WO2025199647A1

WO2025199647A1 - C4'-modified nucleoside analogues and synthetic methods

Info

Publication number: WO2025199647A1
Application number: PCT/CA2025/050436
Authority: WO
Inventors: Michael MEANWELL; Thirupathi NULIGONDA
Original assignee: University of Alberta
Current assignee: University of Alberta
Priority date: 2024-03-27
Filing date: 2025-03-27
Publication date: 2025-10-02
Anticipated expiration: 2026-09-27

Abstract

Disclosed are methods of synthesizing a modified nucleoside by providing a chiral intermediate scaffold molecule where a ketone and a dialkyl acetal provide two points for structural diversification while simultaneously serving to enable ribose ring formation, with a intramolecular trans-acetalization reaction to construct the modified ribose core, followed by glycosylation to produce a C4ʹ-modified nucleoside. Also disclosed are novel C4ʹ-modified nucleoside analogues.

Description

C4 -MODIFIED NUCLEOSIDE ANALOGUES AND SYNTHETIC METHODS

TECHNICAL FIELD

[1] The present invention relates to methods of synthesizing nucleoside analogue compounds, as well as novel C4'-modified nucleoside analogues.

BACKGROUND OF THE ART

[2] Nucleoside analogues are an important class of small molecules for the advancement of modern medicine with widespread uses as life-saving treatments for cancer and viral infections [1- 5], Within this domain, C4'-modified nucleoside analogues are currently of interest for the development of antiviral small molecules and oligonucleotide-based therapeutics [6-16], New clinical candidates include CL-197 with nanomolar activity (EC50 = 0.9 nM) and low cytotoxicity for treating HIV-1 infection [7], Islatravir includes an ethynyl modification at C4' which is essential for increased binding interactions in the hydrophobic pocket of the HIV-1 reverse transcriptase (RT) and for modulating sugar ring conformation to hinder DNA synthesis [6],

[3] However, C4'-modified nucleoside analogues remain difficult to synthesize. Traditional semi-synthetic approaches are hampered by iterative protection/deprotection sequences that ultimately lead to lengthy 9-16 step sequences with low-modularity and poor atom-economy. Such processes are not amenable for diverse library generation in drug discovery as the four contiguous stereocenters that constitute the ribose core of the nucleoside pose a unique synthetic challenge for the efficient manipulation of the ribose backbone in any semi-synthetic strategy [8-16], In fact, a single synthetic route is rarely able to incorporate both different C4'-modifications and nucleobases. In recent years, Merck Inc. has invested considerable resources in addressing some of these shortcomings with the aim of advancing improved process routes for Islatravir [9-13],

[4] To date, Merck has reported five separate synthesis with each relying on one or more biocatalytic steps. Their efforts culminated in the development of a 6-step biocatalytic approach involving the bioengineering of five novel enzymes for the synthesis of Islatravir (see 2 — 3).12 More recently, Kaspar reported an enzymatic transglycosylation of 4 '-methyluridine (4), which itself is prepared in 10 steps from uridine, to access a collection of 4'-methyl nucleoside analogues [14], While innovative biocatalytic approaches have become powerful tools for the synthesis of certain analogues, they are inherently substrate specific which limits their utility for diverse library generation in medicinal chemistry. For this reason, protocols strictly using de novo chemical synthesis are desired due to their potential for increased modularity and are overall far more accessible to practitioners [15-19], In 2020, a collaborative effort between the Britton Lab and Merck resulted in a significantly shorter 4 step de novo synthesis of protected C4'-modified nucleoside analogues[15] Though this advance represented an improvement over the current paradigm, it generates C4'- modified nucleoside analogues in poor-to-good enantioselectivities (66-90 %ee), is only applicable to a very small subset of nucleobases, and affords the lyxose configuration which is less desirable in drug discovery than the canonical D-ribose configuration. Currently, there is no general platform that provides direct access to libraries of naturally configured C4' -modified nucleoside analogues.

[5] C4 ' -modified nucleoside analogues continue to attract global attention for their use in antiviral drug development and oligonucleotide-based therapeutics. However, current approaches to C4'- modified nucleoside analogues still involve lengthy, non-modular routes that are unamenable to library synthesis.

SUMMARY

[6] The present disclosure provides a modular process to synthesize a C4'-modified nucleoside analogue through an intramolecular trans-acetalization of readily assembled polyhydroxylated frameworks. Overall, the reduction in step-count compares favorably to biocatalytic approaches and can enable new opportunities in drug design around this popular chemotype. Generally, the methods described herein comprise a strategy centered on a chiral intermediate scaffold molecule where a ketone and a dialkyl acetal provide two points for structural diversification while simultaneously serving to enable ribose ring formation. The methods comprise a unique intramolecular trans- acetalization reaction to construct the modified ribose core. Subsequent glycosylation provides rapid entry to several C4'-modified nucleoside analogues.

[7] In one aspect, disclosed is a method of synthesizing a modified nucleoside, comprising the steps of

(a) reacting a chiral intermediate comprising an aldol adduct comprising a ketone moiety, a dialkyl acetal moiety and a protecting group with a nucleophilic organometallic compound comprising a modifier R to produce a syn-diol intermediate bearing the modifier R;

(b) selectively deprotecting the dialkyl acetal moiety and trans-acetalizing the syn-diol intermediate to produce a ribose intermediate;

(c) peracylating the ribose intermediate to produce peracyl ribose; and

(d) glycosylating the peracetal ribose with a nucleobase to produce a nucleoside bearing the modifier at position c4’.

The C4' -modification is introduced with the modifier R, which can be any desired or suitable modification. The nucleobase can be any natural or modified purine or pyrimidine base. The glycosylation can result in a C-linked or an N-linked nucleoside.

[8] In some embodiments, the method comprises a first step comprising reacting an aldehyde with a ketone to produce the chiral intermediate.

[9] In some embodiments, the dialkyl acetal moiety is dimethyl acetal, diethyl acetal or diisopropyl acetal.

[10] In some embodiments, the protecting group is an acetonide moiety.

[11] In some embodiments, the chiral intermediate is compound 6 having the structure:

[12] In some embodiments, the nucleophilic organometallic compound is a Grignard reagent, which is R-Mg-X where R is the modifier and X is a halogen, such as Br. Alternatively, the nucleophilic organometallic compound can be an organolithium compound.

[13] In some embodiments, step (b) is performed reacting the syn-diol intermediate (10) with lutidine and trimethyl silyl trifluoromethanesulfonate (TMSOTf). The molar ratio of TMSOTf to lutidine can be between about 1 : 1 to about 3: 1. The reaction temperature is low enough to prevent decomposition of the reactants and/or products, preferably between about -30° C and about 0° C. The reaction is quenched with water or an alkaline solution, such as a saturated sodium bicarbonate solution. The lutidine can be 2,6-lutidine.

[14] In some embodiments, in step (c), the ribose intermediate (13) is peracetylated to produce peracetyl ribose.

[15] In some embodiments, R is a saturated or unsaturated aliphatic substituent, which is straight, branched or cyclic, or R comprises a substituted or unsubstituted aromatic ring. R can be alkyl or cycloalkyl having between 1 and 16 carbon atoms or can be substituted or unsubstituted aryl, heteroaryl, or benzyl. Preferably, R is or comprises methyl, ethyl, propyl, isopropyl, vinyl, hexynyl, dodecyl, cyclopropyl, deuteromethyl, phenyl, or benzyl.

[16] In some embodiments, the nucleobase comprises substituted or unsubstituted purine, cytosine, adenine, thymine, uracil or guanine, or analogues thereof. Preferably, the nucleobase comprises a nucleobase selected from the group consisting of methyl-lH-l,2,4-triazole-3-carboxylate, 5-hydroxy- lH-imidazole-4-carboxamide, purine, 6-chloro-purine, uracil, 5-fluoro-uracil, 5-bromo-uracil, 5- chloro-uracil, 5-iodo-uracil, 5-trifluoromethyl-uracil, cytosine, 5 -bromo-cytosine, 5-methyl-cytosine, 5 -aza-cytosine, adenine, 2-fluoro-adenine, 2-chloro-adenine, 6-methoxy-adenine, and thymine.

[17] In another aspect, disclosed are novel compounds 18, 26-32, 36-39, 41, ent-37, 42-50, 52-58.

DESCRIPTION OF THE DRAWINGS

[18] The drawings are referred to in the description below and form part of this specification. Figure 1 shows a schematic of one example of a synthetic scheme described herein where the modifier is methyl and the nucleobase is thymine. Figure 2 shows exemplary nucleosides with compatible C4'- modifications and nucleobases. Figure 3 shows different nucleoside analogues synthesized with a process described herein. Figure 4 shows additional exemplary nucleosides with compatible C4’ modifications and nucleobases.

DETAILED DESCRIPTION

[19] In the following description, certain specific details are set forth in order to provide a thorough understanding of various embodiments of the disclosure. However, one skilled in the art will understand that the disclosure may be practiced without these details. Before the present invention is described further, it is to be understood that this invention is not limited to particular embodiments described, and as such may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting, since the scope of the present invention will be limited only by the appended claims.

[20] Where a range of values is provided, it is understood that each intervening value, to the tenth of the unit of the lower limit unless the context clearly dictates otherwise, between the upper and lower limit of that range and any other stated or intervening value in that stated range is encompassed within the invention, as well as any subrange encompassed within the stated range which can be envisioned by one skilled in the art.

[21] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, the preferred methods and materials are now described. All publications mentioned herein are incorporated herein by reference to disclose and describe the methods and/or materials in connection with which the publications are cited.

[22] It must be noted that as used herein and in the appended claims, the singular forms "a", "and", and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "a cell" includes a plurality of cells and reference to "a polypeptide" includes reference to one or more polypeptides and equivalents thereof known to those skilled in the art, and so forth. It is further noted that the claims may be drafted to exclude any optional element. As such, this statement is intended to serve as an antecedent basis for use of such exclusive terminology as "solely", "only" and the like in connection with the recitation of claim elements, or use of a "negative" limitation.

[23] “About” is used to provide flexibility to a numerical range endpoint by providing that a given value may be “slightly above” or “slightly below” the endpoint without affecting the desired result. For example, the term “about” would indicate a range surrounding that explicit value. If “X” were the value, “about X” would indicate a value from ±1% to ±10%, preferably a value from ±1% to ±5%, and more preferably, a value from ±1% to ±3%. Thus, “about X” is to teach and provide written description support for a claim limitation of, e.g., ±100%, ±5%, or 3%.

[24] The use herein of the terms “including,” “comprising,” or “having,” and variations thereof, is meant to encompass the elements listed thereafter and equivalents thereof as well as additional elements. Embodiments recited as “including,” “comprising,” or “having” certain elements are also contemplated as “consisting essentially of’ and “consisting of’ those certain elements. As used herein, “and/or” refers to and encompasses any and all possible combinations of one or more of the associated listed items, as well as the lack of combinations where the phrase is interpreted in the alternative (“or”).

[25] Recitation of ranges of values herein is merely intended to serve as a shorthand method of referring individually to each separate value falling within the range, unless otherwise indicated herein, and each separate value is incorporated into the specification as if it were individually recited herein. For example, if a concentration range is stated as 1% to 50%, it is intended that values such as 2% to 40%, 10% to 30%, or 1% to 3%, etc., are expressly enumerated in this specification. These are only examples of what is specifically intended, and all possible combinations of numerical values between and including the lowest value and the highest value enumerated are to be considered to be expressly stated in this disclosure.

[26] All publications and patents cited in this specification are herein incorporated by reference as if each individual publication or patent were specifically and individually indicated to be incorporated by reference and are incorporated herein by reference to disclose and describe the methods and/or materials in connection with which the publications are cited. The citation of any publication is for its disclosure prior to the filing date and should not be construed as an admission that the publication is relevant prior art. Further, the dates of publication provided may be different from the actual publication dates which may need to be independently confirmed.

[27] As will be apparent to those of skill in the art upon reading this disclosure, each of the individual embodiments described and illustrated herein has discrete components and features which may be readily separated from or combined with the features of any of the other several embodiments without departing from the scope or spirit of the present invention. Any recited method can be carried out in the order of events recited or in any other order which is logically possible.

[28] As used herein, the term “nucleobase” or “base” includes naturally occurring or modified purine or pyrimidine bases which includes, but is not limited to, adenine, N⁶-alkylpurines, N⁶- acylpurines (wherein acyl is C(O)(alkyl, aryl, alkylaryl, or arylalkyl), N⁶ -benzylpurine, N⁶- halopurine, N⁶-vinylpurine, N⁶-acetylenic purine, N⁶-acyl purine, N⁶ -hydroxyalkyl purine, N⁶- allcylaminopurine, N⁶-thioallcyl purine, N⁶-alkylpurines, N⁶-alkyl-6-thiopurines, thymine, cytosine, 5-fluorocytosine, 5-methylcytosine, 6-azapyrimidine, including 6-azacytosine, 2- and/or 4- mercaptopyrmidine, uracil, 5-halouracil, including 5-fluorouracil, C⁵-alkylpyrimidines, C⁵- benzylpyrimidines, C⁵-halopyrimidines, C⁵-vinylpyrimidine, C⁵-acetylenic pyrimidine, C⁵-acyl pyrimidine, C⁵-hydroxyalkyl purine, C⁵-amidopyrimidine, C⁵-cyanopyrimidine, C⁵-iodopyrimidine, C⁶-lodo-pyrimidine, C⁵ — Br-vinyl pyrimidine, C⁶ — Br-vinyl pyrimidine, C⁵-nitropyrimidine, C⁵- amino-pyrimidine, N²-alkylpurines, N²-alkyl-6-thiopurines, 5-azacytidinyl, 5-azauracilyl, triazol opyridinyl, imidazolopyridinyl, pyrrol opyrimidinyl, and pyrazolopyrimidinyl. Purine bases include, but are not limited to, guanine, adenine, hypoxanthine, 2,6-diaminopurine, and 6- chloropurine. Nitrogen-containing 5 membered heteroaromatics include, but are not limited to, imidazole and triazole. Functional oxygen and nitrogen groups on the base can be protected as necessary or desired. Suitable protecting groups are well known to those skilled in the art, and include trimethylsilyl, dimethylhexylsilyl, t-butyldimethylsilyl, and t-butyldiphenylsilyl, trityl, alkyl groups, and acyl groups such as acetyl, propionyl, and benzoyl.

[29] The term “nucleoside” means a compound comprising a nucleobase linked to a pentose sugar, and includes C-linked and N-linked nucleosides. A C4’-modified nucleoside bears a modifying group on the C4 atom, and includes, but is not limited to, all those modifiers described or illustrated herein.

[30] In some embodiments, disclosed is a method of synthesizing a modified nucleoside, comprising the steps of:

(a) reacting a chiral intermediate comprising an aldol adduct comprising a ketone moiety, a dialkyl acetal moiety and a protecting group with a nucleophilic organometallic compound reagent comprising a modifier R to produce a syn-diol intermediate bearing the modifier R;

(c) peracylating the ribose intermediate to produce peracyl ribose; and

[31] In some embodiments, the method comprises a first step comprising reacting an aldehyde with a ketone, with a proline catalyst, to produce the chiral intermediate. The D or L stereoisomer of the resulting nucleoside may be selected by use of a D-proline or an L-proline.

[32] In some embodiments, the dialkyl acetal moiety is dimethyl acetal, diethyl acetal or diisopropyl acetal.

[33] In some embodiments, the protecting group is an acetonide moiety.

[34] In some embodiments, the chiral intermediate is

[35] In some embodiments, the nucleophilic organometallic compound is a Grignard reagent is R- Mg-X where R is the modifier and X is a halogen, such as Cl, F, I or Br. Alternatively, the nucleophilic organometallic compound can be an organolithium compound, as is known in the art.

[36] In some embodiments, step (b) is performed reacting the syn-diol intermediate (10) with lutidine and trimethyl silyl trifluoromethanesulfonate (TMSOTf). The molar ratio of TMSOTf to lutidine can be between about 1 : 1 to about 3: 1. The reaction temperature is low enough to prevent decomposition of the reactants and/or products, preferably between about -30° C and about 0° C. The reaction is quenched with water or an alkaline solution, such as a saturated sodium bicarbonate solution. The lutidine can be 2,6-lutidine. [37] In some embodiments, in step (c), the ribose intermediate (13) is peracetylated to produce peracetyl ribose.

[38] In some embodiments, without limitation, R is H, a saturated or unsaturated aliphatic substituent, which is straight, branched or cyclic, or R comprises a substituted or unsubstituted aromatic ring. R can be alkyl or cycloalkyl having between 1 and 16 carbon atoms or can be substituted or unsubstituted aryl or benzyl. In some embodiments, R is or comprises methyl, ethyl, propyl, vinyl, hexynyl, isopropyl, dodecyl, cyclopropyl, deuteromethyl, phenyl, or benzyl.

[39] In some embodiments, the nucleobase comprises substituted or unsubstituted purine, cytosine, adenine, thymine, uracil or guanine, or analogues thereof. Preferably, the nucleobase comprises a nucleobase selected from the group consisting of methyl-lH-l,2,4-triazole-3-carboxylate, 5-hydroxy- lH-imidazole-4-carboxamide, purine, 6-chloro-purine, uracil, 5-fluoro-uracil, 5-bromo-uracil, 5- chloro-uracil, 5-iodo-uracil, 5-trifluoromethy-luracil, cytosine, 5 -bromo-cytosine, 5-methyl-cytosine, 5 -aza-cytosine, adenine, 2-fluoro-adenine, 2-chloro-adenine, 6-methoxy-adenine and thymine.

[40] As shown in Figure 1, a preferred embodiment is disclosed which begins with production of the central chiral building block 6 by way of a proline-catalyzed enantioselective aldol reaction between 2,2-dimethoxyacetaldehyde 8 and 2,2-dimethyl-l,3-dioxan-5-one 9 to produce the chiral intermediate 6 [20,21], Use of L-proline will result in a D-nucleoside , while use of a D-proline will result in an L-nucleoside.

[41] In a second step, a 1,2-addition of the ketone functionality in 6 with a Grignard reagent for the introduction of the eventual C4' -modification. The Grignard reagent comprises a R-Mg-X where R is the modifier and X is halogen. For example, the Grignard reagent may comprise methylmagnesium bromide. With MeMgBr, this step results in the .sj7/-diol intermediate 10, with good yield and good diastereoselectivity (5: 1).

[42] The ,sj77-diol intermediate 10 then undergoes an intramolecular //z//7s-acetalization cascade using TMSOTf and a lutidine, such as 2, 6-lutidine, to construct the modified ribose core 13. In preferred embodiments, this one-pot sequence relies on the selective deprotection of the dimethyl acetal over the acetonide to unveil the oxocarbenium 11 required for ribose ring formation. This approach is contrasted with traditional conditions such as catalytic amounts of BrOnsted acids (e.g., AcOH, HC1, TsOH, TFA) which are unselective and lead to either decomposition of the starting material 10 or a complex mixture of unidentifiable by-products. Other reported conditions such as InC13/H2O [22], I2/acetone [23] and TBSOTf/collidine [24,25] are also unsuccessful.

[43] It was found that preferred conditions of temperature and stoichiometry of TMSOTf and 2,6- lutidine were required for the desired transformation. It is preferred to have an excess of TMSOTf to lutidine, preferably in a range of about 1 : 1 to about 3: 1. Further, the reactants and/or the products may tend to decompose at higher temperatures. Therefore, in preferred embodiments, the reaction takes place at a temperature in the range of about 0° C to about -30°. Further, the reaction may be quenched with water or an alkaline solution, such as a saturated solution of sodium bicarbonate.

[44] In one example, TMSOTf (2 equiv.) and lutidine (1 equiv.) at -10°C followed by a water quench cleanly affords the desired product 13 via a cascade of intramolecular trans-acetalizations. Without restriction to a theory, using plastic molecular models, it is believed that acetonide migration is required for cyclization to occur as it allows for optimal orbital alignment of the oxocarbenium TI* orbital with the incoming tertiary alcohol nucleophile. Without migration, the trajectory of the tertiary alcohol is restricted.

[45] The intermediate 13 is then subjected to ring-opening promoted by TESOTf and peracetylated to produce peracetal ribose intermediate 14, which can then be glycosylated with a chosen nucleobase. For example, Vorbriiggen glycosylation of intermediate 14 with thymine gives 4'-methylthymidine (15, P-anomer only).

[46] Thus, the synthesis is accomplished in only five total steps, or four steps if the chiral intermediate is pre-made or sourced commercially. This may be viewed in stark contrast to the previous shortest chemical synthesis of 4'-methylthymidine 15 of 13 steps [26],

[47] Embodiments of this synthesis include the use of many different organometallic compounds and nucleobases for the generation of high-value 4'-modified nucleoside analogues. Examples may be seen in Figures 2 and 4. For example, this process may comprise the use of an array of topical 4'- modifications including methyl (15, 18-23), ethyl (24, 25), allyl (26, 27), tri deuteromethyl (28-31), vinyl (32), and ethynyl (33, 34), that can be conveniently attached with a chosen nucleobase. d'analogues of natural products adenosine (20, 26, 28), thymidine (15, 31, 32), and cytidine (22, 25, 29, 34) were produced. Non-canonical nucleobases that are in high demand in drug discovery such as 6- methoxy-adenine (18), 2-chloro-adenine (23), 6-chloro-adenine (27), iodouracil (19, 30), and 2- fluoro-adenine (21, 33) were also incorporated in excellent overall yields. In all cases, these new syntheses represent roughly a 2-3 fold improvement in step-count over the previous shortest syntheses for the various nucleosides. For instance, nucleoside 22 was synthesized in 14 steps via a semisynthetic approach starting from diacetone-D-glucose [27], nucleoside 21 in 16 steps [28], Even in comparison to routes that employed newly bioengineered enzymes, our sequence proved to be over 2-fold shorter (i.e., 15, 19, 20, and 23). For example, nucleoside 23 was previously synthesized in 11 steps using a biocatalytic trans-glycosylation of 4'-methyluridine with 2-chloroadenine [14],

[48] Figure 4 shows other Grignards for the 1,2-addition and nucleobases for the glycosylation. As shown in Table 2, 6 undergoes productive 1,2-additions with isopropyl, dodecyl, benzyl, and phenyl magnesium bromides, as well as with hexynyl lithium. However, only isopropyl, dodecyl, benzyl, and phenyl derivatives were compatible with downstream reactions in the process. 8 additional nucleobases including 5 -aza-cytosine, 5 -bromo-cytosine, 5-methyl-cytosine, 5-fluoro-uracil, 5- chloro-uracil, 5-bromo-uracil, 5-iodo-uracil, and 5-trifluoromethyl-uracil were demonstrated to engage in successful glycosylations with a variety of C4'-modified glycosyl donors (6). Figure 3 shows examples (42-58), whose preparations can be found in the examples below, containing these new functionalizations.

[49] The novel compounds (including 18, 26 - 32, 42-58) synthesized with this expedited route map well onto structures previously disclosed in the recent literature and serve to highlight the utility of the disclosed process for exploring chemical space around this valuable chemotype. These novel compounds can be analogues of existing therapeutic compounds, and can provide alternate efficacy, or may be useful intermediate compounds. While a few analogues (i.e., 26 and 27) were obtained in lower overall yields, it is important to recognize that chemical synthesis in medicinal chemistry prioritizes routes for their efficiency and the structural diversity they can access. Furthermore, during pandemic emergencies the ability to rapidly generate and identify antivirals becomes even more important in fighting waves of infection and viral mutations.

[50] It is believed that C4' -modified nucleoside analogues have not been previously synthesized presumably owing to the lengthy routes that would be required to make them. Embodiments of the process disclosed herein were used to synthesize Ribavirin and Mizoribine analogues. Ribavirin (34) is a broad-spectrum antiviral that is listed as an essential medicine by the World Health Organization (WHO) [31] while Mizoribine (35) is a natural product approved in Japan for use as an immunosuppressant during renal transplantation [32], Uniquely, these nucleoside analogues contain unusual triazole and imidazole nucleobases respectively. Their C4' analogues (Figure 3; 36-39) were synthesized in just 5 steps and with modest to good overall yields.

[51] In some embodiments, the process comprises steps to synthesize C-linked nucleosides, which offer much improved metabolic stability compared to their TV-linked counterparts. Using a modified protocol adapted from a recent report by Li and coworkers [19], Surprisingly, using 40 instead of 14 as the glycosyl donor in the C-glycosylation afforded 41 in modest yield. 40 was generated through an alternate ring-opening/activation of 13 using stoichiometric acetic anhydride in the presence of TESOTf. Finally, by performing the initial aldol step with D-proline instead of L-proline we made 4’-ethyl analogue (ent-37) of Levovirin, an L-nucleoside and investigational HCV antiviral [33],

[52] In summary, disclosed is a modular 5-step de novo synthesis of C4'-modified nucleoside analogues. This short sequence relies on an intramolecular trans-acetalizati on cascade to enable broad diversification via Grignard additions and Vorbriiggen glycosylations. Given the robustness of this protocol, many C4'-modifications and nucleobases are compatible to support medicinal chemistry efforts. In all cases, the described process is 2-3 fold shorter than the previous shortest syntheses reported for each analogue.

EXAMPLES

[53] The following working examples provide a further understanding of the methods of the present invention. These examples are of illustrative purposes, and are not meant to limit the scope of the invention. Equivalent, similar or suitable solvents, reagents or reaction conditions may be substituted for those particular solvents, reagents or reaction conditions described without departing from the general scope of the method.

[54] All reactions described were performed at ambient temperature and atmosphere unless otherwise specified. Column chromatography was carried out with 230-400 mesh silica gel (E. Merck, Silica Gel 60). Concentration and removal of trace solvents was done via an IKA rotary evaporator using an IKA RC2 Green Basic circulating cooler and an IKA VacStar pump.

[55] Nuclear magnetic resonance (NMR) spectra were recorded using deuterochloroform (CDC13), deuteromethanol (CD3OD), deuterium oxide (D2O), deuteroacetonitrile (CD3CN) or hexadeuterodimethyl sulfoxide ((CD3)2SO) as the solvent. Signal positions (6) are given in parts per million from tetramethylsilane (60) and were measured relative to the signal of the solvent (’ H NMR: CDC13: 6 7.26; CD3CN: 6 1.96; acetone-d6: 6 2.05; dmso-d6: 2.50; CD3OD: 3.31; D2O: 4.79; ¹³C NMR: CDC13: 6 77.16, acetone-d6: 6 29.84, dmso-d6: 6 39.52, CD3OD: 49.00, CD3CN: 8 1.32 & 118.26 ). Coupling constants (J values) are given in Hertz (Hz) and are reported to the nearest 0.1 Hz. ¹ H NMR spectral data are tabulated in the order: multiplicity (5, singlet; d, doublet; t, triplet; q, quartet; sept, septet; m, multiplet; br broad), coupling constants, number of protons. NMR spectra were recorded on either 400, 500, 600, or 700 MHz spectrometers.

[56] High-resolution mass spectra were recorded using either electron impact (El), electrospray ionization (ESI) or DART techniques by the mass spec lab at the University of Alberta.

[57] Single Crystal XRD were recorded using Bruker D8 Venture/PHOTON II instrument by the SC- XRD lab at the University of Alberta.

