] - and: R ９ This includes hydrogen, methyl, ethyl, benzyl, acetyl, propionyl, butyryl, isobutyryl, pivaloyl, benzoyl, t-butyldimethylsilyl, triisopropylsilyl, or (t-butyl)diphenylsilyl group, -(C=O)A1,L １ , or PX: m is an integer in the range of 2 to 10; R １０ is, L １ -(C=O)-A １ , or -(C=O)-W １ - (CH ２ ) ｐ -W ２ And: W １ is, -(CH ２ )-, -(NH)-, or -(NH)-(C=O)-; W ２ is -O-PX or -[NH(C=O)]-A １ And; p is an integer in the range of 2 to 10; R １１ is hydrogen, methyl, ethyl, or benzyl; R １２ and R １３ They came together, Ketalbridge-(CH ２ ) ２ -, or -CH ２ - [C(CH ３ ) ２ ]-CH ２ - forms; However, R １ , R ２ , and only one of Y is L １ Or it is PX, or L １ [or including PX]; (b) A step of selectively removing one of the protecting groups of formula I to form a reactive hydroxyl group; (c) Providing a first monomer subunit comprising an activated phosphorus group and a protected hydroxyl group, and reacting the activated phosphorus group of the first monomer subunit with the reactive hydroxyl group of the compound of formula I to form a monomer-functionalized solid support comprising a phosphite group; (d) The step of treating the monomer-functionalized solid support with a capping agent and / or the monomer-functionalized solid support with an oxidizing solution or a sulfurizing agent to convert the phosphyte triester groups to phospho triesters or phosphothioate triesters, thereby forming an oxidized or sulfurized functionalized solid support; (e) A method comprising the steps of forming an oligomer-functionalized solid support by repeating steps (b), (c), and (d) once or more as needed on the oxidized or sulfurized functionalized solid support, wherein each time steps (b), (c), and (d) are repeated, the monomer subunits are the same or different.

11. The method according to claim 10, further comprising the steps of deprotecting the oligomer-functionalized solid support of step (e), cleaving the oligomer-functionalized solid support to form an oligomer compound separated from the solid phase material, wherein the cleavage results in the formation of terminal hydroxyl groups on the oligomer compound at the cleavage sites.

12. R １ or R ２ The method according to claim 10, wherein one of them is L1.

13. R ７ The method according to claim 10, wherein is PX.

14. The method according to claim 10, wherein the activated phosphorus group comprises a phosphoramidite, an H-phosphonate, or a phosphate triester.

15. The method according to claim 11, wherein the oligomer compound is an oligonucleotide and optionally contains a non-natural sugar-modified nucleotide residue, a non-natural base-modified nucleotide residue, or a non-nucleotide monomer unit.

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