Synthesis of 2,3-dideoxy-2-fluoro-2,3-endo-methylene-and 2,3-dideoxy-2-fluoro-3-c-hydroxymethyl-2,3-endo-methylene-pentofuranoses and their use in the preparation of conformationally locked bicyclic nucleosides

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Article

Synthesis of 2,3-Dideoxy-2-fluoro-2,3-endo-methylene- and 2,3-Dideoxy-2-

fluoro-3-C-hydroxymethyl-2,3-endo-methylene-pentofuranoses and Their

Use in the Preparation of Conformationally Locked Bicyclic Nucleosides

Rob Clarkson, Zofia Komsta, BENJAMIN A MAYES, Adel Moussa, Montserrat Shelbourne,

Alistair Stewart, Andrew J Tyrrell, Laura L Wallis, and Alexander C. Weymouth-Wilson

J. Org. Chem., Just Accepted Manuscript • Publication Date (Web): 23 Jan 2015

Downloaded from http://pubs.acs.org on January 27, 2015

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Synthesis of 2,3-Dideoxy-2-fluoro-2,3-endo-methylene- and 2,3-

Dideoxy-2-fluoro-3-C-hydroxymethyl-2,3-endo-methylene-

pentofuranoses and Their Use in the Preparation of Conformationally

Locked Bicyclic Nucleosides

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†

,†

,‡

‡

†

Rob Clarkson, Zofia Komsta,* Benjamin A. Mayes,* Adel Moussa, Montserrat Shelbourne,

‡

†

Alistair Stewart, Andrew J. Tyrrell, Laura L. Wallis, Alexander C. Weymouth-Wilson

†

Dextra, Science and Technology Centre, Earley Gate, Whiteknights Road, Reading, RG6 6BZ,

UK and Idenix Pharmaceuticals, 320 Bent Street, Cambridge, MA 02141, USA.

‡

E-mail: zofia.komsta@dextrauk.com (Z. Komsta), mayes.ben@idenix.com (B. Mayes)

ABSTRACT

Construction of protected 2,3-dideoxy-2-fluoro-2,3-endo-methylene-pentofuranoses from D-

glyceraldehyde and 2,3-dideoxy-2-fluoro-3-C-hydroxymethyl-2,3-endo-methylene-pentofuranoses

from D-isoascorbic acid, via Simmons-Smith type stereoselective cyclopropanations on the

respective fluoroallyl alcohols is described. Synthesis of the corresponding conformationally

locked sugar modified uridine and guanosine nucleosides was achieved via Vorbrüggen or

Mitsunobu methodologies. Stereochemical confirmation of the novel nucleosides was performed on

the basis of 2D NOESY NMR experiments. Analysis of 2′,3′-dideoxy-2′-fluoro-3′-C-

hydroxymethyl-2′,3′-endo-methylene-uridine by X-ray crystallography yielded the principal

o

conformational parameters and indicated the furanoid ring adopted a E/ T , East pucker. The

1

uridine and guanosine nucleosides were found to be inactive in the hepatitis C virus (HCV) cell-

based replicon assay, which was corroborated on examination of the corresponding nucleoside

triphosphates against the HCV NS5B polymerase.

INTRODUCTION

Structurally diverse sugar modified nucleosides continue to be investigated for their

pharmacological potential, upon which ostensibly minor compositional or configurational

alterations may have a substantial impact. The substitution of hydrogen or a hydroxyl group in the

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furanose ring by fluorine is one such modification which, either alone or in combination with other

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features, has provided a range of biologically active nucleosides, exemplified by the naturally

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occurring 4′-fluorinated antibiotic nucleocidin 1, the 2′-fluorinated antitumor agents gemcitabine

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and clofarabine 3 and by the first direct acting antiviral approved for the treatment of chronic

hepatitis C virus (HCV) infection, sofosbuvir 4, a monophosphate prodrug of 2′-deoxy-2′-fluoro-2′-

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methyluridine (Figure 1A).

Sugar modification by introduction of a fused ring system to provide conformational restriction is

well established: the resultant ‘locked’ bicyclic nucleosides have been exploited in the context of

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antisense oligonucleotides, short interfering RNA and in the design of potential antiviral agents.

With respect to herpes simplex virus (HSV) and human immunodeficiency virus (HIV), Marquez

et al. have described the divergent preferences, South and North respectively (as described by the

pseudorotational cycle), of various host and viral kinases versus cellular DNA- and viral RNA-

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dependent DNA polymerases (reverse transcriptase) with regard to sugar ring conformation. Based

on structural similarities between the catalytic domains of the HIV reverse transcriptase and HCV

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RNA-dependent RNA polymerase (RdRp), these sugar conformational preferences are anticipated

to be broadly similar. Interestingly, in the case of HCV, examples of both Southern-type bicyclic

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systems, bearing a 3′,4′-oxetane (C2′-endo) 5, and Northern-type 2′-O,4′-C-methylene bridged

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ribonucleoside analogs (C3′-endo) 6, have demonstrated activity at the nucleoside triphosphate

(NTP) level against the HCV NS5B RdRp, however both suffered from inefficient phosphorylation

cascades (Figure 1B).

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′,3′-Dideoxy-2′,3′-exo- and 2′,3′-endo-methylene nucleosides (2′,3′-cyclopropane nucleosides)

are a relatively underexplored class of ring fused sugar modified system, wherein their respective

O4′-exo (West) and O4′-endo (East) furanose ring conformations represent intermediates between

the conventional C3′-endo (North) and C2′-endo (South) pseudorotational cycle antipodes (Figure

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B). Of the three 2′,3′-dideoxy-2′,3′-exo-methylene (α-cyclopropane) nucleosides with natural

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pyrimidine bases, only the cytidine 7 was found to possess weak inhibitory activity against HIV,

whereas the analogs bearing a 2′,3′-cyclopropane in the β-orientation (2′,3′-endo-methylene) 8 were

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found to be devoid of anti-HIV activity. No further furanose functionalization has been reported

for the 2′,3′-endo-methylene nucleosides beyond these initial reports.

In contrast, sugar modified nucleosides incorporating a 3′-C-hydroxymethyl group have been

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investigated with respect to a broad range of potential therapeutic applications; as antiviral (HIV),

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antimicrobial (Mycobacterium tuberculosis) and anticancer (lymphoblastic leukemia) agents (9-

11 Figure 1C). Furthermore, combination of a 3′-C-hydroxymethyl and 2′-arabino-fluoro

substitution provided a pyrimidine nucleoside (12) with antiviral activity against both HIV and

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hepatitis B virus (HBV).

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Notwithstanding the recent regulatory approval of sofosbuvir for the treatment of chronic HCV,

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the search for novel sugar modified nucleoside inhibitors of HCV NS5B RdRp continues. In this

regard, and as part of a wider program to investigate novel, functionalized nucleosides with a high

degree of structural diversity, the synthesis of a series of bicyclic systems was undertaken based on

the 2′,3′-dideoxy-2′,3′-endo-methylene-2′-fluoro motif, either with or without a 3′-C-

hydroxymethyl moiety 13-16, which were anticipated to adopt a relatively atypical East sugar ring

conformation (Figure 1D). The synthesis and structural confirmation of the respective uridine and

guanosine analogs thereof are described herein, along with their antiviral activity against HCV.

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Figure 1. (A) Examples of therapeutic nucleosides bearing sugar ring fluorine substitutions;

nucleocidin 1, gemcitabine 2, clofarabine 3, sofosbuvir 4. (B) Examples of conformationally

restricted bicyclic nucleosides; 3′,4′-oxetane 5, 2′-O,4′-C-methylene bridged 6, 2′,3′-dideoxy-2′,3′-

exo-methylene 7, 2′,3′-dideoxy-2′,3′-endo-methylene 8. (C) Examples of biologically active 3′-

deoxy-3′-C-hydroxymethyl nucleosides; antiviral 9, antimicrobial 10, anticancer 11, antiviral 12.

(D) Target uridine and guanosine 2′,3′-dideoxy-2′,3′-endo-methylene-2′-fluoro nucleosides 13-16.

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RESULTS AND DISCUSSION

Uridine and guanosine nucleosides 13, 14 and 15, 16 were approached using a similar synthetic

strategy, via the respective bicyclic pentofuranoses bearing either 2-fluoro-2,3-endo-methylene or

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-fluoro-3-C-hydroxymethyl-2,3-endo-methylene substituents (Figure 2). Analogous construction

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of a [3.1.0]-bicyclic system was reported for the non-fluorinated 2,3-dideoxy derivative.

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The key transformations en route to the endo-methylene furanoses c were determined to be the

stereoselective Simmons-Smith cyclopropanations of vinyl fluorides a. Subsequent glycosylations

with persilylated nucleobases were then anticipated to provide the conformationally locked target

nucleosides.

Figure 2. Synthetic strategy towards bicyclic nucleosides d via 2-fluoro-2,3-endo-methylene-

pentofuranose scaffold c.

Synthesis of 13 and 14 began with the formation of fluoroallyl alcohol 18a, in two steps from D-

glyceraldehyde following literature procedures.²⁰^,²¹(Scheme 1). Initially, work focused on

optimization of the precedented Simmons-Smith type cyclopropanation of the benzylated

2

0b,22

derivative 18b.

The reaction resulted in the formation of the desired cyclopropane 19a in a

(>2.5

highly diastereoselective manner, however, the requirement for a large excess of ZnEt

2

equivalents), irreproducible yields, modest conversions (30–40%) and a complex impurity profile

rendered this method unsuitable for scale up. Therefore, the trifluoroacetic acid activated reagent

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developed by Shi was investigated, which proved to be more reliable and efficient, requiring only

.1 equivalents of ZnEt for the complete consumption of the starting material. On 33 g scale 19a

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was obtained in 59% yield as a single diastereoisomer. It was, however, observed that the

benzylated fluoroallyl alcohol 18b slowly degraded upon storage at room temperature and thermal

analysis by differential scanning calorimetry (DSC) revealed a sharp exothermic onset at 71 ºC. In

pursuit of a safer synthesis, cyclopropanation of the more thermally stable silyl protected derivative

24

1

8c was investigated. Furukawa’s conditions with 1.5 eq of ZnEt

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reproducibly furnished 19b in

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8% yield on 10 g scale. The Shi modification also proved to be reliable, but lower yielding (48%)

due to the partial removal of the TBDPS protecting group.

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In similar fashion to the non-fluorinated analog, alcohol 20, obtained by deprotection of 19a or

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9b, was transformed into the 2-fluorolactol 21 (anomeric ratio β/α = 1/8, determined by H NMR

spectroscopy) in 50% yield over three steps (Scheme 1).

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Scheme 1

Conditions: (a) triethyl 2-fluoro-2-phosphonoacetate, NaHMDS, THF, −78 °C (47%); (b) DIBAL-

H, Et

45 °C (92%, 2 steps); (e) ZnEt2, TFA, CH

°C (58% of 19b); (g) H , Pd/C, MeOH, rt (84%); (h) TBAF, THF, rt (99%); (i) i. (COCl)

DMSO, CH Cl , −78 °C, then NEt

CH Cl (50%, 3 steps).

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O, −78 °C; (c) BnBr, NaH, DMF, 0 °C to rt (85%, 2 steps); (d) TBDPSCl, imidazole, THF,

2

I

2, CH

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Cl

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, 0 °C (59% of 19a); (f) ZnEt2, CH

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I

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Cl

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, −78 °C to rt, ii. 0.1 M aq HCl, 1,4-dioxane, iii. TBDPSCl, py,

2

Post acetylation of the anomeric hydroxyl, the acetate 22 was coupled with silylated uracil under

Vorbrüggen conditions in anhydrous CH CN, using TMSOTf as an activator (Scheme 2). The

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3

reaction required elevated temperatures, possibly due to both the deactivating effect of the fluorine

on the anomeric position and steric hindrance imparted by the bulky TBDPS group and the -

cyclopropane moiety. The desired nucleoside 23 was obtained in a favorable 8:1 mixture of β:α

anomers, however, in a disappointingly low 12% yield. Attempts to improve the reaction of the

glycosyl acetate 22 by varying the temperature, activator and solvent were unsuccessful, prompting

investigation of alternate coupling approaches. Treatment of the lactol 21 with methanesulfonyl

chloride and triethylamine resulted in the clean formation of the α-chloro sugar 24, rather than the

mesylate 25 (Scheme 2). The formation of an anomeric chloride byproduct upon anomeric

mesylation has been similarly observed with 2-deoxy-2-fluoro-ribose and arabinose derivatives.

The chloride 24 was also formed directly from the acetate 22 via reaction with HCl/Et

although 24 was stable to aqueous workup and prolonged storage at 2-8 C without significant

decomposition, it was observed to readily hydrolyze on silica gel to lactol 21. Mesylation of the

anomeric position was achieved by treatment of lactol 21 with Ms

2

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2

O and

o

2

O and NEt , however, the

3

product 25 was significantly less stable than the respective chloride and almost completely

hydrolyzed upon subjecting to an aqueous workup (pH neutral). Interestingly, in contrast to the

reported non-fluorinated analog, neither the chloride nor the mesylate (in both cases the resulting

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material was used in crude form) appeared to form the desired nucleoside when treated with

silylated uracil in an S 2-type or Vorbrüggen-type condensation.

N

Scheme 2

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Conditions: (a) Ac

β:α = 8:1); (c) MsCl, NEt

NEt , CH Cl , 0 °C (crude 25) (f) persilylated uracil, CHCl

2

O, py (99%, β:α = 1:8); (b) persilylated uracil, TMSOTf, CH

, CH Cl , 0 °C (crude 24); (d) HCl, 1,4-dioxane (crude 24); (e) Ms

, rt.