II. General Procedures

One-Step Preparation of Starting Material 6

Aldol adduct 6 was prepared according to previously published literature.¹ To a solution of ketone 9 (5.10 mL, 38.4 mmol, 1 equiv.) in DMF (19.2 mL) at 0°C was added L-proline (0.885 g, 7.68 mmol, 0.2 equiv). The reaction mixture was then allowed to stir at this temperature for 30 minutes after which time 2,2-dimethoxyacetaldehyde solution (60% by weight in water, 8) (5.80 mL, 38.4 mmol, 1 equiv) was added. The reaction mixture was then left to stir at ~ 4°C for 72 hrs. After completion, as monitored by TLC, the reaction mixture was diluted with EtOAc and water. The aqueous layer was washed 3 times with EtOAc and 3 times with CH2CI2. The aqueous layer was checked by TLC to ensure extraction was complete. The organic layer was then separated, combined, dried with Na2SC>4, filtered, and concentrated under reduced pressure. The crude reaction mixture was then purified with flash column chromatography (10% — 50% EtOAc in hexanes) to afford a single diastereomer of aldol adduct 6 as light yellow oil (4.67 g, 52%). The %ee was confirmed to be 93- 100%ee. All data for 6 matched previous reports.¹

Data for 6: ¹ H NMR (600 MHz, CDCh): 64.69 (d, J = 6.8 Hz, 1H), 4.48 (m, 1H), 4.38 (dd, J = 16.8, 1.5 Hz, 1H), 4.12 (dd, J = 6.7, 2.6 Hz, 1H), 4.03 (d, J = 16.8 Hz, 1H), 3.48 (s, 3H), 3.43 (s, 3H), 1.51 (s, 6H); ¹³C NMR (176 MHz, CDCh): 8 206.4, 103.2, 100.4, 76.0, 71.1, 67.0, 55.2, 54.2, 24.8, 22.9.

HRMS (ESI): Expected mass [M+Na⁺]: 257.0996; found: 257.0994.

General Procedure A: Grignard Addition to Aldol Adduct 6

To a solution of 6 (1 equiv.) in THF or CH2CI2 (0.35 M) at -78°C was added (dropwise) a solution of the Grignard reagent (3 or 4 equiv). The reaction mixture was allowed to stir at -78°C for 2 hrs and then was gradually warmed overnight to room temperature. After completion, as monitored by TLC, the reaction mixture was cooled to 0°C and was slowly quenched via dropwise addition of saturated ammonium chloride solution. The reaction mixture was filtered to remove solids under vacuum filtration. The filtrate was then diluted with dichloromethane and washed three times water. The organic layer was then separated, dried with Na2SC>4, filtered, and concentrated under reduced pressure. The crude reaction mixture was then purified with flash column chromatography to afford a sj’/i-10S (40-72%).

General Procedure B: Intramolecular Bis-Transacetalization

To a solution of s n-lOS (1 equiv.) and 2,6-lutidine (1 equiv.) in CH2CI2 (0.08 M) at -10°C was added dropwise TMSOTf (2 equiv.) in a glass syringe. The reaction mixture was allowed to stir at - 10°C for 1.5 hrs and then water (1/3 volume of CH2CI2 solvent) was added. The reaction was allowed to stirred at room temperature for 30 minutes. After completion, as monitored by TLC, the reaction mixture was diluted with CH2CI2 and washed twice with water. The organic layer was then separated, dried with Na2SO4, filtered, and concentrated under reduced pressure. The crude reaction mixture was then purified with flash column chromatography to afford a 13S (52%-75%).

General Procedure C: Ring Opening- Acetylation

To a solution of 13S (1 equiv.) in Ac2O:CH2Ch (0.20 M; 1 : 1 mixture) at -5°C was added (dropwise) TESOTf (0.625 equiv). The reaction mixture was allowed to stir at -5°C for 2 hrs. After completion, as monitored by TLC, the reaction mixture was then diluted with dichloromethane and washed three times with saturated sodium bicarbonate solution. The organic layer was then separated, dried with Na2SO4, filtered, and concentrated under reduced pressure. The crude reaction mixture was then purified with flash column chromatography to afford a 14S (61%-74%). In some cases, the a-anomer was also formed. The a-anomer can be combined with 14S for the next step without any change in results of the subsequent glycosylation step.

General Procedure D: Glycosylation workup

14S 15S

To a solution of nucleobase (1 equiv.) in dry MeCN (0.10 M) at 0°C was added dropwise BSA (3 equiv) and TMSOTf (2 equiv). The reaction mixture was then heated to 60°C for 2 hrs. The reaction mixture was then cooled to 0°C and the sugar 14S (1 equiv) in 1, 2-DCE (0.30 M) was added to the reaction mixture. The reaction mixture was then heated to 60°C and left stirring at this temperature overnight. After completion, as monitored by TLC, the reaction mixture was concentrated under reduced pressure and the crude reaction mixture was purified with flash column chromatography to afford PA-15S (70-100%). PA-15S was then dissolved in a solution of ammonia in methanol and stirred for 16 hours. The reaction mixture was then concentrated under reduced pressure to afford pure 15S (100%). The acetamide by-product was removed by blowing a steady stream of air over the concentrated sample overnight. III. Preparation and Characterization of Nucleoside Analogues

Preparation of 4'-Methyl Nucleoside Analogues (15, 18-23)

Grignard Addition

Following the General Procedure A with methylmagnesium bromide (3 M in Et2O, 10.0 mL, 30.0 mmol, 3 equiv) and 6 (2.34 g, 10.0 mmol, 1 equiv.) in THF (10 mL) afforded 10 as a colorless oil (1.72 g, 69%) after purification by flash column chromatography (20 — 25% EtOAc in Hexanes).

Ry = 0.6 (70% EtOAc in Hexanes)

Data for 10: ¹ H NMR (400 MHz, CDCh): 6 4.38 (d, J= 1.0 Hz, 1H), 3.77 - 3.75 (m, 1H), 3.71 (d, J = 11.6 Hz, 1H), 3.53 (s, 2H), 3.55 - 3.49 (m, 4H), 3.45 (s, 2H), 3.43 (d, J= 5.6 Hz, 1H), 2.82 (d, J= 3.6 Hz, 1H), 1.44 (s, 3H), 1.38 (s, 3H), 1.36 (s, 3H); ¹³C NMR (125 MHz, CDCh): 6 103.2, 99.0, 73.0, 72.9, 70.1, 67.4, 57.2, 55.6, 28.9, 20.2, 19.2.

HRMS (ESI): Expected mass [M+Na⁺]: 273.1309; found: 273.1307.

[a]²⁰r> = -11.6 (c 3.1, CHCh)

Intramolecular Bis- Transacetalization

Following General Procedure B with TMSOTf (1.82 mL, 10.0 mmol, 2 equiv.), 2,6-lutidine (0.58 mL, 5.0 mmol, 1 equiv.), and 10 (1.25 g, 5.00 mmol, 1 equiv.) in CH2CI2 (38.0 mL) afforded 13 as a colorless liquid (0.613 g, 66%) after purification by flash column (5 — 7% EtOAc in Hexanes).

Ry = 0.5 (30% EtOAc in Hexanes)

H NMR (600 MHz, CDCh): 6 5.35 (s, 1H, H-l), 4.33 (d, J= 5.5 Hz, 1H, H-2), 4.11 (d, J= 5.5 Hz, 1H, H-3), 3.29 (d, J= 7.1 Hz, 1H, 5-Ha), 3.18 (d, J= 7.1 Hz, 1H, 5-Hb), 1.52 (s, 3H, CH₃), 1.46 (s, 3H, CH₃), 1.30 (s, 3H, CH₃); ¹³C NMR (125 MHz, CDCh): 6 112.3, 100.2, 83.8, 82.3, 80.8, 68.3, 26.2, 25.7, 11.9.

HRMS (DART-MS): Expected mass [M+H⁺]: 187.0965; found: 187.0963.

[a]²⁰/ = -49.3 (c 0.3, CHCh)

Ring Opening-Acetylation

Following General Procedure C with TESOTf (0.426 mL, 1.87 mmol, 0.625 equiv) and 13 (0.558 g, 3.00 mmol, 1 equiv.) in Ac2O:CH2Ch (6.0 mL: 6.0 mL) afforded 14 as a yellow oil (0.726 g, 73%) after purification by flash column chromatography (15 — 20% EtOAc in Hexanes). Data matched previous reports.

Ry = 0.6 (50% EtOAc in Hexanes)

^XH NMR (600 MHz, CDCh): 8 6.16 (s, 1H, H-1), 5.40 (m, 2H, H-2 and H-3), 4.13 (d, J= 12.2, 1H, H-5a), 4.05 (d, J= 11.9 Hz, 1H, H-5b), 2.12 (s, 3H, Ac-CH₃), 2.09 (s, 9H, 3x Ac-CH₃), 1.32 (s, 3H, 4-CH₃); ¹³C NMR (125 MHz, (CDCh): 6 170.4, 169.6, 169.4, 169.3, 97.6, 84.5, 75.2, 71.8, 68.5, 21.22, 20.9, 20.6, 20.5, 19.6.

HRMS (ESI): Expected mass [M+Na⁺]: 355.1000; found: 355.0999.

Preparation of 15 - Glycosylation

Following General Procedure D with BSA (73.0 pL, 0.17 mmol, 3 equiv), TMSOTf (36.0 pL, 0.20 mmol, 2 equiv) and thymine (12.6 mg, 0.100 mmol, 1 equiv.) in dry MeCN (0.50 mL) then sugar 14 (33.2 mg, 0.100 mmol, 1 equiv) in 1, 2-DCE (0.50 mL) afforded 15A as a white solid (34.0 mg, 81%) after purification by flash column chromatography (2-3% MeOH in CH2CI2). Data matched previous reports.

R, = 0.4 (5% Methanol in CH2CI2)

Data for 15A: H NMR (600 MHz, CD3OD): 6 7.47 (d, J= 1.1 Hz, 1H, HetH), 5.95 (d, J= 5.3 Hz, 1H, H-l), 5.57 - 5.53 (m, 1H, H-2), 5.51 (d, = 6.4 Hz, 1H, H-3), 4.21 (s, 2H, H-5a and H-5b), 2.13 (s, 3H, AC-CH₃), 2.13 (s, 3H, Ac-CH₃), 2.07 (s, 3H, Ac-CH₃), 1.89 (d, J= 0.9 Hz, 3H, HetCH₃), 1.35 (s, 3H, 4-CH₃); ¹³C NMR (125 MHZ, CD3OD): 5 5 171.9, 171.3, 171.2, 166.1, 152.2, 138.3, 112.1, 89.1, 84.8, 74.4, 72.5, 68.5, 20.7, 20.3, 20.3, 18.6, 12.4.

HRMS (ESI): Expected mass [M+Na⁺]: 421.1218; found: 421.1200

Stereochemical Assignment for 15A

Analysis of 2D ROESY of nucleoside 15A supported the indicated stereochemistry. Basic Workup

15A 15

Following General Procedure D (work-up) with 15A (19.9 mg, 0.0500 mmol) in ammonia in methanol (7 N, 0.40 mL) afforded 15 (13.6 mg, 100%) as a white solid. The spectral data matched previous reports. ^[2]

Ry = 0.3 (10% Methanol in CH2CI2)

Data for 15: ¹ H NMR (600 MHz, D2O): 6 7.69 (s, 1H, HetH), 6.00 (d, J= 6.5 Hz, 1H, H-l), 4.53 (dd, J= 6.2 Hz, 1H, H-2), 4.24 (d, J= 5.8 Hz, 1H, H-3), 3.68 - 3.63 (m, 2H, H-5a and H-5b), 1.92 (s, 3H, HetH), 1.30 (s, 3H, 4-CH₃); ¹³C NMR (125 MHz, D2O); 6 167.4, 153.0, 138.4, 112.8, 88.6, 88.3, 74.3, 72.4, 67.2, 18.3, 12.5.

HRMS (ESI): Expected mass [M+Na⁺]: 295.0901; found: 295.0898.

Stereochemical Assignment for 15

Analysis of 2D ROESY of nucleoside 15 supported the indicated stereochemistry.

Preparation of 18 - Glycosylation

14 18

Following General Procedure D with BSA (73.0 pL, 0.170 mmol, 3 equiv), TMSOTf (36.0 pL, 0.200 mmol, 2 equiv), and 6-chloro-purine (15.4 mg, 0.100 mmol, 1 equiv.) in dry MeCN (0.50 mL) then the sugar 14 (33.2 mg, 0.100 mmol, 1 equiv) in 1, 2-DCE (0.50 mL) afforded 18A as a white solid (35.0 mg, 84%) after purification by flash column chromatography (2 -3% MeOH in CH2CI2).

Rf = 0.5 (5% Methanol in CH2CI2)

Melting point = 56-58°C

Data for 18A: H NMR (500 MHz, CDCh): 6 8.77 (s, 1H, HetH), 8.30 (s, 1H, HetH), 6.24 (d, J = 5.8 Hz, 1H, H-l), 6.07 (dd, J= 5.8 Hz, 1H, H-2), 5.69 (d, J= 5.8 Hz, 1H, H-3), 4.25 (s, 2H, H-5a and H-5b), 2.18 (s, 3H, Ac-CH₃), 2.11 (s, 3H, Ac-CH₃), 2.05 (s, 3H, Ac-CH₃), 1.40 (s, 3H, 4-CH₃); ¹³C NMR (125 MHz, CDCI3): 6 170.2, 169.5, 169.3, 152.4, 151.7, 151.5, 143.7, 132.4, 86.1, 84.9, 73.7, 71.7, 67.7, 20.9, 20.6, 20.5, 18.9.

HRMS (ESI): Expected mass [M+Na⁺]: 449.0834; found: 449.0834.

[a]²⁰r> = -9.0 (c 1.6, MeOH)

Stereochemical Assignment for 18A

Analysis of 2D ROESY of nucleoside 18A supported the indicated stereochemistry.

Basic Workup

1.0 pL of NaOMe solution (25% w/v solution in methanol) was added to 18A (21.3 mg, 0.0500 mmol) in 0.20 mL methanol, and allowed to stir at room temperature overnight. After completion of the reaction, as monitored by TLC, the reaction mixture quenched with water and extracted 3 times with EtOAc. Combined organic layers were dried on Na2SC>4 and concentrated under reduced pressure to afford 18 (14.7 mg, 100%) as a white solid.

R, = 0.4 (10% Methanol in CH2CI2)

Melting point = 145-147°C

Data for 18: ¹ H NMR (600 MHz, CD3OD): 8 'H NMR 8.52 (s, 1H, HetH), 8.49 (s, 1H, HetH), 6.03 (d, J= 7.1 Hz, 1H, H-l), 4.95 (dd, J= 7.0, 5.5 Hz, 1H, H-2), 4.26 (d, J = 5.4 Hz, 1H, H-3), 4.20 (s, 3H, Het-OCH₃), 3.69 (d, J= 12.0 Hz, 1H, H-5a), 3.58 (d, J= 12.0 Hz, 1H, H-5b), 1.28 (s, 3H, 4- CH₃); ¹³C NMR (125 MHz, CD3OD): 6 162.5, 153.1, 152.5, 144.2, 123.0, 90.7, 89.8, 75.9, 73.8, 69.1, 54.9, 18.8.

HRMS (ESI): Expected mass [M+Na⁺]: 319.1013; found: 319.1013.

[a]²⁰r> = -44.2 (c 0.19, CHCI3)

Stereochemical Assignment for 18

Analysis of 2D ROESY of nucleoside 18 supported the indicated stereochemistry.

Preparation of 19 - Glycosylation

14 19A

Following General Procedure D with BSA (73.0 pL, 0.170 mmol, 3 equiv), TMSOTf (36.0 pL, 0.200 mmol, 2 equiv) and 5-iodouracil (23.7 mg, 0.100 mmol, 1 equiv.) in dry MeCN (0.50 mL) then sugar 14 (33.2 mg, 0.100 mmol, 1 equiv) in 1, 2-DCE (0.50 mL) afforded 19A as a white solid (39.0 mg, 78%) after purification by flash column chromatography (2-3% MeOH in CH2CI2).

R, = 0.4 (5% Methanol in CH2CI2)

Melting point = 68-70°C

Data for 19A: ¹ H NMR (700 MHz, CDCh): 6 'H NMR 8.36 (bs, 1H, HetNH), 7.94 (s, 1H, HetH), 6.12 (d, J= 5.6 Hz, 1H, H-l), 5.41 (m, 2H, H-2 and H-3), 4.26 (d, J= 12.2 Hz, 1H, H-5a), 4.12 (d, J = 12.2 Hz, 1H, H-5b), 2.26 (s, 3H, Ac-CH₃), 2.16 (s, 3H, Ac-CH₃), 2.09 (s, 3H, Ac-CH₃), 1.34 (s, 3H, 4-CH₃); ¹³C NMR (125 MHZ, CDCh): 6 ¹³C NMR 170.0, 169.7, 169.5, 159.6, 150.2, 143.9, 86.1, 84.3, 73.6, 71.2, 69.7, 68.0, 21.3, 20.6, 20.5, 18.8.

HRMS (ESI): Expected mass [M+Na⁺]: 533.0027; found: 533.0027.

[a]²⁰r> = -16.9 (c 0.19, CHCh)

Stereochemical Assignment for 19 A

Analysis of 2D ROESY of nucleoside 19A supported the indicated stereochemistry.

Basic workup

19A 19

Following General Procedure D (work-up) with 19A (25.5 mg, 0.0500 mmol) in ammonia in methanol (7 N, 0.40 mL) afforded 19 (19.1 mg, 100%) as a white solid.

R, = 0.3 (10% Methanol in CH2CI2)

Data for 19: ¹ H NMR (600 MHz, CD3OD): 8 8.59 (s, 1H, HetH), 5.92 (d, J= 5.9 Hz, 1H, H-l), 4.34 (dd, J = 5.8 Hz, 1H, H-2), 4.14 (d, J = 5.7 Hz, 1H, H-3), 3.59 - 3.54 (m, 2H, H-5a and H-5b), 1.22 (s, 3H, 4-CH₃); ¹³C NMR (125 MHz, CD3OD): 6 162.9, 152.5, 147.7, 90.0, 88.8, 76.5, 73.0, 68.6, 68.0, 18.8.

HRMS (ESI): Expected mass [M+Na⁺]: 382.9746; found: 382.9744.

Stereochemical Assignment for 19

Analysis of 2D ROESY of nucleoside 19 supported the indicated stereochemistry.

Preparation of 20 - Glycosylation

14 20A

Following General Procedure D with BSA (73.0 pL, 0.170 mmol, 3 equiv), TMSOTf (36.0 pL, 0.200 mmol, 2 equiv) and adenine (13.5 mg, 0.100 mmol, 1 equiv.) in dry MeCN (0.50 mL) then sugar 14 (33.2 mg, 0.100 mmol, 1 equiv) in 1, 2-DCE (0.50 mL) afforded afford a 20A as a white solid (30.0 mg, 74%) after purification by flash column chromatography (3-4% MeOH in CH2CI2).

R, = 0.5 (10% Methanol in CH2CI2)

Melting point = 83-85°C

Data for 20A: H NMR (600 MHz, CD3OD): 6 8.24 (s, 1H, HetH), 8.22 (s, 1H, HetH), 6.25 (d, J = 5.4 Hz, 1H, H-l), 6.15 (dd, J= 5.7 Hz, 1H, H-2), 5.79 (d, J= 5.7 Hz, 1H, H-3), 4.32 (d, J= 11.8 Hz, 1H, H-5a), 4.21 (d, J= 11.8 Hz, 1H, H-5b), 2.17 (s, 3H, Ac-CH₃), 2.05 (s, 3H, Ac-CH₃), 2.04 (s, 3H, AC-CH₃), 1.40 (s, 3H, 4-CH₃); ¹³C NMR (125 MHz, CD3OD): 8 172.0, 171.2, 171.1, 157.4, 154.1, 150.6, 141.4, 120.5, 87.2, 85.5, 74.8, 72.9, 68.5, 20.6, 20.34, 20.27, 18.9.

HRMS (ESI): Expected mass [M+Na⁺]: 430.1333; found: 430.1335.

[a]²⁰r> = -38.8 (c 0.13, MeOH)

Stereochemical Assignment for 20A

Analysis of 2D ROESY of nucleoside 20A supported the indicated stereochemistry.

Basic workup

Following General Procedure D (work-up) with 20A (20.3 mg, 0.0500 mmol) in ammonia in methanol (7 N, 0.40 mL) afforded 20 (14.0 mg, 100%) as a white solid. Data matched previous reports.

R, = 0.5 (20% Methanol in CH2CI2)

Data for 20: ¹ H NMR (600 MHz, CD3OD): 6 8.26 (s, 1H, HetH), 8.18 (s, 1H, HetH), 5.91 (d, J= 7.4 Hz, 1H, H-l), 4.95 (dd, J= 7.4, 5.4 Hz, 1H, H-2), 4.23 (d, J= 5.4 Hz, 1H, H-3), 3.69 (d, J= 12.1 Hz, 1H, H-5a), 3.56 (d, J= 12.1 Hz, 1H, H-5b), 1.27 (s, 3H, 4-CH₃); ¹³C NMR (125 MHz, CD3OD): 6 157.7, 153.4, 149.9, 142.3, 121.2, 90.9, 89.9, 75.6, 74.0, 69.4, 18.8.

HRMS (ESI): Expected mass [M+Na⁺]: 304.1016; found: 304.1016.

Stereochemical Assignment for 20

Analysis of 2D ROESY of nucleoside 20 supported the indicated stereochemistry.

Preparation of 21 - Glycosylation

14 21A

Following General Procedure D, BSA (73.0 pL, 0.170 mmol, 3 equiv), TMSOTf (36.0 pL, 0.200 mmol, 2 equiv), and 2-fluoro-adenine (15.3 mg, 0.100 mmol, 1 equiv.) in dry MeCN (0.50 mL) then sugar 14 (33.2 mg, 0.100 mmol, 1 equiv) in 1, 2-DCE (0.50 mL) afforded 21A as a white solid (30.0 mg, 72%) after purification by flash column chromatography (3-4% MeOH in CH2CI2).

R, = 0.6 (10% Methanol in CH2CI2)

Melting point = 161-163°C Data for 21A: ' H NMR (400 MHz, CD3OD + CDCI3): 6 7.99 (s, 1H, HetH), 6.09 (d, J= 5.8 Hz, 1H, H-l), 5.93 (dd, = 5.9 Hz, 1H, H-2), 5.64 (d, J= 5.9 Hz, 1H, H-3), 4.24 - 4.17 (m, 2H, H-5a and H- 5b), 2.16 (s, 3H, Ac-CH₃), 2.10 (s, 3H, Ac-CH₃), 2.04 (s, 3H, Ac-CH₃), 1.36 (s, 3H, 4-CH₃); ¹³C NMR (125 MHz, CD3OD + CDCI3): 6 171.2, 170.3, 170.2, 159.6 (d,J= 211 Hz), 157.8, 151.3, 151.2, 139.6, 85.9, 84.8, 73.9, 71.9, 67.9, 20.8, 20.53, 20.47, 18.8; ¹⁹F NMR (376 MHz, CD3OD + CDCI3) 8 -50.7.

HRMS (ESI): Expected mass [M+Na⁺]: 448.1239; found: 448.1237.

[a]²⁰o = -10.3 (c 0.26, MeOH).

Stereochemical Assignment for 21 A

Analysis of 2D ROESY of nucleoside 21A supported the indicated stereochemistry.

Basic workup

Following General Procedure D (work-up) with 21A (21.2 mg, 0.0500 mmol) in ammonia in methanol (7 N, 0.40 mL) afforded 21 (14.9 mg, 100%) as a white solid. Data matched previous reports.³

Rf = 0.5 (20% Methanol in CH2CI2)

Data for 21A: H NMR (400 MHz, CD3OD): 6 8.22 (s, 1H, HetH), 5.85 (d, J = 7.2 Hz, 1H, H-l), 4.88 - 4.83 (m, 1H, H-2), 4.22 (d, J= 5.4 Hz, 1H, H-3), 3.67 (d, J= 12.0 Hz, 1H, H5a), 3.55 (d, J = 12.0 Hz, 1H, H5b), 1.26 (s, 3H, 4-CH₃); ¹³C NMR (125 MHz, CD3OD): 6 160.3 (d, J = 209.7 Hz), 159.2 (d, = 20.6 Hz), 151.6 (d, J= 19.2 Hz), 142.1 (d, J= 2.5 Hz), 119.1 (d, J= 3.9 Hz), 90.3, 89.5, 75.6, 73.7, 69.1, 18.8; ¹⁹F NMR (376 MHz, CD3OD) 6 -53.6.

HRMS (ESI): Expected mass [M+Na⁺]: 322.0922; found: 322.0930.

Stereochemical Assignment for 21

Analysis of 2D ROESY of nucleoside 21 supported the indicated stereochemistry.

Preparation of 22 - Glycosylation

Following General Procedure D with BSA (73.0 pL, 0.170 mmol, 3 equiv), TMSOTf (36.0 pL, 0.200 mmol, 2 equiv), and cytosine (11.1 mg, 0.100 mmol, 1 equiv.) in dry MeCN (0.50 mL) then sugar 14 (33.2 mg, 0.100 mmol, 1 equiv) in 1, 2-DCE (0.50 mL) afforded 22A as a white solid (30.0 mg, 79%) after purification by flash column chromatography (4-5% MeOH in CH2CI2).

R, = 0.4 (10% Methanol in CH2CI2)

Melting point = 78-80°C

Data for 22A: H NMR (500 MHz, CD3OD): 6 7.67 (d, J= 7.5 Hz, 1H, HetH), 6.01 (d, J= 4.8 Hz, 1H, H-l), 5.93 (d, J= 7.5 Hz, 1H, HetH), 5.54 - 5.49 (m, 2H, H-2 and H-3), 4.23 - 4.18 (m, 2H, H- 5a and H-5b), 2.12 (s, 3H, Ac-CH₃), 2.11 (s, 3H, Ac-CH₃), 2.07 (s, 3H, Ac-CH₃), 1.36 (s, 3H, 4- CH₃); ¹³C NMR (125 MHz, CD3OD): 8 172.0, 171.3, 171.2, 167.7, 158.0, 143.0, 96.6, 90.0, 84.9, 75.1, 72.6, 68.7, 20.7, 20.4, 20.3, 18.8.

HRMS (ESI): Expected mass [M+Na⁺]: 406.1221; found: 406.1222.

[a]²⁰r> = -0.61 (c 1.51, CHCI3)

Stereochemical Assignment for 22 A

Analysis of 2D ROESY of nucleoside 22A supported the indicated stereochemistry.

Basic workup

Following General Procedure D (work-up) with 22A (19.1 mg, 0.0500 mmol) in ammonia in methanol (7 N, 0.40 mL) afforded 22 (12.8 mg, 100%) as a white solid. Data matched previous reports.

R, = 0.6 (20% Methanol in CH2CI2) Data for 22: H NMR (400 MHz, CD3OD): 8 8.00 (d, J = 7.5 Hz, 1H, HetH), 5.90 - 5.88 (m, 2H, HetH and H-l), 4.31 (dd, J = 5.6 Hz, 1H, H-2), 4.14 (d, J = 5.8 Hz, 1H, H-3), 3.59 - 3.51 (m, 2H, H5a and H5b), 1.23 (s, 3H, 4-CH₃); ¹³C NMR (125 MHz, CD3OD): 6 167.6, 158.8, 143.6, 96.1, 91.7, 88.4, 76.5, 72.7, 68.1, 18.7.

HRMS (ESI): Expected mass [M+Na⁺]: 280.0904; found: 280.0904.

Stereochemical Assignment for 22

Analysis of 2D ROESY of nucleoside 22 supported the indicated stereochemistry.

Preparation of 23 - Glycosylation

14 23A

Following General Procedure D, BSA (73.0 pL, 0.170 mmol, 3 equiv), TMSOTf (36.0 pL, 0.200 mmol, 2 equiv), and 2-chloro-adenine (16.9 mg, 0.100 mmol, 1 equiv) in dry MeCN (0.50 mL) then sugar 14 (33.2 mg, 0.100 mmol, 1 equiv) in 1, 2-DCE (0.50 mL) afforded 23A as a white solid (33.0 mg, 75%) after purification by flash column chromatography (2 — 3 % MeOH in CH2CI2).