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CN, 80 °C (12%,

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O,

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When lactol 21 was treated with MsCl in the presence of both NEt

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and pyridine, gradually a

multicomponent mixture was formed, consisting mainly of chloride 24 and pyridinium-type adduct

26 (Scheme 3). After aqueous CuSO

coupled with silylated uracil under Vorbrüggen-type conditions using TMSOTf as an activator in

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workup to remove excess pyridine, the crude mixture was

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anhydrous 1,2-DCE, providing desired uridine 23 in 45% yield (over two steps) as a 4:1 β:α

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mixture of anomers. Approximately 20% of the glycosylpyridinium adduct 26 bearing the triflate

counter-anion was also isolated from the reaction.²⁸

Scheme 3

Conditions: (a) i. MsCl, NEt

45% over 2 steps, β:α = 4:1).

3

, py, CH

2

Cl

2

, rt, ii. persilylated uracil, TMSOTf, 1,2-DCE, 90 °C

(

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Anomers of the 5′-silyl protected uridine analog 23 were inseparable at this stage using traditional

purification techniques, however, the clean β-anomer was isolated via recrystallization after

switching protecting groups to the more crystalline 5′-p-methoxybenzoyl ester 27 (Scheme 4).

Target 2′,3′-dideoxy-2′,3′-endo-methylene-2′-fluorouridine 13 was obtained in 92% yield after

deprotection using NaOMe in MeOH. Stereochemical investigation was performed by 2D NOESY

NMR spectroscopy and NOE correlations from H-1′ to H-4′, H-3′ to H-4′ and correlation between

one of the methylene protons of the cyclopropane ring and the uracil H-6 proton indicated their

respective close spatial relationships and confirmed the desired 2′,3′-endo-methylene and β-

anomeric configuration (Figure 3).

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Scheme 4

Conditions: (a) TBAF, THF, rt; (b) p-MeOBzCl, py (80% over 2 steps, β:α = 4:1); (c)

crystallization from EtOAc to obtain pure β (73% recovery of β anomer); (d) NaOMe, MeOH, rt

(92%).

Figure 3. Confirmation of 13 stereochemistry by 2D NOESY NMR spectroscopy (400 MHz). NOE

correlations reported from extracted 1D data sets.

Synthesis of the corresponding 2′,3′-dideoxy-2′,3′-endo-methylene-2′-fluoroguanosine 14 was

initially attempted under conditions previously successful for the uridine analog. Thus, lactol 21

was treated with MsCl, NEt and pyridine, and after aqueous workup the crude mixture was reacted

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29

with silylated 2-N-acetyl-6-O-diphenylcarbamoylguanine (Robins’ reagent ) in the presence of

TMSOTf. The desired guanosine product was obtained in only 12% yield as a 1.3:1 mixture of β:α

anomers. Alternatively, utilizing the acetate donor 22 under analogous conditions did not furnish

any nucleoside. Installation of the purine moiety was, however, more efficiently achieved via

condensation of lactol 21 (1:8 mixture of β:α anomers) with N-Boc-2-amino-6-chloropurine under

Mitsunobu conditions. Purine nucleoside 28 was obtained in 58% yield as an 8:1 mixture of β:α

3

0

anomers. Treatment of 28 with the sodium salt of 3-hydroxypropionitrile to effect S Ar

N

displacement of the chloride moiety followed by β-elimination of the acrylonitrile resulted in

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formation of the protected guanosine 29 in moderate yield (55%). Conveniently, the TBDPS

protecting group was also cleanly removed under the reaction conditions and, after isolation, 29

was obtained in a 10:1 β:α anomeric mixture. Removal of the Boc protection in 29 was

accomplished with AcOH, however, it was necessary to stop the reaction prior to completion to

minimize acid-induced product degradation. Alternative deprotection conditions were also

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investigated using HCl/MeOH and TFA/H O mixtures, however, in all cases low product yields

2

and/or poor recovery of starting material were obtained. After removal of the Boc moiety,

sequential desalting using basic resin and trituration with MeOH provided guanosine 14 as the

clean β anomer (16% yield of pure β-anomer over 2 steps). As with uridine analog 13, the

stereochemical assignment of the final compound was made on the basis of 2D NOESY NMR

spectroscopy (Figure 4). Correlation between H-1′ to H-4′, H-3′ to H-4′ and between one of the

methylene protons of the cyclopropane ring and the guanine H-8 proton confirmed the required

2

′,3′-endo-methylene and β-anomeric configuration.

Scheme 5

Conditions: (a) PPh , 2-NHBoc-6-Cl-purine, DIAD, THF, rt (β:α = 8:1, 58%); (b) NaH, 3-

3

hydroxypropionitrile, THF 0 °C to rt (β:α = 10:1, 55%); (c) i. AcOH, 90 °C (β:α = 10:1, 34% of 14

and 28% recovery of 29), ii. trituration from MeOH to obtain pure β (47% recovery).

Figure 4. Confirmation of 14 stereochemistry by 2D NOESY NMR spectroscopy (400 MHz). NOE

correlations reported from extracted 1D data sets.

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Synthesis of the 3′-C-hydroxymethyl analogs 15 and 16 started with readily available D-

isoascorbic acid, which was transformed into the ethyl ester 30 in three steps according to

3

1

published procedures (Scheme 6). Reduction to diol 31 was achieved cleanly with NaBH in

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EtOH at room temperature, conditions to which most esters remain unreactive, presumably

32

facilitated by close proximity of the hydroxyl group to the alkoxycarbonyl in 30. The crude diol

3

1 was treated with Bu SnO in refluxing MeOH and the resulting stannylene acetal was opened

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with BnBr in the presence of TBAI to effect benzyl protection with 4:1 selectivity for the desired

primary over secondary hydroxyl. The appropriately protected D-erythritol 32 was isolated in

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0% yield over six steps from D-isoascorbic acid, with only a single purification step required.

Oxidation to the corresponding D-erythrulose 33 was initially performed using Dess-Martin

periodinane then switched to the more cost effective Swern conditions on larger scale (Scheme 6).

Subsequent formation of the fluorinated alkene isomers 34 was achieved via Horner–Wadsworth–

Emmons (HWE) olefination with triethyl 2-fluoro-2-phosphonoacetate.

ratio of geometric isomers obtained was established at the subsequent allyl alcohol 35, vide infra.

Accordingly, it was determined that selectivity of the HWE reaction favored (Z)-34 over (E)-34.

The best selectivity of ~1.8:1 (Z):(E) was achieved when the least chelating potassium base

2

0,34

The exact identity and

(KHMDS) was used, in comparison with 2.8:1 for NaHMDS and 4:1 for n-BuLi. After workup, the

crude 34 mixture was telescoped. The next step, ester reduction, was performed using NaBH , LiCl

and EtOH in THF. The cis/trans isomers were separated at this stage using column chromatography

and the desired (E)-35 was isolated in 24% yield over three steps (47% for the (Z)-35 isomer)

4

.

Scheme 6

+

Conditions: (a) Amberlyst 120 H , acetone, reflux then NEt

EtI, MeCN, reflux; (d) NaBH

reflux (30% yield from D-isoascorbic acid); (f) (COCl)

3

; (b) H

2

O

2

, K

2

CO

3

, H O, 0°C to rt; (c)

2

4

, EtOH, rt; e) i. Bu

2

SnO, MeOH, reflux; ii. BnBr, TBAI, PhMe,

2

, DMSO, CH

2

Cl

2

, −78 °C, then NEt

3

, −78

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°

C to rt; (g) triethyl 2-fluoro-2-phosphonoacetate, KHMDS, THF, −78 °C (1.8:1 Z:E ratio); (h)

NaBH

4

, LiCl, EtOH, THF, rt (47% (Z)-35 and 24% (E)-35 over 3 steps).

To confirm the desired trans stereochemistry of the minor isomer (E)-35, and by extension

precursor 34, the compound was cyclized in a three step process, which would have been sterically

unfeasible with the isomeric alkene (Z)-35 (Scheme 7). The alcohol was first oxidized using Dess-

Martin periodinane and then treated with acid to remove the acetonide. Protection as the t-

butyldiphenylsilyl (TBDPS) ethers was then accomplished using imidazole in THF. Two cyclic

systems were isolated, the di-silylated furanose 36b (17% yield over 3 steps, 4:1 mixture of β:α

0

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2

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8

9

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9

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9

0

3

6

37

anomers) and the di-silylated pyranose 36a (60% yield over 3 steps, 6:1 mixture of anomers ), thus

establishing the identities of the respective geometric isomers at the prior olefination.

Furanose 36b is a potential substrate for cyclopropanation. In recent work we have demonstrated

the possibility of high yielding and stereoselective Simmons-Smith-type cyclopropanation

3

8

performed at the non-fluorinated 2,3-positions of a carbohydrate furanose ring system. The endo-

stereoselectivity of the reaction was attributed to chelation of the zinc carbenoid species to the

oxygen atoms at C5OBn and/or C1OBn, directing the methylene group onto the same (β) face of

the ring. Fluorine-substituted olefins are generally regarded as deactivated substrates for Simmons-

Smith reactions due to their reduced electron density.²⁰^aTherefore, the presence of the 2-fluoro

substituent as well as the sterically hindered environment caused by the large silyl groups in 36b

was anticipated to be problematic for the successful installation of a methylene moiety. The

standard Furukawa (ZnEt

cyclopropanation and only a reaction performed using the procedure established by Denmark,

with ZnEt and chloroiodomethane in 1,2-DCE, gave the bicyclic product 37 as a single undesired

exo-diastereoisomer in a good yield (55%). Stereochemical investigation was performed by 2D

NOESY NMR spectroscopy and NOE correlation between H-4 and the one of the CH protons of

24

23

2

/CH

2

I

2

) and Shi (ZnEt

2

/CH

2

I

2

/CF

3

CO

2

H) conditions did not promote

3

9

2

the cyclopropane ring (NOE of 1.2% at 400 MHz) indicated their close spatial relationship. It is

feasible that the high stereoselectivity of the reaction was driven by the steric hindrance and lack of

oxygen chelation on the β-face of the ring. In contrast, the pyranose derivative 36a appeared

unreactive to any of the aforementioned cyclopropanation conditions.

Scheme 7

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41

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44

45

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49

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Conditions: (a) Dess-Martin periodinane, CH

imidazole, THF, rt (over 3 steps: 1:6 mixture of β:α; 36b 17%, 4:1 mixture of β:α and 36a 60%, 6:1

mixture of anomers); (d) 1 M ZnEt in hexanes CH Cl , 0 °C to rt (47% for 38a dr = 50:10

and 10% for 38b); (e) TBDPSCl, imidazole, THF, rt (over 3 steps: endo-37 50%) (f) 1 M ZnEt in

hexanes, ClCH 1,2-DCE, −10 °C to rt (55%, only β anomer isolated).

2

Cl

2

, rt; (b) 0.1 M aq HCl, 1,4-dioxane; (c) TBDPSCl,

2

,

2

I

2, CH

2

I

2 ,

Based on the formation of the undesired exo-methylene on cyclopropanation of dihydrofuran

6b, the methodology utilized previously in the synthesis of lactol 21 was pursued. Thus, the

3

alkene (E)-35 was protected with TBDPSCl in pyridine and then submitted to various

2

4

39

cyclopropanation conditions; Furukawa (ZnEt

2

/CH

2

I

2

), Denmark (ZnEt

2

/ICH Cl) and Shi

2

3

(ZnEt

2

/CH

2

I

2

/CF

3

CO

2

H). None of the tested reactions were successful and only unchanged

starting material was recovered in each case. It was, however, observed that reaction of the

unprotected allyl alcohol (E)-35 under Furukawa’s conditions did lead to formation of the

40

cyclopropane 38a with 5:1 diastereoselectivity, favoring the desired (2R,3R) product (Scheme 7).

Unexpectedly, aqueous quenching of the reaction was accompanied by significant methylation of

the hydroxyl group in both the product as well as the unreacted alkene. To minimize formation of

the methyl ethers, a reverse quenching procedure was implemented. The reaction mixture was

transferred via cannula into a vigorously stirred biphasic mixture of EtOAc and aqueous NH

4

Cl.

The yield of the desired cyclopropane 38a ranged between 35–47%, with formation of the methyl

4

1

ether 38b at 10–20% and recovery of the starting material (E)-35 at 10–20%. After oxidation of

8a to the corresponding aldehyde using Dess-Martin periodinane and acid catalyzed removal of

the acetonide, the ring was closed exclusively to the di-silylated furanose endo-37 using TBDPSCl

3

4

2

and imidazole in THF (1:6 β:α anomer ratio) in good yield (50% over three steps). To the best of

our knowledge these are the first reported examples of such 2-fluoro-3-C-hydroxymethyl

functionalized bicyclic furanoses (Scheme 7): the presented methodologies allow access to both

2

′,3′-endo- and exo-methylene diastereoisomers of 37.

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Unsurprisingly, the sterically hindered di-TBDPS furanoses endo-37 and exo-37 did not readily

undergo nucleosidation using a variety of solvents and Lewis acids, providing only low yield of the

corresponding uridine derivatives (<10%, as judged by NMR spectroscopy, not isolated). It was

anticipated that peracetylated derivative 41 would be a preferable substrate (Scheme 8).

Accordingly, oxidation of the alcohol 38a and acetonide removal were followed by acetylation with

0

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9

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8

9

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2

3

4

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7

8

9

0

43

Ac

2

O in pyridine. The diacetylated sugar 39 was obtained exclusively in its furanose form as an

inseparable 1:7 β:α mixture of anomers in 88% yield over three steps. Removal of the 3-C-

hydroxymethyl benzyl protection via hydrogenolysis and subsequent acetylation furnished the

desired triacetate 41 (91% over 2 steps). The triacetate successfully underwent nucleosidation with

silylated uracil under Vorbrüggen conditions,²⁵giving the acetylated uridine derivative 42 in

excellent yield (84%) as a 10:1 mixture of β:α anomers. The pure β-anomer was isolated via

recrystallization from EtOAc/n-heptane and after deprotection with methanolic ammonia the

desired nucleoside 15 was isolated in 78% yield. 2D NOESY NMR spectroscopy indicated

correlation between H-1′ to H-4′ and from one of the methylene protons of the cyclopropane ring to

the uracil H-6 proton, demonstrating the desired 2′,3′-endo-methylene and β-anomeric

configuration, which was confirmed by X-ray crystallography (Figures 5 and 7).

Scheme 8

Conditions: (a) Dess-Martin periodinane, CH

°C to rt (88% over 3 steps, 1:7 β:α mixture of anomers); (d) H

py, 0 °C to rt (91% over 2 steps, 1:7 β:α mixture of anomers); (f) i. persilylated uracil, TMSOTf,

CH CN, 50 °C (84%), ii. recrystallization from EtOAc/n-heptane to obtain clean β-anomer (72%);

g) NH , MeOH, rt (78%).

2

Cl

2

, rt; (b) 0.1 M aq HCl, 1,4-dioxane; (c) Ac

2

O, py,

0

2

, 10% Pd/C, MeOH, rt; (e) Ac

2

O,

3

(

3

Figure 5. Confirmation of 15 stereochemistry by 2D NOESY NMR spectroscopy (400 MHz). NOE

correlations reported from extracted 1D data sets.

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Synthesis of the guanosine analog 16 was initially attempted under Vorbrüggen conditions,

2

9

whereby acetate 41 was treated with silylated 2-N-acetyl-6-O-diphenylcarbamoylguanine and

TMSOTf in anhydrous acetonitrile. As in the case of the corresponding acetate 22 (Scheme 2)

without the 3-C-hydroxymethyl substitution, the reaction resulted mainly in decomposition of the

starting material and the desired protected nucleoside was isolated in low yield (20%), as an

inseparable 1:1 mixture of anomers. Thus, a Mitsunobu approach was employed, which had been

successfully implemented in the prior synthesis of guanosine 14 (Scheme 5). The anomeric acetate

41 was therefore hydrolyzed and the resulting lactol 43 (1:6 mixture of β:α) was coupled with N-

Boc-2-amino-6-chloropurine in the presence of PPh and DIAD in THF (Scheme 9). The desired

protected nucleoside 44 was isolated as an inseparable 5:1 mixture of β:α anomers in 46% yield.

Removal of the N-Boc protection was attempted under various acidic conditions (MeOH/HCl,

10

11

12

13

14

15

16

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19

20

21

22

23

24

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28

29

30

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32

33

34

35

36

37

38

39

40

41

42

43

44

45

46

47

48

49

50

51

52

53

54

55

56

57

58

59

60

3

AcOH/H

material was observed. The deprotection was however, cleanly achieved in good yield (76% yield,

:1 β:α anomer ratio) using excess TMSOTf (8 equivalents) in CH Cl , followed by aqueous

NaHCO workup. Interestingly, when the reaction was performed in the presence of 2,6-lutidine or

2

O, HCO

2

H/H

2

O and TFA/H

2

O/CH

2

Cl

2

) but in each case significant decomposition of the

5

2

3

triethylamine according to literature precedent,⁴⁴the conversion rate slowed significantly.

Transformation of the chloropurine base in 45 to guanine and concomitant deacetylation was

achieved by treatment with 2-mercaptoethanol and NaOMe in refluxing methanol giving the

desired nucleoside 16 in 79% yield. This method gave superior yield (79% vs 50%) in comparison

30

with the reaction performed with the sodium salt of 3-hydroxypropionitrile, which was previously

utilized with the analogous guanosine derivative 28 (Scheme 5). Due to the very low solubility of

the final product in multiple solvents and solvent mixtures, separation of the anomers was

troublesome. An analytically pure sample of 16 was isolated by preparative HPLC. Stereochemical

assignment of the final guanosine nucleoside 16 was made on the basis of 2D NOESY NMR

spectroscopy. Correlations from H-1′ to H-4′ and from one of the methylene protons of the

cyclopropane ring to the guanine H-8 proton were observed, confirming the desired 2′,3′-endo-

methylene and β-anomeric configuration (Figure 6).

Scheme 9

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6

Conditions: a) H

THF, rt (46%, 5:1 β:α); (c) TMSOTf, CH

NaOMe, MeOH, reflux (79%, 5:1 β:α); (e) anomer separation by HPLC.

2

O, TMSOTf, CH

3

CN (84%, 1:6 β:α); (b) 2-NHBoc-6-Cl-purine, DIAD, PPh

3

,

2

Cl , 0 °C (76%, 5:1 β:α); (d) 2-mercaptoethanol,

2

0

1

2

3

4

5

6

7

8

9

0

1

2

3

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8

9

0

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8

9

0

1

2

3

4

5

6

7

8

9

0

1

2

3

4

5

6

7

8

9

0

Figure 6. Confirmation of 16 stereochemistry by 2D NOESY NMR spectroscopy (400 MHz). NOE

correlations reported from extracted 1D data sets.

In anticipation of improved solubility and therefore potentially easier anomer separation at earlier

stages of the synthesis, the route to 16 was modified by starting with the benzyl protected furanose

3

9. Hydrolysis of the anomeric acetate 39 furnished the desired lactol 46 in good yield (69%, 1:6

β:α anomer ratio) but was accompanied by formation of significant amount of 1,1′-α,α′-linked

disaccharide 47 (18% yield) (Scheme 10). The nucleoside 48 was obtained via coupling of the

lactol with 2-NHBoc-6-Cl-purine under Mitsunobu conditions (Scheme 11). Notably, the anomeric

ratio was higher than in case of the fully acetylated derivative 44 (7:1 vs 5:1 of β:α) (Scheme 9).

Synthesis of the partially protected guanosine 49 was achieved on treatment of the chloropurine 48

with 2-mercaptoethanol and NaOMe in good yield (66%, >30:1 β:α). On removal of the Boc group

with TMSOTf in CH

product 16 was observed. With a large excess of TMSOTf (20 equivalents) followed by treatment

of the crude mixture with solid NaHCO and MeOH, the amount of the debenzylated material

Cl

2 2

, partial deprotection of the benzyl moiety and formation of the target

3

significantly increased. Isolation of the clean β-anomer 16 (30% yield) could be accomplished by

sequential chromatographic purification on normal and reverse phase silica gel. Benzyl protected

nucleoside 50 was isolated in 40% yield and was further subjected to dilute hydrogenolysis using

4

5

10% Pd/C in EtOAc:iPrOH:H O 5:3:1, allowing isolation of further guanosine 16 in 30% yield.

2

Scheme 10

Conditions: a) H

2

O, TMSOTf, CH CN (46 69%, 1:6 β:α; and 47 18%, single α-α′-linked).

3

Scheme 11

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Conditions: a) 2-NHBoc-6-Cl-purine, DIAD, PPh

3

, THF, rt (50%, 7:1 β:α); (b) 2-mercaptoethanol,

NaOMe, MeOH, reflux (66%, >30:1 β:α); (c) TMSOTf (18 eq), CH

2

Cl

2

, 0 °C then NaHCO and

3

MeOH (50 40% β; and 16 30% β); (d) Pd/C, H

2

, EtOAc, i-PrOH, H

2

O, rt (30%).

Stereochemical assignment of the final compounds 13, 14, 15 and 16 were made on the basis of 1D

and 2D NMR spectroscopy and NOESY experiments (see Figures 4, 3, 5 and 6).

The X-ray crystallographic structure of nucleoside 15 is illustrated in Figure 7: the principal

conformational parameters obtained therefrom are presented in Table 1. Analysis of the solid state

structure revealed that the cyclopropane ring is not a perfect equilateral triangle, with the C3′–CH

bond being 2.6% and 2.0% longer than the C2′F–CH and C3′–C2′F bonds respectively. The

cyclopropane ring is inclined at an angle of 116.7° to the mean plane of the furanose ring. The

conformation around the glycosyl bond is anti with a torsion angle χ of −153.4° (C2–N1–C1′–O4′).

In contrast to the analogous 2′,3′-deoxy-2′,3′-endo-methylene systems, the conformation around

the C4′–C5′ bond is synclinal (–sc) with a torsion angle γ of −71.41° (C3′–C4′–C5′–O5′): an

intramolecular hydrogen bond is evident between O5′H and the C3′-hydroxymethyl group. As

anticipated, the furanoid ring adopts an almost East pucker with a pseudorotational angle P of 99.6°

2

1

4

o

and a maximum puckering amplitude v

conformations.

m

of 35.3° placing it midway between E and T

1

8

b,46,47,48

Table 1. Major conformational and geometric parameters from X-ray structure of uridine 15.

Uridine 15

Bond lengths:

C1′–C2′

C2′–C3′

C3′–C4′

1.504 (Å)

1.493 (Å)

1.513 (Å)

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C2′–CH

C3′– CH

C1′–N1

2

1.484 (Å)

1.523 (Å)

1.467 (Å)

2

Bond angles:

C1′–C2′–C3′

C2′–C3′–C4′

C1′–C2′–CH

C2′–C3′–CH

C3′–C2′–CH

106.93°

104.53°

119.72°

58.94°

61.54°

59.52°

0

1

2

3

4

5

6

7

8

9

0

1

2

3

4

5

6

7

8

9

0

1

2

3

4

5

6

7

8

9

0

1

2

3

4

5

6

7

8

9

0

1

2

3

4

5

6

7

8

9

0

2

C2′–CH

2

–C3′

CH –C3′–C4′

2

113.73°

Torsion angles:

C1′–C2′–C3′–C4′ (v

C2′–C3′–C4′–O4′ (v

C3′–C4′–O4′–C1′ (v

C4′–O4′–C1′–C2′ (v

O4′–C1′–C2′–C3′ (v

C3′–C4′–C5′–O5′ (γ)

C2–N1–C1′–O4′ (χ)

Phase angle of

pseudorotation (P)^a

Maximum puckering

amplitude (v

)^b

2

)

−5.89°

−14.87°

31.95°

−35.23°

24.89°

3

)

4

0

1

−71.41°

−153.37°

9

3

9.6°

5.3°

m

a

Calculated from: tan P = [(v

4

+ v

1

) – (v

3

+ v

0

)]/3.077v

2

, as v < 0, 180° is added to the calculated

2

b

/cosP.⁴²

value of P. Calculated from: v

m

= v

2

Figure 7 ORTEP drawing of the X-ray crystallographic structure of the uridine derivative 15.

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′-Fluoro-2′,3′-endo-methylene nucleosides 13, 14, 15 and 16 were evaluated in a whole cell-

based HCV replicon assay: neither anti-HCV activity (EC50 >100 M) nor cytotoxicity (CC50 >100

M) was observed in vitro. In order to determine whether the lack of activity in the replicon was

due to a failure of cellular kinases to recognize these nucleosides as substrates for conversion to the

49



10

11

12

13

14

15

16

17

18

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35

36

37

38

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44

45

46

47

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49

50

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respective NTPs, or due to the lack of activity of the NTPs themselves against the RdRp, 13-TP,

50

1

4-TP and 15-TP were evaluated against the purified HCV NS5B 1b wild type polymerase. All

three NTPs were found to be inactive (IC50 >100 M) indicating that these highly functionalized,

fused sugar ring systems were not incorporated by the HCV RdRp, presumably either due to the

resultant unnatural East conformational or additional stereoelectronic deficiencies.

CONCLUSION

Two conformationally locked sugar modified bicyclic nucleoside systems were investigated based

on a 2′,3′-dideoxy-2′,3′-endo-methylene-2′-fluoro motif. Synthesis of the first example of a 2,3-

dideoxy-2,3-endo-methylene-pentofuranose featuring a 2-fluoro group is described. Access to two

novel 2-fluoro-3-C-hydroxymethyl-pentofuranoses bearing either 2,3-endo- or exo-methylene

moieties is provided. Uridine and guanosine nucleosides of the respective endo-methylene systems

were structurally confirmed and the 3′-C-hydroxymethyl-uridine analog was determined by X-ray

o

crystallography to adopt an East sugar ring conformation ( E/ T ). Anti-HCV activity was

1

evaluated and the nucleosides were found to be inactive in a whole cell replicon assay and as their

respective NTPs against the HCV NS5B polymerase.

EXPERIMENTAL

General Experimental Reactions requiring anhydrous conditions were conducted in oven dried

apparatus under dry argon atmosphere, utilizing commercially available dry solvents and reagents.

All common reagents (including Dess-Martin periodinane) were purchased from commercial

1

13

19

sources and used without further purification. H, C and F NMR spectra were recorded on a 400

19

MHz Fourier transformation spectrometer using an internal deuterium lock. F NMR spectra were

1

recorded with H decoupling. Spectra were obtained from samples prepared in 5 mm diameter

tubes in CDCl

3

, CD

3

OD or DMSO-d . Multiplicities are as quoted: s = singlet, d = doublet, t =

6

triplet, q = quartet, m = multiplet, bs = broad singlet, app = apparent. Coupling constants (J) are

reported in Hz. Signal assignments are based on COSY, DEPT, HSQC and HMBC spectra. Melting

points were not corrected. HRMS spectra were obtained using electrospray ionisation (ESI).