R, = 0.38 (5% MeOH in CH2CI2)

Melting point = 65°C

Data for 23A: ^XH NMR (500 MHz, CDCI3): 6 7.97 (s, 1H, HetH), 6.20 (d, = 6.4 Hz, 1H, H-l), 6.17 (s, 2H, HetNH₂), 5.91 - 5.89 (m, 1H, H-2), 5.63 (d, J= 5.7 Hz, 1H, H-3), 4.25 (d, J= 12.0 Hz, 1H, H-5a), 4.19 (d, J= 12.0 Hz, 1H, H-5b), 2.18 (s, 3H, Ac-CH₃), 2.14 (s, 3H, Ac-CH₃), 2.05 (s, 3H, Ac- CH₃), 1.37 (s, 3H, 4-CH₃); ¹³C NMR (125 MHz, CDCI3): 6 170.2, 169.54, 169.46, 156.3, 154.6, 151.1, 138.9, 118.9, 85.0, 84.5, 74.0, 71.9, 67.9, 21.0, 20.6, 20.5, 18.9.

HRMS (ESI): Expected mass [M+Na⁺]: 464.0943; found: 464.0943.

[a]²⁰D = +5.1 (c 0.16, CHCI3)

Stereochemical Assignment for 23 A

Analysis of 2D ROESY of nucleoside 23A supported the indicated stereochemistry.

Basic workup

Following General Procedure D (work-up) with 23A (22.0 mg, 0.0500 mmol) in ammonia in methanol (7 N in methanol, 0.40 mL) afforded 23 (15.8 mg, 100%) as a white solid.

Rf = 0.65 (20% MeOH in CH2CI2)

Melting point = 243 °C

Data for 23: ¹ H NMR (500 MHz, CD3OD): 6 8.24 (s, 1H, HetH), 5.87 (d, J= 7.2 Hz, 1H, H-l), 4.88 (dd, J= 7.2, 5.4 Hz, 1H, H-2), 4.23 (d, J = 5.4 Hz, 1H, H-3), 3.69 (d, J = 12.1 Hz, 1H, H-5a), 3.56 (d, J= 12.1 Hz, lH, H-5b), 1.27 (s, 3H, 4-CH₃); ¹³C NMR (125 MHz, CD3OD): 6 158.3, 155.0, 151.2, 142.4, 120.0, 90.6, 89.7, 75.6, 73.8, 69.2, 18.8.

HRMS (ESI): Expected mass [M+H⁺]: 316.0807; found: 316.0808.

[a]²% = -32.5 (c 0.55, MeOH)

Stereochemical Assignment for 23

Analysis of 2D ROESY of nucleoside 23 supported the indicated stereochemistry.

Preparation of 4'-Ethyl Nucleoside Analogues (23-25)

Grignard Addition

Following the General Procedure A with ethyl magnesium bromide (3 M in diethyl ether, 14.2 mL, 42.7 mmol, 4 equiv) and 6 (2.50 g, 10.7 mmol, 1 equiv.) in dry THF (30.5 mL) afforded 10A as a light yellow oil (1.52 g, 54%) after purification by flash column chromatography (10 — 60% EtOAc in hexanes).

Ry = 0.6 (60% EtOAc in Hexanes)

Data for 10A: H NMR (600 MHz, CDCI3): 64.40 (d, J= 1.2 Hz, 1H), 3.86 (dd, J= 9.8, 4.1 Hz, 1H), 3.82 (d, J= 9.8, 1H), 3.76 (d, J= 11.6 Hz, 1H), 3.56 (s, 3H), 3.55 (dd, J= 11.1, 1.0 Hz, 1H), 3.48 (s, 3H) 2.75 (d, J= 4.0 Hz, 1H); 2.02 (m, 1H), 1.61 (m, 1H), 1.48 (s, 3H), 1.38 (s, 3H), 1.01 (dd, = 7.5 Hz, 3H); ¹³C NMR (126 MHz, CDCh): 6 103.2, 98.9, 73.7, 72.2, 68.9, 65.2, 57.2, 55.5, 28.8, 23.6, 19.3, 6.8.

HRMS (ESI): Expected mass [M+Na⁺]: 287.1465; found: 287.1466.

[a]²⁰r> = -39.8 (c 1.01, CHCh)

Intramolecular Bis- Transacetalization

Following General Procedure B with TMSOTf (0.960 mL, 5.20 mmol, 2 equiv.), 2,6-lutidine (306 pL, 2.60 mmol, 1 equiv.), and 10A (0.700 g, 2.60 mmol, 1 equiv.) in dry CH2CI2 (33.0 mL) afforded 13A as a light yellow oil (0.350 g, 75%) after purification by flash column chromatography (10 — 30% EtOAc in hexanes).

Ry = 0.6 (30% EtOAc in Hexanes)

Data for 13A: H NMR (400 MHz, CDCh): 6 5.38 (s, 1H, H-l), 4.35 (d, J= 5.2 Hz, 1H, H-2), 4.25 (d, J =5.2, 1H, H-3), 3.32 (d, J = 7.2 Hz, 1H, 5-Ha), 3.28 (d, J= 7.2 Hz, 1H, 5-Hb), 2.11 (m, 1H, 4- CHaHbCHi), 1.95 (m, 1H, 4-CHaHbCH₃), 1.47 (s, 3H, CH₃), 1.33 (s, 3H, CH₃), 1.04 (dd, J= 7.6 Hz, 3H, 4-CH₂CH₃) ; ¹³C NMR (126 MHz, CDCh): 6 112.2, 99.9, 87.1, 81.9, 79.2, 66.5, 26.1, 25.6, 19.8, 8.68.

HRMS (ESI): Expected mass [M+H⁺]: 201.1121; found: 201.1122

[a]²⁰r> = -16.7 (c 1.55, CHCh)

Ring Opening-Acetylation

Following General Procedure C with TESOTf (0.237 mL, 1.47 mmol, 0.625 equiv) and 13A (335 mg, 1.68 mmol, 1 equiv.) in Ac2O:CH2Ch (4.2 mL: 4.2 mL) afforded 14A as a yellow oil (429 mg, 74%) after purification by flash column chromatography (0% — 30% EtOAc in hexanes).

R./ = 0.6 (50% EtOAc in Hexanes)

Data for 14A: H NMR (400 MHz, CDCh): 6 6.19 (s, 1H, H-l), 5.50 (d, J = 5.5 Hz, 1H, H-3), 5.42 (dd, J= 5.5, 1.5 Hz, 1H, H-2), 4.15 (s, 2H, 5-Ha and 5-Hb), 2.12-2.09 (12H, 4x Ac-CH₃), 1.82 (m, 1H, 4-CHaHbCH₃), 1.68 (m, 1H, 4-CHaHbCH3), 0.97 (dd, J = 7.2, 3H, 4-CHaHbCH₃); ¹³C NMR (126 MHz, (CDCh): 6 170.3, 169.4, 169.29, 169.25, 97.7, 86.3, 75.2, 72.4, 66.0, 25.4, 21.1, 20.8, 20.5, 20.4, 7.5. HRMS (ESI): Expected mass [M+Na⁺]: 369.1156; found: 369.1155.

[a]²⁰r> = -27.2 (c 1.61, CHC1₃)

Preparation of 24 - Glycosylation

14A 24A

Following General Procedure D with BSA (84.7 pL, 0.347 mmol, 3 equiv), TMSOTf (41.9 pL, 0.231 mmol, 2 equiv), and 2-fluoro-adenine (17.7 mg, 0.116 mmol, 1 equiv.) in dry MeCN (1.16 mL) then sugar 14A (40.0 mg, 0.116 mmol, 1 equiv) in 1,2-DCE (0.40 mL) afforded 24A as a white powder (57.1 mg, 97%) after purification by flash column chromatography (0 — 10% MeOH in CH2CI2).

R, = 0.6 (10% Methanol in CH2CI2)

Melting point = 165-167°C

Data for 24A: H NMR (600 MHz, MeOD): 6 8.17 (s, 1H, HetH), 6.11 (m, 2H, H-l and H-2), 5.75 (d, J= 5.3 Hz, 1H, H-3), 4.36 (d, J= 12.0 Hz, 1H, H-5a), 4.27 (d, J= 12.0 Hz, 1H, H-5b), 2.17 (s, 3H, Ac-CH₃), 2.08 (s, 3H, Ac-CH₃), 2.02 (s, 3H, Ac-CH₃), 1.95 (m, 1H, 4-CHaHbH₃), 1.75 (m, 1H, 4-CHaHbH₃ ), 0.99 (dd, J= 7.6 Hz, 1H, 4-CHaHbH₃); ¹³C NMR (126 MHz, MeOD): 8 172.1, 171.2, 171.1, 160.6 (d, J= 206 Hz), 159.3 (d, J= 21.9 Hz), 152.2 (d, J= 19.5 Hz), 141.4, 118.8, 87.4, 86.9, 74.3, 73.3, 66.3, 25.8, 20.7, 20.4, 20.2, 7.8. ¹⁹F NMR (376 MHz, MeOD): 6 -52.7

HRMS (ESI): Expected mass [M+Na⁺]: 462.1395; found: 462.1391.

[a]²% = -10.1 (c 0.82, MeOH)

Following General Procedure D (work-up) with 24A (46.0 mg, 0.105 mmol) in ammonia in methanol (7 N, 1.55 mL) afforded 24 (32.8 mg, 100%) as a white solid. Data matched previous reports.³

R = 0.5 (20% Methanol in CH2CI2)

Data for 24: ¹ H NMR (400 MHz, MeOD): 6 8.21 (s, 1H, HetH), 5.81 (d, J= 7.6 Hz, 1H, H-l), 4.91 (dd, J = 7.6, 5.4 Hz, 1H, H-2), 4.23 (d, J= 5.4 Hz, 1H, H-3), 3.72 (d, J= 12.3 Hz, 1H, H-5a), 3.64 (d, J = 12.3 1H, H-5b), 1.77 (m, 2H, 4-CH₂CH₃), 0.97 (dd, J = 7.2 Hz, 3H, 4-CH₂CH₃); ¹³C NMR (176 MHz, MeOD): 6 160.2 (d, J = 209 Hz), 159.2 (d, J= 20.1 Hz), 151.6 (d, J = 18.9 Hz), 142.3, 119.2, 91.4, 90.3, 75.4, 73.9, 66.7, 26.2, 8.6; ¹⁹F NMR (376 MHz, MeOD): 6 -53.8

HRMS (ESI): Expected mass [M+Na⁺]: 336.1079; found: 336.1080.

Stereochemical Assignment for 24

Analysis of 2D ROESY of nucleoside 24 supported the indicated stereochemistry.

Preparation of 25 - Glycosylation

Following General Procedure D with BSA (42.4 pL, 0.175 mmol, 3 equiv), TMSOTf (20.4 pL, 0.115 mmol, 2 equiv), and cytosine (6.4 mg, 0.058 mmol, 1 equiv.) in dry MeCN (0.58 mL) then sugar 14A (20 mg, 0.058 mmol, 1 equiv) in 1, 2-DCE (0.20 mL) afforded 25A as a yellow oil (23 mg, 100%) after purification by flash column chromatography (0% — 10% MeOH in CH2CI2).

R, = 0.4 (10% Methanol in CH2CI2)

Melting point = 89-91 °C

Data for 25A: H NMR (600 MHz, MeOD): 8 7.69 (d, J= 7.6 Hz, 1H, HetH), 6.04 (d, J= 5.0 Hz, 1H, H-l), 5.96 (d, J= 7.3 Hz, 1H, HetH), 5.55 (m, 2H, H-2 and H-3), 4.27 (d, J= 11.9 Hz, 1H, H- 5a), 4.24 (d, J= 11.9 Hz, 1H, H-5b), 2.12 (s, 3H, Ac-CH₃), 2.11 (s, 3H, Ac-CH₃), 2.04 (s, 3H, Ac- CH₃), 1.89 (m, 1H, 4-CHaHbH₃), 1.72 (m, 1H, 4-CHaHbH₃), 0.98 (dd, J = 7.4 Hz, 3H, 4-CHaHbH₃); ¹³C NMR (126 MHz, MeOD): 6 172.0, 171.3, 171.2, 167.5, 157.9, 143.0, 96.9, 89.4, 86.7, 74.9, 73.3, 66.7, 25.7, 20.8, 20.38, 20.37, 7.7.

HRMS (ESI): Expected mass [M+Na⁺]: 420.1377; found: 420.1377.

[a]²⁰r> = +23.0 (c 1.51, CHC1₃)

Basic workup

25A 25

Following General Procedure D (work-up step) with 25A (22.9 mg, 0.0580 mmol) in a solution of ammonia in methanol (7 N, 0.80 mL) afforded 25 (15.3 mg, 98%) as a light yellow oil.

Rf = 0.6 (20% Methanol in CH2CI2)

Data for 25: ¹ H NMR (600 MHz, MeOD): 6 7.97 (d, J= 7.5 Hz, 1H, HetH), 5.91 (m, 2H, HetH and H-l), 4.37 (m, 1H, H-2), 4.17 (d, J= 5.8 Hz, 1H, H-3), 3.63 (d, J= 11.8 Hz, 1H, H-5a), 3.58 (d, J = 11.8 Hz, 1H, H-5b), 1.81 (m, 1H, 4-CHaHbCH₃), 1.68 (m, 1H, 4-CHaHbCH₃), 0.94 (dd, J= 7.6 Hz, 3H, 4-CH2CH3); ¹³C NMR (126 MHz, MeOD): 6 167.6, 159.0, 143.7, 96.4, 91.2, 90.2, 76.4, 73.3, 65.8, 25.9, 8.4.

HRMS (ESI): Expected mass [M+Na⁺]: 294.1060; found: 294.1058.

Stereochemical Assignment for 25

Analysis of 2D ROESY of nucleoside 25 supported the indicated stereochemistry.

Preparation of 4'-Allyl Nucleoside Analogues (26 and 27)

Grignard Addition

Following the General Procedure A with allyl magnesium bromide (1.0 M in Et2O, 15.0 mL, 10.0 mmol, 3 equiv) and 6 (1.17 g, 5.00 mmol, 1 equiv.) in dry CH2CI2 (15.0 mL) afforded 10B as a colorless oil (0.552 g, 40%) after purification by flash column chromatography (20 — 25% EtOAc in hexanes).

R./ = 0.43 (50% EtOAc in Hexanes)

Data for 10B:^JH NMR (500 MHz, CDC1₃): 8 5.99 - 5.91 (m, 1H), 5.18 - 5.13 (m, 2H), 4.36 (d, J = 1.5 Hz, 1H), 3.85 - 3.80 (m, 2H), 3.70 (d, J= 11.5 Hz, 1H), 3.53 - 3.50 (m, 4H), 3.44 (s, 4H), 2.92 (d, J = 3.8 Hz, 1H), 2.69 (dd, J= 14.0, 8.4 Hz, 1H), 2.37 (ddd, J = 14.0, 6.1, 1.3 Hz, 1H), 1.43 (s, 3H), 1.36 (s, 3H); ¹³C NMR (125 MHz, CDC1₃): 6 133.4, 118.5, 103.3, 99.1, 73.4, 72.2, 68.8, 66.0, 57.2, 55.6, 36.2, 28.8, 19.4. HRMS (ESI): Expected mass [M+Na⁺]: 299.1465; found: 299.1462.

[a]²⁰r> = -41.8 (c 0.73, CHCh)

Intramolecular Bis- Transacetalization

Following General Procedure B with TMSOTf (0.728 mL, 4.00 mmol, 2 equiv.), 2,6-lutidine (0.232 mL, 2.00 mmol, 1 equiv.), and 10B (0.552 g, 2.00 mmol, 1 equiv.) in CH2CI2 (15.0 mL) afforded 13B as a colorless liquid (0.255 g, 60%) after purification by flash column chromatography (5 — 7% EtOAc in hexanes).

R./ = 0.61 (20% EtOAc in Hexanes)

Data for 13B: ¹ H NMR (400 MHz, CDCh): 6 5.88 - 5.77 (m, 1H, 4-CH₂CHCH₂), 5.37 (s, 1H, H-l),

5.25 - 5.11 (m, 2H, 4-CH₂CHCH₂), 4.34 (d, J= 5.5 Hz, 1H, H-2), 4.20 (d, J = 5.5 Hz, 1H, H-03),

3.26 (m, 2H, 5-Ha and 5-Hb), 2.79 (dd, J= 14.0, 7.4 Hz, 1H, 4-CHaHbCHCH₂), 2.68 (dd, J= 14.0, 7.3 Hz, 1H, 4-CHaHbCHCH₂), 1.46 (s, 3H, CH₃), 1.31 (s, 3H, CH₃); ¹³C NMR (176 MHz, CDCh): 6 130.7, 118.2, 111.5, 99.2, 84.7, 81.1, 78.6, 65.6, 30.5, 25.2, 24.7.

HRMS (DART-MS): Expected mass [M+H⁺]: 213.1121; found: 213.1118.

[a]²⁰r> = -21.4 (c 0.55, CHCh)

Ring Opening-Acetylation

Following General Procedure C with TESOTf (0.141 mL, 0.625 mmol, 0.625 equiv) and 13B (0.212 g, 1.00 mmol, 1 equiv.) in Ac2O:CH2Ch (2.0 mL: 2.0 mL) afforded 14B as a yellow oil (0.251 g, 70%) after purification by flash column chromatography (15 — 20% EtOAc in hexanes).

Ry = 0.54 (50% EtOAc in Hexanes)

Data for 14B: H NMR (500 MHz, CDCh): 6 6.21 (d, J= 1.6 Hz, 1H, H-l), 5.86 - 5.78 (m, 1H, 4- CH2CHCH2), 5.50 (d, J = 5.4 Hz, H-3), 5.42 (dd, J = 5.4, 1.6 Hz, 1H, H-2), 5.14 - 5.09 (m, 2H, 4- CH2CHCH2), 4.14 (d, J= 11.7 Hz, 1H, 5-Ha), 4.09 (d, J= 11.7 Hz, 1H, 5-Hb), 2.59 (dddd, J= 14.5, 6.1, 1.4 Hz, 1H, 4-CHaHbCHCH₂), 2.41 (dd, J = 14.5, 8.3 Hz, 1H, 4-CHaHbCHCH₂), 2.11 (s, 3H, ACCH₃), 2.09 (s, 6H, 2x AcCH₃), 2.08 (s, 3H, AcCH₃); ¹³C NMR (125 MHz, (CDCh): 6 170.3, 169.5, 169.4 (2C), 132.0, 119.2, 97.8, 85.5, 75.2, 72.2, 66.5, 37.8, 21.2, 21.0, 20.7, 20.6.

HRMS (ESI): Expected mass [M+Na⁺]: 381.1156; found: 381.1156.

[a]²⁰z) = 17.4 (c 1.13, CHCh)

Preparation of 26 - Glycosylation

Following General Procedure D with BSA (73.0 pL, 0.170 mmol, 3 equiv), TMSOTf (36.0 pL, 0.200 mmol, 2 equiv), and adenine (13.5 mg, 0.100 mmol, 1 equiv) in dry MeCN (0.50 mL) then 14B (35.8 mg, 0.100 mmol, 1 equiv) in 1, 2-DCE (0.50 mL) afforded 26A as a white solid (26.0 mg, 60%) after purification by flash column chromatography (1 — 2 % MeOH in CH2CI2).

Rz = 0.50 (5% Methanol in CH2CI2)

Melting point = 65°C

Data for 26A: H NMR (500 MHz, CDCh): 6 8.37 (s, 1H, HetH), 8.00 (s, 1H, HetH), 6.25 (d, J = 6.8 Hz, 1H, H-l), 6.11 (dd, J= 6.8, 5.5 Hz, 1H, H-2), 5.81 - 5.71 (m, 1H, 4-CH₂CHCHaHb), 5.72 (d, J= 5.5 Hz, 1H, H-3), 5.64 (s, 2H, HetNH₂), 5.19 - 5.15 (m, 2H, 4-CH₂CHCHaHb), 4.31 (d, J= 12.1 Hz, 1H, H-5a), 4.25 (d, J = 12.1 Hz, 1H, H-5b), 2.67 (ddd, J = 13.2, 5.8, 3.2 Hz, 1H, 4- CHaHbCHCH₂), 2.47 (dd, J= 14.6, 8.0 Hz, 1H, 4-CHaHbCHCH₂), 2.19 (s, 3H, Ac-CH₃), 2.15 (s, 3H, Ac-CH₃), 2.02 (s, 3H, Ac-CH₃); ¹³C NMR (125 MHz, CDCh): 6 170.2, 169.4, 169.3, 155.6, 153.5, 150.2, 138.9, 131.2, 120.1, 120.0, 85.4, 84.9, 73.5, 72.1, 66.6, 37.4, 21.0, 20.7, 20.5.

HRMS (ESI): Expected mass [M+H⁺]: 434.1670; found: 434.1668.

[a]²% = -37.8 (c 0.40, CHCh)

Stereochemical Assignment for 26A

Analysis of 2D ROESY of nucleoside 26A supported the indicated stereochemistry.

Basic workup

Following General Procedure D (work-up) with 26A (21.6 mg, 0.0500 mmol) in ammonia in methanol (7 N in methanol, 0.40 mL) afforded 26 (15.4 mg, 100%) as a white solid.

Rf = 0.46 (20% Methanol in CH2CI2)

Melting point = 101°C

Data for 26: ¹ H NMR (700 MHz, CD3OD): 6 8.26 (s, 1H, HetH), 8.18 (s, 1H, HetH), 5.98 - 5.93 (m, 1H, 4-CH₂CHCHaHb), 5.91 (d, J = 7.7 Hz, 1H, H-l), 5.10 (dd, J = 17.2, 1.3 Hz, 1H, 4- CH₂CHCHaHb), 5.05 (d, J = 10.2 Hz, 1H, 4-CH₂CHCHaHb), 4.98 (dd, J= 7.5, 5.5 Hz, 1H, H-2), 4.26 (d, J= 5.2 Hz, 1H, H-3), 3.71 (d, J= 12.3 Hz, 1H, 5-Ha), 3.61 (d, J= 12.3 Hz, 1H, 5-Hb), 2.53 (m, 2H, 4-CH₂CHCHaHb); ¹³C NMR (125 MHz, CD3OD): 6 157.7, 153.3, 149.9, 142.4, 135.2, 121.2, 118.2, 90.9, 90.7, 75.4, 73.9, 67.8, 38.8.

HRMS (ESI): Expected mass [M+H⁺]: 308.1353; found: 308.1355.

[a]²⁰o = -31.5 (c 0.27, MeOH)

Stereochemical Assignment for 26

Analysis of 2D ROESY of nucleoside 26 supported the indicated stereochemistry.

Preparation of 27 - Glycosylation

Following General Procedure D with BSA (73.0 pL, 0.170 mmol, 3 equiv), TMSOTf (36.0 pL, 0.200 mmol, 2 equiv), and 6-chloropurine (15.5 mg, 0.100 mmol, 1 equiv) in dry MeCN (0.50 mL) then sugar 14B (35.8 mg, 0.100 mmol, 1 equiv) in 1, 2-DCE (0.50 mL) afforded 27A as a white solid (40.7 mg, 90%) after purification by flash column chromatography (1 — 2 % MeOH in CH2CI2). R, = 0.70 (5% Methanol in CH2CI2)

Melting point = 61 °C

Data for 27A: H NMR (500 MHz, CDCh): 6 8.77 (s, 1H, HetH), 8.32 (s, 1H, HetH), 6.29 (d, J = 6.7 Hz, 1H, H-l), 6.13 (dd, J= 6.5, 5.7 Hz, 1H, H-2), 5.80 - 5.73 (m, 1H, 4-CH₂CHCHaHb), 5.71 (d, J= 5.6 Hz, 1H, H-3), 5.19 - 5.16 (m, 2H, 4-CH₂CHCHaHb), 4.32 (d, J= 12.2 Hz, 1H, H-5a), 4.26 (d, J= 12.2 Hz, 1H, H-5b), 2.67 (dd, J= 14.6, 6.5 Hz, 1H, 4-CHaHbCHCH₂), 2.48 (dd, J= 14.6, 8.0 Hz, 1H, 4-CHaHbCHCH₂), 2.20 (s, 3H, Ac-CH₃), 2.13 (s, 3H, Ac-CH₃), 2.02 (s, 3H, Ac-CH₃); ¹³C NMR (125 MHz, CDCh): 6 170.1, 169.4, 169.2, 152.4, 151.7, 151.6, 143.6, 132.4, 130.9, 120.3, 85.9, 85.7, 73.4, 72.0, 66.4, 37.3, 21.0, 20.6, 20.4.

HRMS (ESI): Expected mass [M+Na⁺]: 475.0991; found: 475.0997.

[a]²⁰r> = -53.0 (c 0.16, CHCh)

Stereochemical Assignment for 27 A

Analysis of 2D ROESY of nucleoside 27A supported the indicated stereochemistry

Deprotection of 27 A

1.0 pL of NaOMe solution (25% w/v solution in methanol) was added to 27A (22.6 mg, 0.0500 mmol) in 0.20 mL methanol, and allowed to stir at room temperature overnight. After completion of the reaction, as monitored by TLC, the reaction mixture quench with water and extracted 3 times with EtOAc. Combined organic layers were dried on Na2SC>4 and concentrated under reduced pressure and the crude reaction mixture was purified with flash column chromatography (3 : 3 : 4 solution of EtOAc : acetone : hexane) to afford 27 (13.0 mg, 80%) as a white solid.

R, = 0.45 (10% Methanol in CH2CI2)

Melting point = 98°C

Data for 27: ¹ H NMR (500 MHz, CD3OD): 8 8.82 (s, 1H, HetH), 8.75 (s, 1H, HetH), 6.14 (d, J= 7.2 Hz, 1H, H-l), 5.94 (dddd, J= 17.4, 10.2, 7.2 Hz, 1H, 4-CHaCHbCHCHaHb), 5.16 - 5.04 (m, 2H, 4- CHaCHbCHCHaHb), 4.98 (dd, J= 7.2, 5.3 Hz, 1H, H-2), 4.32 (d, J= 5.3 Hz, 1H, H-3), 3.71 (d, J= 12.0 Hz, 1H, H5a), 3.65 (d, J = 12.0 Hz, 1H, H5b), 2.60 (dd, J = 14.2, 7.1 Hz, 1H, 4- CHaCHbCHCHaHb), 2.52 (dd, J= 14.2, 7.4 Hz, 1H, 4-CHaCHbCHCHaHb).; ¹³C NMR (125 MHz, CD3OD): 6 152.94, 152.91, 151.7, 147.4, 135.0, 133.2, 118.3, 90.7, 90.2, 76.2, 73.7, 66.8, 38.3.

HRMS (ESI): Expected mass [M+Na⁺]: 349.0674; found: 349.0673. [a]²⁰o = -61.7 (c 0.53, MeOH)

Stereochemical Assignment for 27

Analysis of 2D ROESY of nucleoside 27 supported the indicated stereochemistry.

Preparation of S5 - Glycosylation

Following General Procedure D with BSA (73.0 pL, 0.170 mmol, 3 equiv), TMSOTf (36.0 pL, 0.200 mmol, 2 equiv), and thymine (12.6 mg, 0.100 mmol, 1 equiv.) in dry MeCN (0.50 mL) then sugar 14B (35.8 mg, 0.100 mmol, 1 equiv) in 1, 2-DCE (0.50 mL) afforded S5A as white solid (36.0 mg, 85%) after purification by flash column chromatography (2 — 3 % MeOH in CH2CI2).

Ry = 0.54 (5% Methanol in CH2CI2)

Melting point = 50°C

Data for S5A: H NMR (500 MHz, CDCh): 6 8.87 (s, 1H, HetNH), 7.24 (d, J= 1.2 Hz, 1H, HetH), 6.21 (d, J= 6.9 Hz, 1H, H-l), 5.78 - 5.70 (m, 1H, 4-CH2CHCH2), 5.48 - 5.42 (m, 2H, H-2 and H-3), 5.16 (m, 2H, 4-CH2CHCH2), 4.27 (d, J= 12.2 Hz, 1H, H-5a), 4.13 (d, J= 12.2 Hz, 1H, H-5b), 2.63 - 2.58 (m, 1H, 4-CHaHbCHCH₂), 2.39 (dd, J= 14.6, 8.0 Hz, 1H, 4-CHaHbCHCH₂), 2.18 (s, 3H, AC-CH₃), 2.16 (s, 3H, AC-CH₃), 2.05 (s, 3H, Ac-CH₃), 1.93 (d, J= 1.2 Hz, 3H, HetCH₃); ¹³C NMR (125 MHz, CDCh): 6 169.9, 169.7, 169.5, 163.3, 150.7, 134.6, 131.0, 120.2, 112.2, 85.0, 84.6, 72.6, 71.6, 66.9, 37.3, 21.0, 20.6, 20.5, 12.9.