Optical rotations were recorded using a light source at λ= 589 nm.Crystallographic data for the

nucleoside 15 have been deposited at the Cambridge Crystallographic Data Centre as

supplementary publication no. CCDC 1018857.

β-D-2′,3′-Dideoxy-2′-fluoro-2′,3′-endo-methyleneuridine (13)

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To a solution of 27 (140 mg, 0.37 mmol, β anomer) in MeOH (15 mL) at rt was added NaOMe in

MeOH (25% w/w) to obtain pH ~12. The mixture was stirred for 2 h and solid CO

achieve pH 7. The crude mixture was concentrated onto silica and purified by column

chromatography (SiO , EtOAc/MeOH/H O gradient) to give clean 13 as a white amorphous solid

(83 mg, 0.34 mmol, 92% yield).

2

was added to

2

0

1

2

3

4

5

6

7

8

9

0

1

2

3

4

5

6

7

8

9

0

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2

3

4

5

6

7

8

9

0

1

2

3

4

5

6

7

8

9

0

1

2

3

4

5

6

7

8

9

0

¹H NMR (400 MHz, MeOD): δ = 7.67 (1H, d, J = 8.2, C6H), 6.41 (1H, d, J = 2.9, C1′H), 5.63

(

2

1H, app d, J = 8.2, C5H), 4.34 (1H, dt, J = 3.4, 5.4 Hz, C4′H), 3.48 (2H, d, J = 5.4, C5′H

2

), 2.12–

1

3

.05 (1H, m, C3′H), 1.50–1.36 (2H, m, CFCH

2

); C NMR (100 MHz, MeOD): δ = 165.8 (C4=O),

152.5 (C2=O), 141.9 (C6H), 103.1 (C5H), 84.9 (d, J = 248, C2′F), 84.7 (d, J = 28 Hz, C1′H), 79.4

19

1

(

C4′H), 62.5 (C5′H

CDCl δ = −210.9; (IR) νmax(cm ): 3300, 1698, 1462, 1381, 1265, 1214, 1115, 1054; HRMS (ESI-

TOF) m/z: (M+H) calcd for C10

2

), 22.9 (d, J = 7, C3′H), 10.2 (d, J = 11, CFCH ); F{ H} NMR (376 MHz,

2

-1

3

+

21

H12FN

2

O

4

243.0781; found 243.0770; [α]

D

+19.4 (c 0.6, MeOH).

β-D-2′,3′-Dideoxy-2′-fluoro-2′,3′-endo-methyleneguanosine (14)

Nucleoside 29 (1.0 g, 2.62 mmol) was dissolved in AcOH (20 mL) and stirred at 90 °C for 8 h. The

mixture was concentrated in vacuo, concentrated onto silica gel and purified by column

chromatography (SiO

2

, EtOAc:MeOH:H O) to give product 14 as an acetic acid salt (10:1 mixture

2

of β:α anomers, 306 mg, 0.90 mmol, 34%). Starting material 29 was also isolated (280 mg, 0.73

mmol, 28%). The product was re-purified using reverse phase column chromatography (30 g C18

column, H

2

O:CH CN 0→5% gradient) and then de-salted using Dowex-Marathon free base resin to

3

give 150 mg of free amine 14 (10:1 mixture of β:α anomers) as a white amorphous solid. The

product was triturated 3 times from MeOH to give 70 mg (0.24 mmol) of clean β 14 as off-white

amorphous solid.

¹H NMR (400 MHz, d

.30 (1H, d, J = 3.2, C1′H), 4.83 (1H, t, J = 5.7, C5′OH), 4.39 (1H, dt, J = 3.2, 5.7, C4′H), 3.4–3.36

2H, m, C5′H ), 2.34–2.28 (1H, m, C3′H), 1.76 (1H, app q, J = 5.4, CFCH ), 1.60–1.51 (1H, m,

-DMSO): δ = 156.7 (C), 154.0 (C), 151.6 (C), 134.5 (C8H),

), 22.3 (d, J =

-DMSO) δ = −208.8; (IR) νmax(cm^-

-DMSO): δ = 10.69 (1H, bs, NH), 7.98 (1H, s, C8H), 6.56 (2H, bs, NH ),

6

2

6

(

2

A

H

B

1

3

CFCH

A

H

B

); C NMR (100 MHz, d

6

1

16.4 (C), 84.4 (d, J = 247, C2′F), 81.5 (d, J = 27, C1′H), 79.1 (C4′H), 60.6 (C5′H

2

1

9

1

7, C3′H), 9.7 (d, J = 11, CFCH

2

); F{ H} NMR (376 MHz, d

6

1

)

: 3307, 3134, 1691, 1639, 1596, 1533, 1482, 1365, 1174, 1046, 1022; HRMS (ESI-TOF) m/z:

+

21

(M+Na) calcd for C11

H12FN

5

NaO

3

304.0816; found: 304.0810; [α]

D

−20.0 (c 0.1, DMSO).

β-D-2′,3′-Dideoxy-3′-C-hydroxymethyl-2′-fluoro-2′,3′-endo-methyleneuridine (15)

To a solution of the acetylated uridine 42 (150 mg, 0.45 mmol) in MeOH (8 mL) was added 7N

methanolic NH

was concentrated in vacuo. The desired nucleoside 15 was isolated by column chromatography

(SiO , EtOAc:MeOH:H O gradient) in 78% yield (95 mg, 0.35 mmol) as white amorphous solid.

The product was recrystallized from CHCl :MeOH to give needles, mp: 85–87 ˚C.

3

(2.4 mL, 16.80 mmol) and the mixture was stirred for 16 h at rt. The crude mixture

2

3

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¹H NMR (400 MHz, MeOD): δ = 7.77 (1H, d, J = 8.2, C6H), 6.50 (1H, d, J = 2.7, C1′H), 5.72 (1H,

d, J = 8.2, C5H), 4.47 (1H, t, J = 5.4, C4′H), 3.84 (1H, d, J = 12.3, CH

CH ), 3.72–3.63 (2H, m, C5′H ), 1.71 (1H, app. t, J = 7.7, CFCH

.8, CFCH ); C NMR (100 MHz, MeOD): δ = 165.8 (C4=O), 152.5 (C2=O), 141.8 (C6),

A

H

B

), 3.77 (1H, d, J = 12.3,

A

H

B

2

A

H

B

), 1.44 (1H, ddd, J = 0.8,

1

3

7

A

H

B

103.2 (C5), 86.0 (d, J = 250.0, C2′F), 84.3 (d, J = 27.0, C1′H), 81.0 (C4′H), 62.0 (d, J = 20,

10

11

12

13

14

15

16

17

18

19

20

21

22

23

24

25

26

27

28

29

30

31

32

33

34

35

36

37

38

39

40

41

42

43

44

45

46

47

48

49

50

51

52

53

54

55

56

57

58

59

60

19

C5′H

2

), 60.5 (d, J = 5.0, CH

2

), 34.2 (d, J = 8.0, C3′), 13.8 (d, J = 11, CFCH

2

); F NMR (376

-1

MHz, MeOD): δ −215.58 (1F, CF); (IR) νmax(cm ): 3370, 2942, 2883, 1680, 1461, 1380, 1264,

+

1029; HRMS (ESI-TOF) m/z: (M+Na ) calcd for C11

H13FN

2

NaO 295.0701; found 295.0711;

5

21

[

α]

D

+31.7 (0.69, MeOH);

β-D-2′,3′-Dideoxy-3′-C-hydroxymethyl-2′-fluoro-2′,3′-endo-methyleneguanosine (16)

To a solution of compound 45 (81 mg, 0.20 mmol) in anhydrous MeOH (1.5 mL) was added 2-

mercaptoethanol (0.55 µL, 0.78 mmol) followed by sodium methoxide (42.2 mg, 0.78 mmol) at rt.

The mixture was stirred for 5 h at 66 °C, then cooled to rt and neutralised by the addition of solid

CO

chromatography (SiO

9%, mixture of 5:1 β:α anomers). The pure β anomer (white amorphous solid) was isolated using

reverse phase HPLC.

2

. The mixture was concentrated in vacuo and the residue was purified by column

2

, EtOAc:water:methanol) to give 16 as a white solid (48 mg, 0.15 mmol,

3

5

7

¹H NMR (400 MHz, d

6.26 (1H, d, J = 3.6 C1′H), 4.98 (1H, br s, OH), 4.85 (1H, br s, OH), 4.41 (1H, app t, J = 5.4,

C4′H), 3.77 (1H, d, J = 12.2, (C3′)CH ), 3.62 (1H, d, J = 12.2, (C3′)CH ), 3.56 (1H, dd, J =

.7, 11.8, C5′H ), 3.45 (1H, dd, J = 6.5, 11.8, C5′H ), 1.88 (1H, app t, J = 7.5, CFCH ),

1.46 (1H, dd, J = 7.8, 18.2, CFCH ); C NMR (100 MHz, d -DMSO) δ = 156.8 (C), 154.1 (C),

51.6 (C), 134.5 (C8H), 116.3 (C), 85.6 (d, J = 248, C2′F), 81.3 (d, J = 26, C1′H), 79.4 (C′4H),

-DMSO) δ = 10.79 (1H, br s, NH), 7.97 (1H, s, CH8), 6.61 (2H, br s, NH ),

6

2

A

H

B

A

H

B

4

A

H

B

A

H

B

A

H

B

1

3

A

H

B

6

1

6

1

9

1

0.4 (C5′H

2

), 58.5 ((C3′)CH

2

), 33.3 (d, J = 8, C3′), 13.2 (d, J = 11, CFCH ); F{ H} NMR (376

2

-1

MHz, d

6

-DMSO) −213.8; (IR) νmax(cm ): 3309, 3115, 2929, 1687, 1603, 1531, 1362, 1029; HRMS

+

(ESI-TOF) m/z: (M+Na ) calcd for C12

H14FN

5

NaO 334.0922; found 334.0912.

4

(S,E)-3-(2,2-dimethyl-1,3-dioxolan-4-yl)-2-fluoroprop-2-en-1-ol (18a)

Alcohol 18a was prepared in two steps from D-glyceraldehyde according to the literature

2

0,21

procedures.

¹H NMR (400 MHz, CDCl

4.32 (1H, dd, J = 1.5, 6.5, CH

OCH ), 3.62 (1H, app. t, J = 7.6 , OCH

C(CH

The spectroscopic data for this compound was unavailable in the literature.

): δ = 5.27 (1H, dd, J = 8.9, 19.2, CHCF), 4.77–4.71 (1H, m, OCH),

OH), 4.27 (1H, dd, J = 2.6, 6.5, CH OH ), 4.13 (1H, dd, J = 6.0, 8.3,

), 2.22 (1H, br s, OH), 1.43 (3H, s, C(CH ), 1.39 (3H, s,

): δ =161.7 (d, J = 256, CF), 109.7 (C(CH ), 107.5 (d, J =

), 57.8 (d, J = 31, CH

3

2

3 2

)

1

3

)

2

); C NMR (100 MHz, CDCl

3

3 2

)

2

2, CHCF), 70.9 (d, J = 13, CHO), 69.7 (d, J = 3, OCH

2

OH), 26.7

19

-1

(C(CH

3

)

2

), 25.8 (C(CH

3

)

2

); F NMR (376 MHz, CDCl

3

) δ = −105.4 (1F, m, CF); (IR) νmax(cm ):

19

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2

3

4

5

6

3

420, 2990, 2940, 2880, 1700, 1450, 1370,1290, 1150, 1050, 1020; HRMS (ESI-TOF) m/z:

+

21

(

M+Na ) calcd for C

(S,E)-4-(3-(Benzyloxy)-2-fluoroprop-1-en-1-yl)-2,2-dimethyl-1,3-dioxolane (18b)

Benzylated alcohol 18b was prepared from 18a according to the literature procedure and the H and

8

H

13FNaO

3

199.0741; found 199.0745; [α]

D

−18.0° (c 1.1, CHCl

3

).

1

0

1

2

3

4

5

6

7

8

9

0

1

2

3

4

5

6

7

8

9

0

1

2

3

4

5

6

7

8

9

0

1

2

3

4

5

6

7

8

9

0

1

2

3

4

5

6

7

8

9

0

13

20a

C NMR data agreed with those published in the literature.

(S,E)-4-(3-(tert-Butyldiphenylsilyloxy)-2-fluoroprop-1-en-1-yl)-2,2-dimethyl-1,3-dioxolane

(18c)

To a solution of alcohol 18a (5.2 g, 29.5 mmol) in anhydrous THF (80 mL) was added imidazole

(5.0 g, 74 mmol), followed by tert-butyl(chloro)diphenylsilane (11.5 mL, 44 mmol). After stirring

for 10 min at rt the slurry was stirred at 45 °C for 1 h. The reaction was quenched by the addition of

MeOH (50 mL), concentrated and the residue purified by column chromatography (SiO , EtOAc/n-

heptane) to afford the alkene 18c as a yellow oil (11.2 g, 27.0 mmol, 92% over 2 steps).