HRMS (ESI): Expected mass [M+Na⁺]: 447.1374; found: 447.1378.

[a]²⁰r> = -41.6 (c 0.10, CHCh)

Stereochemical Assignment for S5A

Analysis of 2D ROESY of nucleoside S5A supported the indicated stereochemistry.

Basic workup

Following General Procedure D (work-up step) with S5A (21.2 mg, 0.0500 mmol) in ammonia in methanol (7 N in methanol, 0.40 mL) afforded S5 (14.9 mg, 100%) as a white solid.

R, = 0.44 (10% Methanol in CH2CI2)

Melting point = 140°C

Data for S5: H NMR (500 MHz, CD3OD): 6 7.85 (d, J = 1.1 Hz, 1H, HetH), 5.96 (d, J = 7.1 Hz, 1H, H-l), 5.94 - 5.88 (m, 1H, 4-CHaCHbCHCHaHb), 5.10 - 5.03 (m, 2H, 4-CHaCHbCHCHaHb), 4.40 (dd, J= 7.0, 5.7 Hz, 1H, H-2), 4.17 (d, J= 5.6 Hz, 1H, H-3), 3.62 - 3.56 (m, 2H, H5a and H5b), 2.53 (dd, J = 14.3, 7.0 Hz, 1H, 4-CHaCHbCHCHaHb), 2.41 (dd, J = 14.3, 7.5 Hz, 1H, 4- CHaCHbCHCHaHb), 1.88 (d, J = 1.1 Hz, 3H, Het-CHs); ¹³C NMR (125 MHz, CD3OD): 8 166.3, 153.0, 138.6, 135.1, 118.1, 111.8, 89.0, 75.5, 73.3, 66.7, 38.4, 12.4.

HRMS (ESI): Expected mass [M+Na⁺]: 321.1057; found: 321.1056.

[a]²⁰o = -47.2 (c 0.62, MeOH)

Stereochemical Assignment for S5

Analysis of 2D ROESY of nucleoside S5 supported the indicated stereochemistry.

Also the recrystallization in acetonitrile : hexane allowed for the relative stereochemistry to be assigned using single X-ray crystallography (see X-ray structures, CCDC NO: 2333367).

Preparation of 4 -CD.; Nucleoside Analogues (28 - 31)

Grignard Addition

Following the General Procedure A with trideuteromethyl magnesium iodide (1 M in Et2O, 15.0 mL, 15.0 mmol, 3 equiv), and 6 (1.17 g, 5.00 mmol, 1 equiv) in dry CH2CI2 (15.0 mL) afforded 10C as a colorless oil (0.886 g, 70%) after purification by flash column chromatography (20 — 25% EtOAc in hexanes).

R./ = 0.32 (50% EtOAc in Hexanes)

Data for 10C: ¹ H NMR (400 MHz, CDCI3): 64.37 (d, J= 1.5 Hz, 1H), 3.79 - 3.74 (m, 2H), 3.70 (d, J= 11.2 Hz, 1H), 3.54 (s, 1H), 3.52 (s, 3H), 3.48 - 3.44 (m, 4H), 2.87 (d, J = 3.9 Hz, 1H), 1.43 (s, 3H), 1.35 (s, 3H); ¹³C NMR (125 MHz, CDCh): 6 103.3, 99.0, 73.0, 72.9, 70.1, 67.2, 57.3, 55.6, 29.0, 19.2.

HRMS (ESI): Expected mass [M+Na⁺]: 276.1497; found: 276.1497.

[a]²⁰r> = -15.4 (c 1.97, CHCh)

Intramolecular Bis- Transacetalization

Following General Procedure B with TMSOTf (0.728 mL, 4.00 mmol, 2 equiv.), 2,6-lutidine (0.232 mL, 2.00 mmol, 1 equiv.), and 10C (0.506 g, 2.00 mmol, 1 equiv.) in CH2CI2 (15.0 mL) afforded 13C as colorless liquid (0.246 g, 65%) after purification by flash column chromatography (5 — 7% EtOAc in hexanes).

R./ = 0.38 (20% EtOAc in Hexanes)

Data for 13C: H NMR (500 MHz, CDCh): 6 5.36 (s, 1H, H-l), 4.34 (d, J= 5.5 Hz, 1H, H-2), 4.11 (d, J= 5.5 Hz, 1H, H-3), 3.29 (d, J= 7.1 Hz, 1H, H-5a), 3.19 (d, J= 7.1 Hz, 1H, H-5b), 1.46 (s, 3H, CH₃), 1.31 (s, 3H, CH₃); ¹³C NMR (125 MHz, CDCh): 6 112.3, 100.3, 83.8, 82.4, 80.7, 68.3, 26.2, 25.7.

HRMS (DART-MS): Expected mass [M+H⁺]: 190.1153; found: 190.1151.

[a]²⁰/ = -26.0 (c 0.3, CHCh)

Ring Opening-Acetylation

Following General Procedure C with TESOTf (0.141 mL, 0.625 mmol, 0.625 equiv) and 13C (189.2 mg, 1.00 mmol, 1 equiv.) in Ac2O:CH2Ch (2.0 mL: 2.0 mL) afforded 14C as yellow oil (0.234 g, 70%) after purification by flash column chromatography (15 — 20% EtOAc in hexanes).

R./ = 0.57 (50% EtOAc in Hexanes)

Data for 14C: H NMR (500 MHz, CDCh): 6 6.16 (d, J= 0.8 Hz, 1H, H-l), 5.42 - 5.39 (m, 2H, H- 2 and H-3), 4.13 (d, J= 11.7 Hz, 1H, H-5a), 4.05 (d, J = 11.7 Hz, 1H, H-5b), 2.12 (s, 3H, AcCH₃), 2.09 (s, 9H, 3x ACCH₃); ¹³C NMR (125 MHz, CDCh): 6 170.5, 169.6, 169.42, 169.39, 97.6, 84.4, 75.3, 71.8, 68.5, 21.2, 21.0, 20.7, 20.6.

HRMS (ESI): Expected mass [M+Na⁺]: 358.1188; found: 358.1190.

[a]²⁰D = -7.1 (c 0.75, CHCh)

Preparation of 28 - Glycosylation

13C 28A

Following General Procedure D with BSA (73.0 pL, 0.170 mmol, 3 equiv), TMSOTf (36.0 pL, 0.200 mmol, 2 equiv), and adenine (13.5 mg, 0.100 mmol, 1 equiv) in dry MeCN (0.50 mL) then sugar 14C (33.5 mg, 0.100 mmol, 1 equiv) in 1, 2-DCE (0.50 mL) afforded 28A as a white solid (34.1 mg, 85%) after purification by flash column chromatography (2 — 4 % MeOH in CH2CI2).

R, = 0.28 (5% Methanol in CH2CI2)

Melting point = 91 °C

Data for 28A: H NMR (500 MHz, CDCh): 6 8.37 (s, 1H, HetH), 7.98 (s, 1H, HetH), 6.21 (d, J = 6.1 Hz, 1H, H-l), 6.07 (dd, J= 5.9 Hz, 1H, H-2), 5.71 (d, J= 5.7 Hz, 1H, H-3), 5.63 (s, 2H, HetNH₂), 4.28 - 4.21 (m, 2H, H-5a and H-5b), 2.17 (s, 3H, Ac-CH₃), 2.12 (s, 3H, Ac-CH₃), 2.05 (s, 3H, Ac- CH₃).; ¹³C NMR (125 MHZ, CDCh): 6 170.3, 169.5, 169.4, 155.6, 153.5, 150.1, 139.0, 120.2, 85.3, 84.3, 73.8, 71.9, 67.9, 21.0, 20.6, 20.5.

HRMS (ESI): Expected mass [M+H⁺]: 411.1702; found: 411.1702.

[a]²% = -14.6 (c 0.38, CHCh)

Stereochemical Assignment for 28A

Analysis of 2D ROESY of nucleoside 28A supported the indicated stereochemistry.

Basic workup Following General Procedure D (work-up) with 28A (20.5 mg, 0.0500 mmol) in a solution of ammonia in methanol (7 N in methanol, 0.40 mL) afforded 28 (14.2 mg, 100%) as a white solid.

R, = 0.43 (20% Methanol in CH2CI2)

Melting point = 102°C

Data for 28: ¹ H NMR (500 MHz, CD3OD): 6 8.26 (s, 1H, HetH), 8.18 (s, 1H, HetH), 5.91 (d, J= 7.4 Hz, 1H, H-l), 4.95 (dd, J= 7.4, 5.4 Hz, 1H, H-2), 4.23 (d, J= 5.4 Hz, 1H, H-3), 3.69 (d, J= 12.1 Hz, 1H, H-5a), 3.56 (d, J= 12.1 Hz, 1H, H-5b); ¹³C NMR (125 MHz, CD3OD): 6 157.7, 153.4, 149.9, 142.3, 121.2, 90.9, 89.7, 75.6, 74.0, 69.3.

HRMS (ESI): Expected mass [M+H⁺]: 285.1385; found: 285.1383.

[a]²⁰o = -41.2 (c 0.24, MeOH)

Stereochemical Assignment for 28

Analysis of 2D ROESY of nucleoside 28 supported the indicated stereochemistry.

Preparation of 29 - Glycosylation

Following General Procedure D with BSA (73.0 pL, 0.170 mmol, 3 equiv), TMSOTf (36.0 pL, 0.200 mmol, 2 equiv), and cytosine (23.8 mg, 0.100 mmol, 1 equiv) in dry MeCN (0.50 mL) then sugar 14C (33.5 mg, 0.100 mmol, 1 equiv) in 1, 2-DCE (0.50 mL) afforded 29A as white solid (30.9 mg, 80%) after purification by flash column chromatography (2 — 5 % MeOH in CH2CI2).

R, = 0.48 (10% Methanol in CH2CI2)

Melting point = 101°C

Data for 29A: H NMR (500 MHz, CD3OD): 8 7.68 (d, J= 7.5 Hz, 1H, HetH), 6.01 (d, J= 4.8 Hz, 1H, H-l), 5.93 (d, J= 7.5 Hz, 1H, HetH), 5.52 (m, 2H, H-2 and H-3), 4.23 - 4.17 (m, 2H, H-5a and H-5b), 2.12 (s, 6H, 2x Ac-CH₃), 2.07 (s, 3H, Ac-CH₃); ¹³C NMR (125 MHz, CD3OD): 6 172.0, 171.3, 171.2, 167.7, 158.0, 143.0, 96.6, 90.0, 84.7, 75.1, 72.6, 68.6, 20.7, 20.4, 20.3.

HRMS (ESI): Expected mass [M+Na⁺]: 409.1409; found: 409.1406.

[a]²⁰r> = +42.5 (c 0.32, CHCI3)

Stereochemical Assignment for 29 A

Analysis of 2D ROESY of nucleoside 29A supported the indicated stereochemistry. Basic workup

29A 29

Following General Procedure D (work-up) with 29A (19.3 mg, 0.0500 mmol) in ammonia in methanol (7 N in methanol, 0.40 mL) afforded 29 (13.0 mg, 100%) as a white solid.

R, = 0.23 (30% Methanol in CH2CI2)

Melting point = 110°C

Data for 29:¹H NMR (500 MHz, CD3OD): 6 8.00 (d, J= 7.5 Hz, 1H, HetH), 5.90 - 5.88 (m, 2H, HetH and H-l), 4.31 (dd, J= 5.6 Hz, 1H, H-2), 4.14 (d, J= 5.8 Hz, 1H, H-3), 3.57 (d, J= 11.8 Hz, 1H, H-5a), 3.53 (d, J= 11.8 Hz, 1H, H-5b); ¹³C NMR (125 MHz, CD3OD): 6 167.6, 158.8, 143.6, 96.1, 91.7, 88.3, 76.6, 72.7, 68.0.

HRMS (ESI): Expected mass [M+H⁺]: 261.1273; found: 261.1273.

[a]²⁰o = +7.7 (c 0.60, MeOH)

Stereochemical Assignment for 29

Analysis of 2D ROESY of nucleoside 29 supported the indicated stereochemistry.

Preparation of 30 - Glycosylation

Following General Procedure D with BSA (73.0 pL, 0.170 mmol, 3 equiv), TMSOTf (36.0 pL, 0.200 mmol, 2 equiv), and 5-iodouracil (23.8 mg, 0.100 mmol, 1 equiv) in dry MeCN (0.50 mL) then sugar 14C (33.5 mg, 0.100 mmol, 1 equiv) in 1, 2-DCE (0.50 mL) afforded 30A as a white solid (43.6 mg, 85%) after purification by flash column chromatography (2 — 5 % MeOH in CH2CI2).

R, = 0.42 (5% Methanol in CH2CI2)

Melting point = 73 °C Data for 30A: H NMR (500 MHz, CDCh): 6 9.16 (s, 1H, HetNH), 7.94 (s, 1H, HetH), 6.13 - 6.10 (m, 1H, H-l), 5.42 - 5.39 (m, 2H, H-2 and H-3), 4.24 (d, J= 12.2 Hz, 1H, H-5a), 4.12 - 4.09 (d, J= 12.2 Hz, 1H, H-5b), 2.25 (s, 3H, Ac-CH₃), 2.15 (s, 3H, Ac-CH₃), 2.08 (s, 3H, Ac-CH₃); ¹³C NMR (125 MHz, CDCh): 6 170.0, 169.6, 169.5, 159.5, 150.1, 143.8, 86.0, 84.1, 73.6, 71.2, 69.7, 67.9, 21.2, 20.52, 20.48.

HRMS (ESI): Expected mass [M-H]- : 512.0251; found: 512.0252.

[a]²⁰r> = -11.8 (c 0.36, CHCh)

Stereochemical Assignment for 30A

Analysis of 2D ROESY of nucleoside 30A supported the indicated stereochemistry

Also the recrystallization in acetonitrile : hexane allowed for the relative stereochemistry to be assigned using single X-ray crystallography (see X-ray structures, CCDC NO: 2333368).

Basic workup

30A 30

Following General Procedure D (work-up) with 30A (25.6 mg, 0.0500 mmol) in ammonia in methanol (7 N in methanol, 0.40 mL) afforded 30 (19.3 mg, 100%) as a white solid.

Rf = 0.58 (20% Methanol in CH2CI2)

Melting point = 221 °C

Data for 30: H NMR (500 MHz, DMSO-de): 6 8.47 (s, 1H, HetH), 5.78 (d, J = 6.6 Hz, 1H, H-l), 5.34 (dd, J= 4.9 Hz, 1H, 5-OH), 5.28 (d, J= 6.1 Hz, 1H, 2-OH), 4.99 (d, J= 5.2 Hz, 1H, 3-OH), 4.21 (m, 1H, H-2), 3.93 (dd, J= 5.2 Hz, 1H, H-3), 3.43 - 3.37 (m, 2H, H-5a and H-5b); ¹³C NMR (125 MHz, DMSO-de): 6 160.4, 150.6, 145.4, 87.0, 86.8, 74.3, 71.4, 69.6, 66.5.

HRMS (ESI): Expected mass [M-H]~: 385.9934; found: 385.9935.

[a]²⁰o = -15.8 (c 0.96, DMSO)

Stereochemical Assignment for 30

Analysis of 2D ROESY of nucleoside 30 supported the indicated stereochemistry.

Preparation of 31 - Glycosylation

14C 31A

Following General Procedure D with BSA (73.0 pL, 0.170 mmol, 3 equiv), TMSOTf (36.0 pL, 0.200 mmol, 2 equiv), and thymine (12.6 mg, 0.100 mmol, 1 equiv.) in dry MeCN (0.50 mL) then sugar 14C (33.5 mg, 0.100 mmol, 1 equiv) in 1, 2-DCE (0.50 mL) afforded 31A as a white solid (36.1 mg, 90%) after purification by flash column chromatography (3 — 6 % MeOH in CH2CI2).

R, = 0.45 (5% Methanol in CH2CI2)

Melting point = 57°C

Data for 31A: H NMR (500 MHz, CD3OD): 6 7.47 (d, J= 1.2 Hz, 1H, HetH), 5.95 (d, J= 5.3 Hz, 1H, H-l), 5.56 (dd, J= 6.3, 5.3 Hz, 1H, H-2), 5.51 (d, J= 6.4 Hz, 1H, H-3), 4.21 (s, 2H, H-5a and H- 5b), 2.13 (s, 6H, 2x Ac-CH₃), 2.07 (s, 3H, Ac-CH₃), 1.89 (d, J= 1.2 Hz, 3H); ¹³C NMR (125 MHz, CD3OD): 8 171.9, 171.3, 171.2, 166.1, 152.2, 138.3, 112.1, 89.1, 84.7, 74.5, 72.6, 68.5, 20.8, 20.33, 20.32, 12.4.

HRMS (ESI): Expected mass [M+Na⁺]: 424.1406; found: 424.1411.

[a]²⁰r> = -3.0 (c 0.46, CHCI3)

Stereochemical Assignment for 31A

Analysis of 2D ROESY of nucleoside 31A supported the indicated stereochemistry.

Basic workup

Following General Procedure D (work-up) with 31A (20.1 mg, 0.0500 mmol) in ammonia in methanol (7 N in methanol, 0.20 mL) afforded 31 (13.7 mg, 100%) as a white solid.

R, = 0.65 (20% Methanol in CH2CI2)

Melting point = 235°C

Data for 31: H NMR (500 MHz, DMSO-de) 6 7.74 (d, J = 1.1 Hz, 1H, HetH), 5.81 (d, J = 7.2 Hz, 1H, H-l), 5.18 (m, 2H), 4.97 (d, J= 5.0 Hz, 1H), 4.21 (m, 1H, H-2), 3.92 (dd, J= 5.2 Hz, 1H, H-3), 3.41 (dd, J= 11.5, 5.4 Hz, 1H, H-5a), 3.35 (dd, J = 11.5, 5.2 Hz, 1H, H-5b), 1.78 (d, J= 0.9 Hz, HetCH₃); ¹³C NMR (125 MHz, DMSO-de) 6 163.7, 151.0, 136.5, 109.4, 86.1, 86.0, 73.4, 71.4, 66.7, 12.2.

HRMS (ESI): Expected mass [M-H]‘: 274.1124; found: 274.1124.

[a]²⁰D = -7.3 (c 0.21, MeOH)

Stereochemical Assignment for 31

Analysis of 2D ROESY of nucleoside 31 supported the indicated Stereochemistry

Preparation of 4'-Vinyl Nucleoside Analogues (32)

Grignard Addition

Following the General Procedure A with vinyl magnesium bromide (0.70 M in THF, 28.5 mL, 20.0 mmol, 4 equiv) and 6 (1.17 g, 5.00 mmol, 1 equiv.) in dry CH2CI2 (15.0 mL) afforded 10D as a colorless oil (0.524 g, 40%) after purification by flash column chromatography (20 — 25% EtOAc in hexanes).

R./ = 0.43 (50% EtOAc in Hexanes)

Data for 10D: H NMR (400 MHz, CDCh): 6 6.48 (dd, J= 17.5, 11.0 Hz, 1H), 5.53 (dd, J= 17.5, 1.8 Hz, 1H), 5.33 (dd, J = 11.0, 1.7 Hz, 1H), 4.40 (d, J= 1.7 Hz, 1H), 8 3.87 (dd, J = 10.2, 4.0 Hz, 2H), 3.80 (s, 1H), 3.76 (ddd, J= 9.4, 4.6, 1.7 Hz, 1H), 3.59 (d, J= l l.l Hz, 1H), 3.47 (s, 3H), 3.55 (s, 3H) 2.66 (d, J = 4.6 Hz, 1H), 1.52 (s, 3H), 1.44 (s, 3H); ¹³C NMR (125 MHz, CDCh): 6 138.0, 114.6, 103.1, 99.3, 73.2, 73.1, 69.9, 69.4, 57.2, 55.7, 28.9, 19.1.

HRMS (ESI): Expected mass [M+Na⁺]: 285.1309; found: 285.1306.

[a]²% = -17.6 (c 1.8, CHCh)

Intramolecular Bis- Transacetalization Following General Procedure B with TMSOTf (0.728 mL, 4.00 mmol, 2 equiv.), 2,6-lutidine (0.232 mL, 2.00 mmol, 1 equiv.), and 10D (0.524 g, 2.00 mmol, 1 equiv.) in CH2CI2 (15.0 mL) afforded 13D as a colorless liquid (0.257 g, 65%) after purification by flash column chromatography (5 — 7% EtOAc in hexanes).

Ry = 0.49 (20% EtOAc in Hexanes)

Data for 13D: H NMR (400 MHz, CDCh): 'HNMR 8 6.14 (dd, J= 17.8, 11.2 Hz, 1H, 4-CHCH₂), 5.48 (m, 2H, 4-CHCH₂), 5.44 (s, 1H, H-l), 4.38 (d, J= 5.4 Hz, 1H, H-2), 4.28 (d, J= 5.4 Hz, 1H, H- 3), 3.41 (d, J= 7.1 Hz, 1H, H-5a), 3.34 (d, J= 7.1 Hz, 1H, H-5b), 1.46 (s, 3H, CH₃), 1.30 (s, 3H, CH₃); ¹³C NMR (125 MHz, CDCh): 6 128.8, 119.9, 112.6, 100.4, 86.2, 82.2, 81.2, 66.9, 26.2, 25.7.

HRMS (DART-MS): Expected mass [M+H⁺]: 199.0965; found: 199.0971.

[a]²⁰r> = -3.9 (c 0.17, CHCh)

Ring Opening-Acetylation

Following General Procedure C with TESOTf (0.141 mL, 0.625 mmol, 0.625 equiv) and 13D (0.198 g, 1.00 mmol, 1 equiv.) in Ac2O:CH2Ch (2.0 mL: 2.0 mL) afforded 14D as a yellow oil (0.241 g, 70%) after purification by flash column chromatography (15 — 20% EtOAc in hexanes).

R./ = 0.70 (50% EtOAc in Hexanes)

Data for 14D: H NMR (600 MHz, CDCh): 6 6.26 (d, J= 1.1 Hz, 1H), 5.77 (dd, J= 17.3, 11.0 Hz, 1H), 5.53 - 5.49 (m, 2H), 5.36 (dd, J= 5.2, 1.4 Hz, 1H, H-2), 5.32 - 5.30 (m, 1H), 4.23 (d, J= 11.9 Hz, 1H), 4.11 (d, J= 11.9 Hz, 1H), 2.11 (s, 3H), 2.09 (s, 3H), 2.06 (d, J= 1.8 Hz, 6H).; ¹³C NMR (125 MHz, (CDCh): 6 170.4, 169.6, 169.5, 169.3, 133.3, 117.6, 97.7, 85.9, 74.9, 71.9, 66.5, 21.2, 21.0, 20.64, 20.58.

HRMS (ESI): Expected mass [M+Na⁺]: 367.1000; found: 367.1000.

[a]²⁰r> = -25.8 (c 0.30, CHCh)

Preparation of 32 - Glycosylation Following General Procedure D with BSA (73.0 pL, 0.170 mmol, 3 equiv), TMSOTf (36.0 pL, 0.200 mmol, 2 equiv), and thymine (12.6 mg, 0.100 mmol, 1 equiv.) in dry MeCN (0.50 mL) then sugar 14D (34.4 mg, 0.100 mmol, 1 equiv) in 1, 2-DCE (0.50 mL) afforded 32A as a white solid (33.6 mg, 85%) after purification by flash column chromatography (3 — 6 % MeOH in CH2CI2).

Ry = 0.50 (5% Methanol in CH2CI2)

Melting point = 70°C

Data for 32A: H NMR (500 MHz, CD3OD): 8 7.52 (d, J= 1.1 Hz, 1H, HetH), 5.97 (d, J= 5.2 Hz, 1H, H-l), 5.91 (dd, J= 17.3, 10.9 Hz, 1H, 4-CHCH₂), 5.63 (d, J= 6.3 Hz, 1H, H-3), 5.59 - 5.49 (m, 2H, H, H-2 and 4-CHCHaHb), 5.39 (dd, J = 10.9, 1.2 Hz, 1H, 4-CHCHaHb), 4.27 (d, J= 12.0 Hz, 1H, 5-Ha), 4.18 (d, J= 12.0 Hz, 1H, 5-Hb), 2.12 (s, 3H, Ac-CH₃), 2.10 (s, 3H, Ac-CH₃), 2.04 (s, 3H, Ac-CH₃), 1.91 (d, J = 1.1 Hz, 3H, HetCH₃); ¹³C NMR (125 MHz, CD3OD): 6 171.9, 171.4, 171.1, 166.1, 152.2, 138.4, 134.0, 117.9, 112.2, 89.4, 86.8, 74.1, 72.5, 67.0, 20.8, 20.4, 20.3, 12.4.

HRMS (ESI): Expected mass [M-H]’: 409.1253; found: 409.1255.

[a]²⁰r> = -47.4 (c 1.1, CHC1₃)

Stereochemical Assignment for 32A

Analysis of 2D ROESY of nucleoside 32A supported the indicated stereochemistry.

Basic workup

32A 32

Following General Procedure D (work-up) with 32A (20.5 mg, 0.0500 mmol) in ammonia in methanol (7 N in methanol, 0.40 mL) afforded 32 (14.2 mg, 100%) as a white solid.

Ry = 0.58 (20% Methanol in CH2CI2)

Melting point = 149°C

Data for 32: H NMR (500 MHz, CD3OD) 6 7.86 (d, J= 1.2 Hz, 1H, HetH), 5.96 (m, 2H, H-l and 4-CHCHaHb), 5.43 (dd, J = 17.4, 1.8 Hz, 1H, 4-CHCHaHb), 5.23 (dd, J = 11.0, 1.8 Hz, 1H, 4- CHCHaHb), 4.34 - 4.30 (m, 2H, H-2 and H-3), 3.65 (d, J= 11.8 Hz, 1H, 5-Ha), 3.48 (d, J= 11.9 Hz, 1H, H-5b), 1.90 (d, J= 1.2 Hz, 3H, HetCH₃).; ¹³C NMR (125 MHz, CD3OD) 6 166.4, 152.8, 138.6, 136.5, 115.7, 111.7, 90.3, 89.6, 75.4, 72.9, 66.9, 12.4.

HRMS (ESI): Expected mass [M-H]': 283.0936; found: 283.0939.

[a]²⁰o = -40.2 (c 0.86, MeOH) Stereochemical Assignment for 32

Analysis of 2D ROESY of nucleoside 32 supported the indicated stereochemistry.

Preparation of 4'-Alkynyl Nucleoside Analogues (33 and 34)

IMS protected Alkynyl lithium Addition

To a solution of trimethyl silyl acetylene (2.07 mL, 15.0 mmol, 3 equiv.) in THF (0.35 M) at -78°C was added (dropwise) a solution of the n-BuLi (1.6 M in Hexane, 15.0 mmol, 3 equiv.). The reaction mixture was allowed to stir at 0°C for 2 hrs and then the solution of 6 (1.17 g, 5.00 mmol, 1 equiv.) in THF (10.0 mL) was added to it at -78°C. The reaction mixture was gradually warmed overnight to room temperature. After completion, as monitored by TLC, the reaction mixture was cooled to 0°C and was slowly quenched via dropwise addition of saturated ammonium chloride solution. The reaction mixture was filtered to remove solids under vacuum filtration. The filtrate was then diluted with EtOAc and washed three times with water. The organic layer was then separated, dried with Na2SC>4, filtered, and concentrated under reduced pressure. The crude reaction mixture was then purified with flash column chromatography (20 — 25 % of EtOAc in Hexanes) to afford a 10E as a colorless oil (1.19 g, 72%).

R = 0.6 (60% EtOAc in Hexanes)

Data for 10E: H NMR (600 MHz, CDCh): 6 4.43 (d, J= 1.8 Hz, 1H), 4.15 (s, 1H), 4.11 (ddd, J = 9.0, 4.7, 1.8 Hz, 1H), 3.89 (d, J= 11.2 Hz, 1H), 3.75 (dd, J= 14.0, 10.1 Hz, 2H), 3.53 (s, 3H), 3.50 (s, 3 H), 2.75 (d, = 4.7 Hz, 1H), 1.45 (s, 3H), 1.41 (s, 3H), 0.19 (s, 9H); ¹³C NMR (125 MHz, CDCh): 6 105.3, 103.1, 99.4, 90.5, 74.0, 72.9, 68.5, 66.8, 57.3, 55.9, 28.5, 19.5, 0.2.

HRMS (ESI): Expected mass [M+Na⁺]: 355.1547; found: 355.1545.

[a]²⁰/ = -34.8 (c 0.86, CHCh)

Intramolecular Bis- Transacetalization Following General Procedure B with TMSOTf (0.728 mL, 4.00 mmol, 2 equiv.), 2,6-lutidine (0.232 mL, 2.00 mmol, 1 equiv.), and 10E (0.665 g, 2.00 mmol, 1 equiv.) in CH2CI2 (15.0 mL) afforded 13E as colorless liquid (0.279 g, 52%) after purification by flash column chromatography (5 — 7% EtOAc in hexanes).

Ry = 0.6 (30% EtOAc in Hexanes)

Data for 10E: H NMR (400 MHz, CDCh): 6 5.40 (s, 1H, H-l), 4.35 (d, J= 5.4 Hz, 1H, H-2), 4.30 (d, J= 5.4 Hz, 1H, H-3), 3.43 (s, 2H, H-5a and H-5b), 1.50 (s, 3H, CH₃), 1.32 (s, 3H, CH₃), 0.21 (s, 9H, TMS); ¹³C NMR (125 MHz, CDCh): 6 113.0, 100.4, 96.2, 95.1, 81.9, 81.5, 79.0, 68.1, 26.2, 25.8, -0.1.

HRMS (DART-MS): Expected mass [M+H⁺]: 269.1204; found: 269.1200.

[a]²⁰/ = +32.6 (c 0.29, CHCh)

Ring Opening-Acetylation

Following General Procedure C with TESOTf (0.142 mL, 0.620 mmol, 0.625 equiv) and 13E (0.268 g, 1.00 mmol, 1 equiv.) in Ac2O:CH2Ch (2.0 mL: 2.0 mL) afforded 14E as a yellow oil (0.252 g, 61%) after purification by flash column chromatography (10 — 15% EtOAc in Hexanes).

R./ = 0.7 (50% EtOAc in Hexanes)

Data for 14E: H NMR (600 MHz, CDCh): 6 6.26 (d, J= 1.4 Hz, 1H, H-l), 5.48 (d, J= 5.0 Hz, 1H, H-3), 5.36 (dd, J= 5.0, 1.5 Hz, 1H, H-2), 4.39 (d, J= 11.9 Hz, 1H, H-5a), 4.20 (d, J= 11.9 Hz, 1H, H-5b), 2.13 (s, 3H, Ac-CH₃), 2.11 (s, 3H, Ac-CH₃), 2.10 (s, 3H, Ac-CH₃), 2.09 (s, 3H, Ac-CH₃), 0.18 (s, 9H, TMS); ¹³C NMR (125 MHz, CDCh): 6 169.9, 169.4, 169.3, 168.8, 98.4, 97.6, 94.8, 81.1, 74.5, 70.8, 66.4, 21.0, 20.8, 20.5, 20.5, -0.3.

HRMS (ESI): Expected mass [M+Na⁺]: 437.1238; found: 437.1241.

[a]²⁰o = -97.7 (c 2.07, CHCh)

Preparation of 33 - Glycosylation

Following General Procedure D with BSA (73.0 pL, 0.170 mmol, 3 equiv), TMSOTf (36.0 pL, 0.200 mmol, 2 equiv), and adenine (15.3 mg, 0.100 mmol, 1 equiv.) in dry MeCN (0.50 mL) then sugar 14E (41.4 mg, 0.100 mmol, 1 equiv) in 1, 2-DCE (0.50 mL) afforded 33A as a white solid (39.0 mg, 78%) after purification by flash column chromatography (3-4% MeOH in CH2CI2).

R, = 0.6 (10% Methanol in CH2CI2)

Melting point = 158-160°C

Data for 33A: ' H NMR (400 MHz, CDCI3): 6 7.89 (s, 1H, HetH), 6.20 (d, J= 6.1 Hz, 1H, H-l), 6.13 (bs, 2H, HetNH₂), 5.85 (dd, J= 6.1 Hz, 1H, H-2), 5.73 (d, J= 6.1 Hz, 1H, H-3), 4.46 (d, J= 12.1 Hz, 1H, H-5a), 4.38 (d, J= 12.1 Hz, 1H, H-5b), 2.17 (s, 3H, Ac-CH₃), 2.16 (s, 3H, Ac-CH₃), 2.07 (s, 3H, AC-CH₃), 0.20 (s, 9H, TMS); ¹³C NMR (125 MHz, CDCI3): 6 170.1, 169.5, 169.2, 159.3 (d, J= 211 Hz), 157.4, 157.3, 139.4, 118.5, 97.6, 96.2, 85.8, 81.2, 72.7, 70.5, 66.0, 20.9, 20.8, 20.5, -0.2; ¹⁹F NMR (376 MHz, CDC13) 8 -49.8.

HRMS (ESI): Expected mass [M+Na⁺]: 530.1478; found: 530.1487.

[a]²⁰r> = -26.0 (c 1.06, CHCI3)

Stereochemical Assignment for 33 A

Analysis of 2D ROESY of nucleoside 33A supported the indicated stereochemistry.

Following General Procedure D (work-up) with 33A (25.3 mg, 0.0500 mmol) in ammonia in methanol (7 N, 0.40 mL) afforded 33 (15.4 mg, 100%) as a white solid. The spectral data matched previous reports. ^[4]

R, = 0.5 (15% Methanol in CH2CI2) Data for 33: ¹ H NMR (600 MHz, CD₃0D): 6 8.23 (s, 1H, HetH), 5.98 (d, J= 6.3 Hz, 1H, H-l), 4.78 (dd, J= 6.0 Hz, 1H, H-2), 4.38 (d, J= 5.6 Hz, 1H, H-3), 3.87 (d, J= 12.2 Hz, 1H, H-5a), 3.75 (d, J=

12.2 Hz, 1H, H-5b), 3.10 (s, 1H, 4-CCH); ¹³C NMR (125 MHz, (CD3OD); 6 160.4 (d, J= 209 Hz),

159.2 (d, J= 20 Hz), 151.6 (d, J= 19 Hz), 141.9 (d, J= 2 Hz), 119.0 (d, J= 3 Hz), 90.7, 86.6, 80.6, 78.9, 74.8, 73.0, 67.7; ¹⁹F NMR (376 MHz, DMSO-de) 6 -52.0.

HRMS (ESI): Expected mass [M+Na⁺]: 332.0766; found: 332.0761.

Stereochemical Assignment for 33

Analysis of 2D ROESY of nucleoside 33 supported the indicated stereochemistry.

Preparation of 34 - Glycosylation

Following General Procedure D with BSA (73.0 pL, 0.170 mmol, 3 equiv), TMSOTf (36.0 pL, 0.200 mmol, 2 equiv), and cytosine (11.1 mg, 0.100 mmol, 1 equiv.) in dry MeCN (0.50 mL) then sugar 14E (41.4 mg, 0.100 mmol, 1 equiv) in 1, 2-DCE (0.50 mL) afforded 34A as a white solid (35.0 mg, 77%) after purification by flash column chromatography (3-4% MeOH in CH2CI2).

Rf = 0.5 (10% Methanol in CH2CI2)

Melting point = 102-104°C

Data for 34A: H NMR (600 MHz, CD3OD): 8 7.63 (d, J= 7.5 Hz, 1H, HetH), 6.01 (d, J= 4.6 Hz, 1H, H-l), 5.92 (d, J= 7.5 Hz, 1H, HetH), 5.58 (d, J= 6.6 Hz, 1H, H-2), 5.51 (dd, J= 5.6 Hz, 1H, H- 3), 4.44 (d, J= 11.9 Hz, 1H, H-5a), 4.28 (d, J= 11.9 Hz, 1H, H-5b), 2.12 (s, 3H, Ac-CH₃), 2.11 (s, 3H, AC-CH₃), 2.09 (s, 3H, Ac-CH₃), 0.21 (s, 9H, TMS); ¹³C NMR (125 MHz, CD3OD): 6 171.7, 171.3, 170.8, 167.8, 157.7, 143.8, 99.9, 96.8, 96.0, 91.5, 81.4, 74.0, 71.7, 66.6, 20.7, 20.6, 20.4, -0.3.

HRMS (ESI): Expected mass [M+H⁺]: 466.1640; found: 466.1639.

[a]²⁰r> = -11.7 (c 0.31, CHCI3)

Stereochemical Assignment for 34A

Analysis of 2D ROESY of nucleoside 34A supported the indicated stereochemistry.

Basic workup

Following General Procedure D (work-up) with 34A (23.2 mg, 0.0500 mmol) in ammonia in methanol (7 N, 0.40 mL) afforded 34 (13.3 mg, 100%) as a white solid. Data matched previous reports. ^[4]

R, = 0.4 (15% Methanol in CH2CI2)

Data for 34: ¹ H NMR (400 MHz, CD3OD): 6 7.89 (d, J= 7.5 Hz, 1H, HetH), 5.99 (d, J= 3.7 Hz, 1H, H-l), 5.89 (d, J= 7.5 Hz, 1H, HetH), 4.27 - 4.23 (m, 2H, H-2 and H-3), 3.81 (d, J= 12.1 Hz, 1H, H- 5a), 3.73 (d, J = 12.1 Hz, 1H, H-5b), 3.05 (s, 1H, CC-H); ¹³C NMR (125 MHz, CD3OD); 6 176.4, 167.6, 158.4, 143.5, 96.3, 92.8, 85.2, 78.9, 75.4, 71.7, 66.5.

HRMS (ESI): Expected mass [M+H⁺]: 268.0928; found: 268.0927.

Stereochemical Assignment for 34

Analysis of 2D ROESY of nucleoside 34 supported the indicated stereochemistry.

Preparation of Ribavirin Analogues (36, 37)

Preparation of 36 - Glycosylation

Following General Procedure D with BSA (73.0 pL, 0.170 mmol, 3 equiv), TMSOTf (36.0 pL, 0.200 mmol, 2 equiv), and methyl-lH-l,2,4-triazole-3-carboxylate (12.7 mg, 0.100 mmol, 1 equiv.) in dry MeCN (0.50 mL) then sugar 14 (33.2 mg, 0.100 mmol, 1 equiv) in 1, 2-DCE (0.50 mL) afforded 36A as a white solid (19.0 mg, 47%) after purification by flash column chromatography (50-60% EtOAc in Hexanes).

R, = 0.4 (5% Methanol in CH2CI2)

Melting point = 75-77°C

Data for 36A: ¹ H NMR (400 MHz, CDCI3): 8 8.38 (s, 1H, HetH), 6.04 (d, J= 4.3 Hz, 1H, H-l), 5.91 (dd, J = 5.4, 4.4 Hz, 1H, H-2), 5.62 (d, J= 5.6 Hz, 1H, H-3), 4.25 (d, J = 12.1 Hz, 1H, H-5a), 4.10 (d, J= 12.1 Hz, 1H, H-5b), 3.98 (s, 3H, HetOCH₃), 2.14 (s, 3H, Ac-CH₃), 2.12 (s, 3H, Ac-CH₃), 2.10 (s, 3H, AC-CH₃), 1.37 (s, 3H, 4-CH₃); ¹³C NMR (125 MHz, CDCh): 6 170.3, 169.3, 169.1, 159.9, 155.6, 144.5, 89.7, 86.0, 75.0, 71.4, 67.2, 52.9, 24.8, 20.8, 20.5, 19.2.

HRMS (ESI): Expected mass [M+Na⁺]: 422.1170; found: 422.1169.

[a]²⁰o = -28.9 (c 0.66, MeOH)

Stereochemical Assignment for 36A

Analysis of 2D ROESY of nucleoside 36A supported the indicated stereochemistry.

Basic workup

Following General Procedure D (work-up) with 36A (19.0 mg) in ammonia in methanol (7 N, 0.40 mL) afforded 36 (12.3 mg, 100%) as a white solid.

Rf = 0.4 (20% Methanol in CH2CI2)

Melting point = 106-108°C

Data for 36: ¹ H NMR (400 MHz, CD3OD): 6 8.75 (s, 1H, HetH), 5.89 (d, J= 5.2 Hz, 1H, H-l), 4.75 (dd, J= 5.3 Hz, 1H, H-2), 4.25 (d, J= 5.3 Hz, 1H, H-3), 3.60 (d, J= 11.8 Hz, 1H, H-5a), 3.52 (d, J= 11.8 Hz, lH, H-5b), 1.30 (s, 3H, 4-CHJ); ¹³C NMR (125 MHz, CD3OD): 6 163.3, 158.1, 146.6, 93.6, 89.9, 77.1, 73.3, 68.1, 18.7.

HRMS (ESI): Expected mass [M+Na⁺]: 281.0856; found: 281.0857. a]²⁰/ = -39.4 (c 0.78, MeOH)

Stereochemical Assignment for 36

Analysis of 2D ROESY of nucleoside 36 supported the indicated stereochemistry.

Preparation of 37 - Glycosylation

Following General Procedure D with BSA (0.422 mL, 1.73 mmol, 3 equiv), TMSOTf (0.209 mL, 1.16 mmol, 2 equiv), and methyl-lH-l,2,4-triazole-3-carboxylate (74.0 mg, 0. 0.578 mmol, 1 equiv.) in dry MeCN (5.80 mL) then 14A (0.200 g, 0.578 mmol, 1 equiv) in 1, 2-DCE (1.90 mL) afforded 37A as a white solid (938 mg, 40%) after purification by flash column chromatography (0— >10% MeOH in CH2CI2).

R, = 0.43 (5% Methanol in CH2CI2)

Melting point = 102-104°C

Data for 37A: H NMR (400 MHz, CD3CN): 8 8.49 (s, 1H, HetH), 6.10 (d, J = 4.7 Hz, 1H, H-l), 5.90, m, 1H, H-2), 5.63 (d, J = 5.8 Hz, 1H, H-3), 4.31 (d, J = 12.2 Hz, 1H, H-5a), 4.05 (d, J= 12.2 Hz, 1H, H-5b), 3.91 (s, 3H, HetOCH₃), 2.11 (s, 3H, Ac-CH₃), 2.04 (s, 3H, Ac-CH₃), 2.02 (s, 3H, Ac- CH₃), 1.90 (m, 1H, 4-CHaHbCH₃), 1.73 (m, 1H, 4-CHaHbCH₃), 0.95 (dd, J = 7.6 Hz, 3H, 4- CH₂CH₃); ¹³C NMR (125 MHz, CD₃OD): 6 172.2, 171.1, 170.9, 161.2, 156.3, 147.6, 90.3, 88.8, 75.6, 73.5, 66.2, 53.1, 26.3, 20.8, 20.3, 20.2, 7.8.

HRMS (ESI): Expected mass [M+Na⁺]: 436.1327; found: 436.1329. a]²⁰r> = -27.4 (c 1.33, MeOH)

Stereochemical Assignment for 37 A

Analysis of 2D ROESY of nucleoside 37A supported the indicated stereochemistry.

Basic workup

Following General Procedure D (work-up) with 37A (40.0 mg, 0.0970 mmol) in ammonia in methanol (7 N, 1.33 mL) afforded 37 as a white solid (26.4 mg, 100%).

R, = 0.4 (20% Methanol in CH2CI2) Melting point = 109-111°C

Data for 37: ¹ H NMR (400 MHz, CD₃OD): 6 8.72 (s, 1H, HetH), 5.84 (d, J= 6.3 Hz, 1H, H-l), 4.83 (m, 1H, H-2), 4.22 (d, J= 5.2 Hz, 1H, H-3), 3.64 (m, 2H, H-5a and H-5b), 1.81 (m, 2H, 4-CH₂CH₃), 0.96 (dd, J= 7.7 Hz, 3H, 4-CH2CH3); ¹³C NMR (125 MHz, (CD3OD): 6 163.4, 158.3, 146.7, 93.5, 91.9, 77.0, 73.7, 65.2, 25.4, 8.3.

HRMS (ESI): Expected mass [M+Na⁺]: 295.1013; found: 295.1015. a]²⁰/ = -30.7 (c 0.28, MeOH)

Stereochemical Assignment for 37

Analysis of 2D ROESY of nucleoside 37 supported the indicated stereochemistry.

Preparation of Mizoribine Analogues (38, 39)

Preparation of 38 - Glycosylation

Following General Procedure D with BSA (73.0 pL, 0.170 mmol, 3 equiv), TMSOTf (36.0 pL, 0.200 mmol, 2 equiv), and 5-hydroxy-lH-imidazole-4-carboxamide (12.7 mg, 0.100 mmol, 1 equiv) in dry MeCN (0.50 mL) then sugar 14C (33.5 mg, 0.100 mmol, 1 equiv) in 1, 2-DCE (0.50 mL) afforded a pink solid (28.1 mg, 70%) after purification by flash column chromatography (5 — 7 % MeOH in CH2CI2).

Rf = 0.43 (10% Methanol in CH2CI2)

Melting point = 81 °C

Data for 38A: H NMR (500 MHz, CDCh): 6 8.03 (s, 1H, HetH), 6.62 (s, 1H, H-l), 5.62 (dd, J = 5.9, 1.8 Hz, 1H, H-2), 5.35 (d, = 5.9 Hz, 1H, H-3), 4.33 (d, J= 12.4 Hz, 1H, H-5a), 4.06 (d, J= 12.4 Hz, 1H, H-5b), 2.18 (s, 3H, Ac-CH₃), 2.16 (s, 3H, Ac-CH₃), 2.06 (s, 3H, Ac-CH₃); ¹³C NMR (125 MHz, CDCh): 6 170.2, 169.4, 169.0, 162.3, 159.0, 125.9, 101.8, 89.9, 84.8, 75.7, 68.8, 66.8, 21.0, 20.7, 20.5.

HRMS (ESI): Expected mass [M+H⁺]: 403.1539; found: 403.1545.

[a]²⁰r> = -31.6 (c 0.20, CHCh)

Stereochemical Assignment for 38A

Analysis of 2D ROESY of nucleoside 38A supported the indicated Stereochemistry Basic workup

38A 38

Following General Procedure D (work-up) with 38A (20.1 mg, 0.0500 mmol) in ammonia in methanol (7 N in methanol, 0.20 mL) afforded 38 (13.8 mg, 100%) as a brownish solid.

Rf = 0.23 (30% Methanol in CH2CI2)

Melting point = 160°C

Data for 38: H NMR (500 MHz, CD3OD): 6 8.62 (s, 1H, HetH), 6.38 (s, 1H, H-l), 4.26 - 4.23 (m, 2H, H-2 and H-3), 3.64 (d, J= 12.0 Hz, 1H, H-5a), 3.57 (d, J= 12.0 Hz, 1H, H-5b); ¹³C NMR (125 MHz, CD3OD): 8 165.2, 160.0, 127.2, 101.3, 93.7, 89.3, 78.7, 70.5, 66.7.

HRMS (ESI): Expected mass [M+Na⁺]: 299.1041; found: 299.1041.

[a]²% = +1.13 (c 0.45, MeOH)

Stereochemical Assignment for 38

Analysis of 2D ROESY of nucleoside 38 supported the indicated stereochemistry.

Preparation of 39 - Glycosylation

Following General Procedure D with BSA (73.0 pL, 0.170 mmol, 3 equiv), TMSOTf (36.0 pL, 0.200 mmol, 2 equiv), and 5-hydroxy-lH-imidazole-4-carboxamide (12.7 mg, 0.100 mmol, 1 equiv) in dry MeCN (0.50 mL) then sugar 14B (35.8 mg, 0.100 mmol, 1 equiv) in 1, 2-DCE (0.50 mL) afforded 39A as pink solid (29.7 mg, 70%) after purification by flash column chromatography (5 — 7 % MeOH in CH2CI2). Rf = 0.43 (10% Methanol in CH2CI2)

Melting point = 101°C

Data for 39A: ¹ H NMR (500 MHz, CDCI3): 6 7.98 (s, 1H, HetH), 6.72 (d, J= 2.3 Hz, 1H, H-l), 5.90 - 5.81 (m, 1H, 4-CH2CHCH2), 5.63 (dd, J = 6.0, 2.5 Hz, 1H, H-2), 5.45 (d, J = 6.0 Hz, 1H, H-3), 5.21 - 5.17 (m, 2H, 4-CH₂CHCH₂), 4.31 (d, J= 12.5 Hz, 1H, H-5a), 4.17 (d, J= 12.5 Hz, 1H, H-5b), 2.83 (dd, J= 14.8, 6.1 Hz, 1H, 4-CHaHbCHCH₂), 2.41 (dd, J= 14.8, 8.5 Hz, 1H, 4-CHaHbCHCH₂), 2.16 (s, 3H, Ac-CH₃), 2.15 (s, 3H, Ac-CH₃), 2.07 (s, 3H, Ac-CH₃); ¹³C NMR (125 MHz, CDCI3): 6 170.1, 169.3, 169.1, 162.3, 158.9, 131.2, 126.2, 120.0, 101.9, 89.5, 85.8, 75.5, 69.3, 65.5, 37.5, 21.0, 20.7, 20.5.

HRMS (ESI): Expected mass [M+Na⁺]: 448.1327; found: 448.1326.

[a]²⁰r> = -34.0 (c 0.35, CHCI3)

Stereochemical Assignment for 39 A

Analysis of 2D ROESY of nucleoside 39A supported the indicated stereochemistry.

Basic workup

39A 39

Following General Procedure D (work-up) with 39A (21.3 mg, 0.0500 mmol) in ammonia in methanol (7 N in methanol, 0.40 mL) afforded 39 (14.9 mg, 100%) as a brownish solid.

Rf = 0.23 (30% Methanol in CH2CI2)

Melting point = 156°C

Data for 39: ¹ H NMR (500 MHz, CD3OD): 8 8.56 (s, 1H, HetH), 6.43 (d, J= 2.0 Hz, 1H, H-l), 5.98 - 5.93 (m, 1H, 4-CH₂CHCHaHb), 5.11 - 5.05 (m, 2H, 4-CH₂CHCHaHb), 4.31 - 4.29 (m, 2H, H-2 and H-3), 3.74 (d, J = 12.0 Hz, 1H, H-5a), 3.56 (d, J= 12.0 Hz, 1H, 5-Hb), 2.76 (dd, J= 14.5, 6.6 Hz, 1H, 4-CHaHbCHCHaHb), 2.39 (dd, J= 14.5, 7.9 Hz, 1H, 4-CHaHbCHCHaHb); ¹³C NMR (125 MHz, CD3OD): 6 165.3, 160.2, 134.9, 127.5, 118.1, 101.4, 93.5, 90.3, 78.8, 71.3, 64.7, 37.9.

HRMS (ESI): Expected mass [M+H⁺]: 300.1190; found: 300.1188.

[a]²% = -8.0 (c 0.57, CHCI3)

Stereochemical Assignment for 39

Analysis of 2D ROESY of nucleoside 39 supported the indicated stereochemistry. Preparation of C-linked Nucleoside 41

Ring opening-acetylation

To a solution of 13 (0.186 g, 1.00 mmol, 1 equiv.) (which was prepared by general procedure A followed by general procedure B) in CH2CI2 (0.20 M) at -5°C was added (dropwise) AC2O (0.470 mL, 5.00 mmol, 5 equiv.) and TESOTf (0.141 mL, 0.625 mmol, 0.625 equiv.). The reaction mixture was allowed to stir at -5°C for one hour. After completion, as monitored by TLC, the reaction mixture was then diluted with dichloromethane and washed three times with saturated sodium bicarbonate solution. The organic layer was then separated, dried with Na2SO4, filtered, and concentrated under reduced pressure. The crude reaction mixture was then purified with flash column chromatography (10 — 15% EtOAc in Hexanes) to afford a 40 as a colorless oil (0.198 g, 69%).

Rf = 0.65 (50% EtOAc in Hexanes)

Data for 40: 'H NMR (500 MHz, CDCh) 6 6.15 (s, 1H, H-l), 4.79 (d, J= 6.0 Hz, 1H, H-2), 4.56 (d, J = 6.0 Hz, 1H, H-3), 4.10 (d, J= 11.4 Hz, 1H, H-5a), 3.98 (d, J= 11.4 Hz, 1H, H-5b), 2.10 (s, 3H, AC-CH₃), 2.05 (s, 3H, Ac-CH₃), 1.52 (s, 3H, CH₃), 1.37 (s, 3H, CH₃), 1.35 (s, 3H, CH₃). ¹³C NMR (125 MHz, CDCh) 6 170.6, 169.7, 113.4, 101.6, 88.0, 86.4, 82.5, 67.9, 26.4, 25.2, 21.3, 21.0, 18.6.

HRMS (ESI): Expected mass [M+Na⁺]: 311.1101; found: 311.1099.

Preparation of 41 — Ni-catalyzed cross-coupling

A Schlenk tube was charged with 40 (28.8 mg, 0.100 mmol, 1 equiv.), 4-iodo anisole (46.8 mg, 0.200 mmol, 2 equiv.), Ni(acac)2 (2.5 mg, 0.010 mmol, 0.1 equiv.), Zn (13.0 mg, 0.200 mmol, 2 equiv.), Ligand (2,2':6',2"-Terpyridine, 2.3 mg, 0.01 mmol, 0.1 equiv.). Schlenk tube was evacuated and backfilled three times with Ar, and THF 0.5 mL was added with syringe. Reaction mixture was cooled down to 0 °C and TMSBr (0.0527 mL, 0.400 mmol, 4 equiv.) was added to it. After stirring for one hour at 0 °C, the reaction mixture was warm to r.t which was left for overnight. After completion of the reaction, as monitored by TLC, the reaction mixture was quenched with NaHCCh solution and extracted with EtOAc (6 mL x 3). Organic layers were dried over Na2SO4, and concentrated under reduced pressure and crude was passed through the short pad of silica which was used for the next step.

41A (16.8 mg, 0.0500 mmol) was dissolved in a solution of HC1 in methanol (3.0 M, 0.40 mL) and allowed to stir at room temperature for one hour. After completion of the reaction, as monitored by TLC, the reaction mixture was concentrated under reduced pressure. The crude reaction mixture was purified via column chromatography (10-15% EtOAc in Hexanes) to afford 41 (9.9 mg, 39%) as a as a light-yellow oil.

Ry = 0.3 (90% EtOAc in Hexanes)

Data for 41: ¹ H NMR (400 MHz, CDCh): 6 7.32 (d, J= 8.6 Hz, 2H, ArH), 6.90 (d, J= 8.7 Hz, 2H, ArH), 4.73 (d, J= 7.0 Hz, 1H, H-l), 4.23 (d, J = 4.5 Hz, 1H), 4.11 (m, 1H), 3.81 (s, 3H, ArOCH₃), 3.63 - 3.59 (m, 2H, 5-Ha and 5-Hb) 2.51 (d, J = 4.2 Hz, 1H), 2.45 (d, J= 5.9 Hz, 1H), 1.91 - 1.88 (m, 1H), 1.31 (s, 3H, 4-CH₃); ¹³C NMR (125 MHz, (CDCh); 6 159.7, 131.7, 127.7, 114.3, 85.0, 82.9, 77.8, 73.1, 68.3, 55.5, 17.4.