2

¹H NMR (400 MHz, CDCl

9.5, 19.2, CHCF), 4.48-4.45 (1H, m, OCH), 4.31 (1H, dd, J = 13.5, 26.3 ( J

) δ = 7.69–7.65 (4H, m, ArH), 7.47–7.37 (6H, m, ArH), 5.17 (1H, dd, J

3

=

H

,

F

), CFCH

OSi), 3.89 (1H, dd, J = 6.0, 8.2, OCH ), 3.47 (1H,

), 1.29 (3H, d, J = 0.4, C(CH ), 1.06 (9H, s,

) δ = 161.2 (d, J = 260, CF), 135.63, (2 × Ar-CH), 135.61,

(2 × Ar-CH), 132.9 (Ar-C), 132.7 (Ar-C), 130.0 (2 × Ar-CH), 129.9 (2 × Ar-CH), 127.9 (2 × Ar-

CH), 127.8 (2 × Ar-CH), 109.3 (C(CH ), 107.4 (d, J = 21.8 CHCF), 70.8 (d, J = 13.2, OCH),

9.7 (d, J = 2.7, OCH ), 58.9 (d, J = 31.0, CFCH

(C(CH ), 19.2 SiC(CH

A

H

B

OSi),

3

4.26 (1H, dd, J = 13.5, 20.6 ( J

dd, J = 7.5, 8.2, OCH ), 1.37 (3H, s, C(CH

); C NMR (100 MHz, CDCl

H

,

F

), CFCH

H

A B

2

3

)

2

)

3 2

13

C(CH

3

)

3

)

3 2

6

2

), 26.7 (C(CH

3

)

2

), 26.7 (C(CH

3

)

3

), 25.8

19

-1

3

)

2

3

)

3

; F NMR (376 MHz, CDCl

3

) δ = −104.3 (1F, m, CF); (IR) νmax(cm ):

3

070, 3050, 2985, 2930, 2860, 1700, 1470, 1430, 1380, 1370, 1110, 1060; HRMS (ESI-TOF) m/z:

+

21

(

M+Na ) calcd for C24

(S)-4-((1S,2R)-2-((Benzyloxy)methyl)-2-fluorocyclopropyl)-2,2-dimethyl-1,3-dioxolane (19a)

To 138.0 mL of ZnEt (1M in hexane, 138.0 mmol) in anhydrous CH Cl (138 mL) at 0 °C was

added TFA (9.6 mL, 125.0 mmol) in anhydrous CH Cl (30 mL) drop-wise over 40 min. The

reaction was stirred for 30 min and then a solution of CH (10.9 mL, 135.0 mmol) in anhydrous

CH Cl (100 mL) was added over 15 min. After a further 30 min stirring alkene 18b (33.0 g, 125.0

H31FO

3

SiNa 437.1919; found 437.1914; [α]

D

−8.0° (c 1.0, CHCl

3

).

2

I

2 2

2

mmol) in anhydrous CH Cl (100.0 mL) was added over 20 min. The reaction was stirred at 0 °C

for 30 min and then warmed to 15 °C over 1 h, before re-cooling to 3 °C and stirring for a further

16 h. The reaction was quenched by the addition of sat. aq. NH Cl (300 mL) over 30 min at 3°C.

The aqueous layer was separated and extracted with CH Cl (2 × 500 mL), the combined organic

extracts were dried over MgSO , filtered and evaporated to dryness. Purification by column

2

4

2

4

chromatography (SiO

The H and C NMR data agreed with those published in the literature.

2

, EtOAC:n-heptane) gave 20.7 g (73.8 mmol, 59%) of 19a as a yellow oil.

1

13

20b

20

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(

S)-4-((1S,2R)-2-((tert-Butyldiphenylsilyloxy)methyl)-2-fluorocyclopropyl)-2,2-dimethyl-1,3-

dioxolane (19b)

To the alkene 18c (10.0 g, 24 mmol) in anhydrous CH

2

Cl

2

(185 mL) at 2 °C was added ZnEt (15%

2

wt. in toluene, 30.0 mL, 36 mmol) drop-wise over 15 min. The reaction was stirred for a further 15

min at 2 °C and then a solution of CH (2.9 mL, 36 mmol) in anhydrous CH Cl (15 mL) was

added drop-wise over 15 min. The reaction was allowed to warm to rt and stirred for 3 days. The

reaction mixture was re-cooled to 0 °C and sat. aq. NH Cl (300 mL) was added slowly to quench

the reaction. The aqueous layer was extracted with CH Cl (2 × 500 mL) and the combined organic

layers dried over MgSO , filtered and concentrated. The residue purified by column

chromatography (SiO , EtOAc:n-heptane) to give 6.0 g of 19b (14 mmol, 58%) as a yellow oil.

2

I

2

10

11

12

13

14

15

16

17

18

19

20

21

22

23

24

25

26

27

28

29

30

31

32

33

34

35

36

37

38

39

40

41

42

43

44

45

46

47

48

49

50

51

52

53

54

55

56

57

58

59

60

4

2

4

2

¹H NMR (400 MHz, CDCl

) δ = 7.73–7.67 (4H, m, Ar-H), 7.43–7.36 (6H, m, Ar-H), 4.27 (1H,

3

ddd, J = 0.9, 12.7, 16.4, CFCH

CH

.63–1.55 (1H, m, CH=CF), 1.43 (3H, s, C(CH

CFCH CH), 1.08 (9H, s, C(CH ), 0.75 (1H, dd, J = 8.8, 7.2, CFCH

MHz, CDCl ) δ = 135.6 (2 × Ar-CH), 133.4 (Ar-C), 132.9 (Ar-C), 127.9 (4 × Ar-CH), 127.8 (4 ×

Ar-CH), 109.3 (C(CH ), 80.1 (d, J = 210, CF), 75.4 (d, J = 2, OCH), 70.0 (OCH ), 65.1 (d, J =

21, CH OH), 26.9 ((CH

A

H

B

O), 4.21 (1H, dd, J = 5.7, 7.8, CH

A

H

B

O), 3.86 (1H, t, J = 7.8,

3

A

H

B

O), 3.80 (1H, app q, J = 7.2, CHO), 3.67 (1H, dd, J = 12.7, 32.8 ( J

H

,

F

), CFCH

A

H O),

B

1

3

)

2

), 1.33 (3H, s, C(CH

3

) ), 1.29–1.20 (1H, m,

H CH); C NMR (100

B

2

1

3

A

H

B

3

)

3

A

3

)

2

3

)

3

19

CSi), 26.8 (CH

3

), 25.8 (CH

3

), 25.0 (d, J = 12, CHCF), 19.3 (SiC(CH

3 2

)

1

-1

1

2

3.7 (d, J = 11, CFCH

2

); F{ H} NMR (376 MHz, CDCl

957, 2858, 1428, 1107, 1067, 848, 701; [α]

3

) = −177.5; (IR) νmax(cm ): 2985, 2957,

2

1

D

−3.8 (c 1.0, CHCl ).

3

((1R,2S)-2-((S)-2,2-Dimethyl-1,3-dioxolan-4-yl)-1-fluorocyclopropyl)methanol (20)

Method A: To 10% Pd/C (5.00 g, 50% wet) was added cyclopropane 19a (7.20 g, 25.7 mmol) in

MeOH (200 mL) under argon. The flask was de-gassed, charged with hydrogen, and then heated to

reflux for 3.5 h. The reaction was cooled to rt and the Pd/C filtered and rinsed with EtOAc (400

mL), followed by CH

column chromatography (SiO

1.6 mmol, 84%).

2

Cl

2

(200 mL). The filtrate was concentrated and the residue purified by

2

, EtOAc:n-heptane) to give the alcohol 20 as a yellow oil (4.10 g,

2

Method B: To cyclopropane 19b (4.86 g, 11.3 mmol) in anhydrous THF (120 mL) was added

TBAF (1M in THF, 13.6 mL, 13.6 mmol) at 5 °C. The reaction was stirred for 16 h and then

concentrated. The residue was purified by column chromatography (SiO , EtOAc/n-heptane) to

give the alcohol 20 as a yellow oil (2.15 g, 11.3 mmol, quant. yield).

2

¹H NMR (400 MHz, CDCl

OCH), 3.79–3.63 (2H, m, CH

) δ = 4.29–4.19 (2H, m, CH

OH and OCH ), 3.93 (1H, app q, J = 6.8,

3

2

OH and OCH ), 2.09 (1H, dd, J = 5.3, 7.4, OH), 1.60–1.50 (1H, m,

2

CHCF), 1.44 (3H, s, C(CH

.86 (1H, m, CFCH

3

)

2

1

), 1.38–1.29 (1H, m, CFCH

A

H

B

CH), 1.36 (3H, s, C(CH

) δ = 109.3 (C(CH ), 81.6 (d, J = 219,

), 63.8 (d, J = 22.5, CH OH), 26.6 (C(CH ),

3

)

2

), 0.92–

3

0

A

H

B

CH); C NMR (100 MHz, CDCl

3

3 2

)

CF), 74.5 (d, J = 2.1, OCH), 69.72 (d, J = 0.8, OCH

2

1

)

3 2

1

9

5.7 (C(CH

3

)

2

), 24.4 (d, J = 11.9, CHCF), 13.4 (d, J = 11.1, CFCH

2

); F{ H} NMR (376 MHz,

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6

-1

CDCl

(

CHCl

pentofuranose (21)

3

) δ = −178.8; (IR) νmax(cm ): 3425, 2987, 2935, 2877, 1455, 1247, 1156, 1050, 843; HRMS

+

21

ESI-TOF) m/z: (M+Na ) calcd for C

9

H15FO

3

Na 213.0897; found 213.0890.[α]

D

−58.7 (c 1.0,

3

).α/β-5-O-tert-Butyldiphenylsilyl-2,3-dideoxy-2-fluoro-2,3-endo-methylene-D-

To oxalyl chloride (3.65 mL, 43 mmol) in anhydrous CH

DMSO (6.89 mL, 97 mmol) in anhydrous CH

alcohol 20 (4.10 g, 22 mmol) in anhydrous CH

reaction was stirred for 45 min and then quenched by the addition of triethylamine (30.0 mL, 215

mmol) over 15 min. The resultant slurry was stirred for 5 min and then slowly warmed to rt.

2

Cl (100 mL) at −78 °C was added

2

0

1

2

3

4

5

6

7

8

9

0

1

2

3

4

5

6

7

8

9

0

1

2

3

4

5

6

7

8

9

0

1

2

3

4

5

6

7

8

9

0

1

2

3

4

5

6

7

8

9

0

2

Cl

2

(50 mL) drop-wise over 15 min. After 10 min

(50 mL) was added drop-wise over 15 min. The

2

Cl

2

CH

2

Cl

2

(50 mL) and H

2

O (150 mL) were added. The aqueous layer was separated and extracted

SO , filtered and

with CH

2

Cl (2 × 100 mL) and the combined organic extracts dried over Na

2

4

concentrated to give a yellow oil, which was used in the next step without further purification. To a

solution of the crude aldehyde in 1,4-dioxane (50 mL) was added 0.1 N HCl (50 mL), ensuring the

pH is 1-2, and the resultant solution was stirred for 16 h at rt. The reaction was adjusted to pH 9–10

with K

CHCl

concentrated. The residue was dissolved in anhydrous CH

68 mmol) was added, followed by TBDPSCl (13.6 mL, 54 mmol). The reaction was stirred at rt

for 16 h, then quenched by the addition of MeOH (20 mL) and then concentrated. The residue was

purified by column chromatography (SiO , EtOAc:n-heptane) to give lactol 21 as a yellow oil (4.15

g, 11 mmol inseparable mixture of anomers, 1:8 β:α, 50 % over 3 steps).

2

CO

(100 mL). The combined organic extracts were dried over Na

Cl

3

and concentrated in vacuo. The residue was triturated with CH

2

Cl

2

(3 × 200 mL) and

3

2

SO , filtered and

4

2

(200 mL) and pyridine (13.6 mL,

1

2

¹H NMR (400 MHz, CDCl

) δ = 7.66–7.64 (4Hα + 4Hβ, m, Ar-H), 7.44–7.25 (6Hα + 6Hβ, m, Ar-

H), 5.66 (1Hβ, ddd, J = 1.0, 4.3, 8.4, C1H), 5.38 (1Hα, dd, J = 2.3, 4.8, C1H), 4.63–4.59 (1Hα, m,

3

C4H), 4.36–4.33 (1Hβ, m, C4H), 3.67 (1Hα + 1Hβ, ddd for α: J = 1.3, 5.0, 10.3, C5H ), 3.48–

.43 (1Hα + 1Hβ, m, C5H ), 3.07 (1Hα, d, J = 4.8, OH), 2.89 (1Hβ, d, J = 8.4, OH), 2.17–2.08

1Hα + 1Hβ, m, C3H), 1.29–1.21 (1Hα + 1Hβ, m, CFCH ), 1.06 (3Hα + 3Hβ, s, C(CH ),

); C NMR (100 MHz, CDCl3, α anomer) δ = 135.61 (Ar-

A

H

B

3

(

0

A

H

B

A

H

B

)

3 3

1

3

.89–0.82 (1Hα + 1Hβ, m, CFCH

A

H

B

CH), 135.59 (Ar-CH), 133.4 (Ar-C), 133.3 (Ar-C), 129.7 (4 × Ar-CH), 127.72 (2 × Ar-CH),

127.71 (2 × Ar-CH), 95.1 (d, J = 19, C1H), 84.4 (d, J = 254, CF), 76.7 (C4H), 63.2 (C5H ), 26.8

); F{ H} NMR (376

2

19

1

(C(CH

3

)

3

), 21.6 (d, J = 7.9, C3H), 19.2 (SiC(CH

3

)

3

), 10.9 (d, J = 11, CFCH

2

-1

MHz, CDCl

3

) δ = −211.5; (IR) νmax(cm ): 3406, 3071, 3049, 2956, 2930, 2857, 1589, 1390, 1234,

1105, 1055, 946, 700; HRMS (ESI-TOF) m/z: (M+Na ) calcd for C22

+

H

27FNaO Si 409.1606; found

3

4

09.1625.