HRMS (ESI): Expected mass [M+Na⁺]: 277.1046; found: 277.1046.

[a]²⁰/> = -25.9 (c 0.10, CHCh)

Stereochemical Assignment for 41

Analysis of 2D ROESY of nucleoside 41 supported the indicated stereochemistry.

IV. Gram Scale Synthesis of Compound 15

6 (5.30 gm) 10 (3.72 gm, 65%)

Following the General Procedure A with methylmagnesium bromide (3 M in Et2O, 22.6 mL, 67.9 mmol, 3 equiv) and 6 (5.30 g, 22.6 mmol, 1 equiv.) in CH2CI2 (60 mL) afforded 10 as a colorless oil (3.72 g, 65%) after purification by flash column chromatography (20 — 25% EtOAc in Hexanes).

10 (3.72 gm) 13 (1.10 gm, 39%)

To a solution of 10 (3.72 g, 1 equiv.) and 2,6-lutidine (1.62 mL, 1 equiv.) in CH2CI2 (110 mL) at - 10°C was added dropwise TMSOTf (5.09 mL, 2 equiv.) in a glass syringe. The reaction mixture was allowed to stir at -10°C for 1.5 hrs and then saturated NaHCOi solution (37 mL) was slowly added at -10°C. The reaction was allowed to be stirred at room temperature for 10 minutes. The reaction mixture was diluted with CH2CI2 and washed twice with water. The organic layer was then separated, dried with Na2SO4, filtered, and concentrated under reduced pressure. The crude reaction mixture was then purified with flash column chromatography (5 — 10% EtOAc in Hexanes) to afford 13 (1.10 g, 39%).

13 (1.10 gm) 14 (1.45 gm, 74%)

Following General Procedure C with TESOTf (0.820 mL, 3.63 mmol, 0.62 equiv) and 13 (1.10 g, 5.85 mmol, 1 equiv.) in Ac2O:CH2Ch (15 mL: 15 mL) afforded 14 as a yellow oil (1.45 g, 74%) after purification by flash column chromatography (15 — 20% EtOAc in Hexanes).

14 (1 .45 gm) 15A (1.55 gm, 89%)

Following General Procedure D with BSA (3.19 mL, 13.1 mmol, 3 equiv), TMSOTf (1.58 mL, 8.74 mmol, 2 equiv) and thymine (0.580 g, 4.37 mmol, 1 equiv.) in dry MeCN (43.7 mL) then sugar 14 (1.45 g, 4.37 mmol, 1 equiv) in 1, 2-DCE (14.6 mL) afforded 15A as a white solid (1.55 g, 89%) after purification by flash column chromatography (2-3% MeOH in CH2CI2).

15A (1 .55 gm) 15 (1.05 gm, 100%)

Following General Procedure D (work-up step) with 15A (1.55 g) in ammonia in methanol (7 N, 28.0 mL) afforded 15 (1.05 g, 100%) as a white solid.

Preparation of 42 - Glycosylation

Following General Procedure D with BSA (73.0 pL, 0.170 mmol, 3 equiv), TMSOTf (36.0 pL, 0.200 mmol, 2 equiv), and adenine (11.1 mg, 0.100 mmol, 1 equiv) in dry MeCN (0.50 mL) then 14B (35.8 mg, 0.100 mmol, 1 equiv) in 1, 2-DCE (0.50 mL) afforded 42A as a white solid (36.8 mg, 90%) after purification by flash column chromatography (2 — 3 % MeOH in CH2CI2).

R, = 0.32 (5% Methanol in CH2CI2)

Data for 42A: H NMR (500 MHz, CD3OD): 6 7.66 (dd, J= 7.5, 0.7 Hz, 1H), 6.09 (d, J= 5.4 Hz, 1H), 5.93 (dd, J= 7.5, 1.2 Hz, 1H), 5.91 - 5.82 (m, 1H), 5.60 - 5.53 (m, 2H), 5.21 - 5.12 (m, 2H), 4.23 (q, J= 11.9 Hz, 2H), 2.70 - 2.65 (m, 1H), 2.47 (dd, J= 14.7, 8.2 Hz, 1H), 2.14 (s, 3H), 2.12 (s, 3H), 2.06 (s, 3H); ¹³C NMR (125 MHz, CD3OD): 6 171.8, 171.2, 171.0, 167.7, 158.0, 158.0, 142.8, 133.0, 119.7, 96.8, 89.4, 85.7, 74.8, 73.0, 67.2, 37.8, 20.7, 20.3, 20.3.

HRMS (ESI): Expected mass [M+H⁺]: 410.1558; found: 410.1562.

[a]²% = -1.80 (c 0.6, CHCI3)

Stereochemical Assignment for 42A

Analysis of 2D ROESY of nucleoside 42A supported the indicated stereochemistry.

Basic workup

Following General Procedure D (work-up) with 42A (20.4 mg, 0.0500 mmol) in ammonia in methanol (7 N in methanol, 0.40 mL) afforded 42 (14.1 mg, 100%) as a white solid.

Rf = 0.40 (10% Methanol in CH2CI2)

Data for 42: ¹ H NMR (500 MHz, CD3OD): 6 7.98 (d, J= 7.5 Hz, 2H), 5.95 - 5.88 (m, 5H), 5.06 (dd, J= 20.2, 14.2 Hz, 4H), 4.37 (dd, J= 8.5, 3.4 Hz, 2H), 4.22 - 4.14 (m, 2H), 3.61 (d, J= 11.8 Hz, 2H), 3.55 (d, J= 11.9 Hz, 2H), 2.57 (dd, J= 14.2, 6.8 Hz, 2H), 2.42 (dd, J= 14.2, 7.6 Hz, 2H); ¹³C NMR (125 MHz, CD3OD): 6 167.5, 158.8, 143.7, 135.1, 118.0, 96.2, 91.3, 89.2, 76.2, 73.0, 66.3, 38.3.

HRMS (ESI): Expected mass [M+H⁺]: 284.1241; found: 284.1237.

[a]²% = -45.96 (c 0.5, MeOH)

Stereochemical Assignment for 42

Analysis of 2D ROESY of nucleoside 42 supported the indicated stereochemistry.

Preparation of 4 '-dodecyl Nucleoside Analogues (43)

Grignard Addition

Following the General Procedure A dodecylmagnesium bromide (3.0 M in Et2O, 2.5 mL, 7.50 mmol, 3 equiv) and 6 (0.585 g, 2.50 mmol, 1 equiv.) in dry CH2CI2 (7.0 mL) afforded 10F as a colorless oil (0.303 g, 30%) after purification by flash column chromatography (20 — 25% EtOAc in hexanes).

R./ = 0.42 (50% EtOAc in Hexanes)

Data for lOF^H NMR (700 MHz, CDCI3): 6 4.38 (d, J= 1.3 Hz, 1H), 3.85 (d, J= 9.6 Hz, 1H), 3.78 (d, = 9.6 Hz, 1H), 3.74 (d, J= 11.6 Hz, 1H), 3.54 (s, 3H), 3.46 (s, 3H), 3.23 (bs, 1H), 2.70 (bs, 1H), 2.01 - 1.95 (m, 1H), 1.45 (s, 3H), 1.36 (s, 3H), 1.33 - 1.20 (m, 22H), 0.87 (d, J= 7.2 Hz, 3H); ¹³C NMR (175 MHz, CDCI3): 6 103.3, 99.0, 73.8, 72.2, 69.1, 65.9, 57.3, 55.6, 32.0, 31.3, 30.5, 29.8, 29.8, 29.8, 29.8, 29.7, 29.5, 28.8, 22.8, 22.5, 19.4, 14.2.

HRMS (DART-MS): Expected mass [M+NH₄ ⁺]: 422.3476; found: 422.3473.

[a]²⁰z) = -14.13 (c 4.0, CHCh).

Intramolecular Bis- Transacetalization

Following General Procedure B with TMSOTf (0.0728 mL, 0.40 mmol, 2 equiv.), 2,6-lutidine (0.0232 mL, 0.2 mmol, 1 equiv.), and 10F (80.9 mg, 0.2 mmol, 1 equiv.) in CH2CI2 (1.50 mL) afforded 13F as a colorless liquid (30.6 mg, 45%) after purification by flash column chromatography (5 — 7% EtOAc in hexanes).

R./ = 0.61 (20% EtOAc in Hexanes)

Data for 13F: ^XH NMR (500 MHz, CDCh): 6 4.32 (d, J= 5.5 Hz, 1H), 4.19 (d, J= 5.5 Hz, 1H), 3.28 (d, J= 7.1 Hz, 1H), 3.25 (d, J= 7.1 Hz, 1H), 2.07 - 2.01 (m, 1H), 1.89 - 1.83 (m, 1H), 1.45 (s, 3H), 1.37 - 1.33 (m, 2H), 1.32 - 1.28 (m, 8H), 1.27 (d, J= 12.1 Hz, 14H), 0.88 (t, J= 7.0 Hz, 3H); ¹³C NMR (125 MHz, CDCh): 6 112.2, 99.9, 86.7, 82.0, 79.6, 66.9, 32.0, 30.3, 29.8, 29.7, 29.6, 29.6, 29.5, 27.0, 26.2, 25.7, 24.5, 22.8, 14.2.

HRMS (DART-MS): Expected mass [M+H⁺]: 341.2686; found: 341.2687.

[a]²⁰r> = -6.14 (c 1.25, CHCh)

Ring Opening-Acetylation

Following General Procedure C with TESOTf (14.1 L, 0.0625 mmol, 0.625 equiv) and 13F (34.0 mg, 0.10 mmol, 1 equiv.) in Ac2O:CH2Ch (0.2 mL: 0.2 mL) afforded 14F as yellow oil (31.5 mg, 65%) after purification by flash column chromatography (25 — 30% EtOAc in hexanes).

R./ = 0.43 (30% EtOAc in Hexanes)

Data for 14F: ^XH NMR (700 MHz, CDCh): 6 6.17 (d, J= 1.6 Hz, 1H), 5.48 (d, J= 5.5 Hz, 1H), 5.41 (dd, J= 5.5, 1.6 Hz, 1H), 4.14 (d, J= 11.7 Hz, 1H), 4.10 (d, J= 11.7 Hz, 1H), 2.10 (s, 3H), 2.09 (s, 3H), 2.08 (s, 6H), 1.73 - 1.69 (m, 1H), 1.63 - 1.59 (m, 1H), 1.33 - 1.18 (m, 20H), 0.87 (t, J= 7.1 Hz, 3H); ¹³C NMR (175 MHz, (CDCh): 6 170.4, 169.5, 169.4, 169.3, 97.9, 86.3, 75.3, 72.5, 66.4, 32.7, 32.0, 30.3, 29.8, 29.7, 29.7, 29.7, 29.6, 29.49, 23.0, 22.8, 21.2, 20.9, 20.6, 20.5, 14.2.

HRMS (DART -MS): Expected mass [M+Na⁺]: 504.3167; found: 504.3167.

[a]²⁰r> = -23.5 (c 2.7, CHCh).

Preparation of 14 - Glycosylation

Following General Procedure D with BSA (73.0 pL, 0.170 mmol, 3 equiv), TMSOTf (36.0 pL, 0.200 mmol, 2 equiv), and cytosine (11.1 mg, 0.100 mmol, 1 equiv) in dry MeCN (0.50 mL) then sugar 14F (48.6 mg, 0.100 mmol, 1 equiv) in 1, 2-DCE (0.50 mL) afforded 43A as a white solid (41.88 mg, 78%) after purification by flash column chromatography (1 — 2 % MeOH in CH2CI2).

Rf = 0.45 (5% Methanol in CH2CI2)

Data for 43A: H NMR (700 MHz, CD3OD): 6 7.70 (d, J = 7.6 Hz, 1H), 6.06 (d, J = 52 Hz, 1H), 5.96 (d, J= 7.5 Hz, 1H), 5.57 - 5.51 (m, 2H), 4.28 (d, J= 11.9 Hz, 1H), 4.24 (d, J= 11.9 Hz, 1H), 2.13 (d, J= 7.9 Hz, 3H), 2.12 (s, 3H), 2.05 (s, 3H), 1.85 - 1.81 (m, 1H), 1.71 - 1.67 (m, 1H), 1.49 (dd, J = 10.3, 6.5 Hz, 1H), 1.38 - 1.24 (m, 19H), 0.90 (t, J = 7.1 Hz, 3H); ¹³C NMR (125 MHz, CD3OD): 6 171.9, 171.2, 171.0, 167.2, 157.5, 142.9, 96.7, 89.1, 86.6, 74.8, 73.2, 67.1, 33.0, 33.0, 31.2, 30.7, 30.7, 30.7, 30.6, 30.5, 30.4, 24.0, 23.7, 20.7, 20.3, 20.3, 14.4.

HRMS (ESI): Expected mass [M+H⁺]: 538.3123; found: 538.3121.

[a]²% = +13.4 (c 1.60, MeOH)

Stereochemical Assignment for 43 A

Analysis of 2D ROESY of nucleoside 43 A supported the indicated stereochemistry.

Deprotection of 43 A

Following General Procedure D (work-up) with 43A (26.8 mg, 0.0500 mmol) in ammonia in methanol (7 N in methanol, 0.40 mL) afforded 43 (20.5 mg, 100%) as a white solid.

R, = 0.40 (10% Methanol in CH2CI2)

Data for 43: ¹ H NMR (500 MHz, CD3OD): 6 7.97 (d, J= 7.5 Hz, 1H), 5.90 (t, J= 7.4 Hz, 2H), 4.39 - 4.32 (m, 1H), 4.16 (d, J= 5.7 Hz, 1H), 3.64 (d, J= 11.7 Hz, 1H), 3.58 (d, J= 11.7 Hz, 1H), 1.79 - 1.71 (m, 1H), 1.63 (ddd, J= 14.1, 10.9, 5.1 Hz, 1H), 1.40 - 1.37 (m, 2H), 1.31 - 1.29 (m, 18H), 0.90 (t, = 7.0 Hz, 3H); ¹³C NMR (125 MHz, CD3OD): 6 167.5, 158.9, 143.6, 96.2, 91.2, 90.1, 76.2, 73.3, 66.2, 33.3, 33.0, 31.6, 30.7, 30.7, 30.6, 30.4, 24.6, 23.7, 14.4.

HRMS (ESI): Expected mass [M+Na⁺]: 434.2625; found: 434.2624.

[a]²⁰o = -0.09 (c 1.00, MeOH)

Stereochemical Assignment for 43

Analysis of 2D ROESY of nucleoside 14 supported the indicated stereochemistry.

Also the recrystallization in MeOH : DCM allowed for the relative stereochemistry to be assigned using single X-ray crystallography (see X-ray structures, CCDC NO: 2414494).

Preparation of 4 - Cl).; Nucleoside Analogues (44-45)

Preparation of 44- Glycosylation

Following General Procedure D with BSA (73.0 pL, 0.170 mmol, 3 equiv), TMSOTf (36.0 pL, 0.200 mmol, 2 equiv), and 5-bromocytosine (19.0 mg, 0.100 mmol, 1 equiv.) in dry MeCN (0.50 mL) then sugar 14C (33.5 mg, 0.100 mmol, 1 equiv) in 1, 2-DCE (0.50 mL) afforded 44A as white solid (39.9 mg, 86%) after purification by flash column chromatography (2 — 3 % MeOH in CH2CI2). Rz = 0.57 (5% Methanol in CH2CI2)

Data for 44A: H NMR (700 MHz, CDCI3): 6 7.94 (s, 1H), 7.86 (s, 1H), 6.21 - 6.17 (m, 1H), 5.72 (s, 1H), 5.42 - 5.38 (m, 2H), 4.23 (d, J= 12.2 Hz, 1H), 4.11 (d, J= 12.2 Hz, 1H), 2.21 (s, 3H), 2.11 (s, 3H), 2.09 (s, 3H); ¹³C NMR (125 MHz, CDCI3): 6 170.0, 169.5, 169.5, 162.2, 154.3, 140.9, 88.1, 87.0, 83.8, 74.4, 70.7, 67.6, 21.09, 20.6, 20.5.

HRMS (ESI): Expected mass [M+Na⁺]: 487.0516; found: 487.0514.

[a]²⁰r> = -10.9 (c 0.50, CHCI3)

Stereochemical Assignment for 44 A

Analysis of 2D ROESY of nucleoside 44A supported the indicated stereochemistry.

Basic workup

Following General Procedure D (work-up step) with 44A (23.2 mg, 0.050 mmol) in ammonia in methanol (7 N in methanol, 0.40 mL) afforded 44 (16.9 mg, 100%) as a white solid.

R, = 0.45 (15% Methanol in CH2CI2)

Data for 44: ¹ H NMR (500 MHz, CD3OD): 6 8.51 (s, 1H), 5.89 (d, J= 4.8 Hz, 1H), 4.30 - 4.24 (m, 1H), 4.16 (d, J= 5.7 Hz, 1H), 3.59 (d, J= 11.7 Hz, 1H), 3.55 (d, J= 11.7 Hz, 1H); ¹³C NMR (125 MHz, CD3OD): 6 164.0, 157.5, 144.2, 91.8, 88.8, 88.7, 77.1, 72.2, 67.6.

HRMS (ESI): Expected mass [M+Na⁺]: 361.0197; found: 361.0195.

[a]²⁰o = -4.73 (c 0.8, MeOH)

Stereochemical Assignment for 44

Analysis of 2D ROESY of nucleoside 44 supported the indicated stereochemistry.

Preparation of 45 - Glycosylation

14C 45A

Following General Procedure D with BSA (73.0 pL, 0.170 mmol, 3 equiv), TMSOTf (36.0 pL, 0.200 mmol, 2 equiv), and 5-methylcytosine (12.5 mg, 0.100 mmol, 1 equiv.) in dry MeCN (0.50 mL) then sugar 14C (33.5 mg, 0.100 mmol, 1 equiv) in 1, 2-DCE (0.50 mL) afforded 45A as white solid (38.0 mg, 85%) after purification by flash column chromatography (2 — 3 % MeOH in CH2CI2).

Ry = 0.44 (4% Methanol in CH2CI2)

Data for 45A: ¹ H NMR (700 MHz, CDCh): 6 7.30 (d, J= 0.9 Hz, 1H), 6.26 (d, J= 5.7 Hz, 1H), 5.43 - 5.38 (m, 2H), 4.22 (d, J= 12.0 Hz, 1H), 4.13 (d, J= 11.9 Hz, 1H), 2.16 (s, 3H), 2.12 (s, 3H), 2.06 (s, 3H), 1.95 (d, J= 0.8 Hz, 3H); ¹³C NMR (175 MHz, CDCh): 6 172.6, 170.1, 169.7, 169.6, 165.5, 155.8, 138.0, 102.5, 86.5, 83.1, 73.8, 71.3, 68.0, 21.0, 20.6, 20.6, 13.5.

HRMS (ESI): Expected mass [M+Na⁺]: 423.1562; found: 423.1562.

[a]²⁰o = +18.3 (c 0.60, CHCh)

Stereochemical Assignment for 45 A

Analysis of 2D ROESY of nucleoside 45A supported the indicated stereochemistry.

Basic workup

Following General Procedure D (work-up step) with 45A (20.0 mg, 0.0500 mmol) in ammonia in methanol (7 N in methanol, 0.40 mL) afforded 45 (13.7 mg, 100%) as a white solid.

Ry = 0.55 (15% Methanol in CH2CI2)

Data for 45: ¹ H NMR (500 MHz, CD3OD): 6 7.85 (d, J= 0.9 Hz, 1H), 5.89 (d, J= 5.5 Hz, 1H), 4.32 (t, J= 5.7 Hz, 1H), 4.15 (d, J= 5.8 Hz, 1H), 3.58 (d, J= 11.8 Hz, 1H), 3.54 (d, J= 11.8 Hz, 1H), 1.96 (d, J= 0.8 Hz, 3H); ¹³C NMR (125 MHz, CD3OD): 6 167.2, 158.7, 141.0, 104.3, 91.5, 88.2, 76.4, 72.6, 68.0, 13.2.

HRMS (ESI): Expected mass [M+Na⁺]: 297.1249; found: 297.1249. [a]²⁰/ = + l . l 5 (c 0.4, MeOH)

Stereochemical Assignment for 45

Analysis of 2D ROESY of nucleoside 45 supported the indicated stereochemistry.

Preparation of 4'-Ethyl Nucleoside Analogues (46-48)

Preparation of 46 - Glycosylation

14A 46A

Following General Procedure D with BSA (73.0 pL, 0.170 mmol, 3 equiv), TMSOTf (36.0 pL, 0.200 mmol, 2 equiv), and 5-methylcytosine (12.5 mg, 0.100 mmol, 1 equiv.) in dry MeCN (0.50 mL) then sugar 14A (34.6 mg, 0.100 mmol, 1 equiv) in 1, 2-DCE (0.50 mL) afforded 46A as white solid (36.5 mg, 89%) after purification by flash column chromatography (2 — 3 % MeOH in CH2CI2).

Rz = 0.41 (4% Methanol in CH2CI2)

Data for 46A: ¹ H NMR (500 MHz, CDCh): 6 7.27 (d, J= 0.9 Hz, 1H), 6.29 (d, J= 6.7 Hz, 1H), 5.47 (d, = 6.0 Hz, 1H), 5.41 - 5.37 (m, 1H), 4.24 (d, J= 11.9 Hz, 1H), 4.17 (d, J= 11.9 Hz, lH), 2.16 (s, 3H), 2.11 (s, 3H), 2.02 (s, 3H), 1.93 (d, J = 0.8 Hz, 3H), 1.89 - 1.83 (m, 1H), 1.67 - 1.60 (m, 1H), 0.94 (t, J = 7.5 Hz, 3H); ¹³C NMR (125 MHz, CDCh): 6 170.2, 169.7, 169.6, 165.6, 155.9, 137.6, 102.8, 85.5, 84.8, 73.4, 71.7, 66.3, 25.1, 21.0, 20.6, 13.5, 7.6.

HRMS (ESI): Expected mass [M+H]⁺: 412.1714; found: 412.1716.

[a]²⁰r> = +10.47 (c 0.60, CHCh)

Stereochemical Assignment for 46A

Analysis of 2D ROESY of nucleoside 46A supported the indicated stereochemistry.

Basic workup

46A 46

Following General Procedure D (work-up) with 46A (20.5 mg, 0.050 mmol) in ammonia in methanol (7 N, 0.40 mL) afforded 46 (14.2 mg, 100%) as a white solid.

R, = 0.40 (15% Methanol in CH2CI2)

Data for 46: ¹ H NMR (500 MHz, CD3OD): 6 7.80 (d, J= 0.9 Hz, 1H), 5.88 (d, J= 6.6 Hz, 1H), 4.38 (t, J = 6.1 Hz, 1H), 4.18 (d, J = 5.7 Hz, 1H), 3.66 (d, J = 11.7 Hz, 1H), 3.59 (d, J = 11.7 Hz, 1H), 1.96 (s, 3H), 1.86 - 1.78 (m, 1H), 1.71 - 1.64 (m, 1H), 0.95 (t, J= 7.6 Hz, 3H); ¹³C NMR (125 MHz, MeOD): 6 167.2, 158.9, 141.1, 104.5, 91.2, 90.0, 76.1, 73.2, 65.8, 25.8, 13.2, 8.3.

HRMS (ESI): Expected mass [M+Na⁺]: 308.1217; found: 308.1214.

[a]²⁰r> = -11.26 (c 1.90, CHCI3)

Stereochemical Assignment for 46

Analysis of 2D ROESY of nucleoside 46 supported the indicated stereochemistry.

Preparation of 47 - Glycosylation

Following General Procedure D with BSA (73.0 pL, 0.170 mmol, 3 equiv), TMSOTf (36.0 pL, 0.200 mmol, 2 equiv), and 5-bromocytosine (19.0 mg, 0.100 mmol, 1 equiv.) in dry MeCN (0.50 mL) then sugar 10A (34.6 mg, 0.100 mmol, 1 equiv) in 1, 2-DCE (0.50 mL) afforded 47A as white solid (40.9 mg, 86%) after purification by flash column chromatography (2 — 3 % MeOH in CH2CI2).

R, = 0.38 (5% Methanol in CH2CI2)

Data for 47A: ¹ H NMR (500 MHz, CDCh): 8 7.84 (s, 1H), 6.23 (d, J= 6.0 Hz, 1H), 5.46 (d, J= 6.0 Hz, 1H), 5.39 (t, J= 6.0 Hz, 1H), 4.25 (d, J= 12.1 Hz, 1H), 4.19 (d, J= 12.1 Hz, 1H), 2.21 (s, 3H), 2.11 (s, 3H), 2.06 (s, 3H), 1.89 - 1.85 (m, 1H), 1.68 - 1.61 (m, 1H), 0.96 (t, J= 7.6 Hz, 3H); ¹³C NMR (125 MHz, CDCI3): 6 170.1, 169.6, 169.5, 162.0, 154.3, 140.7, 88.3, 86.3, 85.7, 74.1, 71.3, 66.3, 25.3, 21.1, 20.5, 20.5, 7.6. HRMS (ESI): Expected mass [M+Na]⁺: 498.0482; found: 498.0482.

[a]²⁰r> = -14.4 (c 0.45, CHCh)

Stereochemical Assignment for 47 A

Analysis of 2D ROESY of nucleoside 47 A supported the indicated stereochemistry.

Basic workup

Following General Procedure D (work-up step) with 47A (23.8 mg, 0.050 mmol) in a solution of ammonia in methanol (7 N, 0.40 mL) afforded 47 (17.5 mg, 100%) as a white solid.

Ry = 0.46 (15% Methanol in CH2CI2)

Data for 47: H NMR (500 MHz, MeOD): 6 8.44 (s, 1H), 5.90 (d, J= 6.0 Hz, 1H), 4.35 (d, J= 5.7 Hz, 1H), 4.20 (dd, J = 2.8, 1.7 Hz, 1H), 3.68 (d, J= 11.6 Hz, 1H), 3.60 (d, J= 11.6 Hz, 1H), 1.87 - 1.80 (m, 1H), 1.70 - 1.63 (m, 1H), 0.95 (t, J= 7.6 Hz, 3H); ¹³C NMR (125MHz, MeOD): 6 164.0, 157.6, 144.3, 91.4, 90.6, 89.0, 76.9, 73.0, 65.6, 25.9, 8.4.

HRMS (ESI): Expected mass [M+Na⁺]: 372.0166; found: 372.0172.

[a]²⁰/ = -1.73 (c 1.9, MeOH)

Stereochemical Assignment for 47

Analysis of 2D ROESY of nucleoside 47 supported the indicated stereochemistry.

Preparation of 48 - Glycosylation

Following General Procedure D with BSA (73.0 pL, 0.170 mmol, 3 equiv), TMSOTf (36.0 pL, 0.200 mmol, 2 equiv), and 5-azacytosine (11.2 mg, 0.100 mmol, 1 equiv.) in dry MeCN (0.50 mL) then sugar 10A (34.6 mg, 0.100 mmol, 1 equiv) in 1, 2-DCE (0.50 mL) afforded 48A as white solid (35.8 mg, 90%) after purification by flash column chromatography (2 — 3 % MeOH in CH2CI2). Ry = 0.45 (5% Methanol in CH2CI2)

Data for 48A: ¹ H NMR (500 MHz, CDCI3): 6 6 8.23 (s, 1H), 7.39 (s, 1H), 6.07 - 6.01 (m, 2H), 5.56

- 5.50 (m, 2H), 4.26 - 4.17 (m, 2H), 2.13 (s, 3H), 2.12 (s, 3H), 2.06 (s, 3H), 1.91 - 1.83 (m, 1H), 1.70

- 1.62 (m, 1H), 0.96 (t, J= 7.5 Hz, 3H); ¹³C NMR (125 MHz, CDCI3): 6 170.3, 169.6, 169.5, 165.9, 155.7, 153.3, 86.8, 86.1, 74.0, 71.5, 65.8, 25.1, 20.9, 20.5, 20.5, 7.6.