α/β-1-O-Acetyl-5-O-tert-butyldiphenylsilyl-2,3-dideoxy-2-fluoro-2,3-endo-methylene-D-

pentofuranose (22)

To the solution of lactol 21 (125 mg, 0.32 mmol) in pyridine (2.5 mL) was added Ac

.30 mmol) drop-wise at 0 °C and the mixture was gradually warmed out to rt and stirred for 16 h.

MeOH (0.5 mL) was added drop-wise and the mixture concentrated in vacuo. The residue was

2

O (0.5 mL,

5

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8

9

purified by column chromatography (SiO

quantitative yield (138 g, 0.32 mmol, mixture of anomers, 1:8 β:α).

2

, acetone:n-heptane) to give 22 as a colorless oil in

¹H NMR (400 MHz, CDCl

Ar-H), 6.32 (1H, d, J = 2.3, C1H), 4.62–4.58 (1H, m, C4H), 3.69 (1H, ddd, J = 1.5, 4.8, 10.4,

C5H ), 3.44 (1H, dd, J = 7.3, 10.4, C5H ), 2.20–2.13 (1H, m, C3H), 2.14 (3H, s, COCH ),

1.34–1.26 (1H, m, CFCH ), 1.06 (9H, s, C(CH ); C NMR

100 MHz, CDCl ) δ (major α anomer) = 170.0 (CO), 135.59 (2 × Ar-CH), 135.58 (2 × Ar-CH),

33.3 (Ar-C), 133.2 (Ar-C), 129.8 (2 × Ar-CH), 127.75 (2 × Ar-CH), 127.74 (2 × Ar-CH), 94.0 (d,

J = 18, C1H), 82.9 (d, J = 254, CF), 78.5 (C4H), 62.8 (d, J = 2.9, C5H ), 26.8 (C(CH ), 21.7 (d, J

); F{ H} NMR (376

): δ (major, α anomer) = 7.67–7.64 (4H, m, Ar-H), 7.43–7.35 (6H, m,

3

A

H

B

A

H

B

3

10

11

12

13

14

15

16

17

18

19

20

21

22

23

24

25

26

27

28

29

30

31

32

33

34

35

36

37

38

39

40

41

42

43

44

45

46

47

48

49

50

51

52

53

54

55

56

57

58

59

60

1

3

A

H

B

3

)

3

), 0.90–0.85 (1H, m, CFCH

A

H

B

(

3

1

2

)

3 3

19

1

=

7.7, C3H), 21.2 (COCH

3

), 19.2 (SiC(CH

3

)

3

), 10.7 (d, J = 10.8, CFCH

2

-1

MHz, CDCl

3

) δ = −212.4; (IR) νmax(cm ): 3071, 3016, 2969, 2931, 2858, 1752, 1427, 1363, 1217,

+

1104, 1007, 970, 700; HRMS (ESI-TOF) m/z: (M+Na ) calcd for C24

H

29FO SiNa 451.1717; found

4

51.1722.

β/α-D-5′-O-tert-Butyldiphenylsilyl-2′,3′-dideoxy-2′-fluoro-2′,3′-endo-methyleneuridine (23)

To a solution of lactol 21 (1.04 g, 2.69 mmol)) in anhydrous CH Cl (100 mL) at 0 °C was added

NEt (4.3 mL) followed by MsCl (0.96 mL, 12.40 mmol). The mixture was stirred at 0 °C for 1 h,

2

3

then 15 min at rt. Pyridine (5.02 mL) was added followed by an additional portion of MsCl (2.4

mL, 31.01 mmol) and the mixture was stirred for 30 min at rt. The mixture was diluted with

CH

2

Cl

2

(100 mL) and washed with 10% aqueous CuSO

4

solution (300 mL). The aqueous phase

SO ,

was extracted with CH

2

Cl (2 × 50 mL) and the combined organic layers dried over Na

2

4

filtered and concentrated in vacuo to give yellow oil. (The product was unstable on silica gel). The

residue was dissolved in anhydrous 1,2-DCE (70 mL) and added to the silylated uracil (11.24 mmol

,

see the general method for silylation of uracil below), followed by the drop-wise addition of

TMSOTf (1.12 mL, ~2 min addition) at rt. The mixture was stirred for 10 min then transferred to a

pre-heated oil bath at 90 °C and stirred for 1 h 10 min. The mixture was then cooled to rt and

quenched with sat. aq. NaHCO

mL), the combined organic layers dried over Na

reaction mixture was purified by column chromatography (SiO

give 0.58 g (1.22 mmol, 45% yield over 2 steps) of a 4:1 mixture of β:α anomers of the nucleoside

3

(100 mL). The aqueous layer was extracted with CHCl

SO , filtered and concentrated in vacuo. The crude

, n-heptane:acetone, 050%) to

3

(3 × 100

2

4

2

5

1

23 (glass solid). The pyridinium adduct 26 was isolated in 11% yield (177 mg, 0.30 mmol, α

anomer, amorphous off-white solid).

General procedure for silylation of uracil: Uracil (1.26 g, 11.24 mmol) was treated with HMDS

(

2

50 mL) in the presence of (NH

.5 h. Excess HMDS was evaporated under reduced pressure at 50 °C to give a cloudy oil, which

was directly used in the nucleosidation reaction.

4

)

2

SO

4

(140 mg, 1.06 mmol) under argon and stirred at 130 °C for

¹H NMR (400 MHz, CDCl

): δ (minor, α anomer) = 9.12 (1H, bs, NH), 7.66–7.63 (4H, m, 4 × Ar-

3

H), 7.47-7.37 (6H, m, 6 × Ar-H), 7.29 (1H, dd, J = 2.0, 8.1, C6H), 6.30 (1H, app s, C1′H), 5.79

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8

9

1

2

3

4

5

6

(

1H, dd, J = 1.7, 8.1, C5H), 4.62–4.58 (1H, m, C4′H), 3.68 (1H, ddd, J = 1.3, 5.0, 10.5, C5′H

A

H ),

B

3

.43 (1H, dd, J = 7.2, 10.4, C5′H

CFCH ), 1.06 (9H, s, C(CH

CDCl ): δ (minor, α anomer) = 162.8 (C4=O), 150.7 (C2=O), 140.6 (C6H), 135.6 (4 × Ar-CH),

133.02 (Ar-C), 132.96 (Ar-C), 129.97 (Ar-CH), 129.94 (Ar-CH), 127.9 (2 × Ar-CH), 127.8 (2 ×

A

H ), 2.42–2.37 (1H, m, C3′H), 1.48 (1H, ddd, J = 7.2, 10.0, 17.4,

B

1

3

A

H

B

3

)

3

, 0.97 (1H, app q, J = 5.4, CFCH

A

H ); C NMR (100 MHz,

B

3

0

1

2

3

4

5

6

7

8

9

0

1

2

3

4

5

6

7

8

9

0

1

2

3

4

5

6

7

8

9

0

1

2

3

4

5

6

7

8

9

0

1

2

3

4

5

6

7

8

9

0

Ar-CH), 103.0 (C5H), 83.4 (d, J = 28, C1′H), 81.8 (d, J = 255, C2′F), 79.1 (C4′H), 62.9 (C5′H

2

),

1

2

6.8 ((CH

MHz, CDCl

m, 6 × Ar-H and C6H), 6.52 (1H, d, J = 2.8, C1′H), 5.66 (1H, d, J = 8.2, C5H), 4.49 (1H, m,

C4′H), 3.72 (1H, ddd, J = 0.8, 4.5, 10.8, C5′H ), 3.62 (1H, dd, J = 5.8, 10.9, C5′H ), 2.19–

), 1.06 (9H, s, C(CH , 1.31–1.26 (1H, m

3

)

3

C), 24.0 (d, J = 7, C3′H), 19.2 (C(CH

3

)

3

), 11.5 (d, J = 11, CFCH

2

); H NMR (400

3

): δ (major, β anomer) = 8.82 (1H, bs, NH), 7.66–7.63 (4H, m, Ar-H), 7.47–7.35 (7H,

A

H

B

A

H

B

2.12 (1H, m, C3′H), 1.47–1.40 (1H, m, CFCH

A

H

B

3 3

)

13

CFCH

1

A

H

B

); C NMR (100 MHz, CDCl

3

): δ (major, β anomer) = 162.4 (C4=O), 150.3 (C2=O),

39.3 (C6H), 135.6 (4 × Ar-CH), 132.9 (Ar-C), 132.8 (Ar-C), 129.99 (Ar-CH), 129.98 (Ar-CH)

27.85 (2 × Ar-CH), 127.81 (2 × Ar-CH), 102.6 (C5H), 83.5 (d, J = 27, C1′H), 83.5 (d, J = 250,

C2′F), 77.6 (C4′H), 63.1 (C5′H

= 11, CFCH

2

4

2

), 26.8 ((CH

); F{ H} NMR (376 MHz, CDCl

931, 2858, 1681, 1457, 1141, 701; HRMS (ESI-TOF) m/z: (M+H) calcd for C26

81.1959; found: 481.1960.

3

)

3

C), 22.3 (d, J = 7, C3′H), 19.2 (C(CH

3

)

3

), 10.0 (d, J

19

1

-1

2

3

) δ = −206.4 (β), −208.8 (α); (IR) νmax(cm ): 3070,

+

H30FN

2

4

O Si

α/β-5-O-tert-Butyldiphenylsilyl-2,3-dideoxy-2-fluoro-2,3-endo-methylene-D-pentofuranosyl

pyridin-1-ium triflate (26)

¹H NMR (400 MHz, CDCl

7

): δ = 9.14 (2H, app d, J = 6.6, 2 × Ar-H), 8.77 (1H, ddd, J = 1.3, 2.6,

3

.8, Ar-H), 8.27 (2H, app t, J = 7.2, 2 × Ar-H), 7.73–7.70 (4H, m, 4 × Ar-H), 7.51-7.43 (6H, m, 6

Ar-H), 6.69 (1H, d, J = 1.7, C1′H), 5.23–5.19 (1H, m, C4′H), 5.84 (1H, dd, J = 1.0, 10.8,

×

C5′H

.5, 10.2, 18.0, CFCH

100 MHz, CDCl

A

H

B

), 3.73 (1H, dd, J = 6.0, 10.8, C5′H

A

H

B

), 2.76–2.71 (1H, m, C3′H), 1.80 (1H, ddd, J =

13

7

(

A

H

B

), 1.34 (1H, app q, J = 6.0, CFCH

A

H

B

), 1.09 (9H, s, C(CH

3

)

3

); C NMR

3

): δ = 149.2 (Ar-CH), 143.4 (2 × Ar-CH), 136.8 (2 × Ar-CH), 136.7 (2 × Ar-

CH), 134.12 (2 × Ar-C), 131.18 (Ar-CH), 131.15 (Ar-CH), 129.6 (2 × Ar-CH), 128.97 (2 × Ar-

CH), 128.96 (2 × Ar-CH), 97.2 (d, J = 17, C1′H), 86.3 (d, J = 257, C2′F), 83.7 (C4′H), 64.2

(C5′H

for CF

ν

2

), 27.3 ((CH

3

)

3

C), 24.5 (d, J = 8, C3′H), 20.0 (C(CH

3

)

3

), 13.1 (d, J = 11, CFCH

2

), (signal

19

1

3

-

was not observed); F{ H} NMR (376 MHz, CDCl

max(cm ):3133, 2932, 2859, 1739, 1259, 1155, 1112, 1030; HRMS (ESI-TOF) m/z: (M) calcd for

3

) δ = −80.1 (TfO), −206.6; (IR)

1

+

−

C

27

H

31FNO

2

Si 448.2108; found: 448.2108; (ESI-TOF) m/z: (TfO) calcd for CO

3

F S 148.9520;

21

found: 148.9516; [α]

D

−69.6 (c 1.1, CHCl ).

3

β-D-5′-O-p-Methoxybenzoyl-2′,3′-dideoxy-2′-fluoro-2′,3′-endo-methyleneuridine (27)

To a solution of nucleoside 23 (4:1 β:α mixture, 390 mg, 0.81 mmol) in THF (10 mL) was added a

solution of TBAF (1 mL, 1M in THF, 1 mmol) drop-wise at 0 °C and the mixture was allowed to

gradually warm up to rt over 3 h. The reaction mixture was concentrated in vacuo. The residue was

dissolved in pyridine (8 mL) and solid para-methoxybenzoyl chloride (273 mg, 1.60 mmol) was

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added in one portion at rt. After 4 h an additional portion of para-methoxybenzoyl chloride (273

mg, 1.60 mmol) was added and the reaction stirred for 16 h. The reaction mixture was quenched

with MeOH (2 mL) and concentrated in vacuo. The residue was concentrated onto silica and

purified by column chromatography (SiO , n-heptane:acetone) to give 245 mg (0.65 mmol, 80%

2

yield over two steps) of ester 27 as a yellow solid (4:1 β:α mixture). The product was recrystallized

from EtOAc to give 135 mg of clean β anomer as colorless needle clusters, mp. 230–232 °C.

10

11

12

13

14

15

16

17

18

19

20

21

22

23

24

25

26

27

28

29

30

31

32

33

34

35

36

37

38

39

40

41

42

43

44

45

46

47

48

49

50

51

52

53

54

55

56

57

58

59

60

¹H NMR (400 MHz, CDCl

1H, d, J = 8.2, C6H), 7.00–6.91 (2H, m, 2 × Ar-H), 6.55 (1H, d, J = 2.8, C1′H), 5.75 (1H, dd, J =

2.4, 8.2, C5H), 4.74 (1H, app q, J = 5.1, C4′H), 4.38 (1H, dd, J = 5.7, 12, C5′H ), 4.34 (1H, dd,

J = 5.0, 12, C5′H ), 3.87 (3H, s, OCH ), 2.24–2.17 (1H, m, C3′H), 1.65–1.55 (1H, m,

CFCH ), 1.41 (1H, app q, J = 6.9, CFCH

165.9 (C), 163.8 (C), 162.1 (C), 150.2 (C2=O), 139.1 (C6H), 131.8 (2 × Ar-CH), 121.7 (Ar-C),

13.8 (2 × Ar-CH), 102.9 (C5H), 83.5 (d, J = 27, C1′H), 82.9 (d, J = 251, C2′F), 75.4 (C4′H), 63.1

): δ (β anomer) = 8.23 (1H, bs, NH), 8.00-7.96 (2H, m, 2 × Ar-H), 7.47

3

(

A

H

B

A

H

B

3

13

A

H

B

A

H

B

); C NMR (100 MHz, CDCl ): δ (β anomer) =

3

1

19

1

(

C5′H

2

), 55.5 (CH

3

O), 21.8 (d, J = 7, C3′H), 10.1 (d, J = 11, CFCH ); F{ H} NMR (376 MHz,

2

-1

CDCl

3

) δ = −209.35; (IR) νmax(cm ): 3116, 2361, 2341, 1685, 1607, 1458, 1257, 1170; HRMS

+

21

(ESI-TOF) m/z: (M+H) calcd for C18

H18FN

2

O

6

377.1140; found: 377.1162; [α]

D

+10.3 (c 0.2,

CHCl ).

3

2-tert-Butyloxycarbonylamino-9-(5′-O-tert-butyldiphenylsilyl-2′,3′-dideoxy-2′-fluoro-2′,3′-

endo-methylene-β/α-D-pentofuranosyl)-6-chloro-9H-purine (28)

To a solution of lactol 21 (100 mg, 0.26 mmol) in anhydrous THF (5 mL) was added PPh (122

3

mg, 0.47 mmol) followed by N-Boc-2-amino-6-chloropurine (125 mg, 0.47 mmol) at rt. The

mixture was stirred for 10 min and DIAD was added drop-wise (92 μL, 0.47 mmol). The mixture

was stirred for 4 h and concentrated in vacuo. The residue was purified by column chromatography

(

SiO

¹H NMR (400 MHz, CDCl

.50 (1H, bs, NH), 7.45–7.36 (6H, m, ArH), 6.62 (1H, dd, J = 1.1, 4.0 Hz, C1′H), 4.60–4.56 (1H,

m, C4′H), 3.75 (1H, ddd, J = 0.9, 4.9, 10.7, C5′CH ), 3.64 (1H, dd, J = 6.2, 10.7, C5′H ),

), 1.52 (9H, s, (CH CSi), 1.27–1.24 (1H,

2

, n-heptane:acetone) to give 28 as a mixture of 8:1 β:α anomers (98 mg, 0.15 mmol, 58%).

3

): δ (major, β anomer) = 8.07 (1H, s, C8H), 7.67–7.63 (4H, m, ArH),

7

A

H

B

A

H

B

2.30–2.23 (1H, m, C3′H), 1.58–1.51 (1H, m, CFCH

m, CFCH

A

H

B

)

3 3

13

A

H

B

), 1.06 (9H, s, (CH

3

)

3

C); C NMR (100 MHz, CDCl

3

): δ (major, β anomer) = 153.1

(

C=O Boc), 152.7 (C), 151.5 (C), 149.9 (C), 141.4 (C8H), 135.5 (4 × Ar-CH), 133.0 (Ar-C), 132.9

(Ar-C), 129.9 (2 × Ar-CH), 127.8 (4 × Ar-CH), 84.1 (d, J = 251, C2′F), 83.4 (d, J = 27, C1′H),

1.7 ((CH CO), 78.1 (C4′H), 63.0 (C5′H ), 28.2 ((CH CSi) 26.8 ((CH CO), 23.1 (d, J = 7,

C3′H), 19.2 ((CH CSi), 10.6 (d, J = 11, CFCH ); F{ H} NMR (376 MHz, CDCl ) δ = −206.6

8

3

)

3

2

3

1

)

3

)

3 3

19

3

)

3

2

3

-1

(α anomer), −209.5 (β anomer); (IR) νmax(cm ): 2932, 1751, 1572, 1449, 1135, 1111, 1076; HRMS

+

(ESI-TOF) m/z: (M+H) calcd for C32

H38ClFN

5

4

O Si 638.2366; found: 638.2363.

N-tert-Butyloxycarbonyl-β/α-D-2′,3′-dideoxy-2′-fluoro-2′,3′-endo-methyleneguanosine (29)

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4

5

6

To a suspension of NaH (60% in mineral oil, 94 mg, 2.35 mmol) in anhydrous THF (2 mL) at −78

°

−

C was added 3-hydroxypropionitrile (162 μL, 2.35 mmol) drop-wise. The mixture was stirred at

78 °C for 20 min then 1 h at 0 °C. A solution of 28 (300 mg, 0.47 mmol) in anhydrous THF (2

mL) was added drop-wise and the mixture stirred at 0 °C for 2 h and then further 3h at rt. The

reaction was quenched at 0 °C with MeOH and concentrated in vacuo. The residue was

0

1

2

3

4

5

6

7

8

9

0

1

2

3

4

5

6

7

8

9

0

1

2

3

4

5

6

7

8

9

0

1

2

3

4

5

6

7

8

9

0

1

2

3

4

5

6

7

8

9

0

concentrated onto silica gel and purified by column chromatography (SiO

2

, CHCl :MeOH,

3

0

→10%) to give 29 as a white amorphous solid (11:1 mixture of β:α anomers, 100 mg, 0.26 mmol,

55% yield).

¹H NMR (400 MHz, d

-DMSO) δ (major, β anomer) = 11.16 (2H, bs, NH and NHBoc), 8.24 (1H,

6

s, C8H), 6.38 (1H, d, J = 3.4, C1′H), 4.85 (1H, t, J = 5.2, C5′OH), 4.43 (1H, app dt, J = 3.3, 5.7,

C4′H), 3.44–3.42 (2H, m, C5′H ), 2.36–2.33 (1H, m, C3′H), 1.76 (1H, app q, J = 6.8, CFCH ),

1.60–1.51 (1H, m, CFCH ), 1.50 (9H, s, (CH -DMSO): δ (major,

β anomer) = 155.0 (C6=O), 153.8 (C=O Boc), 149.3 (C), 148.1 (C), 137.0 (C8H), 119.6 (C), 84.6

2

A

H

B

13

A

H

B

3

)

3

C); C NMR (100 MHz, d

6

(

d, J = 247, C2′F), 82.6 (C(CH

3

)

3

), 82.3 (d, J = 28, C1′H), 78.2 (C4′H), 60.6 (C5′H

2

), 27.7

19

1

((CH C), 22.5 (d, J = 7 Hz, C3′H), 9.9 (d, J = 11, CFCH

3

)

3

2

1

); F{ H} NMR (376 MHz, d

6

-DMSO)

-

δ = −206.1 (α anomer), −208.5 (β anomer); (IR) νmax(cm ): 3230, 2979, 2933, 1680, 1606, 1562,

1478, 1455, 1402, 1367, 1244, 1150, 1097, 1052, 784; HRMS (ESI-TOF) m/z: (M+H) calcd for

+

C

16

H21FN

5

O 382.1527; found: 382.1519.

(S)-1-((R)-2,2-Dimethyl-1,3-dioxolan-4-yl)ethane-1,2-diol (31)

32

To a solution of crude ester 30 (157.0 g, 0.77 mol) in EtOH (1.5 L) at 0 °C was added NaBH

4

(58.2 g, 1.54 mol) portion-wise over 30 min. The reaction was stirred at rt for 2 h, then quenched

by the addition of AcOH (90 mL) and MeOH (150 mL). The mixture was stirred for 16 h at room

temperature and concentrated in vacuo. The resulting moist solid was triturated with EtOAc and the

filtrate was concentrated in vacuo to give 31 as a pale brown oil (133.9 g, 0.83 mol). This material

1

13

was used in the next step without further purification. The H and C NMR spectroscopic data

agreed with those published in the literature.³¹

¹H NMR (400 MHz, CDCl

3.79 (1H, dd, J = 3.3, 11.1, HOCH

HOCH ), 2.81 (2H, br. s, 2 × OH), 1.42 (3H, s, OC(CH

100 MHz, CDCl ): δ = 109.2 (C(CH

(HOCH ), 26.5 (C(CH ), 25.3 (C(CH

): δ = 4.10–4.04 (2H, m, HOCH and OCH

), 3.99–3.92 (1H, m, OCH ),

3

2

), 3.75–3.70 (1H, m, HOCH), 3.64 (1H, dd, J = 5.5, 11.1,

13

2

3

)

2

), 1.36 (3H, s, OC(CH

3

)

2

); C NMR

), 63.6

(

3

)

2

), 76.3 (HOCH), 72.2 (HOCH), 65.9 (OCH

2

3

)

2

3

2

) ).

(S)-2-(Benzyloxy)-1-((R)-2,2-dimethyl-1,3-dioxolan-4-yl)ethanol (32)

To a solution of crude 31 (133.9 g, 0.83 mol) in anhydrous toluene (2.6 L) was added dibutyltin

oxide (200 g, 0.83 mol) and the mixture was heated at reflux for 4 h with Dean-Stark apparatus.

After cooling to 40 °C, benzyl bromide (147 mL, 1.24 mol) and TBAI (61.0 g, 0.17 mol) were

added and the reaction heated at reflux overnight. After cooling to rt, the mixture was diluted with

CH

2

Cl

2

(2.6 L) and washed with sat. aq. NaHCO

3

(2.6 L). The aq. layer was then extracted with

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2

3

4

5

6

7

8

9

CH

2

Cl

2

(2 × 1 L). The combined organic layers were washed with 10% NaCl (2.6 L). The organic

phase was dried (Na

2

SO ), filtered and concentrated in vacuo. The resulting 4:1 mixture of

4

diastereoisomers was separated by column chromatography (EtOAc:n-heptane gradient) to give the

desired 32 as a major isomer (84.6 g, 0.34 mol, 30% yield over 6 steps) and its diastereoisomer

(minor isomer, 21.0 g, 7% yield over 6 steps). The H and C NMR spectroscopic data of 32 and

its regioisomer agreed with those published in the literature.³

1

13

3

10

11

12

13

14

15

16

17

18

19

20

21

22

23

24

25

26

27

28

29

30

31

32

33

34

35

36

37

38

39

40

41

42

43

44

45

46

47

48

49

50

51

52

53

54

55

56

57

58

59

60

(

R)-2-(Benzyloxy)-1-(2,2-dimethyl-1,3-dioxolan-4-yl)ethanone (33)

To a solution of oxalyl chloride (12.6 mL, 147.0 mmol) in anhydrous CH

was added a solution of DMSO (23.5 mL, 330.7mmol) in anhydrous CH

2

Cl

2

(100 mL) at −78 °C

(20 mL) drop-wise

2

Cl

2

over 50 min. After 15 min a solution of alcohol 32 (18.5 g, 73.5 mmol) in anhydrous CH

mL) was added drop-wise over 15 min. The reaction was stirred for 90 min at −78 °C and

quenched by the drop-wise addition of Et N (102 mL, 734.8 mmol) over 15 min. The resultant

slurry was stirred for 5 min and gradually warmed to rt. Then CH Cl (150 mL) and H O (150 mL)

were added and the mixture stirred for further 5 min. The aqueous layer was separated and

extracted with CH Cl (150 mL), the combined organic extracts were dried over Na SO , filtered

and concentrated in vacuo at rt to give ketone 33 as a yellow oil, which was used in the next step

without further purification. A small sample of the ketone was purified by column chromatography

(SiO

2

Cl (35

2

3

2

4

, n-heptane:acetone) for analysis.³⁵

2

¹H NMR (400 MHz, CDCl

4

): δ = 7.38–7.27 (5H, m, Ar-H), 4.60–4.56 (3H, m, OCH

OCH Ph), 4.23 (1H, dd, J = 7.9, 8.8, OCH ), 4.02 (1H, dd, J = 5.5, 8.8,

), 1.43 (3H, s, OC(CH ); C NMR (100 MHz, CDCl ): δ = 206.75

),

);

IR) νmax(cm ): 3031, 2988, 2937, 2886, 1734, 1373, 1258, 1214, 1070; HRMS (ESI-TOF) m/z:

Ph and OCH),

3

2

.39 (2H, s, C=OCH

2

13

OCH

2

3

)

2

), 1.36 (3H, s, OC(CH

3

)

2

3

(C=O), 137.03 (Ar-C), 128.6 (2 × Ar-CH), 128.1 (Ar-CH), 128.0 (2 × Ar-CH), 111.0 (C(CH

7

(

)

3 2

9.1 (OCH), 73.5 (OCH Ph), 72.8 (CH OCH Ph), 66.5 (OCH ), 25.9 (C(CH ), 24.9 (C(CH

2

3

)

2

)

3 2

-1

+

21

(M+Na ) calcd for C14

H18NaO

4

273.1097; found: 273.1121; [α]

D

+37.1 (c 1.0, CHCl ).

3

(

S,E)-4-(Benzyloxy)-3-(2,2-dimethyl-1,3-dioxolan-4-yl)-2-fluorobut-2-en-1-ol ((E)-35) and

S,Z)-4-(Benzyloxy)-3-(2,2-dimethyl-1,3-dioxolan-4-yl)-2-fluorobut-2-en-1-ol ((Z)-35)

To a solution of triethyl 2-fluoro-2-phosphonoacetate (21.4 g, 88.2 mmol) in anhydrous THF (148

mL) at −78 °C was added KHMDS (1M in THF, 88 mL, 88.2 mmol) drop-wise over 45 min. The

reaction was stirred at −78 °C for 30 min and a solution of the crude ketone 33 (73.5 mmol) in THF

(

150 mL) was added drop-wise over 40 min maintaining the temperature below −70 °C. The

reaction was stirred at −78 °C for 1 h, then at rt for 90 min. The mixture was then poured onto

vigorously stirred sat. aq. NH Cl (200 mL) and the aqueous layer was extracted with TBME (2 ×

50 mL). The combined organic extracts were dried (Na SO ), filtered and concentrated in vacuo

4

2

4

to give crude alkene 34 as a yellow oil (27.2 g, mixture of (Z):(E) isomers, dr = 18:10). The mixture

was used in the next step without further purification.

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8

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4

5

6

To a solution of crude (E/Z)-34 (73.5 mmol) in anhydrous THF (460 mL) was added anhydrous

LiCl (7.8 g, 187.7 mol) and NaBH

was added drop-wise (166.5 mL). The reaction was allowed to warm to rt and vigorously stirred for

d. The mixture was then diluted with EtOAc (1 L) and quenched by the addition of 10% aq. citric

acid solution (0.7 L). The mixture was then washed successively with 10% aq. citric acid solution

1 L), water (0.5 L) and sat. aq. NaHCO solution (0.5 L). The organic phase was dried (Na SO ),

filtered and concentrated in vacuo. The crude material was purified by column chromatography

(SiO , EtOAc:n-heptane) to give the desired minor isomer (E)-35 (yellow oil, 5.3 g, 18.0 mmol,

4% yield over 3 steps) and the major isomer (Z)-35 (yellow oil, 10.2 g, 34.5 mmol, 47% yield

4

(6.9 g, 183.7 mmol). The mixture was cooled to 0 °C and EtOH

3

0

1

2

3

4

5

6

7

8

9

0

1

2

3

4

5

6

7

8

9

0

1

2

3

4

5

6

7

8

9

0

1

2

3

4

5

6

7

8

9

0

1

2

3

4

5

6

7

8

9

0

(

3

2

4

2

3

5

over 3 steps).

1

(

E)-35: H NMR (400 MHz, CDCl

3

): δ = 7.36–7.25 (5H, m, Ar-H), 4.77 (1H, d, J = 6.4, 8.2,

Ph), 4.38–4.23 (2H, m, HOCH ), 4.19 (1H, dd, J = 2.7,

Ph), 4.12–4.05 (2H, m, CH OC(CH and C=CCH OCH Ph), 3.88 (1H, t, J =

), 2.72 (1H, t, J = 6.5, OH), 1.41 (3H, s, OC(CH ), 1.38 (3H, s, OC(CH );

C NMR (100 MHz, CDCl ): δ = 160.3 (d, J = 259.7, CHC=CF), 137.9 (Ar-C), 128.4 (2 × Ar-

CH), 127.9 (Ar-CH), 127.8 (2 × Ar-CH), 114.4 (d, J = 14.0, CHC=CF), 109.3 (C(CH ), 73.2 (d,

OCH), 4.48 (2H, 2 × d, J = 11.8, OCH

2

1

8

0.8, C=CCH

2

OCH

2

3

)

2

.3, CH OC(CH

2

3

)

2

3

)

2

)

3 2

1

3

)

3 2

J = 7.9, OCH), 72.6 (OCH

C=CCH OCH Ph), 58.0 (d, J = 31.3, CH

MHz, CDCl

2

Ph), 68.5 (d, J = 3.0, CH

2

OC(CH

3

)

2

), 62.5 (d, J = 8.6,

19

2

OH), 26.2 (C(CH

3

)

2

), 25.6 (C(CH

3

)

2

); F NMR (376

-1

3

): δ −104.5 (1F, CF); (IR) νmax(cm ): 3433, 3031, 2934, 2875, 1697, 1454,1371, 1212,

+

1155, 1026, 895; HRMS (ESI-TOF) m/z: (M+Na ) calcd for C16

H21FNaO 319.1316; found

4

21

3

19.1329; [α]

D

+13.1 (c 1.0, CHCl

): δ = 7.38–7.25 (5H, m, Ar-H), 5.12 (1H, m, OCH), 4.52 (2H,

Ph), 4.28–4.14 (2H, m, HOCH ), 4.14–4.01 (3H, m, CH OC(CH and

3

).

1

(

2

Z)-35: H NMR (400 MHz, CDCl

× d, J = 11.7, OCH

3

2

)

3 2

C=CCH

OC(CH

2

OCH

2

Ph), 3.69 (1H, t, J = 8.1, CH

2

OC(CH

3

)

2

), 2.76 (1H, t, J = 6.5, OH), 1.38 (3H,

): δ = 161.3 (d, J = 261.8,

13

3

)

2

), 1.37 (3H, OC(CH

3

)

2

); C NMR (100 MHz, CDCl

3

CHC=CF), 137.4 (Ar-C), 128.6 (2 × Ar-CH), 128.1 (Ar-CH), 128.0 (2 × Ar-CH), 114.7 (d, J =9.8,

CHC=CF), 109.3 (C(CH ), 72.9 (OCH Ph), 70.7 (d, J = 9.4, OCH), 67.7 (d, J = 2.9,

CH OC(CH ), 63.4 (d, J = 8.5, C=CCH OCH

25.4 (C(CH ); F NMR (376 MHz, CDCl

3

)

2

3

)

2

Ph), 58.5 (d, J = 31.0, CH

): δ −109.6 (1F, CF). (IR) νmax(cm ): 3422, 3031,

2

OH), 26.2 (C(CH

3

) ),

2

19

-1

3

)

2

3

+

2

934, 2881, 1696, 1454,1371, 1213, 1154, 1053, 1026, 856; HRMS (ESI-TOF) m/z: (M+Na ) calcd

2

1

for C16

H

21FNaO

4

319.1316; found 319.1326; [α]

D

+22.4 (c 1.0, CHCl

3

).

(((5S)-4-((Benzyloxy)methyl)-3-fluoro-5,6-dihydro-2H-pyran-2,5-diyl)bis(oxy))bis(tert-

butyldiphenylsilane) (36a) and (((5S)-4-((Benzyloxy)methyl)-5-(((tert-

butyldiphenylsilyl)oxy)methyl)-3-fluoro-2,5-dihydrofuran-2-yl)oxy)(tert-butyl)diphenylsilane

36b)

(

To a solution of (E)-35 (3.0 g, 10.1 mmol) in CH

2

Cl (45 mL) at 0 °C was added Dess-Martin

2

periodinane (10.7 g, 25.3 mmol) in one portion. The cooling bath was removed and the reaction

allowed to stir at rt for 3 h. An aqueous 10% solution of Na

2

S

2

O

3

(100 mL) was then added and the

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8

9

mixture vigorously stirred for 1 h. Phases were separated and the aqueous extracted with CH

2

Cl (3

2

×

100 mL). The combined organic extracts were dried (Na

2

SO ), filtered and concentrated in

4

vacuo. The crude aldehyde was dissolved in 1,4-dioxane (47 mL) and 0.5 M HCl (23 mL) was

added, the resultant cloudy solution was stirred on the rotary evaporator at 40 °C for 2 h

occasionally turning on the vacuum to remove produced acetone. The reaction was then cooled to 0

10

11

12

13

14

15

16

17

18

19

20

21

22

23

24

25

26

27

28

29

30

31

32

33

34

35

36

37

38

39

40

41

42

43

44

45

46

47

48

49

50

51

52

53

54

55

56

57

58

59

60

°

C and solid NaHCO

residue was triturated with CH

concentrated in vacuo. The residue was dissolved in anhydrous CH

2.60 g, 38.20 mmol) and TBDPSCl (7.5 mL, 28.65 mmol) were added. The reaction was stirred at

rt for 48 h and then quenched by the addition of water (5 mL). The mixture was diluted with

CH Cl (100 mL) then washed successively with 1 M aq. HCl (70 mL) and sat. aq. NaHCO (70

mL). The combined organic extracts were dried (Na SO ), filtered and concentrated in vacuo. The

crude material was purified by column chromatography (SiO , Acetone/n-heptane) to give a 3.5:1

3

was added to adjust the pH 9–10. The mixture was concentrated and the

Cl (3 × 50 mL). The combined organic washes were filtered and

Cl (70 mL) and imidazole

2

(

2

3

2

4

2

mixture of di-TBDPS protected pyranose 36a and furanose 36b (5.7 g, 7.8 mmol, 77% combined

yield over 3 steps) as colorless oil. Additional column chromatography, eluting with a toluene:n-

heptane gradient (0→100%), allowed these products to be separated. The furanose 36b was

isolated as a 4:1 mixture of β:α anomers (colorless oil), while the pyranose 36a (colorless oil) as a

6

:1 mixture of anomers (the stereochemistry of the major and minor anomers of 36a could not be

assigned from analysis of the NOESY NMR spectroscopy).

1

36a, major anomer, isolated: H NMR (400 MHz, CDCl

3

): δ = 7.71–7.64 (8H, m, ArH), 7.44–7.21

), 4.07

), 3.63 (1H, d, J =

); C NMR (100 MHz, CDCl ) δ =

56.3 (d, J = 267, CF), 138.1 (Ar-C), 136.0 (Ar-CH), 135.9 (Ar-CH), 135.8 (Ar-CH), 133.9 (Ar-

C), 133.0 (Ar-C), 133.0 (Ar-C), 132.9 (Ar-C), 129.80 and 129.79 (Ar-CH), 129.6 (Ar-CH), 128.3

(

17H, m, ArH), 5.24 (1H, app s, C1H), 4.40–4.32 (4H, m, PhCH2, C4H and (C3)CH

1H, ddd, J = 1.4, 3.9, 11.5, (C3)CH ), 3.93 (1H, dd, J = 2.2, 12.6, C5H

), 1.04 (9H, s, C(CH

A

H

B

A

H

B

A

H

B

13

12.6, C5H

A

H

B

3

)

3

), 1.01 (9H, s, C(CH

3

)

3

1

(

Ar-CH), 127.8 (Ar-CH), 127.7 (Ar-CH), 127.58 and 127.57 (Ar-CH), 112.8 (d, J = 5, C3), 87.4

d, J = 37, C1H), 72.3 (CH Ph), 65.0 (d, J = 7, CH4), 64.6 (C5H ), 61.8 (d, J = 5, (C3)CH ), 26.9

), 26.7 (C(CH ), 19.4, (SiC(CH ), 19.3 (SiC(CH ); F{ H} NMR (376 MHz,

2

1

9

1

(C(CH

3

)

3

)

3

)

3

3 3

)

-1

CDCl

1

[α]

3

) δ = −122.6; (IR) νmax(cm ): 3071, 3049, 2999, 2957, 2930, 2858, 1427, 1361, 1111, 1077,

+

026, 700; HRMS (ESI-TOF) m/z: (M+Na ) calcd for C45

H

51FNaO

4

2

Si 753.3202; found 753.3185;

2

1

D

+11.4 (c 1.0 CHCl ).

3

1

36a, minor anomer, isolated: H NMR (400 MHz, CDCl

3

) δ = 7.71–7.63 (8H, m, ArH), 7.45–7.20

17H, m, ArH), 5.07 (1H, app s, C1H), 4.60–4.55 (1H, m, C4H), 4.38 (2H, app br s, PhCH ), 4.36

1H, d, J = 11.6, (C3)CH ), 4.32 (1H, d, J = 11.6, (C3)CH ), 3.83 (1H, dd, J = 9.3, 10.8,

), 1.09 (9H, s, C(CH ), 1.07 (9H, s,

(

2

A

H

B

A

H

B

C5H

A

H

B

), 3.28 (1H, ddd, J = 1.1, 5.4, 10.8, C5H

A

H

B

3 3

)

13

C(CH

3

)

3

); C NMR (100 MHz, CDCl

3

) δ = 155.0 (d, J = 237, CF), 138.2 (Ar-C), 136.0 (Ar-CH),

1

35.9 (Ar-CH), 135.79 (Ar-CH), 135.77 (Ar-CH), 133.9 (Ar-C), 132.9 (Ar-C), 132.8 (Ar-C),

32.6 (Ar-C), 129.89 (Ar-CH), 129.87 (Ar-CH), 129.8 (Ar-CH), 128.2 (Ar-CH), 127.70 (Ar-CH),

27.69 (Ar-CH), 127.64 (Ar-CH), 127.61 (Ar-CH), 127.59 (Ar-CH), 127.5 (Ar-CH), 115.8 (d, J =

29

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Article Doi

Synthesis of 2,3-dideoxy-2-fluoro-2,3-endo-methylene-and 2,3-dideoxy-2-fluoro-3-c-hydroxymethyl-2,3-endo-methylene-pentofuranoses and their use in the preparation of conformationally locked bicyclic nucleosides

DOI: 10.1021/jo502712g

Source and publish data:

Authors:

Article abstract of DOI:10.1021/jo502712g

Full text of DOI:10.1021/jo502712g

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