HRMS (ESI): Expected mass [M+Na⁺]: 421.1330; found: 421.1329.

[a]²⁰/> = +16.2 (c 1.77, CHCh)

Stereochemical Assignment for 48A

Analysis of 2D ROESY of nucleoside 48A supported the indicated stereochemistry.

Basic workup

Following General Procedure D (work-up step) with 48A (19.9 mg, 0.050 mmol) in a solution of ammonia in methanol (7 N, 0.40 mL) afforded 48 (13.6 mg, 100%) as a white solid.

Ry = 0.46 (20% Methanol in CH2CI2)

Data for 48: H NMR (500 MHz, CD3OD): 6 8.62 (s, 1H), 5.78 (d, J= 6.2 Hz, 1H), 4.51 (t, J= 5.9 Hz, 1H), 4.22 (d, J= 5.7 Hz, 1H), 3.67 (d, J = i i.JRz, 1H), 3.59 (d, J= 11.7 Hz, 1H), 1.87 - 1.80 (m, 1H), 1.72 - 1.65 (m, 1H), 0.95 (t, J= 7.6 Hz, 3H); ¹³C NMR (125 MHz, CD3OD): 6 167.7, 158.5, 156.7, 91.5, 90.9, 76.2, 73.1, 65.6, 25.8, 8.3.

HRMS (ESI): Expected mass [M+Na⁺]: 295.1013; found: 295.1012.

[a]²⁰o = +17.05 (c 0.75, MeOH)

Stereochemical Assignment for 48

Analysis of 2D ROESY of nucleoside 48 supported the indicated stereochemistry.

Also the recrystallization in MeOH : DCM allowed for the relative stereochemistry to be assigned using single X-ray crystallography (see X-ray structures, CCDC NO: 2414493).

Preparation of 4'-Methyl Nucleoside Analogues (49-51)

Preparation of 49 - Glycosylation

Following General Procedure D with BSA (73.0 pL, 0.300 mmol, 3 equiv), TMSOTf (36.0 pL, 0.200 mmol, 2 equiv), and 5-azacytosine (12.5 mg, 0.100 mmol, 1 equiv.) in dry MeCN (0.50 mL) then sugar 14 (33.2 mg, 0.100 mmol, 1 equiv) in 1, 2-DCE (0.50 mL) afforded 49A as white solid (32.2 mg, 84%) after purification by flash column chromatography (2 — 3 % MeOH in CH2CI2).

R, = 0.48 (5% Methanol in CH2CI2)

Data for 49A: H NMR (700 MHz, CD3OD): 6 8.30 (s, 1H), 5.80 (d, J= 3.8 Hz, 1H), 5.66 (dd, J = 6.5, 3.8 Hz, 1H), 5.59 (d, J= 6.5 Hz, 1H), 4.26 (d, J= 11.9 Hz, 1H), 4.15 (d, J= 11.9 Hz, 1H), 2.11 (s, 3H), 2.10 (s, 3H), 2.09 (s, 3H), 1.37 (s, 3H); ¹³C NMR (175 MHz, CD3OD): 6 172.6, 171.4, 171.1, 167.8, 158.3, 155.6, 91.4, 85.5, 75.2, 72.3, 68.1, 20.6, 20.4, 20.2, 18.6.

HRMS (ESI): Expected mass [M+Na⁺]: 407.1173; found: 407.1172.

[a]²⁰z) = +19.39 (c 1.9, CHCh)

Stereochemical Assignment for 49 A

Analysis of 2D ROESY of nucleoside 49 A supported the indicated stereochemistry.

Basic workup

Following General Procedure D (work-up step) with 49A (19.2 mg, 0.050 mmol) in a solution of ammonia in methanol (7 N, 0.40 mL) afforded 49 (12.9 mg, 100%) as a white solid.

R, = 0.40 (20% Methanol in CH2CI2)

Data for 49: ¹ H NMR (500 MHz, CD3OD): 6 8.67 (s, 1H), 5.80 (d, J= 5.0 Hz, 1H), 4.46 - 4.42 (m, 1H), 4.20 (d, J= 5.8 Hz, 1H), 3.59 (d, J= 11.8 Hz, 1H), 3.55 (d, J= 11.8 Hz, 1H), 1.24 (s, 3H); ¹³C NMR (125 MHz, CD3OD): 6 167.7, 158.3, 156.6, 91.8, 89.1, 76.5, 72.4, 6.8, 18.6.

HRMS (ESI): Expected mass [M+Na⁺]: 281.0856; found: 281.0856. [a]²⁰o = +1.66 (c 0.35, MeOH)

Stereochemical Assignment for 49

Analysis of 2D ROESY of nucleoside 49 supported the indicated stereochemistry.

Preparation of 50 - Glycosylation

Following General Procedure D with BSA (73.0 pL, 0.300 mmol, 3 equiv), TMSOTf (36.0 pL, 0.200 mmol, 2 equiv), and 5-bromocytosine (19.0 mg, 0.100 mmol, 1 equiv.) in dry MeCN (0.50 mL) then sugar 14 (33.2 mg, 0.100 mmol, 1 equiv) in 1, 2-DCE (0.50 mL) afforded 50A as white solid (41.1 mg, 89%) after purification by flash column chromatography (2 — 3 % MeOH in CH2CI2).

Rz = 0.57 (5% Methanol in CH2CI2)

Data for 50A: H NMR (500 MHz, CD3OD): 8 8.02 (s, 1H), 5.98 (d, J= 4.4 Hz, 1H), 5.52 (dt, J = 13.8, 5.4 Hz, 2H), 4.25 (d, J = 12.0 Hz, 1H), 4.19 (d, J = 12.0 Hz, 1H), 2.16 (s, 3H), 2.11 (s, 3H), 2.08 (s, 3H), 1.36 (s, 3H); ¹³C NMR (125 MHz, CD3OD): 6 171.8, 171.2, 171.2, 164.2, 156.7, 143.4, 90.3, 89.2, 85.4, 75.4, 72.2, 68.4, 20.8, 20.3, 20.3, 18.8.

HRMS (ESI): Expected mass [M+Na⁺]: 484.0326; found: 484.0330.

[a]²⁰r> = -39.96 (c 1.17, CHCh)

Stereochemical Assignment for 50A

Analysis of 2D ROESY of nucleoside 50A supported the indicated stereochemistry.

Basic workup Following General Procedure D (work-up step) with 50A (23.1 mg, 0.050 mmol) in a solution of ammonia in methanol (7 N, 0.40 mL) afforded 50 (16.8 mg, 100%) as a white solid.

Ry = 0.45 (15% Methanol in CH2CI2)

Data for 50: H NMR (500 MHz, CD3OD): 6 8.50 (s, 1H), 5.90 (d, J= 4.9 Hz, 1H), 4.31 - 4.26 (m, 1H), 4.17 (d, J = 5.7 Hz, 1H), 3.60 (d, J= 11.7 Hz, 1H), 3.56 (d, J= 11.7 Hz, 1H), 1.24 (s, 3H); ¹³C NMR (125MHz, CD3OD): 6 164.0, 157.5, 144.2, 91.7, 88.8, 88.8, 77.1, 72.2, 67.6, 18.8.

HRMS (ESI): Expected mass [M+Na⁺]: 358.0009; found: 358.0007.

[a]²⁰/ = -4.36 (c 2.07, MeOH)

Stereochemical Assignment for 50

Analysis of 2D ROESY of nucleoside 50 supported the indicated stereochemistry.

Preparation of 51 - Glycosylation

Following General Procedure D with BSA (73.0 pL, 0.300 mmol, 3 equiv), TMSOTf (36.0 pL, 0.200 mmol, 2 equiv), and 5-methylcytosine (12.5 mg, 0.100 mmol, 1 equiv.) in dry MeCN (0.50 mL) then sugar 14 (33.2 mg, 0.100 mmol, 1 equiv) in 1, 2-DCE (0.50 mL) afforded 51A as white solid (35.7 mg, 94%) after purification by flash column chromatography (1 — 2 % MeOH in CH2CI2).

Ry = 0.38 (4% Methanol in CH2CI2)

Data for 51A: ¹ H NMR (500 MHz, CDCI3): 6 7.27 (s, 1H), 6.21 (d, J= 5.1 Hz, 1H), 5.43 - 5.37 (m, 2H), 4.21 (d, J= 11.9 Hz, 1H), 4.12 (d, J= 11.9 Hz, 1H), 2.14 (s, 3H), 2.11 (s, 3H), 2.05 (s, 3H), 1.93 (s, 3H), 1.32 (s, 3H); ¹³C NMR (125 MHz, CDCI3): 6 170.1, 169.6, 169.5, 165.5, 155.6, 137.8, 102.8, 86.6, 83.2, 73.8, 71.2, 67.9, 20.9, 20.6, 20.5, 18.7, 13.5.

HRMS (ESI): Expected mass [M+Na⁺]: 420.1377; found: 420.1376.

[a]²⁰r> = +18.35 (c 3.1, CHCI3)

Stereochemical Assignment for 51 A

Analysis of 2D ROESY of nucleoside 51A supported the indicated stereochemistry.

Basic workup

Following General Procedure D (work-up step) with 51A (19.8 mg, 0.050 mmol) in a solution of ammonia in methanol (7 N, 0.40 mL) afforded 51 (13.5 mg, 100%) as a white solid.

Rf = 0.45 (15% Methanol in CH2CI2)

Data for 51: ¹ H NMR (500 MHz, CD3OD): 8 77.85 (d, J= 0.9 Hz, 1H), 5.94 - 5.86 (m, 1H), 4.36 - 4.28 (m, 1H), 4.16 (d, J= 5.8 Hz, 1H), 3.59 (d, J= 11.8 Hz, 1H), 3.55 (d, J= 11.8 Hz, 1H), 1.95 (d, = 0.9 Hz, 3H), 1.23 (s, 3H); ¹³C NMR (125 MHz, CD3OD): 6 167.2, 158.7, 140.9, 104.4, 91.3, 88.3, 76.4, 72.6, 68.0, 18.7, 13.2.

HRMS (ESI): Expected mass [M+H]⁺: 272.1241; found: 272.1241.

[a]²⁰/ = 3.90 (c 0.20, CHCh)

Stereochemical Assignment for 51

Analysis of 2D ROESY of nucleoside 51 supported the indicated stereochemistry.

Preparation of 4'-Phenyl Nucleoside Analogues (52)

Grignard Addition

Following the General Procedure A with phenyl magnesium bromide (3 M in Et20, 2.5 mL, 7.50 mmol, 3 equiv), and 6 (0.585 g, 2.5 mmol, 1 equiv) in dry CH2CI2 (7.0 mL) afforded 10G as a colorless oil (0.240 g, 30%) after purification by flash column chromatography (30 — 35% EtOAc in hexanes).

R./ = 0.40 (40% EtOAc in Hexanes)

Data for 10G: H NMR (600 MHz, CDCh): 6 7.83 (dd, J= 8.4, 1.2 Hz, 2H), 7.35 (t, J= 7.6 Hz, 2H), 7.29 - 7.28 (m, 1H), 4.35 (d, J= 1.8 Hz, 1H), 4.13 (d, = 9.6 Hz, 1H), 4.03 (s, 1H), 4.01 (d, J= 11.6 Hz, 1H), 3.78 (d, J = 11.6 Hz, 1H), 3.53 (s, 3H), 3.49 (d, J= 9.6 Hz, 1H), 3.35 (s, 3H), 2.54 (d, J = 2.7 Hz, 1H), 1.59 (s, 3H), 1.54 (s, 3H); ¹³C NMR (125 MHz, CDCh): 6 143.0, 127.9, 127.2, 126.3, 103.2, 99.8, 74.3, 72.4, 70.6, 70.3, 57.2, 55.9, 28.0, 19.7.

HRMS ((DART-MS): Expected mass [M+NH₄]⁺: 330.1911; found: 330.1911. [a]²⁰/> = -9.37 (c 0.38, CHCh)

Intramolecular Bis- Transacetalization

Following General Procedure B with TMSOTf (0.072 mL, 0.40 mmol, 2 equiv.), 2,6-lutidine (0.0232 mL, 0.20 mmol, 1 equiv.), and 10G (0.062 g, 0.20 mmol, 1 equiv.) in CH2CI2 (1.50 mL) afforded 13G as colorless liquid (0.030 g, 62%) after purification by flash column chromatography (5 — 7% EtOAc in hexanes).

R./ = 0.55 (20% EtOAc in Hexanes)

Data for 13G: H NMR (600 MHz, CDCh): 6 7.41 (d, J= 4.4 Hz, 4H), 7.37 (dt, J= 8.4, 4.1 Hz, 1H), 5.57 (s, 1H), 4.52 (d, J= 5.4 Hz, 1H), 4.49 (d, J= 5.4 Hz, 1H), 3.72 (d, J= 7.0 Hz, 1H), 3.43 (d, J= 7.0 Hz, 1H), 1.45 (s, 3H), 1.29 (s, 3H); ¹³C NMR (125 MHz, CDCh): 6 133.4, 128.6, 128.5, 126.3, 112.5, 100.5, 87.6, 82.4, 81.5, 68.6, 26.2, 25.6.

HRMS (DART-MS): Expected mass [M+NH4]⁺: 266.1387; found: 266.1387.

[a]²⁰o = +9.39 (c 0.69, CHCh)

Ring Opening-Acetylation

Following General Procedure C with TESOTf (14.1 |1L, 0.0625 mmol, 0.625 equiv) and 13G (24.8 mg, 0.10 mmol, 1 equiv.) in Ac2O:CH2Ch (0.2 mL: 0.2 mL) afforded 14G as yellow oil (26.0 mg, 66%) after purification by flash column chromatography (15 — 20% EtOAc in hexanes).

R./ = 0.55 (50% EtOAc in Hexanes)

Data for 14G: ¹ H NMR (400 MHz, CDCh): 6 7.36 - 7.33 (m, 3H), 7.33 - 7.27 (m, 2H), 6.46 (d, J= 2.8 Hz, 1H), 5.78 (d, J= 5.1 Hz, 1H), 5.54 (dd, J= 5.1, 2.9 Hz, 1H), 4.49 (d, J= 12.2 Hz, 1H), 4.41 (d, J = 12.2 Hz, 1H), 2.16 (s, 3H), 2.08 (s, 3H), 1.91 (s, 3H), 1.77 (s, 3H); ¹³C NMR (125 MHz, CDCh): 6 170.3, 169.5, 169.4, 136.9, 128.0, 127.9, 126.1, 98.0, 88.7, 75.2, 72.5, 67.8, 21.2, 21.0, 20.3, 20.3.

HRMS (ESI): Expected mass [M+Na⁺]: 417.1156; found: 417.1154.

[_a]2o_n = .49 22 (c 0.49, CHCh) Preparation of 52 - Glycosylation

14G 52A

Following General Procedure D with B SA (36.5 pL, 0.150 mmol, 3 equiv), TMSOTf (18.0 pL, 0.100 mmol, 2 equiv), and adenine (6.3 mg, 0.05 mmol, 1 equiv) in dry MeCN (0.25 mL) then sugar 14G (19.7 mg, 0.05 mmol, 1 equiv) in 1, 2-DCE (0.25 mL) afforded 52A as a white solid (317.9 mg, 78%) after purification by flash column chromatography (2 — 3 % MeOH in CH2CI2).

Ry = 0.40 (4% Methanol in CH2CI2)

Data for 52A: H NMR (600 MHz, CDCh): 6 8.89 (s, 1H), 7.51 (d, J= 1.2 Hz, 1H), 7.39 - 7.29 (m, 5H), 6.46 (d, J= 8.1 Hz, 1H), 5.87 (d, J= 5.2 Hz, 1H), 5.60 (dd, J= 8.1, 5.2 Hz, 1H), 4.72 (d, J = 12.4 Hz, 1H), 4.14 (d, J= 12.4 Hz, 1H), 2.24 (s, 3H), 1.99 (s, 3H), 1.99 (d, J = 1.1 Hz, 3H), 1.72 (s, 3H); ¹³C NMR (125 MHz, CDCh): 6 169.8, 169.7, 169.4, 163.3, 150.9, 136.39, 134.4, 128.5, 128.5, 125.5, 112.4, 87.9, 84.4, 72.4, 72.2, 69.2, 21.0, 20.4, 20.2, 12.9.

HRMS (ESI): Expected mass [M+Na⁺]: 483.1374; found: 483.1371.

[a]²⁰o = -60.9 (c 0.48, CHCh)

Stereochemical Assignment for 52A

Analysis of 2D ROESY of nucleoside 52A supported the indicated stereochemistry.

Basic workup

Following General Procedure D (work-up) with 52A (23.0 mg, 0.0500 mmol) in a solution of ammonia in methanol (7 N in methanol, 0.40 mL) afforded 52 (16.7 mg, 100%) as a white solid.

Ry = 0.46 (15% Methanol in CH2CI2)

Data for 52: H NMR (600 MHz, CD3OD): 6 8.06 (s, 1H), 7.42 (dd, J= 8.3, 1.1 Hz, 2H), 7.33 (t, J = 7.6 Hz, 2H), 7.23 (dd, J= 10.7, 4.0 Hz, 1H), 6.12 (d, J= 7.4 Hz, 1H), 4.62 (dd, J= 7.4, 5.3 Hz, 1H), 4.51 (d, J= 5.3 Hz, 1H), 3.91 (d, J= 11.9 Hz, 1H), 3.56 (d, J= 11.9 Hz, 1H), 1.93 (s, 3H); ¹³C NMR (125 MHz, CD3OD): 6 166.3, 153.1, 140.7, 138.7, 128.7, 128.0, 127.2, 111.8, 92.3, 89.0, 75.5, 74.3, 69.8, 12.4.

HRMS (ESI): Expected mass [M+Na⁺]: 357.1057; found: 357.1055.

[a]²⁰o = -22.52 (c 0.61, DMSO)

Stereochemical Assignment for 52

Analysis of 2D ROESY of nucleoside 52 supported the indicated stereochemistry.

Preparation of 4'-Benzyl Nucleoside Analogues (53-57)

Grignard Addition

Following the General Procedure A with benzyl magnesium bromide (1 M in Et2O, 15.0 mL, 15.0 mmol, 3 equiv), and 6 (1.17 g, 5.00 mmol, 1 equiv) in dry CH2CI2 (15.0 mL) afforded 10H as a colorless oil (0.798 g, 49%) after purification by flash column chromatography (20 — 25% EtOAc in hexanes).

Ry = 0.55 (50% EtOAc in Hexanes)

Data for 10H: H NMR (400 MHz, CDCh): 6 7.34 - 7.26 (m, 5H), 4.44 (d, J= 1.7 Hz, 1H), 3.99 (dd, J= 9.5, 1.6 Hz, 1H), 3.93 (d, J= 9.5 Hz, 1H), 3.57 (s, 3H), 3.50 (s, 3H), 3.37 (d, J= 6.9 Hz, 2H), 3.29 (d, J= 13.5 Hz, 1H), 2.88 (d, J= 13.5 Hz, 1H), 1.48 (s, 6H); ¹³C NMR (125 MHz, CDCh): 6 137.0, 131.0, 128.1, 126.3, 103.3, 99.2, 73.7, 72.4, 69.0, 65.0, 57.3, 55.7, 37.3, 29.4, 19.2.

HRMS (ESI): Expected mass [M+Na⁺]: 349.1622; found: 349.1622.

[a]²⁰r> = -14.4 (c 1.73, CHCh)

Intramolecular Bis- Transacetalization

Following General Procedure B with TMSOTf (0.728 mL, 4.00 mmol, 2 equiv.), 2,6-lutidine (0.232 mL, 2.00 mmol, 1 equiv.), and 10H (0.652 g, 2.00 mmol, 1 equiv.) in CH2CI2 (15.0 mL) afforded 13H as colorless liquid (0.267 g, 51%) after purification by flash column chromatography (5 — 7% EtOAc in hexanes). R./ = 0.50 (20% EtOAc in Hexanes)

Data for 13H: H NMR (600 MHz, CDCh): 6 7.35 - 7.26 (m, 5H), 5.41 (s, 1H), 4.32 (d, J= 5.5 Hz, 1H), 4.14 (d, J= 5.5 Hz, 1H), 3.45 (d, J= 13.5 Hz, 1H), 3.31 (d, J = 1.3 Hz, 1H), 3.17 (d, J= 13.5 Hz, 1H), 3.06 (d, J= 7.3 Hz, 1H), 1.56 (s, 3H), 1.35 (s, 3H); ¹³C NMR (125 MHz, CDCh): 6 135.7, 129.7, 128.7, 127.0, 112.4, 100.2, 86.6, 82.0, 79.4, 66.6, 33.5, 26.3, 25.8.

HRMS (DART-MS): Expected mass [M+NH₄]⁺: 263.1278.; found: 263.1276.

[a]²⁰/ = +28.04 (c 1.05, CHCh)

Ring Opening-Acetylation

Following General Procedure C with TESOTf (0.141 mL, 0.625 mmol, 0.625 equiv) and 13H (262.3 mg, 1.00 mmol, 1 equiv.) in Ac2O:CH2Ch (2.0 mL: 2.0 mL) afforded 14H as yellow oil (273.6 mg, 67%) after purification by flash column chromatography (15 — 20% EtOAc in hexanes).

R./ = 0.57 (40% EtOAc in Hexanes)

Data for 14H: H NMR (600 MHz, CDCh): 6 7.29 - 7.26 (m, 2H), 7.24 -7.23(m, 3H), 6.30 (d, J = 1.5 Hz, 1H), 5.59 (d, J= 5.3 Hz, 1H), 5.44 (dd, J = 5.3, 1.6 Hz, 1H), 3.98 (d, J= 11.6 Hz, 1H), 3.92 (d, J= 11.6 Hz, 1H), 3.16 (d, J= 14.4 Hz, 1H), 2.86 (d, J= 14.4 Hz, 1H), 2.14 (s, 3H), 2.10 (s, 3H), 2.07 (s, 3H), 2.07 (s, 3H); ¹³C NMR (125 MHz, CDCh): 6 170.2, 169.4, 169.3, 169.2, 135.2, 130.8, 128.3, 127.0, 97.7, 85.3, 75.2, 72.6, 66.0, 38.7, 21.2, 20.9, 20.6, 20.6.

HRMS (ESI): Expected mass [M+Na⁺]: 431.1313; found: 431.1316.

[a]²⁰o = -64.9 (c 0.82, CHCh).

Preparation of 53 - Glycosylation

Following General Procedure D with BSA (73.0 pL, 0.170 mmol, 3 equiv), TMSOTf (36.0 pL, 0.200 mmol, 2 equiv), and thymine (12.6 mg, 0.100 mmol, 1 equiv) in dry MeCN (0.50 mL) then sugar 14H (40.8 mg, 0.100 mmol, 1 equiv) in 1, 2-DCE (0.50 mL) afforded 53A as a white solid (35.5 mg, 75%) after purification by flash column chromatography (2 — 3 % MeOH in CH2CI2). Ry = 0.28 (5% Methanol in CH2CI2)

Data for 53A: ¹ H NMR (600 MHz, CDCh): 6 8.87 (s, 1H), 7.34 - 7.26 (m, 3H), 7.24 (d, J= 1.3 Hz, 1H), 7.24 - 7.21 (m, 2H), 6.37 (d, J= 6.8 Hz, 1H), 5.57 (d, J= 6.0 Hz, 1H), 5.49 (t, J= 6.4 Hz, 1H), 4.20 (d, J= 12.1 Hz, 1H), 3.90 (d, J= 12.1 Hz, 1H), 3.28 (d, J= 14.6 Hz, 1H), 2.80 (d, J= 14.6 Hz, 1H), 2.23 (s, 3H), 2.17 (s, 3H), 2.11 (s, 3H), 1.93 (s, 3H); ¹³C NMR (125 MHz, CDCh): 6 169.8, 169.6, 169.5 163.3, 150.6, 134.6, 134.5, 130.5, 128.6, 127.3, 112.2, 85.1, 84.6, 72.7, 71.8, 66.5, 38.2, 20.9, 20.6, 20.5, 12.8.

HRMS (ESI): Expected mass [M+Na⁺]: 497.1531; found: 497.1532.

[a]²⁰o = -54.6 (c 0.57, CHCh)

Stereochemical Assignment for 53 A

Analysis of 2D ROESY of nucleoside 53A supported the indicated stereochemistry.

Basic workup

Following General Procedure D (work-up) with 53A (23.9 mg, 0.0500 mmol) in a solution of ammonia in methanol (7 N in methanol, 0.40 mL) afforded 53 (17.4 mg, 100%) as a white solid.

Ry = 0.43 (15% Methanol in CH2CI2)

Data for 53: H NMR (600 MHz, CD3OD): 6 6 7.80 (d, J = 1.1 Hz, 1H), 7.28 (d, J = 7.1 Hz, 2H), 7.22 (t, J= 7.5 Hz, 2H), 7.16 (t, J= 7.3 Hz, 1H), 6.09 (d, J= 6.7 Hz, 1H), 4.44 - 4.38 (m, 1H), 4.26 (d, J= 5.6 Hz, 1H), 3.47 (d, J= 11.6 Hz, 1H), 3.35 (d, J= 11.6 Hz, 1H), 3.16 (d, J= 14.3 Hz, 1H), 2.89 (d, J= 14.3 Hz, 1H), 1.85 (s, 3H); ¹³C NMR (125 MHz, CD3OD): 6 166.3, 153.0, 138.6, 138.5, 131.7, 128.9, 127.2, 111.7, 89.2, 89.1, 75.5, 73.3, 66.0, 39.0, 12.3.

HRMS (ESI): Expected mass [M+Na⁺]: 357.1057; found: 357.1055.

[a]²⁰n = -100.9 (c 0.68, MeOH)

Stereochemical Assignment for 53

Analysis of 2D ROESY of nucleoside 53 supported the indicated stereochemistry.

Preparation of 54 - Glycosylation

14H 54A

Following General Procedure D with BSA (73.0 pL, 0.170 mmol, 3 equiv), TMSOTf (36.0 pL, 0.200 mmol, 2 equiv), and 5-chlorouracil (14.6 mg, 0.100 mmol, 1 equiv) in dry MeCN (0.50 mL) then sugar 14H (40.8 mg, 0.100 mmol, 1 equiv) in 1, 2-DCE (0.50 mL) afforded 54A as a white solid (39.5 mg, 80%) after purification by flash column chromatography (2 — 3 % MeOH in CH2CI2).

R, = 0.35 (5% Methanol in CH2CI2)

Data for 54A: H NMR (600 MHz, CDCh): 6 8.95 (s, 1H), 7.75 (s, 1H), 7.33 - 7.26 (m, 3H), 7.21 (d, J= 6.9 Hz, 2H), 6.31 (d, J= 6.1 Hz, 1H), 5.55 (d, J= 6.1 Hz, 1H), 5.46 (t, J= 6.1 Hz, 1H), 4.24 (d, J= 12.3 Hz, 1H), 3.88 (d, J= 12.3 Hz, 1H), 3.28 (d, J= 14.7 Hz, 1H), 2.77 (d, J= 14.7 Hz, 1H), 2.21 (s, 3H), 2.18 (s, 3H), 2.10 (s, 3H); ¹³C NMR (125 MHz, CDCh): 6 169.8, 169.6, 169.5, 158.3, 149.5, 135.9, 134.2, 130.5, 128.7, 127.5, 110.3, 86.1, 85.5, 73.4, 71.6, 66.6, 38.5, 21.0, 20.6, 20.5.

HRMS (ESI): Expected mass [M+Na]⁺ : 517.0984; found: 517.0981.

[a]²% = -51.5 (c 0.29, CHCh)

Stereochemical Assignment for 54A

Analysis of 2D ROESY of nucleoside 54A supported the indicated stereochemistry.

Basic workup

Following General Procedure D (work-up) with 54A (24.7 mg, 0.0500 mmol) in ammonia in methanol (7 N in methanol, 0.40 mL) afforded 54 (18.4 mg, 100%) as a white solid.

R, = 0.45 (15% Methanol in CH2CI2) Data for 54: H NMR (500 MHz, CD3OD): 6 8.38 (s, 1H), 7.28 (d, J= 7.1 Hz, 2H), 7.23 (t, J= 7.4 Hz, 2H), 7.19 - 7.14 (m, 1H), 6.10 (d, J= 6.3 Hz, 1H), 4.41 (t, J= 5.9 Hz, 1H), 4.26 (d, J= 5.6 Hz, 1H), 3.49 (d, J= 11.6 Hz, 1H), 3.38 (d, J= 11.5 Hz, 1H), 3.18 (d, J = 14.3 Hz, 1H), 2.90 (d, J= 14.3 Hz, 1H); ¹³C NMR (125 MHz, CD3OD): 6 161.4, 151.9, 139.7, 138.4, 131.6, 128.9, 127.2, 109.6, 89.8, 89.7, 76.3, 73.3, 66.0, 39.2.

HRMS (ESI): Expected mass [M+Na]⁺: 391.0667; found: 391.0665.

[a]²⁰o = -68.4 (c 0.63, MeOH)

Stereochemical Assignment for 54

Analysis of 2D ROESY of nucleoside 54 supported the indicated stereochemistry.

Preparation of 55 - Glycosylation

Following General Procedure D with BSA (73.0 pL, 0.170 mmol, 3 equiv), TMSOTf (36.0 pL, 0.200 mmol, 2 equiv), and 5-bromouracil (19.0 mg, 0.100 mmol, 1 equiv) in dry MeCN (0.50 mL) then sugar 14H (40.8 mg, 0.100 mmol, 1 equiv) in 1, 2-DCE (0.50 mL) afforded 55A as white solid (38.8 mg, 72%) after purification by flash column chromatography (2 — 3 % MeOH in CH2CI2).

Ry = 0.35 (5% Methanol in CH2CI2)

Data for 55A: H NMR (500 MHz, CD3OD): 8 76 9.17 (s, 1H), 7.89 (s, 1H), 7.36 - 7.30 (m, 3H), 7.27 - 7.22 (m, 2H), 6.36 (d, J= 6.2 Hz, 1H), 5.59 (d, J = 6.1 Hz, 1H), 5.51 (t, J = 6.1 Hz, 1H), 4.29 (d, J= 12.2 Hz, 1H), 3.92 (d, J= 12.3 Hz, 1H), 3.33 (d, J= 14.7 Hz, 1H), 2.81 (d, J= 14.7 Hz, 1H), 2.25 (s, 3H), 2.24 (s, 3H), 2.15 (s, 3H); ¹³C NMR (125 MHz, CD3OD): 6 169.8, 169.6, 169.5, 158.4, 149.8, 138.5, 134.3, 130.5, 128.7, 127.5, 98.1, 86.0, 85.5, 73.4, 71.6, 66.7, 38.4, 21.0, 20.6, 20.5. HRMS (ESI): Expected mass [M+Na⁺]: 561.0479; found: 561.0482.

[a]²⁰r> = -61.2 (c 0.51, CHCI3)

Stereochemical Assignment for 55A

Analysis of 2D ROESY of nucleoside 55A supported the indicated stereochemistry.

Basic workup

Following General Procedure D (work-up) with 55A (26.9 mg, 0.0500 mmol) in ammonia in methanol (7 N in methanol, 0.40 mL) afforded 55 (20.6 mg, 100%) as a white solid.

Rf = 0.45 (15% Methanol in CH2CI2)

Data for 55 ' H NMR (600 MHz, CD3OD): 6 8.49 (s, 1H), 7.28 (d, J= 7.3 Hz, 2H), 7.23 (dd, J= 7.8, 7.3 Hz, 2H), 7.17 (t, J = 7.3 Hz, 1H), 6.09 (d, J = 6.2 Hz, 1H), 4.41 (t, J = 5.9 Hz, 1H), 4.26 (d, J = 5.5 Hz, 1H), 3.49 (d, J= 11.5 Hz, 1H), 3.38 (d, J= 11.6 Hz, 1H), 3.18 (d, J= 14.3 Hz, 1H), 2.90 (d, J= 14.3 Hz, 1H); ¹³C NMR (125 MHz, CD3OD): 6 161.5, 152.1, 142.4, 138.4, 131.6, 128.9, 127.2, 97.4, 89.8, 89.8, 76.3, 73.3, 66.0, 39.2.

HRMS (ESI): Expected mass [M+H⁺]: 371.1214; found: 371.1214.

[a]²% = -56.6 (c 1.06, MeOH)

Stereochemical Assignment for 55

Analysis of 2D ROESY of nucleoside 55 supported the indicated stereochemistry.

Preparation of 56 - Glycosylation

14H 56A

Following General Procedure D with BSA (73.0 pL, 0.170 mmol, 3 equiv), TMSOTf (36.0 pL, 0.200 mmol, 2 equiv), and 5-fluorouracil (13.0 mg, 0.100 mmol, 1 equiv) in dry MeCN (0.50 mL) then sugar 14H (40.8 mg, 0.100 mmol, 1 equiv) in 1, 2-DCE (0.50 mL) afforded 56A as a white solid (35.3 mg, 74%) after purification by flash column chromatography (2 — 3 % MeOH in CH2CI2).

Rf = 0.40 (5% Methanol in CH2CI2)

Data for 56A: H NMR (500 MHz, CDCh): 6 9.24 (d, J= 4.2 Hz, 1H), 7.60 (d, J= 6.1 Hz, 1H), 7.32 - 7.25 (m, 3H), 7.22 - 7.19 (m, 2H), 6.32 (dd, J= 6.3, 1.5 Hz, 1H), 5.53 (d, J= 6.0 Hz, 1H), 5.45 (t, = 6.1 Hz, 1H), 4.23 (d, J= 12.2 Hz, 1H), 3.87 (d, J= 12.2 Hz, 1H), 3.28 (d, J= 14.7 Hz, 1H), 2.77 (d, J= 14.7 Hz, 1H), 2.21 (s, 3H), 2.15 (s, 3H), 2.10 (s, 3H); ¹³C NMR (175 MHz, CDCI3): 6 169.80, 169.65, 169.52, 156.45 (d, J= 27.1 Hz), 149.28, 141.07 (d, J= 239.5 Hz), 134.32, 130.50, 128.70, 127.48, 123.19 (d, J= 34.6 Hz), 85.91, 85.38, 73.14, 71.67, 66.65, 38.48, 20.92, 20.66, 20.54.

HRMS (DART-MS): Expected mass [M+NH₄]⁺: 479.1460; found: 479.1459.

[a]²⁰o = -45.29 (c 0.62, CHCh).

Stereochemical Assignment for 56A

Analysis of 2D ROESY of nucleoside 56A supported the indicated stereochemistry.

Basic workup

Following General Procedure D (work-up) with 56A (23.9 mg, 0.0500 mmol) in ammonia in methanol (7 N in methanol, 0.40 mL) afforded 56 (17.6 mg, 100%) as a white solid.

Rf = 0.40 (15% Methanol in CH2CI2)

Data for 56: ¹ H NMR (600 MHz, CD₃OD): 6 8.22 (d, J= 6.9 Hz, 1H), 7.29 - 7.26 (m, 2H), 7.23 (dd, J = 10.2, 4.8 Hz, 2H), 7.17 (dd, J= 8.2, 6.3 Hz, 1H), 6.10 (dd, J= 6.4, 1.7 Hz, 1H), 4.39 (t, J= 6.0 Hz, 1H), 4.25 (d, J= 5.6 Hz, 1H), 3.49 (d, J= 11.6 Hz, 1H), 3.37 (d, J= 11.6 Hz, 1H), 3.17 (d, J = 14.3 Hz, 1H), 2.89 (d, J = 14.3 Hz, 1H); ¹³C NMR (125 MHz, CD3OD): 6 159.9 (d, J = 25.8 Hz), 151.8, 141.9 (d, J= 233.4 Hz), 138.5, 131.6, 128.9, 127.2, 126.4 (d, J= 34.9 Hz), 89.6, 89.5, 76.1, 73.3, 66.0, 39.2.

HRMS (ESI): Expected mass [M-H]~: 375.0963; found: 375.0958.

[a]²⁰r> = -82.0 (c 1.11, MeOH)

Stereochemical Assignment for 56

Analysis of 2D ROESY of nucleoside 56 supported the indicated stereochemistry.

Preparation of 57 - Glycosylation 5-trifluoromethyluracil

OAc OAc 0°C to 60°C

14H 57A

Following General Procedure D with BSA (73.0 pL, 0.170 mmol, 3 equiv), TMSOTf (36.0 pL, 0.200 mmol, 2 equiv), and 5-(Trifluoromethyl)uracil (18.0 mg, 0.100 mmol, 1 equiv.) in dry MeCN (0.50 mL) then sugar 14H (40.8 mg, 0.100 mmol, 1 equiv) in 1, 2-DCE (0.50 mL) afforded 57A as a white solid (41.1 mg, 78%) after purification by flash column chromatography (2 — 3 % MeOH in CH2CI2).

Rf = 0.40 (5% Methanol in CH2CI2)

Data for 57A: H NMR (500 MHz, CDCI3): 6 9.01 (s, 1H), 8.02 (s, 1H), 7.34 - 7.27 (m, 3H), 7.21 (d, J= 6.7 Hz, 2H), 6.29 (d, J= 6.1 Hz, 1H), 5.54 (d, J= 6.1 Hz, 1H), 5.48 (t, J= 6.1 Hz, 1H), 4.32 (d, J= 12.4 Hz, 1H), 3.84 (d, J= 12.4 Hz, 1H), 3.29 (d, J= 14.7 Hz, 1H), 2.79 (d, J= 14.7 Hz, 1H), 2.21 (s, 3H), 2.11 (s, 3H), 2.11 (s, 3H); ¹³C NMR (125 MHz, CD3OD): 66 169.9, 169.6, 169.4, 157.8, 149.4, 140.1 (q, J= 6.3 Hz), 134.1, 130.4, 128.7, 127.5, 121.7 (q, J = 270.3 Hz), 106.5 (q, J = 33.4 Hz), 86.5, 85.9, 73.5, 71.5, 66.6, 38.5, 20.6, 20.6, 20.5.

HRMS (ESI): Expected mass [M+Na⁺]: 551.1248; found: 551.1240.

[a]²⁰r> = -53.1 (c 0.59, CHCI3)

Stereochemical Assignment for 57 A

Analysis of 2D ROESY of nucleoside 57A supported the indicated stereochemistry.

Preparation of 4'-iso propyl Nucleoside Analogues (58)

Grignard Addition

Following the General Procedure A isopropyl magnesium bromide (1.3 M in Et2O, 11.5 mL, 15.0 mmol, 3 equiv) and 6 (1.17 g, 5.00 mmol, 1 equiv.) in dry CH2CI2 (15.0 mL) afforded 101 as a colorless oil (0.158 g, 11%) after purification by flash column chromatography (20 — 25% EtOAc in hexanes).

R./ = 0.50 (60% EtOAc in Hexanes)

Data for 101: H NMR (600 MHz, CDCI3): 6 4.43 (d, J= 1.5 Hz, 1H), 4.00 - 3.95 (m, 1H), 3.92 - 3.85 (m, 2H), 3.54 (s, 3H), 3.47 (s, 3H), 2.96 (s, 1H), 2.73 (d, J= 4.6 Hz, 1H), 2.18 - 2.10 (m, 1H), 1.44 (s, 3H), 1.40 (d, = 5.9 Hz, 1H), 1.33 (s, 3H), 1.16 (d, J= 7.0 Hz, 3H), 1.03 (d, J = 6.8 Hz, 3H); ¹³C NMR (125 MHz, CDCh): 6 103.3, 99.3, 74.4, 71.9, 70.6, 64.7, 57.3, 55.7, 32.3, 27.3, 20.2, 18.7, 17.4.

HRMS (ESI): Expected mass [M+Na⁺]: 301.1622; found: 301.1620.

[a]²⁰r> = -3.72 (c 2.04, CHCh)

Intramolecular Bis- Transacetalization

Following General Procedure B with TMSOTf (0.182 mL, 1.00 mmol, 2 equiv.), 2,6-lutidine (0.058 mL, 0.50 mmol, 1 equiv.), and 101 (0.126 g, 0.50 mmol, 1 equiv.) in CH2CI2 (3.80 mL) afforded 131 as colorless liquid (0.0378 g, 39%) after purification by flash column chromatography (5 — 7% EtOAc in hexanes).

Ry = 0.45 (30% EtOAc in Hexanes)

Data for 131: H NMR (600 MHz, CDCh): 6 5.35 (s, 1H), 4.33 (d, J= 5.5 Hz, 1H), 4.29 (d, J= 5.5 Hz, 1H), 3.34 (d, J= 7.2 Hz, 1H), 3.18 (d, J= 7.2 Hz, 1H), 2.55 - 2.48 (m, 1H), 1.45 (s, 3H), 1.30 (s, 3H), 1.08 (d, = 7.0 Hz, 3H), 1.03 (d, J = 7.1 Hz, 3H); ¹³C NMR (125 MHz, CDCh): 6 112.3, 100.0, 89.6, 82.1, 79.5, 63.4, 26.1, 25.6, 25.6, 18.3, 16.8.

HRMS (DART -MS): Expected mass [M+H⁺]: 215.1278; found: 215.1275.

[a]²⁰z) = -l 1.6 (c 0.36, CHCh)

Ring Opening-Acetylation

Following General Procedure C with TESOTf (28.2 |1L, 0.125 mmol, 0.625 equiv) and 131 (42.8 mg, 0.20 mmol, 1 equiv.) in Ac2O:CH2Ch (0.4 mL: 0.4 mL) afforded 141 as yellow oil (45.3 mg, 63%) after purification by flash column chromatography (25 — 30% EtOAc in hexanes).

R./ = 0.60 (50% EtOAc in Hexanes)

Data for 141 (mixture of a, anomers): ¹ H NMR (600 MHz, CDCh): 6 6.36 (d, J= 4.8 Hz), 6.20 (d, J= 3.6 Hz), 5.61 (dd, J= 5.4, 3.6 Hz), 5.55 - 5.52 (m), 5.49 (d, J= 5.9 Hz), 4.28 (d, J= 12.1 Hz), 4.12 (dt, J= 14.3, 7.8 Hz), 2.37 (dt, J= 14.0, 7.0 Hz), 2.28 (dt, J= 14.1, 7.1 Hz), 2.15 (s), 2.13 (s), 2.13 (s), 2.12 (s), 2.08 (s), 2.08 (s), 2.06 (s), 2.02 (s), 1.04 (t, J= 6.9 Hz), 0.89 (t, J= 7.2 Hz); ¹³C NMR (125 MHz, CDCh): 6 170.5, 170.4, 169.9, 169.8, 169.8, 169.6, 169.3, 169.3, 98.5, 94.0, 89.5, 89.2, 75.5, 73.2, 71.3, 70.7, 64.8, 64.7, 32.0, 31.6, 21.3, 21.2, 21.1, 21.1, 20.7, 20.6, 20.5, 20.4, 18.3, 17.8, 17.1, 17.1.

HRMS (ESI): Expected mass [M+Na⁺]: 383.1313; found: 383.1315.

[a]²⁰o = +37.39 (c 0.88, CHCh)

Preparation of 58 - Glycosylation

Following General Procedure D with BSA (73.0 pL, 0.170 mmol, 3 equiv), TMSOTf (36.0 pL, 0.200 mmol, 2 equiv), and adenine (13.5 mg, 0.100 mmol, 1 equiv) in dry MeCN (0.50 mL) then sugar 141 (36.0 mg, 0.100 mmol, 1 equiv) in 1, 2-DCE (0.50 mL) afforded 58A as a white solid (25.3 mg, 82%) after purification by flash column chromatography (2 — 3 % MeOH in CH2CI2).

Rf = 0.38 (5% Methanol in CH2CI2)

Data for 58A: H NMR (700 MHz, CDCh): 6 8.36 (s, 1H), 8.09 (s, 1H), 6.27 (d, J = 8.3 Hz, 1H), 6.16 (dd, J= 8.3, 5.0 Hz, 1H), 6.02 (bs, 2H), 5.65 (d, J= 5.0 Hz, 1H), 4.44 (d, J= 12.2 Hz, 1H), 4.23 (d, J= 12.2 Hz, 1H), 2.43-2.39 (m, 1H), 2.23 (s, 3H), 2.21 (s, 3H), 1.95 (s, 3H), 1.05 (d, J= 7.0 Hz, 3H), 0.92 (d, J = 6.9 Hz, 3H); ¹³C NMR (175 MHz, CDCh): 6 170.3, 169.5, 169.4, 155.0, 152.2, 150.3, 138.8, 119.9, 88.4, 83.9, 73.7, 72.7, 65.1, 31.8, 21.2, 20.7, 20.4, 17.9, 16.9.

HRMS (ESI): Expected mass [M+Na⁺]: 458.1646; found: 458.1640.

[a]²% = -0.28 (c 0.50, CHCh)

Stereochemical Assignment for 58A

Analysis of 2D ROESY of nucleoside 58A supported the indicated stereochemistry.

Basic workup

Following General Procedure D (work-up) with 58A (21.7 mg, 0.0500 mmol) in a solution of ammonia in methanol (7 N in methanol, 0.40 mL) afforded 58 (15.4 mg, 100%) as a white solid.

Ry = 0.43 (15% Methanol in CH2CI2)

Data for 58: ^XH NMR (500 MHz, CD3OD): 6 8.24 (s, 1H), 8.18 (s, 1H), 5.84 (d, J= 8.1 Hz, 1H), 5.05 (dd, J= 8.1, 5.1 Hz, 1H), 4.27 (d, J = 5.1 Hz, 1H), 3.80 (d, J = 12.3 Hz, 1H), 3.72 (d, J= 12.3 Hz, 1H), 2.43 -2.38 (m, 1H), 1.05 (d, J = 6.9 Hz, 3H), 0.99 (d, J= 7.1 Hz, 3H); ¹³C NMR (125 MHz, CD3OD): 6 157.7, 153.2, 149.7, 142.6, 121.3, 93.6, 91.0, 75.4, 74.2, 64.2, 32.3, 18.9, 17.4.

HRMS (ESI): Expected mass [M+Na⁺]: 332.1329; found: 332.1335.

[a]²⁰o = -23.3 (c 0.49, MeOH)

Stereochemical Assignment for 58

Analysis of 2D ROESY of nucleoside 58 supported the indicated stereochemistry.

Preparation of 4 '-hexynyl derived acetal (13 J)

Grignard Addition

To a solution of w-Hexyne (1.70 mL, 15.0 mmol, 3 equiv.) in THF (0.20 M) at -78°C was added (dropwise) a solution of the w-BuLi (1.6 M in Hexane, 15.0 mmol, 3 equiv.). The reaction mixture was allowed to stir at 0°C for 2 hrs and then the solution of 6 (1.17 g, 5.00 mmol, 1 equiv.) in THF (10.0 mL) was added to it at -78°C. The reaction mixture was gradually warmed overnight to room temperature. After completion, as monitored by TLC, the reaction mixture was cooled to 0°C and was slowly quenched via dropwise addition of saturated ammonium chloride solution. The reaction mixture was filtered to remove solids under vacuum filtration. The filtrate was then diluted with EtOAc and washed three times with water. The organic layer was then separated, dried with Na2SC>4, filtered, and concentrated under reduced pressure. The crude reaction mixture was then purified with flash column chromatography (15 — ► 25 % of EtOAc in Hexanes) to afford a 10 J as a colorless oil (0.379 g, 24%).

R/ = 0.50 (50% EtOAc in Hexanes)

Data for 10J: H NMR (600 MHz, CDCh): 6 4.43 (d, J= 1.8 Hz, 1H), 4.12 (d, J= 9.4 Hz, 1H), 4.05 (s, 1H), 3.86 (d, J= l l.l Hz, 1H), 3.77 (d, J= 11.2 Hz, 1H), 3.73 (d, J= 9.0 Hz, 1H), 3.54 (s, 3H), 3.49 (s, 3H), 2.72 (s, 1H), 2.27 (t, J = 7.1 Hz, 2H), 1.57 - 1.49 (m, 4H), 1.45 (s, 3H), 1.42 (s, 3H), 0.92 (d, J = 13 Hz, 3H); ¹³C NMR (125 MHz, CDCh): 6 103.1, 99.3, 86.5, 79.6, 74.0, 73.1, 68.8, 66.4, 57.2, 55.8, 30.9, 28.5, 22.0, 19.4, 18.7, 13.7.

HRMS (ESI): Expected mass [M+Na⁺]: 339.1778; found: 339.1777.

[a]²⁰o = -24.6 (c 0.32, CHCh)

Intramolecular Bis- Transacetalization

Following General Procedure B with TMSOTf (0.364 mL, 2.00 mmol, 2 equiv.), 2,6-lutidine (0.116 mL, 1.00 mmol, 1 equiv.), and 10J (0.316 g, 1.00 mmol, 1 equiv.) in CH2CI2 (7.60 mL) afforded 13J as colorless liquid (0.078 g, 31%) after purification by flash column chromatography (5 — 7% EtOAc in hexanes). / = 0.60 (30% EtOAc in Hexanes)

Data for 13J: ¹ H NMR (600 MHz, CDCh): 6 5.39 (s, 1H), 4.34 (d, J= 5.4 Hz, 1H), 4.27 (d, J= 5.4 Hz, 1H),

3.42 - 3.37 (m, 2H), 2.32 - 2.39 (m, 2H), 1.55 - 1.52 (m, 2H), 1.50 (s, 3H), 1.43 - 1.39 (m, 2H), 1.31 (s, 3H), 0.91 (t, J= 13 Hz, 3H); ¹³C NMR (125 MHz, CDCh): 6 112.8, 100.2, 91.5, 81.8, 81.5, 79.0, 70.5, 68.1, 30.4, 26.1, 25.7, 22.0, 18.8, 13.7.

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[58] The following references are indicative of the level of skill of one skilled in the art, and the entire contents of each are incorporated herein by reference, for all purposes, where permitted.

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Supplementary References - 1 ( II. General Procedures)

1. Grondal, C., Enders, D. A Direct Organocatalytic Entry to Selectively Protected Aldopentoses and Derivatives. Adv. Synth. Catal. 349, 694 - 702 (2007).

2. Waga, T., Nishizaki, T., Miyakawa, I., Ohrui, H., Meguro. H. Synthesis of 4'-C- Methylnucleosides. Biosci. Biotechnol. Biochem. 57, 1433 - 1438 (1993).

3. Weber, E., Cording-Ley, M. G., Burnie, A. J., Brown, W., Darapaneni, C. M., Paladino, M. (PrimeFour Therapeutics) WO 2023/192499 41, 2023.

4. R. F. Schinazi, F. Amblard, C. Gavegnano, B. Cox, S. Mengshetti (Emory University) WO 2019/133712 Al, 2019.

5. Caravano, A., Sinay, P., Vincent, S. P. 1,4-Anhydrogalactopyraose is not an intermediate of the mutase catalyzed UDP-galactopyranose/furanose interconversion. Bioorg. Med. Chem. Lett.16, 1123- 1125 (2006).

6. Nokami, T., Werz, D. B., Seeberger, P. H. Synthesis and Reactions of 1,4-Anhydrogalactopyranose and 1,4-Anhydroarabinose - Steric and Electronic Limitations. Helv. Chim. Acta. 88, 2823 - 2831 (2005).

7. Boyer, S. EL, Ugarkar, B. G., Erion, M. D. Stereoselective synthesis of 4-C-methyl-2,3,5-tri-O- benzyl-d-ribofuranose and 4-C-methyl-2,3,5-tri-O-benzyl-l-lyxofuranose. Tetrahedron Lett. 44, 4109-4112 (2003). Supplementary References -

Nuligonda, T., Kumar, G., Wang, J. W., Rajapaksha, D., Elayan, I. A., Demir, R., Meanwell, N. J., Wang, S. F., Mahal, L. K., Brown, A., Meanwell, M. W. An enantioselective and modular platform for C4'-modified nucleoside analogue synthesis enabled by intramolecular trans-acetalizations. Nature Communications, 15, 1 - 7 (2024). https://doi.org/10.1038/s41467-024-5152Q-5 (including Supplementary Information)

Claims

1. A method of synthesizing a modified nucleoside, comprising the steps of providing a chiral intermediate scaffold molecule where a ketone and a dialkyl acetal provide two points for structural diversification while simultaneously serving to enable ribose ring formation, with an intramolecular trans-acetalization cascade reaction to construct the modified ribose core, followed by glycosylation to produce a C4' -modi Tied nucleoside.

2. A method of synthesizing a modified nucleoside, comprising the steps of:

(a) reacting a chiral intermediate comprising an aldol adduct comprising a ketone moiety, a dialkyl acetal moiety and a protecting group with a nucleophilic organometallic compoundcomprising a modifier R to produce a syn-diol intermediate bearing the modifier R;

(b) selectively deprotecting the dialkyl acetal moiety and trans-acetalizing the syn-diol intermediate (10) to produce a ribose intermediate;

(c) peracylating the ribose intermediate to produce peracyl ribose; and

(d) glycosylating the peracetal ribose with a nucleobase to produce a nucleoside bearing the modifier at position C4’; wherein the modifier R can be any desired or suitable modification, the nucleobase can be any natural or modified purine or pyrimidine base, and the glycoslyation step results in a C-linked or an N-linked nucleoside.

3. The method of claim 1 which further comprises a first step comprising reacting an aldehyde with a ketone, with a D-proline or an L-proline catalyst, to produce the chiral intermediate.

4. The method of claim 2 or 3, wherein the dialkyl acetal moiety is dimethyl acetal, diethyl acetal or diisopropyl acetal.

5. The method of any one of claims 2-4, wherein the protecting group is an acetonide moiety.

6. The method of claim 5, wherein the chiral intermediate is:

7. The method of any one of claims 2-6, wherein the the nucleophilic organometallic compound is a Grignard reagent or an organolithium reagent.

8. The method of claim 7 wherein the the nucleophilic organometallic compound is a Grignard reagent, which is R-Mg-X where R is the modifier and X is a halogen, such as F, Cl, Br, or I, preferably Br.

9. The method of any one of claims 2-8, wherein step (b) is performed reacting the syn-diol intermediate with lutidine, preferably 2,6-lutidine, and trimethylsilyl trifluoromethanesulfonate (TMSOTf).

10. The method of claim 9 wherein the molar ratio of TMSOTf to lutidine is between about 1 :1 to about 3:1 and/or the reaction temperature is between about -30° C and about 0° C, preferably 2:1 and/or -10° C.

11. The method of claim 9 or 10, wherein the reaction of step (b) is quenched with water or an alkaline solution, such as a saturated sodium bicarbonate solution.

12. The method of any one of claims 2-11, wherein in step (c), the ribose intermediate is peracetylated to produce peracetyl ribose.

13. The method of any one of claims 2-12, wherein R is a saturated or unsaturated aliphatic substituent, which is straight, branched or cyclic, such as alkyl or cycloalkyl having between 1 and 16 carbon atoms, or R comprises a substituted or unsubstituted aromatic ring such substituted or unsubstituted aryl, heteroaryl, or benzyl.

14. The method of claim 13, wherein R is or comprises methyl, ethyl, propyl, isopropyl, vinyl, hexynyl, dodecyl, cyclopropyl, deuteromethyl, phenyl, or benzyl.

15. The method of any one of claims 2-14, wherein the nucleobase selected from the group consisting of methyl-lH-l,2,4-triazole-3-carboxylate, 5-hydroxy-lH-imidazole-4-carboxamide, purine, 6-chloro-purine, uracil, 5-fluoro-uracil, 5-bromo-uracil, 5-chloro-uracil, 5-iodo-uracil, 5- trifluoromethyl-uracil, cytosine, 5-bromo-cytosine, 5-methyl-cytosine, 5-aza-cytosine, adenine, 2- fluoro-adenine, 2-chloro-adenine, 6-methoxy-adenine, and thymine.

16. A compound selected from the group consisting of: