Synthesis and crystal structure of 2′-deoxy-2′-fluoro-4′-thioribonucleosides: substrates for the synthesis of novel modified RNAs

Available online at www.sciencedirect.com

Tetrahedron 64 (2008) 4313e4324

www.elsevier.com/locate/tet

Synthesis and crystal structure of 2⁰-deoxy-2⁰-ﬂuoro-4⁰-thioribonucleosides:

substrates for the synthesis of novel modiﬁed RNAs

a,

Mayumi Takahashi ^a, Shunsuke Daidouji ^a, Motoo Shiro ^b, Noriaki Minakawa ^a,, Akira Matsuda

*

^aGraduate School of Pharmaceutical Sciences, Hokkaido University, Kita-12, Nishi-6, Kita-ku, Sapporo 060-0812, Japan

^bRigaku Corporation, Akishima-shi, Tokyo 196e8666, Japan

Received 4 February 2008; received in revised form 22 February 2008; accepted 22 February 2008

Available online 4 March 2008

Abstract

We report herein the synthesis of appropriately protected 2⁰-deoxy-2⁰-ﬂuoro-4⁰-thiouridine (5), -thiocytidine (7), and -thioadenosine (35) de-

rivatives, substrates for the synthesis of novel modiﬁed RNAs. The synthesis of 5 and 7 was achieved via the reaction of 2,2⁰-O-anhydro-4⁰-thio-

uridine (3) with HF/pyridine in a manner similar to that of its 4⁰-O-congener whereas the synthesis of 35 from 4⁰-thioadenosine derivatives was

unsuccessful. Accordingly, 35 was synthesized via the glycosylation of the ﬂuorinated 4-thiosugar 25 with 6-chloropurine. The X-ray crystal

structural analysis revealed that 2⁰-deoxy-2⁰-ﬂuoro-4⁰-thiocytidine (8) adopted predominately the same C3⁰-endo conformation as 2⁰-deoxy-

2⁰-ﬂuorocytidine.

Keywords: Nucleoside; Fluorination; 2⁰-Deoxy-2⁰-ﬂuoro-4⁰-thionucleoside; X-ray structure

1. Introduction

Chemical modiﬁcation of the 2⁰-position of nucleoside

units is considered to be a highly reliable method for increas-

ing nuclease resistance and hybridization properties of the

resulting RNA molecules.⁷For example, 2⁰-ﬂuoroRNA (2⁰-

FRNA), which consists of 2⁰-deoxy-2⁰-ﬂuoronucleosides, has

the aforementioned favorable properties due to the ﬂuorine

group on the 2⁰-position.⁸In addition, 2⁰-FRNA can be ob-

tained through chemical as well as enzymatic synthesis from

the corresponding nucleoside triphosphates via transcription

by T7 RNA polymerase.⁹Accordingly, a selection of aptamers

by SELEX composed of 2⁰-FRNA was examined, and one of

them has been approved as Macugen^Ò, which is the ﬁrst exam-

ple of a therapeutic aptamer.¹⁰

In view of this information, we planned to synthesize 2⁰-

deoxy-2⁰-ﬂuoro-4⁰-thionucleoside derivatives, a hybrid of 4⁰-

thioribonucleoside and 2⁰-deoxy-2⁰-ﬂuoronucleoside, as new

4⁰-thionucleoside units. The synthesis of the 2⁰-deoxy-2⁰-

ﬂuoro-4⁰-thiouridine and -thiocytidine derivatives 5 and 7 can

be achieved from 2,2⁰-O-anhydro-4⁰-thiouridine (3) in a manner

similar to the one used for its 4⁰-O-congener while the synthesis

of the 2⁰-deoxy-2⁰-ﬂuoro-4⁰-thioadenosine derivative 35

was carried out via condensation between an appropriate

Thus far, a large number of nucleoside derivatives have

been synthesized and incorporated into oligonucleotides

(ONs) for the purpose of applying them to nucleic acid-based

therapeutics including antisense,¹short interfering RNA

(siRNA) strategies,²and aptamers isolated by SELEX technol-

ogy.³In our group, we have been intensely studying the syn-

thesis of a series of 4⁰-thionucleosides⁴and their application

with the aim of developing a functional ON.⁵Among the

4⁰-thionucleic acids investigated, 4⁰-thioRNA, which consists

of 4⁰-thioribonucleosides, seems to be a most promising candi-

date for functional ONs because of its higher nuclease resis-

tance, its hybridization property, and its utility in siRNA

strategy and aptamers.^5,6As a part of our continuing research

project on 4⁰-thioRNA, we envisioned the development of new

4⁰-thionucleoside units to enhance the potential of 4⁰-thioRNA.

* Corresponding authors. Tel.: þ81 11 706 3228; fax: þ81 11 706 4980.

E-mail addresses: noriaki@pharm.hokudai.ac.jp (N. Minakawa), matuda@

pharm.hokudai.ac.jp (A. Matsuda).

doi:10.1016/j.tet.2008.02.071

4314

M. Takahashi et al. / Tetrahedron 64 (2008) 4313e4324

2-ﬂuoro-4-thiosugar and 6-chloropurine. Herein, we wish to re-

port in detail the synthesis of the target compounds. The crystal

structure of 2⁰-deoxy-2⁰-ﬂuoro-4⁰-thiocytidine (8) is also

presented.

at the 3⁰-position of the resulting product was removed by tet-

rabutylammonium ﬂuoride (TBAF) to give 7 in 70% yield in

three steps. Thus compounds 5 and 7, precursors of the phos-

phoramidite unit for the ON synthesis, were prepared effec-

tively. In addition, treatment of 6 with triﬂuoroacetic acid

(TFA) in CH₂Cl₂afforded the 2⁰-deoxy-2⁰-ﬂuoro-4⁰-thiocy-

tidine (8), which was used for structural analysis.

2. Results and discussion

2⁰-Deoxy-2⁰-ﬂuorouridine was ﬁrst prepared in 1961 by

treatment of 2,2⁰-O-anhydrouridine with anhydrous HF.¹¹

This synthetic method was then improved by using HF/pyri-

dine and became a general protocol to afford 2⁰-deoxy-2⁰-ﬂuo-

ropyrimidine nucleosides.^8a,12For the synthesis of the

corresponding purine derivatives, introduction of a ﬂuorine

atom at the 2⁰-position was achieved via an S_N2 type reaction

from arabinofuranosyl purine derivatives.^8a,13With these

methods as examples, the synthesis of 2⁰-deoxy-2⁰-ﬂuoro-4⁰-

thionucleosides was attempted (Scheme 1). Thus, the 4⁰-thio-

uridine derivative 1^4a,5awas converted into the 2,2⁰-O-anhydro

derivative 2 by treatment with triﬂuoromethanesulfonic anhy-

dride (Tf₂O) in the presence of dimethylaminopyridine

(DMAP). The 3⁰,5⁰-O-TIPDS group of 2 was removed by

ammonium ﬂuoride to give 3.¹⁴When 3 was treated with

HF/pyridine in dioxane in a steel container at 125 ^ꢀC, the

2⁰-deoxy-2⁰-ﬂuoro-4⁰-thiouridine (4) was obtained in 88%

yield as is the case of its 4⁰-O-congener.^8aCompound 4 was

then protected with a dimethoxytrityl (DMTr) group to give

5. After acetylation of the 3⁰-hydroxyl group of 5, the resulting

compound was converted into the cytosine derivative 6 by the

usual method. To convert 6 into 7, 6 was heated with benzoic

anhydride in DMF in the absence of an organic amine, such as

triethylamine, which is the usual method for selective protec-

tion of the amino group of the cytosine base.¹⁵However, in

this case, partial deprotection of the 5⁰-O-DMTr group of 7

was observed. Accordingly, 6 was treated with triethylsilyl

chloride (TESCl) to protect the 3⁰-hydroxyl group, followed

by benzoyl chloride (BzCl) in pyridine. Then, the TES group

In order to synthesize the adenosine derivative, preparation

of 4⁰-thioarabinofuranosyl adenine derivatives such as 11 and

13 was ﬁrst examined (Scheme 2). After treatment of the 3⁰,5⁰-

O-TIPDS derivative 9^5awith Tf₂O in pyridine, the resulting

2⁰-O-triﬂate was treated with LiOAc in DMF containing hexa-

methylphosphoramide (HMPA) at room temperature. How-

ever, the desired product 11 was obtained in only 19% yield

along with 49% of 1⁰,2⁰-unsaturated derivative 12. Under the

same conditions, its 4⁰-O-congener afforded the corresponding

arabino-derivative in good yield as reported in the literature.¹⁶

Modiﬁcation of the nucleophile to NaOAc or CsOAc did not

improve the chemical yield of 11. As an alternative method,

oxidation of 10^4dfollowed by reduction of the resulting

2⁰-keto derivative was examined. However, the desired ara-

bino-derivative 13 was obtained in only 28% yield along with

the a-derivative 14. These results would arise from the higher

acidity of the a-hydrogen of 4⁰-thionucleoside (i.e., the H-1⁰

protons) as compared with that of 4⁰-O-congener,¹⁷and would

be a common characteristic of 4⁰-thionucleoside derivatives.^4c

A

O

S

A

+

OAc

TIPDS

B

a,b

c,d

O

S

12

11

TIPDS

O

A^Bz

OH

O

S

9: B = A

10: B = A^Bz

TIPDS

A^Bz

+

TIPDS

OH

O

OH

14

O

13

O

N

Scheme 2. (a) Tf₂O, DMAP, pyridine, CH₂Cl₂; (b) LiOAc, HMPA, DMF; (c)

Ac₂O, DMSO; (d) NaBH₄, MeOH.

NH

N

O

NH

a

c

N

O

N

O

RO

HO

S

Since 11 and 13 were not obtained in sufﬁciently high

yields, condensation between an appropriate 2-ﬂuoro-4-thiosu-

gar derivative and a nucleobase was envisioned as the next tac-

tic. To determine the plausibility of this method, introduction

of a ﬂuorine atom at the 2a-position of the 4-thiosugar was re-

quired. As a ﬂuorination reagent, diethylaminosulfur triﬂuor-

ide (DAST) is widely used, and the reaction generally

proceeds via an S_N2 type pathway. However, in the reaction

of the 4⁰-thionucleoside and the 4-thiosugar derivatives with

DAST, several reported examples suggest that participation

of the sulfur atom may give ﬂuorinated compounds with reten-

tion of the stereochemistry, as depicted in the conversion of 26

into the ﬂuorinated compound 27.^18,19With these previous

results in mind, we anticipated the synthesis of the ﬂuorinated

4-thiosugar, as shown in Scheme 3. The hydroxyl group at the

2-position of 15^4awas inverted by the Mitsunobu reaction to

give 16 as the p-nitrobenzoyl derivative. Deprotection of the

TIPDS

RO

2: R = TIPDS

O

HO

OH

F

1

4

b

3: R = H

d

NHR

N

NH₂

N

O

NH

i

e, f

O

N

O

N

O

DMTrO

g, h

HO

DMTrO

S

HO

6: R = H

HO

F

HO

F

8

5

7: R = Bz

Scheme 1. (a) Tf₂O, DMAP, CH₂Cl₂; (b) NH₄F, MeOH, reﬂux; (c) HF$pyri-

dine, dioxane, 125 ^ꢀC; (d) DMTrCl, pyridine; (e) Ac₂O, Et₃N, DMAP,

CH₃CN; (f) TPSCl, Et₃N, DMAP, CH₃CN, then NH₄OH; (g) TESCl, pyridine,

then BzCl; (h) TBAF, THF; (g) 2% TFA in CH₂Cl₂.

M. Takahashi et al. / Tetrahedron 64 (2008) 4313e4324

4315

O

RO

b

O

HO

S

g

S

c

S

a

f

e

TIPDS

OpNO₂Bz

OR

PMP

O

OH

O

F

RO

O

HO

F

15

16: R = TIPDS

17: R = H

20

22

18: R = pNO₂Bz

19: R = H

21

d

h, i

O

S

TBDPSO

O

TIPDS

O

S

TBDPSO

j

S

k

l

OAc

TIPDS

F

O

PMBzO

F

PMBzO

F

26

27

23

24

25

Scheme 3. (a) p-Nitrobenzoic acid, PPh₃, DIAD, THF; (b) TBAF, AcOH, THF; (c) p-methoxybenzaldehyde dimethylacetal, CSA, DMF; (d) MeNH₂, MeOH; (e)

PBSF, DBU, dioxane, reﬂux; (f) 80% aq AcOH; (g) TIPDSCl₂, pyridine; (h) TBDPSCl, imidazole, DMF; (i) PMBzCl, pyridine; (j) O₃, CH₂Cl₂, ꢁ78 ^ꢀC; (k) Ac₂O,

reﬂux; (l) DAST, CH₂Cl₂, ꢁ15 ^ꢀC.

3,5-O-TIPDS group of 16 by tetrabutylammonium ﬂuoride

(TBAF) afforded 17. To protect the resulting 3- and 5-hy-

droxyl groups, 17 was treated with p-methoxybenzaldehyde

dimethylacetal in the presence of camphorsulfonic acid

(CSA) to give the 3,5-O-p-methoxybenzylidene acetal deriva-

tive 18. To the best our knowledge, 3,5-O-benzylidene acetal

type protecting groups in the furanosides are rare,²⁰and thus

the easy formation of 18 is worth noting. Structural differences

arising from bond lengths and angles between furanosides and

thiofuranosides such as 17 would account for this result. The

p-nitrobenzoyl group of 18 was then removed to give 19, the

substrate for the ﬂuorination. When 19 was heated with per-

ﬂuoro-1-butanesulfonyl ﬂuoride (PBSF)²¹in the presence of

tert-butyldiphenylsilyl (TBDPS) group, a p-methoxybenzoyl

(PMBz) group instead of the expected benzoyl group was intro-

duced at the 3-position to provide more efﬁcient neighboring

group participation and gave 23. To convert 23 into the corre-

sponding 1-O-acetate 25, 23 was at ﬁrst treated with ozone in

CH₂Cl₂at ꢁ78 ^ꢀC to give the sulfoxide 24 quantitatively. Subse-

quent treatment of 24 with Ac₂O under reﬂux gave the desired 25

as a diastereomeric mixture (a/b¼1:1).

Next, the glycosylation of 25 with a nucleobase was exami-

ned. To optimize the reaction conditions (Lewis acid and sol-

vent), the glycosylation with uracil was ﬁrst examined to ease

the detection of a- and b-isomer (Table 1). When SnCl₄was

used as a Lewis acid, decomposition of 25 was observed and

no coupling products were obtained, while the reaction in the

presence of trimethylsilyl triﬂuoromethanesulfonate (TMSOTf)

as a Lewis acid afforded the 4⁰-thiouridine derivatives. Thus,

when 25 was treated with silylated uracil in CH₃CN in the pres-

ence of TMSOTf, 28 was obtained in 77% yield as a diastereo-

meric mixture (a/b¼1:1). In order to improve the b-selectivity,

the reaction solvent was changed to CH₂Cl₂and CCl₄.¹⁸In our

case, however, such attempts resulted in recovery of 25 or partial

deprotection of the TBDPS group at the 5⁰-position to give 29

without improvement of the b-selectivity. To determine whether

the participation came from the 3-O-protecting group in the

glycosylation, we also prepared substrates possessing a

Hunigs base in THF, a ﬂuorinated compound, later conﬁrmed

¨

as 20, was obtained in 29% yield along with the recovered

compound 19 (54%). Therefore, the solvent was changed to

dioxane to increase the reaction temperature. Accordingly,

the ﬂuorinated compound 20 was obtained in 88% yield

when 19 was treated with PBSF in the presence of 1,8-diaza-

bicyclo[5.4.0]undec-7-ene (DBU) in dioxane under reﬂux. To

conﬁrm the stereochemistry of the ﬂuorination, the p-methoxy-

benzylidene acetal group of the ﬂuoro compound (i.e., com-

pound 20) was removed with aqueous acetic acid, and the

resulting product (i.e., compound 21) was converted into

a 3,5-O-TIPDS derivative (i.e., compound 22), whose ¹H

NMR spectrum was not identical with that of 27.¹⁸Hence,

the ﬂuorination on the 2a-position of 19 was achieved by

treatment with PBSF without participation of the sulfur atom

on the 4-position. Since the Mitsunobu reaction to afford 16

also proceeded with inversion of the stereochemistry, the par-

ticipation of the sulfur atom reported so far might be a speciﬁc

phenomenon in the reaction with DAST. Further investigations

will be required for a more detailed discussion.

To install the nucleobase at the C-1 position of the resulting

ﬂuoro-thiosugar, conversion of 21 into an appropriate substrate

for the glycosylation was considered. Watts et al. reported the

synthesis of the 2⁰-ﬂuoro-5-methyl-4⁰-thioarabinouridine deriva-

tive (a/b¼0.7:1) via a Lewis acid catalyzed glycosylation with

assistance of a benzoyl group at the 3-position of the ﬂuoro-

thiosugar.¹⁸With their report as a reference, we prepared the

1-O-acetyl derivative 25, the substrate for the glycosylation, as

shown below. After protection of the 5-OH of 21 with a

Table 1

Glycosylation of silylated uracil with 25

OTMS

O

N

TBDPSO

RO

S

N

NH

OAc

N

OTMS

conditions

O

PMBzO

F

25

28: R = TBDPS

29: R = H

Entry

Conditions

Result

1

2

3

SnCl₄/CH₃CN

TMSOTf/CH₃CN

TMSOTf/CH₂Cl₂

Decomposition

28 (77%, a/b¼1:1)

28 (27%, a/b¼0.8:1)

25 (43%)

4

TMSOTf/CCl₄

28 (12%, a/b¼0.6:1)

29 (27%, a/b¼1.1:1)

4316

M. Takahashi et al. / Tetrahedron 64 (2008) 4313e4324

2,4-dimethoxybenzoyl, a p-nitrobenzoyl, and a methoxymethyl

group instead of the PMBz group of 25, and subjected each of

them to the same reaction. However, all substrates afforded

4⁰-thiouridine derivatives in a similar a/b ratio (from 1:1 to

0.8:1; data not shown), and thus it was concluded that the ex-

pected participation from the 3-O-acyl groups to give the b-se-

lectivity did not occur. Thus far, several examples suggesting

participation from the protecting groups at the 3-position have

been reported to give the glycosylation products in a preferable

b ratio.^18,22Therefore, our results are probably due to the steric

hindrance of the ﬂuorine atom, arising from a larger van der

Waals radius than that of a proton and longer CeF bond length

than that of CeH,²³on the a-face of the thiosugar. In addition,

the 2-ﬂuoro-4-thiosugar derivative is expected to adopt the

C3-endo conformation but not the C2-endo conformation due

to a gauche effect between the electronegative F2 and O4 atoms

(the sugar puckering of 4⁰-thionucleoside derivative will be

described later), and the 3-O-protecting group will locate in

a pseudoequatorial orientation. In this conformation, the 3-

O-protecting group will locate far from its C1 position to afford

preferable b-selectivity (Fig. 1). Although this is, of course, not

the structure of the thionium cation intermediate, this consider-

ation would also be one of the explanations for our results.

The reaction conditions having been ﬁxed (although the b-

selectivity was not satisfactory), we next attempted the glyco-

sylation with 6-chloropurine (Scheme 4). Thus, the thiosugar

25 was treated with silylated 6-chloropurine in CH₃CN in the

presence of TMSOTf at room temperature and the reaction mix-

ture was heated under reﬂux. Upon consumption of 25, the re-

action was quenched and the desired N-9 isomer 31 was

obtained in only 9% yield along with 78% yield of the N-7 iso-

mer 30. Both 30 and 31 were obtained as diastereomeric mix-

tures (a/b¼1:1). A gradual isomerization of 30 to 31 on TLC

analysis was observed on heating with 31, being obtained in

37% yield along with 39% yield of 30 after 16 h of reﬂuxing.

However, further elongation of the reaction time resulted in a re-

duction of the chemical yields of both 30 and 31. Accordingly,

the separated compound 30 was subjected again to the glycosyl-

ation conditions in the presence of the silylated 6-chloropurine

giving 31 in 40% yield. Using these individualized procedures,

we were able to obtain 31 in 52% yield from 25. Then, 31 was

heated in NH₃/EtOH, followed by MeNH₂/MeOH at room tem-

perature to remove the PMBz group, to give 32. When the exo-

cyclic amino group of 32 was benzoylated, the a- and b-isomers

could be separated by ﬂash chromatography to give the desired

b-isomer 33 as the sole product. Deprotection of 33 by TBAF in

THF afforded 34. The resulting 34 was treated with DMTrCl in

pyridine to give the desired 35 as the precursor of the

phosphoramidite unit for the ON synthesis. Likewise, glycosyl-

ation with the silylated 2-amino-6-chloropurine was also

examined to synthesize a 2⁰-deoxy-2⁰-ﬂuoro-4⁰-thioguanosine

derivative. Although the glycosylation with 2-amino-6-chloro-

purine proceeded well to give the corresponding N-7 isomer, no

isomerization to the desired N-9 isomer was achieved under any

conditions examined (data not shown). Therefore, efﬁcient

methods to prepare 2⁰-deoxy-2⁰-ﬂuoro-4⁰-thioguanosine are

still required, and these results will be reported along with the

properties of the corresponding modiﬁed RNA in due course.

Cl

N

a

N

TBDPSO

PMBz

TBDPSO

PMBz

25

+

a

S

N

Cl

O

F

O

F

31

30

b, c

N

NHBz

N

NH₂

d, e

N

S

N

TBDPSO

N

S

N

HO

F

HO

F

33

32

f

NHBz

N

g

HO

DMTrO

S

HO

F

HO

F

34

35

Scheme 4. (a) Silylated 6-chloropurine, TMSOTf, CH₃CN, rt, then reﬂux; (b)

NH₃, EtOH, 80 ^ꢀC; (c) MeNH₂, MeOH; (d) BzCl, pyridine; (e) 1 N NaOH,

EtOH; (f) TBAF, THF; (g) DMTrCl, pyridine.

The structure of 2⁰-deoxy-2⁰-ﬂuoro-4⁰-thiocytidine (8) was

conﬁrmed by X-ray analysis. As can be seen in Figure 2, in-

troduction of the ﬂuorine atom on the a-face of the thiosugar

was conﬁrmed. To date, we and others have reported X-ray

structures of a few 4⁰-thionucleoside derivatives, which were

compared with those of 4⁰-O-congeners.^4b,24In summary, the

overall structures including the sugar puckering and the syn/

anti conformation around the glycosyl bond of the 4⁰-thionu-

cleoside derivatives were essentially similar to the correspond-

ing 4⁰-O-congeners despite a marked conformational change

in the carbohydrate ring. To elucidate the structure of 8, im-

portant geometric parameters for 8 were compared with those

of 2⁰-deoxy-2⁰-ﬂuorocytidine (dfC),²⁵and these data have

been summarized in Table 2. Among the parameters, striking

differences in the bond lengths and angles were observed in

C1⁰eS4⁰and C4⁰eS4⁰, and C4⁰eS4⁰eC1⁰, respectively. Thus,

the bond lengths C1⁰eS4⁰and C4⁰eS4⁰were 1.8287 and

OR

S

OR

S

OR

F

O

F

O

Ar

C2-endo

C3-endo

˚

1.8295 A, respectively, while the reported bond lengths of

˚

dfC were much shorter (i.e., 1.424 and 1.446 A, respectively).

Figure 1. Possible conformation of 2-ﬂuoro-4-thiosugar derivative.

M. Takahashi et al. / Tetrahedron 64 (2008) 4313e4324

4317

markedly. In spite of the partial structural differences between 8

and dfC, their overall structures can be concluded to be similar.

Thus, the cytosine bases are both in the anti conformation with

the glycosidic torsion c (S4⁰eC1⁰eN1eC2)¼ꢁ138.37^ꢀand c

(O4⁰eC1⁰eN1eC2)¼ꢁ150.4^ꢀ. In addition, the C5⁰eC4⁰

bond orientations are both gaucheegauche, i.e., g (O5⁰eC5⁰e

C4⁰eC3⁰)¼51.32^ꢀfor 8 and g (O5⁰eC5⁰eC4⁰eC3⁰)¼51.2^ꢀ

for dfC. Concerning the sugar puckering, every torsion angle

of the sugar ring (n₀en₄) shows some scattering; however, 8

and dfC are both suggested to be in a North-type puckered con-

formation (i.e., C3⁰-endo conformation). Thus, the thiosugar of

8 was estimated to have the pseudorotation phase angle

P¼ꢁ0.9^ꢀand the maximum puckering amplitude n_m¼45.7^ꢀ

while those of the furanose ring in dfC were reported as

P¼22.1^ꢀand n_m¼38.2^ꢀ, respectively.^25,26Torsion angles in-

volving the ﬂuorine atom, each of which is similar, are also

listed in the table. The C3⁰-endo conformation of dfC is ex-

plained by the aforementioned gauche effect between the elec-

tronegative F2⁰and O4⁰atoms (F2⁰eC2⁰eC1⁰eO4⁰fragment).²⁷

Since a sulfur atom is less electronegative than an oxygen atom,

the gauche effect for the F2⁰eC2⁰eC1⁰eS4⁰fragment in 8 is ex-

pected to be weaker compared with that of dfC.²⁸However, at

least in the crystals, this difference was not observed and both

compounds adopted predominately the C3⁰-endo conformation.

Contrary to the crystal structures, the structures in solution were

Figure 2. The crystal structure of 2⁰-deoxy-2⁰-ﬂuoro-4⁰-thiocytidine (8).

The other bond lengths including the glycosidic bond (C1⁰e

N1) were quite similar to those of dfC. In contrast to the

longer bond length of 8, the bond angle C4⁰eS4⁰eC1⁰in

the thiosugar is 95.2^ꢀ, which is 15.1^ꢀless than that of dfC.

The other bond angles in the two sugar moieties do not differ

0

somewhat different. Thus, the coupling constant of J_{1 ,2}of dfC

in its ¹H NMR spectrum was reported as 1.7 Hz in DMSO-d₆,

which supported the predominant C3⁰-endo conformation in so-

lution (the J_{3 ,4}value of dfC was not given in the literature).^8bIn

Table 2

Geometric parameters: bond lengths, angles, and torsion angles that represent

important structural features of 8 and 2⁰-deoxy-2⁰-ﬂuorocytidine (dfC)^a

0

contrast, the J value of 8 in DMSO-d₆was found to be J_{1 ,2}¼4.5

0

and J_{3 ,4}¼5.6 Hz, respectively (see Section 3). Although 8 is es-

timated to prefer the C3⁰-endo conformation from the equation

8

dfC

to calculate the sugar puckering,²⁹the differences in the J_{1 ,2}

˚

Bond lengths (A)

0

C1⁰eN1

1.4699(19)

1.8287(15)

1.533(2)

1.477(4)

values between 8 and dfC should be taken into consideration

in further structural investigations.

b

C1⁰eX4⁰

1.424(4)

1.524(5)

1.513(4)

1.530(5)

1.446(3)

C1⁰eC2⁰

C2⁰eC3⁰

C3⁰eC4⁰

In conclusion, we have synthesized the appropriately pro-

tected 2⁰-deoxy-2⁰-ﬂuoro-4⁰-thiouridine 5, -thiocytidine 7,

and -thioadenosine 35 derivatives as substrates for new modi-

ﬁed RNAs. The synthesis of 5 and 7 was effectively carried

out via 2,2⁰-O-anhydro-4⁰-thiouridine (3) by treatment with

HF/pyridine in a manner similar to that of its 4⁰-O-congener.

Unlike the pyrimidine derivatives, the synthesis of 35 from

the 4⁰-thioadenosine derivatives was unsuccessful. Accord-

ingly, its synthesis was achieved via the glycosylation of the

ﬂuorinated 4-thiosugar 25 with 6-chloropurine. The X-ray

crystal structural analysis of 8 revealed that both 2⁰-deoxy-

2⁰-ﬂuorocytidine (dfC) and 8 adopted predominately the

C3⁰-endo conformation.

1.520(2)

1.539(2)

1.8295(16)

b

C4⁰eX4⁰

Bond angles (^ꢀ)

b

N1eC1⁰eX4⁰

113.85(10)

105.78(9)

107.86(12)

107.44(12)

105.99(10)

95.23(6)

108.4(2)

106.3(2)

103.3(2)

101.5(2)

103.3(2)

110.3(2)

b

X4⁰eC1⁰eC2⁰

C1⁰eC2⁰eC3⁰

C2⁰eC3⁰eC4⁰

b

C3⁰eC4⁰eX4⁰

b

C4⁰eX4⁰eC1⁰

Torsion angles (^ꢀ)

X4⁰eC1⁰eN1eC2^b(c)

O5⁰eC5⁰eC4⁰eC3⁰(g)

C4⁰eX4⁰eC1⁰eC2⁰(n₀)

ꢁ138.37(11)

51.32(17)

13.18(10)

ꢁ150.4(2)

51.2(4)

ꢁ2.9(3)

ꢁ21.4(3)

35.8(3)

b

X4⁰eC1⁰eC2⁰eC3⁰(n₁)

ꢁ35.35(14)

45.72(16)

b

C1⁰eC2⁰eC3⁰eC4⁰(n₂)

3. Experimental section

b

C2⁰eC3⁰eC4⁰eX4⁰(n₃)

ꢁ34.17(14)

11.83(11)

ꢁ37.8(3)

25.8(3)

b

C3⁰eC4⁰eX4⁰eC1⁰(n₄)

3.1. General methods

b

F2⁰eC2⁰eC1⁰eX4⁰

79.68(12)

ꢁ154.85(11)

ꢁ69.03(15)

55.29(16)

91.9(3)

ꢁ149.7(2)

ꢁ77.4(3)

48.2(3)

F2⁰eC2⁰eC1⁰eN1

F2⁰eC2⁰eC3⁰eC4⁰

F2⁰eC2⁰eC3⁰eO3⁰

a

Physical data were measured as follows: melting points are

1

uncorrected. H and ¹³C NMR spectra were recorded at 270,

400, or 500 MHz and 67.5, 100, or 125 MHz instruments in

SDs (standard errors) are given in parentheses.

b

X represents S in the case of 8 and O in the case of dfC.

CDCl₃or DMSO-d₆as the solvent with tetramethylsilane as

4318

M. Takahashi et al. / Tetrahedron 64 (2008) 4313e4324

an internal standard. Chemical shifts are reported in parts per

million (d), and signals are expressed as s (singlet), d (doublet),

t (triplet), q (quartet), m (multiplet), or br (broad). All ex-

changeable protons were detected by the addition of D₂O.

Mass spectra were measured on JEOL JMS-D300 spectrome-

ter. TLC was done on Merck Kieselgel F₂₅₄precoated plates.

Silica gel used for column chromatography was Merck silica

gel 5715. All measurements for X-ray study were made on

a Rigaku RAXIS RAPID imaging plate area detector with

graphite monochromated Cu Ka radiation.

reaction mixture was neutralized with NaHCO₃. The solution

was ﬁltered through a Celite pad, which was washed with

MeOH. The combined ﬁltrate and washings were concentrated

in vacuo. The residue was puriﬁed by a silica gel column, eluted

with MeOH in CHCl₃(5e6%), to give 4 (228 mg, 87%, crystal-

lized from EtOH): mp 137e137.5 ^ꢀC; FAB-LRMS m/z 263

1

(MH^þ); H NMR (DMSO-d₆) d: 11.4 (br s, 1H), 8.09 (d, 1H,

J¼8.0 Hz), 6.07 (dd, 1H, J¼4.8 and 13.4 Hz), 5.77 (d, 1H,

J¼5.4 Hz), 5.69 (d, 1H, J¼8.0 Hz), 5.19 (t, 1H, J¼5.2 Hz),

5.20e5.05 (ddd, 1H, J¼4.8, 8.3, and 50.4 Hz), 4.19e4.12 (m,

1H), 3.73e3.60 (m, 2H), 3.31e3.26 (m, 1H); ¹³C NMR

(DMSO-d₆) d: 162.8, 150.8, 141.1, 102.3, 95.5 (d, J¼228 Hz),

71.2 (d, J¼16.7 Hz), 61.5, 60.5 (d, J¼27.4 Hz), 52.0. Anal.

Calcd for C₉H₁₁FN₂O₄S: C, 41.22; H, 4.23; N, 10.68. Found:

C, 41.12; H, 4.05; N, 10.63.

3.2. 2,2⁰-O-Anhydro-3⁰,5⁰-O-(1,1,3,3-tetraisopropyl-

disiloxane-1,3-diyl)-4⁰-thiouridine (2)

To a solution of 1 (5.8 g, 11.5 mmol) in dry CH₂Cl₂

(120 mL) was added DMAP (5.6 g, 46.0 mmol) followed by

Tf₂O (3.9 mL, 23.0 mmol) at 0 ^ꢀC. After being stirred for

2 h at room temperature, the reaction mixture was quenched

by the addition of saturated aqueous NaHCO₃. The mixture

was concentrated in vacuo, and the residue was partitioned be-

tween AcOEt and H₂O. The separated organic layer was

washed with saturated aqueous NaHCO₃followed by brine.

The organic layer was dried (Na₂SO₄) and concentrated in va-

cuo. The residue was puriﬁed by a silica gel column, eluted

with MeOH in CHCl₃(0e4%), to give 2 (5.2 g, 94%) as

a white foam. An analytical sample was crystallized from

EtOH: mp, 175e176 ^ꢀC; FAB-LRMS m/z 485 (MH^þ); FAB-

HRMS calcd for C₂₁H₃₇N₂O₅SSi₂(MH^þ) 485.1961, found

3.5. 1-[5-O-(4,4⁰-Dimethoxytrityl)-2-deoxy-2-ﬂuoro-4-thio-b-

D-ribofuranosyl]uracil (5)

To a solution of 4 (622 mg, 2.3 mmol) in dry pyridine

(23 mL) was added DMTrCl (1.2 g, 3.5 mmol), and the reac-

tion mixture was stirred for 2 h at room temperature. The re-

action was quenched by the addition of ice. The mixture

was concentrated in vacuo. The residue was diluted with

AcOEt, which was washed with H₂O and saturated aqueous

NaHCO₃, followed by brine. The organic layer was dried

(Na₂SO₄) and concentrated in vacuo. The residue was puriﬁed

by a silica gel column, eluted with MeOH in CHCl₃(0e2%),

to give 5 (1.34 g, quant.) as an ivory foam: FAB-LRMS m/z

565 (MH^þ); FAB-HRMS calcd for C₃₀H₂₉FN₂O₆S (M^þ)

1

485.1975; H NMR (CDCl₃) d: 7.19 (d, 1H, J¼7.6 Hz), 6.09

(d, 1H, J¼7.5 Hz), 5.67 (d, 1H, J¼7.6 Hz), 5.23 (t, 1H,

J¼7.5 Hz), 4.47 (dd, 1H, J¼7.5 and 9.9 Hz), 4.11 (dd, 1H,

J¼2.6 and 12.9 Hz), 3.86 (dd, 1H, J¼2.1 and 12.9 Hz), 3.48

(ddd, 1H, J¼2.1, 2.6, and 12.9 Hz), 1.13e0.93 (m, 28H);

¹³C NMR (CDCl₃) d: 171.7, 158.7, 134.8, 110.5, 87.1, 76.8,

61.5, 58.5, 57.3, 50.7, 18.5, 17.3, 17.2, 17.2, 17.1, 17.0,

16.9, 16.8, 13.8, 13.4, 12.5. Anal. Calcd for C₂₁H₃₆N₂O₅SSi₂:

C, 52.03; H, 7.49; N, 5.78. Found: C, 51.74; H, 7.95; N, 5.31.

1

564.1730, found 564.1728; H NMR (CDCl₃) d: 8.34 (br s,

1H), 7.97 (d, 1H, J¼8.2 Hz), 7.43e7.24 (m, 9H), 6.87e6.84

(m, 4H), 6.20 (dd, 1H, J¼3.3 and 13.0 Hz), 5.47 (d, 1H,

J¼8.2 Hz), 5.04e4.90 (dt, 1H, J¼3.3 and 50.0 Hz), 4.29e

4.20 (m, 1H), 3.79 (s, 6H), 3.61e3.49 (m, 3H), 2.25 (dd,

1H, J¼1.5 and 6.7 Hz); ¹³C NMR (CDCl₃) d: 158.7, 150.2,

144.2, 140.7, 135.0, 134.9, 130.2, 130.1, 128.1, 128.0,

127.2, 113.3, 102.9, 96.6 (d, J¼189 Hz), 87.4, 73.6 (d,

J¼19.1 Hz), 62.2, 55.2, 49.4.

3.3. 2,2⁰-O-Anhydro-4⁰-thiouridine (3)¹⁴

To a solution of 2 (180 mg, 0.37 mmol) in MeOH (4 mL)

was added NH₄F (140 mg, 3.7 mmol), and the reaction mix-

ture was heated under reﬂux for 30 min. The reaction mixture

was cooled to room temperature and concentrated in vacuo.

The residue was puriﬁed by a silica gel column, eluted with

MeOH in CHCl₃(20e30%), to give 3 (90 mg, quant.) as

a white solid: FAB-LRMS m/z 243 (MH^þ); FAB-HRMS calcd

3.6. 1-[5-O-(4,4⁰-Dimethoxytrityl)-2-deoxy-2-ﬂuoro-4-thio-b-

D-ribofuranosyl]cytosine (6)

To a solution of 5 (545 mg, 0.97 mmol) in dry acetonitrile

(10 mL) were added Et₃N (400 mL, 2.9 mmol), Ac₂O (270 mL,

2.9 mmol), and DMAP (11 mg, 0.09 mmol), and the reaction

mixture was stirred for 20 min at room temperature. The reac-

tion was quenched by the addition of ice. The solvent was re-

moved in vacuo and the residue was diluted with AcOEt. The

organic layer was washed with H₂O and saturated aqueous

NaHCO₃followed by brine. The organic layer was dried

(Na₂SO₄) and concentrated in vacuo. The residue was dis-

solved in dry acetonitrile (10 mL), and Et₃N (400 mL,

2.9 mmol), TPSCl (880 mg, 2.9 mmol), and DMAP (350 mg,

2.9 mmol) were added to the solution. The mixture was stirred

for 2 h at room temperature. After the starting material was

consumed, concentrated NH₄OH (28%, 20 mL) was added

1

for C₉H₁₁N₂O₄S (MH^þ) 243.0439, found 243.0444; H NMR

(DMSO-d₆) d: 7.80 (d, 1H, J¼7.5 Hz), 6.18 (d, 1H, J¼7.3 Hz),

5.88e5.84 (m, 2H), 5.35 (d, 1H, J¼7.3 Hz), 5.20 (t, 1H,

J¼5.2 Hz), 4.67e4.66 (m, 1H), 3.41e3.14 (m, 3H).

3.4. 1-(2-Deoxy-2-ﬂuoro-4-thio-b-^D-ribofuranosyl)uracil (4)

To a solution of 3 (250 mg, 1.0 mmol) in MeOH (16 mL) was

added HF/pyridine (70%, 260 mL, 10 mmol), and the reaction

mixture was heated for 48 h at 150 ^ꢀC in a steel container. The

M. Takahashi et al. / Tetrahedron 64 (2008) 4313e4324

4319

and the reaction mixture was kept for 22 h at room tempera-

ture. The whole mixture was concentrated in vacuo. The resi-

due was diluted with H₂O and the aqueous layer was extracted

with CHCl₃(ꢂ3). The combined organic layers were washed

with saturated aqueous NaHCO₃and brine. The organic layer

was dried (Na₂SO₄) and concentrated in vacuo. The residue

was puriﬁed by a silica gel column, eluted with MeOH in

CHCl₃(0e6%), to give 6 (450 mg, 82% in two steps) as a yel-

low foam: FAB-LRMS m/z 564 (MH^þ); FAB-HRMS calcd for

for 1 h at room temperature. The solvent was removed in va-

cuo, and the residue was puriﬁed by a silica gel column, eluted

with MeOH in CHCl₃(0e30%), to give 8 (45 mg, 48% crys-

tallized from H₂O): mp, 159e160.5; FAB-LRMS m/z 262

(MH^þ); ¹H NMR (DMSO-d₆) d: 8.06 (d, 1H, J¼7.5 Hz),

7.27 and 7.24 (each br s, each 1H), 6.16 (dd, 1H, J¼4.5 and

13.9 Hz), 5.78 (d, 1H, J¼7.5 Hz), 5.71 (d, 1H, J¼5.5 Hz),

5.25 (t, 1H, J¼5.3 Hz), 5.07e4.92 (ddd, 1H, J¼3.3, 4.5, and

40.5 Hz), 4.12 (dddd, 1H, J¼3.3, 5.5, 5.6, and 9.1 Hz), 3.73

(ddd, 1H, J¼4.5, 5.3, and 11.7 Hz), 3.63 (dt, 1H, J¼5.3 and

11.7 Hz), 3.31 (ddd, 1H, J¼4.5, 5.3, and 5.6 Hz); ¹³C NMR

(DMSO-d₆) d: 166.2, 157.0, 143.1, 97.0 (d, J¼190.8 Hz),

96.4, 72.1 (d, J¼16.6 Hz), 62.4 (d, J¼27.7 Hz), 62.3, 52.3.

Anal. Calcd for C₉H₁₂FN₃O₃S$2H₂O: C, 36.36; H, 5.42; N,

14.13. Found: C, 36.20; H, 5.42; N, 14.07.

1

C₃₀H₃₁FN₃O₅S (MH^þ) 564.1969, found 564.1962; H NMR

(DMSO-d₆) d: 7.85 (d, 1H, J¼7.4 Hz), 7.40e7.22 (m, 9H),

6.90e6.89 (m, 5H), 6.07 (dd, 1H, J¼2.8 and 14.8 Hz), 5.68

(br s, 2H), 4.95e4.84 (dt, 1H, J¼2.8 and 49.8 Hz), 4.13e

4.08 (m, 1H), 3.73 (s, 6H), 3.44e3.30 (m, 3H); ¹³C NMR

(CDCl₃) d: 165.8, 158.6, 156.7, 144.6, 142.2, 135.6, 135.5,

130.3, 128.3, 128.0, 127.1, 113.3, 97.0 (d, J¼190 Hz), 86.9,

72.6 (d, J¼16.2 Hz), 63.5 (d, J¼29.5 Hz), 55.3, 49.2.

3.9. 9-[2-O-Acetyl-3,5-O-(1,1,3,3-tetraisopropyldisiloxane-

1,3-diyl)-4-thio-b-^D-arabinofuranosyl]adenine (11) and

9-[2-deoxy-3,5-O-(1,1,3,3-tetraisopropyldisiloxane-1,3-diyl)-

4-thio-erythro-pent-1-enofuranosyl]adenine (12)

3.7. N⁴-Benzoyl-1-[5-O-(4,4⁰-dimethoxytrityl)-2-ﬂuoro-4-

thio-b-^D-ribofuranosyl]cytosine (7)

To a solution of 6 (175 mg, 0.31 mmol) in dry pyridine

(3 mL) was added TESCl (68 mL, 0.40 mmol), and the reac-

tion mixture was stirred for 1.5 h at room temperature. After

the starting material was consumed, BzCl (43 mL,

0.37 mmol) was added and the reaction mixture was stirred

for an additional 1.5 h at the same temperature. The reaction

was quenched by the addition of ice and concentrated in va-

cuo. The residue was diluted with AcOEt, which was washed

with H₂O and saturated aqueous NaHCO₃followed by brine.

The organic layer was dried (Na₂SO₄) and concentrated in va-

cuo. The residue was then dissolved in THF (3 mL) and

treated with TBAF (1 M in THF, 0.62 mL, 0.62 mmol) at

0 ^ꢀC. After being stirred for 10 min at the same temperature,

the reaction mixture was concentrated in vacuo, and the mix-

ture was partitioned between AcOEt and H₂O. The separated

organic layer was washed with H₂O and saturated aqueous

NaHCO₃followed by brine. The organic layer was dried

(Na₂SO₄) and concentrated in vacuo. The residue was puriﬁed

by a silica gel column, eluted with MeOH in CHCl₃(0e1%),

to give 7 (144 mg, 70% in three steps) as a white foam: FAB-

LRMS m/z 668 (MH^þ); FAB-HRMS calcd for C₃₇H₃₅FN₃O₆S

To a solution of 9 (500 mg, 0.95 mmol) in dry CH₂Cl₂

(10 mL) were added DMAP (460 mg, 3.8 mmol) and Tf₂O

(240 mL, 1.4 mmol) at 0 ^ꢀC. After being stirred for 40 min at

room temperature, the reaction was quenched by the addition

of ice. The solvent was removed in vacuo and the residue was

diluted with AcOEt. The organic layer was washed with H₂O

and saturated aqueous NaHCO₃followed by brine. The or-

ganic layer was dried (Na₂SO₄) and concentrated in vacuo.

The residue was dissolved in DMF (10 mL) containing

HMPA (4 mL), and LiOAc (470 mg, 5.7 mmol) was added

to the solution. After being stirred for 18 h at room tempera-

ture, the mixture was concentrated in vacuo, and the residue

was partitioned between AcOEt and H₂O. The organic layer

was washed with brine, dried (Na₂SO₄), and concentrated in

vacuo. The residue was puriﬁed by a silica gel column, eluted

with hexane/AcOEt (2:1 to 1:2), to give 11 (100 mg, 19%) as

a yellow foam and 12 (240 mg, 49%) as an orange foam.

Physical data for 11: FAB-LRMS m/z 568 (MH^þ); FAB-

HRMS calcd for C₂₄H₄₂N₅O₅SSi₂(MH^þ) 568.2445, found

1

568.2454; H NMR (CDCl₃) d: 8.40 (s, 1H), 8.31 (s, 1H),

6.12 (d, 1H, J¼6.3 Hz), 5.65 (br s, 2H), 5.48 (dd, 1H, J¼6.3

and 10.3 Hz), 4.65 (t, 1H, J¼10.3 Hz), 4.20 (dd, 1H, J¼3.3

and 12.2 Hz), 4.06 (dd, 1H, J¼2.4 and 12.2 Hz), 3.37 (ddd,

1H, J¼2.4, 3.3, and 10.3 Hz), 1.11e0.98 (m, 28H); ¹³C

NMR (CDCl₃) d: 169.9, 155.6, 153.0, 150.9, 140.6, 119.8,

76.6, 71.8, 59.8, 53.6, 46.4, 20.5, 17.6, 17.5, 17.4, 17.4,

17.1, 17.0, 16.9, 16.7, 13.5, 13.2, 12.4.

Physical data for 12: FAB-LRMS m/z 508 (MH^þ); FAB-

HRMS calcd for C₂₂H₃₈N₅O₃SSi₂(MH^þ) 508.2233, found

1

508.2244; H NMR (CDCl₃) d: 8.44 (s, 1H), 7.92 (s, 1H),

6.39 (d, 1H, J¼2.6 Hz), 5.65 (br s, 2H), 5.64 (dd, 1H, J¼2.6

and 4.4 Hz) 4.17 (dd, 1H, J¼3.7 and 11.0 Hz), 4.12e4.07

(m, 1H), 4.02e3.97 (m, 1H), 1.12e1.01 (m, 28H); ¹³C

NMR (CDCl₃) d: 156.0, 153.8, 149.9, 138.8, 132.3, 120.0,

113.2, 80.9, 65.0, 57.7, 17.7, 17.5, 17.5, 17.4, 17.3, 17.2,

17.0, 16.9, 13.6, 13.1, 12.5.

1

(MH^þ) 668.2231, found 668.2229; H NMR (CDCl₃) d: 9.23

(br s, 1H), 8.64 (d, 1H, J¼8.0 Hz), 7.90 (d, 2H, J¼7.4 Hz),

7.56e7.23 (m, 13H), 6.87e6.85 (m, 4H), 6.10 (d, 1H,

J¼13.1 Hz), 5.18e5.08 (br d, 1H, J¼49.8 Hz), 4.35e4.28

(m, 1H), 3.79 (s, 6H), 3.66e3.52 (m, 3H); ¹³C NMR

(CDCl₃) d: 162.5, 158.5, 155.5, 146.3, 144.0, 135.4, 135.3,

133.0, 132.7, 130.1, 128.7, 128.2, 127.9, 127.7, 127.0,

122.8, 113.1, 96.5 (d, J¼189 Hz), 87.0, 72.4 (d, J¼17.9 Hz),

64.5 (d, J¼29.8 Hz), 61.6, 55.1, 49.2.

3.8. 1-(2-Deoxy-2-ﬂuoro-4-thio-b-^D-ribofuranosyl)-

cytosine (8)

Compound 6 (200 mg, 0.35 mmol) was dissolved in 2%

TFA in CH₂Cl₂(3 mL), and the reaction mixture was stirred

4320

M. Takahashi et al. / Tetrahedron 64 (2008) 4313e4324

3.10. N⁶-Benzoyl-9-[3,5-O-(1,1,3,3-tetraisopropyldisiloxane-

1,3-diyl)-4-thio-b-^D-arabinofuranosyl]adenine (13) and

N⁶-benzoyl-9-[3,5-O-(1,1,3,3-tetraisopropyldisiloxane-

1,3-diyl)-4-thio-a-^D-ribofuranosyl]adenine (14)

J¼8.8 Hz), 5.48 (dd, 1H, J¼8.2 and 15.8 Hz), 4.52 (t, 1H, J¼

8.2 Hz), 4.07 (dd, 1H, J¼3.4 and 12.5 Hz), 3.85 (dd, 1H,

J¼4.9 and 12.5 Hz), 3.35e3.31 (m, 1H), 3.29e3.26 (m, 1H),

2.79 (dd, 1H, J¼8.2 and 10.9 Hz), 1.43e0.93 (m, 28H); ¹³C

NMR (CDCl₃) d: 163.8, 150.4, 134.8, 130.6, 123.4, 79.9,

74.3, 61.7, 48.4, 28.5, 17.5, 17.4, 17.3, 17.1, 13.9, 13.4,

12.9, 12.6.

A mixture of 10 (200 mg, 0.32 mmol) and Ac₂O (1.5 mL)

in DMSO (3 mL) was stirred at room temperature for 2.5 h.

The reaction was quenched by the addition of saturated aque-

ous NaHCO₃. The mixture was partitioned between AcOEt

and H₂O. The separated organic layer was washed with satu-

rated aqueous NaHCO₃followed by brine. The organic layer

was dried (Na₂SO₄) and concentrated in vacuo. The residue

was dissolved in MeOH (6 mL), and NaBH₄(42 mg,

1.1 mmol) was added to the solution. After being stirred for

1 h at room temperature, the reaction mixture was concen-

trated in vacuo, and the residue was partitioned between

AcOEt and H₂O. The organic layer was washed with brine,

dried (Na₂SO₄), and concentrated in vacuo. The residue was

puriﬁed by silica gel column, eluted with MeOH in CHCl₃

(0e2%), to give 13 (56 mg, 28%) as a yellow foam and 14

(110 mg, 53%) as a yellow foam.

3.12. 1,4-Anhydro-2-O-(p-nitrobenzoyl)-4-thio-^D-

arabitol (17)

To a solution of 16 (2.6 g, 4.8 mmol) in THF (50 mL) con-

taining AcOH (550 mL, 9.6 mmol) was added TBAF (1 M in

THF, 9.6 mL, 9.6 mmol) at 0 ^ꢀC. After being stirred for 1 h

at the same temperature, the reaction mixture was concen-

trated in vacuo. The residue was puriﬁed by a silica gel col-

umn, eluted with MeOH in CHCl₃(0e1%), to give 17

(1.4 g, quant.) as a white solid. An analytical sample was crys-

tallized from EtOH/hexane: mp 139 ^ꢀC; FAB-LRMS m/z 299

(M^þ); FAB-HRMS calcd for C₁₂H₁₃NO₆SSi₂(M^þ) 299.046,

found 299.0462; ¹H NMR (DMSO-d₆) d: 8.35 (d, 2H,

J¼8.7 Hz), 8.16 (d, 2H, J¼8.7 Hz), 5.64 (d, 1H, J¼4.9 Hz),

5.30 (dd, 1H, J¼5.0 and 9.6 Hz), 5.00 (t, 1H, J¼5.4 Hz),

4.21 (dd, 1H, J¼4.5 and 9.6 Hz), 3.72e3.66 (m, 1H), 3.44e

3.38 (m, 1H), 3.25e3.18 (m, 2H), 2.90 (dd, 1H, J¼5.0 and

11.6 Hz); ¹³C NMR (DMSO-d₆) d: 163.3, 150.0, 134.6,

130.4, 123.7, 81.0, 76.0, 63.6, 53.4, 31.2. Anal. Calcd for

C₁₂H₁₃NO₆S: C, 48.16; H, 4.38; N, 4.68. Found: C, 48.22;

H, 4.29; N, 4.72.

Physical data for 13: FAB-LRMS m/z 630 (MH^þ); FAB-

HRMS calcd for C₂₉H₄₄N₅O₅SSi₂(MH^þ) 630.2602, found

1

630.2609; H NMR (CDCl₃) d: 9.17 (br s, 1H), 8.69 (s, 1H),

8.56 (s, 1H), 8.01 (d, 2H, J¼7.9 Hz), 7.60e7.50 (m, 3H),

6.09 (d, 1H, J¼6.2 Hz), 4.50e4.47 (m, 1H), 4.38 (t, 1H,

J¼9.2 Hz), 4.20e4.17 (m, 1H), 4.00e3.97 (m, 1H), 3.69 (br

s, 1H), 3.35e3.32 (m, 1H), 1.11e0.98 (m, 28H); ¹³C NMR

(CDCl₃) d: 164.9, 152.6, 152.3, 149.5, 143.3, 133.7, 132.7,

128.8, 128.0, 123.0, 77.3, 74.5, 60.0, 56.4, 47.5, 17.6, 17.5,

17.4, 17.2, 17.1, 17.0, 16.9, 13.5, 13.2, 13.1, 12.6.

Physical data for 14: FAB-LRMS m/z 630 (MH^þ); FAB-

HRMS calcd for C₂₉H₄₄N₅O₅SSi₂(MH^þ) 630.2602, found

1

630.2589; H NMR (CDCl₃) d: 8.99 (br s, 1H), 8.78 (s, 1H),

3.13. 1,4-Anhydro-3,5-O-(p-methoxybenzylidene)-4-thio-

D-arabitol (19)

To a solution of 17 (7.2 g, 24 mmol) in dry DMF (240 mL)

were added CSA (2.8 g, 12 mmol) and p-methoxybenzalde-

hyde dimethylacetal (16 mL, 96 mmol). The reaction mixture

was stirred under reduced pressure (20 mmHg) at 35 ^ꢀC for

7.5 h. The reaction mixture was cooled to room temperature

and the reaction was quenched by the addition of saturated

aqueous NaHCO₃. The whole mixture was concentrated in va-

cuo and the residue was diluted with AcOEt. The organic layer

was washed with H₂O and saturated aqueous NaHCO₃fol-

lowed by brine. The organic layer was dried (Na₂SO₄) and

concentrated in vacuo to give 18. The resulting 18 was dis-

solved in a MeOH solution of MeNH₂(40%, 200 mL) and

the mixture was kept for 3 h at room temperature. The solvent

was removed in vacuo and the residue was coevaporated with

MeOH. The residue was puriﬁed by a silica gel column, eluted

with hexane/AcOEt (7:1e3:2), to give 19 (5.5 g, 85%) as

a white solid. An analytical sample was crystallized from

EtOH/hexane: mp 142 ^ꢀC; FAB-LRMS m/z 269 (MH^þ);

FAB-HRMS calcd for C₁₃H₁₆O₄S (MH^þ) 269.0848, found

8.57 (s, 1H), 8.02 (d, 2H, J¼7.0 Hz), 7.63e7.51 (m, 3H),

6.46 (d, 1H, J¼4.5 Hz), 4.50e4.47 (m, 2H), 4.13 (dd, 1H,

J¼2.8 and 12.3 Hz), 4.04e3.92 (m, 2H), 2.90 (br s, 1H),

1.16e1.00 (m, 28H).

3.11. 1,4-Anhydro-2-O-(p-nitrobenzoyl)-3,5-O-(1,1,3,3-

tetraisopropyldisiloxane-1,3-diyl)-4-thio-^D-arabitol (16)

To a mixture of 15 (1.0 g, 2.5 mmol), p-nitrobenzoic acid

(840 mg, 5.0 mmol), and PPh₃(1.3 g, 5.0 mmol) in dry THF

(20 mL) was added a solution of DIAD (990 mL, 5.0 mmol)

in dry THF (5 mL) dropwise via cannula. After the reaction

mixture was stirred for 20 min at room temperature, the reac-

tion was quenched by the addition of ice. The solvent was re-

moved in vacuo. The residue was partitioned between AcOEt

and H₂O. The separated organic layer was washed with H₂O

and saturated aqueous NaHCO₃followed by brine. The or-

ganic layer was dried (Na₂SO₄) and concentrated in vacuo.

The residue was puriﬁed by a silica gel column, eluted with

hexane/AcOEt (40:1 to 30:1), to give 16 (1.3 g, quant.) as

a yellow oil: FAB-LRMS m/z 541 (M^þ); FAB-HRMS calcd

for C₂₄H₃₉NO₇SSi₂(M^þ) 541.1986, found 541.1978; ¹H

NMR (CDCl₃) d: 8.30 (d, 2H, J¼8.8 Hz), 8.20 (d, 2H,

1

269.0843; H NMR (CDCl₃) d: 7.43 (d, 2H, J¼8.8 Hz), 6.91

(d, 2H, J¼8.8 Hz), 5.53 (s, 1H), 4.40 (dd, 1H, J¼4.3 and

10.4 Hz), 4.33 (ddd, 1H, J¼3.3, 8.2, and 10.4 Hz), 3.89e

3.83 (m, 1H), 3.80 (s, 3H), 3.75e3.70 (m, 1H), 3.30e3.23

(m, 1H), 3.17 (dd, 1H, J¼8.2 and 10.4 Hz), 2.74 (dd, 1H,

M. Takahashi et al. / Tetrahedron 64 (2008) 4313e4324

4321

J¼8.2 and 10.0 Hz), 2.45 (d, 1H, J¼3.3 Hz); ¹³C NMR

(CDCl₃) d: 160.0, 129.4, 127.4, 113.6, 102.1, 87.8, 73.4,

72.0, 55.3, 39.1, 30.2. Anal. Calcd for C₁₃H₁₆O₄S: C, 58.19;

H, 6.01. Found: C, 57.83; H, 5.94.

reaction was quenched by the addition of saturated aqueous

NaHCO₃. The solvent was removed in vacuo, and the residue

was partitioned between AcOEt and H₂O. The separated or-

ganic layer was washed with saturated aqueous NaHCO₃and

brine. The organic layer was dried (Na₂SO₄) and concen-

trated in vacuo. The residue was puriﬁed by a silica gel col-

umn, eluted with hexane/AcOEt (30:1), to give 22 (202 mg,

91%) as a colorless oil: FAB-LRMS m/z 395 (MH^þ); FAB-

HRMS calcd for C₁₇H₃₆FO₃SSi₂(MH^þ) 395.1908, found

395.1908; ¹H NMR (CDCl₃) d: 5.15e5.02 (br d, 1H,

J¼53.4 Hz), 4.17 (ddd, 1H, J¼2.6, 9.9, and 28.4 Hz), 4.07

(dd, 1H, J¼2.8 and 12.5 Hz), 3.93 (dd, 1H, J¼2.0 and

12.5 Hz), 3.56e3.53 (m, 1H), 3.15e2.93 (m, 2H), 1.09e0.98

(m, 28H); ¹³C NMR (CDCl₃) d: 94.6 (d, J¼186.0 Hz),

75.6 (d, J¼18.0 Hz), 58.8, 48.5, 31.2 (d, J¼22.8 Hz),

17.8, 17.7, 17.7, 17.6, 17.6, 17.5, 17.4, 13.9, 13.6, 13.0,

12.9.

3.14. 1,4-Anhydro-2-deoxy-2-ﬂuoro-3,5-O-(p-methoxy-

benzylidene)-4-thio-^D-ribitol (20)

To a solution of 19 (5.0 g, 19 mmol) in dry dioxane

(370 mL) were added DBU (28 mL, 186 mmol) and per-

ﬂuoro-1-butanesulfonyl ﬂuoride (PBSF, 35 mL, 186 mmol)

at 0 ^ꢀC. The reaction mixture was heated under reﬂux for

15 min. The reaction mixture was cooled to room temperature

and the reaction was quenched by the addition of saturated

aqueous NaHCO₃. The solvent was removed in vacuo. The

residue was diluted with AcOEt. The organic layer was

washed with H₂O and saturated aqueous NaHCO₃followed

by brine. The organic layer was dried (Na₂SO₄) and concen-

trated in vacuo. The residue was puriﬁed by a silica gel col-

umn, eluted with hexane/AcOEt (5:1 to 1:1), to give 20

(4.4 g, 88%) as a white solid. An analytical sample was crys-

tallized from EtOH/hexane: mp 131e132 ^ꢀC; FAB-LRMS m/z

271 (MH^þ); ¹H NMR (CDCl₃) d: 7.45 (d, 2H, J¼8.6 Hz), 6.89

(d, 2H, J¼8.6 Hz), 5.53 (s, 1H), 5.40e5.26 (br d, 1H,

J¼53 Hz), 4.58 (dd, 1H, J¼3.7 and 10.2 Hz), 3.91e3.86 (m,

1H), 3.80 (s, 3H), 3.71e3.59 (m, 2H), 3.22e3.03 (m, 2H);

¹³C NMR (CDCl₃) d: 160.0, 129.2, 127.5, 113.6, 102.3, 90.3

(d, J¼185 Hz), 85.8 (d, J¼17.4 Hz), 72.0, 55.3, 38.8, 32.2

(d, J¼23.2 Hz). Anal. Calcd for C₁₃H₁₅FO₃S: C, 57.76; H,

5.59. Found: C, 57.79; H, 5.49.

3.17. 1,4-Anhydro-5-O-(tert-butyldiphenylsilyl)-2-deoxy-

2-ﬂuoro-3-O-(p-methoxybenzoyl)-4-thio-^D-ribitol (23)

To a solution of 21 (550 mg, 3.6 mmol) in DMF (24 mL)

were added imidazole (880 mg, 13 mmol) and TBDPSCl

(1.1 mL, 4.3 mmol), and the reaction mixture was stirred for

2 h at room temperature. The reaction was quenched by the

addition of MeOH. The solution was concentrated in vacuo,

and the residue was partitioned between AcOEt and H₂O.

The separated organic layer was washed with saturated aque-

ous NaHCO₃and brine. The organic layer was dried (Na₂SO₄)

and concentrated in vacuo. The residue was dissolved in dry

pyridine (24 mL), and p-methoxybenzoyl chloride (1.5 mL,

11 mmol) was added to the solution at 0 ^ꢀC. The reaction mix-

ture was stirred for 1.5 h at room temperature. The reaction

was quenched by the addition of ice. The solution was concen-

trated in vacuo, and the residue was partitioned between

AcOEt and H₂O. The separated organic layer was washed

with saturated aqueous NaHCO₃and brine. The organic layer

was dried (Na₂SO₄) and concentrated in vacuo. The residue

was puriﬁed by a silica gel column, eluted with hexane/AcOEt

(10:1), to give 23 (1.9 g, quant. in two steps) as a colorless oil:

FAB-LRMS m/z 525 (MH^þ); FAB-HRMS calcd for

3.15. 1,4-Anhydro-2-deoxy-2-ﬂuoro-4-thio-^D-ribitol (21)

A mixture of 20 (3.0 g, 11.1 mmol) and 80% aqueous

AcOH (100 mL) was heated at 60 ^ꢀC for 1 h. The reaction

mixture was cooled to room temperature and concentrated in

vacuo. The residue was coevaporated with MeOH and puriﬁed

by a silica gel column, eluted with hexane/AcOEt (2:1 to 1:1),

to give 21 (1.4 g, 86%) as an ivory solid. An analytical sample

was crystallized from EtOH: mp 76 ^ꢀC; FAB-LRMS m/z 271

(MH^þ); ¹H NMR (DMSO-d₆) d: 5.41 (d, 1H, J¼6.3 Hz),

5.09e4.96 (dddd, 1H, J¼2.8, 4.0, 6.3, and 53.2 Hz), 4.86 (t,

1H, J¼4.5 Hz), 3.85e3.78 (m, 1H), 3.77e3.73 (m, 1H),

3.39e3.21 (m, 1H), 3.24 (m, 1H), 3.01 (ddd, 1H, J¼4.0,

12.0, and 33.8 Hz), 2.83 (ddd, 1H, J¼2.8, 12.0, and

19.4 Hz); ¹³C NMR (DMSO-d₆) d: 94.8 (d, J¼180 Hz),

77.3, 62.1, 50.0 (d, J¼1.6 Hz), 31.2 (d, J¼21.6 Hz). Anal.

Calcd for C₅H₉FO₂S: C, 39.46; H, 5.96. Found: C, 39.31; H,

5.63.

1

C₂₉H₃₄FO₄SSi (MH^þ) 525.1937, found 525.1937; H NMR

(CDCl₃) d: 7.98 (d, 2H, J¼7.4 Hz), 7.69e7.63 (m, 4H),

7.43e7.30 (m, 6H), 6.92 (d, 2H, J¼8.6 Hz), 5.45e5.34 (m,

2H), 3.89e3.83 (m, 3H), 3.87 (s, 3H), 3.27e3.13 (m, 2H),

1.04 (s, 9H); ¹³C NMR (CDCl₃) d: 165.3, 163.7, 135.6,

132.9, 131.9, 129.7, 127.7, 121.6, 113.6, 92.1 (d,

J¼187 Hz), 76.3 (d, J¼16.7 Hz), 64.4, 55.4, 48.2, 31.6 (d,

J¼21.4 Hz), 26.6, 19.1, 14.1.

3.16. 1,4-Anhydro-2-deoxy-2-ﬂuoro-3,5-O-(1,1,3,3-

3.18. 1,4-Anhydro-5-O-(tert-butyldiphenylsilyl)-2-deoxy-

tetraisopropyldisiloxane-1,3-diyl)-4-thio-^D-ribitol (22)

2-ﬂuoro-3-O-(p-methoxybenzoyl)-4-sulﬁnyl-^D-ribitol (24)

To a solution of 21 (85 mg, 0.56 mmol) in dry pyridine

(5 mL) was added

Ozone gas was bubbled through a solution of 23 (4.7 g,

9.1 mmol) in CH₂Cl₂(60 mL) at ꢁ78 ^ꢀC. After 2 h, argon

gas was bubbled through the reaction mixture to remove ex-

cess ozone. The reaction mixture was allowed to warm to

a solution of TIPDSCl₂(268 mL,

0.84 mmol) in dry pyridine (1 mL) dropwise via cannula at

0 ^ꢀC. After being stirred for 3 h at room temperature, the

4322

M. Takahashi et al. / Tetrahedron 64 (2008) 4313e4324

room temperature and concentrated in vacuo. The residue was

puriﬁed by a silica gel column, eluted with hexane/AcOEt (2:1

to 1:1), to give 24 (4.9 g, quant.) as a diastereomeric mixture

(isomer a/isomer b¼0.8:1): FAB-LRMS m/z 541 (MH^þ);

FAB-HRMS calcd for C₂₉H₃₄FO₅SSi (MH^þ) 541.1881, found

541.1876; ¹H NMR (CDCl₃) d: 8.00 (d, 2H of isomer a,

J¼9.1 Hz), 7.83 (d, 2H of isomer b, J¼9.1 Hz), 7.71e7.21

(m, 10H of isomers a and b), 6.94 (d, 2H of isomer a,

J¼9.1 Hz), 6.89 (d, 2H of isomer b, J¼9.1 Hz), 5.72e5.61

(m, 1H of isomers a and b), 5.57e5.49 (m, 1H of isomers

a and b), 4.43 (dd, 1H of isomer a, J¼2.3 and 11.3 Hz),

4.35 (t, 1H of isomer b, J¼10.3 Hz), 4.02e3.97 (m, 1H of iso-

mers a and b), 3.89 and 3.87 (each s, each 3H of isomers a and

b), 3.86e3.78 (m, 1H of isomer b), 3.62e3.60 (m, 1H of iso-

mer a), 3.50e3.45 (m, 1H of isomer b), 3.35e3.26 (m, 2H of

isomer a), 3.14e3.04 (m, 1H of isomer b), 1.07 and 1.02 (each

s, each 9H of isomers a and b); ¹³C NMR (CDCl₃) d: 165.3,

165.2, 164.0, 163.9, 135.6, 135.5, 135.4, 133.0, 132.5,

132.1, 132.0, 132.0, 132.0, 130.1, 130.0, 129.9, 129.9,

128.0, 127.9, 127.8, 121.0, 120.9, 113.9, 113.7, 92.2 (d, J¼

188.8 Hz), 90.5 (d, J¼185.8 Hz), 77.3, 74.3 (d, J¼17.1 Hz),

73.1, 72.4 (d, J¼17.1 Hz), 63.5, 58.6, 58.0 (d, J¼22.8 Hz),

57.6, 55.6, 55.5, 54.4 (d, J¼20.0 Hz), 29.7, 26.8, 26.7,

19.2.

3.20. 6-Chloro-7-[5-O-(tert-butyldiphenylsilyl)-2-deoxy-2-

ﬂuoro-3-O-(4-methoxybenzoyl)-4-thio-a/b-^D-ribofuranosyl]-

9H-purine (30) and 6-chloro-9-[5-O-(tert-butyldiphenylsilyl)-

2-deoxy-2-ﬂuoro-3-O-(4-methoxybenzoyl)-4-thio-a/b-^D-

ribofuranosyl]-9H-purine (31)

To a suspension of 6-chloropurine (247 mg, 1.6 mmol) in

dry acetonitrile (10 mL) was added HMDS (510 mL,

2.4 mmol), and the mixture was heated under reﬂux until the

reaction mixture turned to be a clear solution to give silylated

6-chloropurine. After the solution was cooled to room temper-

ature, a solution of 25 (238 mg, 0.4 mmol) in dry acetonitrile

(4 mL) and TMSOTf (217 mL, 1.2 mmol) were successively

added to the solution of silylated 6-chloropurine at 0 ^ꢀC. After

being stirred at room temperature for 1 h, the reaction mixture

was heated under reﬂux for 16 h. The reaction was quenched

by the addition of saturated aqueous NaHCO₃, and the reac-

tion mixture was partitioned between AcOEt and H₂O. The

separated organic layer was washed with saturated aqueous

NaHCO₃followed by brine. The organic layer was dried

(Na₂SO₄) and concentrated in vacuo. The residue was puriﬁed

by a silica gel column, eluted with hexane/AcOEt (4:1 to 1:1),

to give a mixture of a/b-anomer (1:1) of 31 (98 mg, 37%) as

a yellow foam and a mixture of a/b-anomer of 30 (104 mg,

39%) as an orange foam.

3.19. 1-O-Acetyl-5-O-(tert-butyldiphenylsilyl)-2-deoxy-2-

ﬂuoro-3-O-(p-methoxybenzoyl)-4-thio-a/b-^D-ribo-

furanose (25)

Physical data for 31: FAB-LRMS m/z 677 (MH^þ); FAB-

HRMS calcd for C₃₄H₃₅FN₄O₄SSi (MH^þ) 677.1820, found

677.1816; ¹H NMR (CDCl₃) d: 8.74 (s, 0.5H), 8.70 (s,

0.5H), 8.69 (d, 0.5H, J¼2.2 Hz), 8.56 (s, 0.5H), 7.98 (d, 1H,

J¼8.6 Hz), 7.88 (d, 1H, J¼9.1 Hz), 7.73e7.28 (m, 10H),

6.93e6.89 (m, 2H), 6.68 (dd, 0.5H, J¼4.0 and 18.3 Hz),

6.37 (dd, 0.5H, J¼4.0 and 13.1 Hz), 5.84e5.47 (m, 2H),

4.40e4.38 (m, 0.5H), 4.09e3.97 (m, 2.5H), 3.86 and 3.85

(each s, 3H), 1.11 and 1.08 (each s, 9H); ¹³C NMR (CDCl₃)

d: 165.1, 165.0, 164.1, 164.1, 152.2, 152.1, 151.9, 151.6,

151.5, 151.2, 145.7, 145.7, 144.0, 135.6, 135.6, 132.6,

132.5, 132.5, 132.3, 132.1, 132.1, 131.7, 130.1, 130.1,

130.0, 130.0, 128.0, 127.9, 127.9, 127.9, 121.0, 120.7,

113.9, 113.9, 93.9 (d, J¼197.9 Hz), 91.2 (d, J¼196.7 Hz),

74.3 (d, J¼15.6 Hz), 73.1 (d, J¼15.6 Hz), 63.4, 63.5, 60.9,

60.6, 60.4, 57.1, 56.9, 55.6, 51.2, 49.9, 26.9, 26.8, 19.3, 19.3.

Physical data for 30: FAB-LRMS m/z 677 (MH^þ); FAB-

HRMS calcd for C₃₄H₃₅FN₄O₄SSi (MH^þ) 677.1821, found

677.1827; ¹H NMR (CDCl₃) d: 9.08 (s, 0.5H), 9.02 (d,

0.5H, J¼1.7 Hz), 8.94 (s, 0.5H), 8.91 (s, 0.5H), 7.95 (d, 1H,

J¼9.1 Hz), 7.83 (d, 1H, J¼9.7 Hz), 7.71e7.35 (m, 10H),

6.96e6.90 (m, 2.5H), 6.71 (dd, 0.5H, J¼2.8 and 10.8 Hz),

5.68e5.45 (m, 2H), 4.28e4.24 (m, 0.5H), 4.08e3.91 (m,

2.5H), 3.89 and 3.86 (each s, 3H), 1.11 and 1.10 (each s,

9H); ¹³C NMR (CDCl₃) d: 165.1, 165.0, 164.2, 162.8, 162.5,

153.0, 152.6, 149.5, 147.7, 142.8, 142.6, 135.7, 135.6,

135.5, 132.5, 132.4, 132.4, 132.1, 130.2, 130.2, 130.1,

130.1, 128.1, 128.0, 122.9, 122.5, 120.7, 120.5, 114.0,

113.9, 95.0 (d, J¼196.7 Hz), 91.4 (d, J¼201.5 Hz), 74.6 (d,

J¼16.7 Hz), 72.2 (d, J¼16.7 Hz), 63.8, 62.8 (d, J¼29.9 Hz),

62.4, 60.3 (d, J¼17.9 Hz), 55.6, 51.6, 49.6, 26.9, 26.8, 19.3,

19.2.

A mixture of 24 (360 mg, 0.67 mmol) and Ac₂O (2.5 mL)

was heated under reﬂux for 40 min. The reaction mixture

was cooled to room temperature and the reaction was

quenched by the addition of ice. The mixture was partitioned

between AcOEt and H₂O. The separated organic layer was

washed with saturated aqueous NaHCO₃followed by brine.

The organic layer was dried (Na₂SO₄) and concentrated in

vacuo. The residue was puriﬁed by a silica gel column,

eluted with hexane/AcOEt (2:1 to 1:1), to give 25

(370 mg, 94%) as a diastereomeric mixture (isomer a/isomer

b¼0.4:1): FAB-LRMS m/z 583 (MH^þ); FAB-HRMS calcd

for C₃₁H₃₆FO₆SSi (MH^þ) 583.1986, found 583.1983; ¹H

NMR (CDCl₃) d: 8.06 (d, 2H of isomer b, J¼8.6 Hz), 7.96

(d, 2H of isomer a, J¼9.0 Hz), 7.73e7.26 (m, 10H of iso-

mers a and b), 6.94e6.92 (m, 2H of isomers a and b),

6.35 (t, 1H of isomer b, J¼4.5 Hz), 6.04 (dd, 1H of isomer

a, J¼1.8 and 9.9 Hz), 5.74e5.71 (m, 1H of isomer b), 5.66e

5.57 (m, 1H of isomer a), 5.40e5.25 (m, 1H of isomers

a and b), 3.90e3.70 (m, 6H of isomers a and b), 2.19 (s,

3H of isomer b), 2.10 (s, 3H of isomer a), 1.06 (s, 9H of iso-

mer b), 1.03 (s, 9H of isomer a); ¹³C NMR (CDCl₃) d:

170.2, 169.4, 165.4, 165.2, 163.9, 163.8, 135.8, 135.7,

135.6, 135.6, 132.9, 132.7, 132.5, 132.3, 132.1, 130.1,

130.0, 129.9, 129.8, 127.9, 127.8, 127.7, 121.8, 121.4,

113.8, 113.7, 93.9 (d, J¼188.3 Hz), 90.2 (d, J¼206.3 Hz),

76.9, 76.8, 74.1 (d, J¼16.9 Hz), 74.0 (d, J¼16.7 Hz), 64.6,

63.2, 58.2, 55.5, 50.5, 50.5, 48.6, 48.6, 26.8, 26.7, 21.1,

21.0, 19.3.

M. Takahashi et al. / Tetrahedron 64 (2008) 4313e4324

4323

3.21. 9-[5-O-(tert-Butyldiphenylsilyl)-2-deoxy-2-ﬂuoro-4-

thio-a/b-^D-ribofuranosyl]adenine (32)

3.72e3.68 (m, 1H), 2.79 (br s, 1H), 1.08 (s, 9H); ¹³C NMR

(CDCl₃) d: 164.6, 152.8, 151.6, 149.6, 141.8, 135.5,

133.4, 132.8, 132.5, 132.3, 130.1, 128.8, 127.9, 123.4, 96.3

(d, J¼192 Hz), 73.7, 64.4, 60.0 (d, J¼28.7 Hz), 51.0, 26.8,

19.2.

Compound 31 (170 mg, 0.24 mmol) was dissolved in etha-

nolic ammonia (saturated at 0 ^ꢀC, 5 mL) and the mixture was

heated at 80 ^ꢀC for 3.5 h in a steel container. The solvent was

removed in vacuo. The residue was dissolved in a MeOH so-

lution of MeNH₂(40%, 5 mL) and the mixture was kept at

room temperature for 1 h. The solvent was removed and

the residue was coevaporated with MeOH. The residue was

puriﬁed by a silica gel column, eluted with MeOH in

CHCl₃(0e3%), to give a mixture of a/b-anomer (1:1) of

32 (110 mg, 88%) as a white foam: FAB-LRMS m/z 524

(MH^þ); FAB-HRMS calcd for C₂₆H₃₁FN₅O₂SSi (MH^þ)

3.23. N⁶-Benzoyl-9-(2-deoxy-2-ﬂuoro-4-thio-b-D-

ribofuranosyl)adenine (34)

To a solution of 33 (270 mg, 0.42 mmol) in THF (4 mL)

was added TBAF (1 M in THF, 0.84 mL, 0.84 mmol) at

0 ^ꢀC. After being stirred for 30 min at the same temperature,

the reaction mixture was concentrated in vacuo. The residue

was puriﬁed by a silica gel column, eluted with MeOH in

CHCl₃(0e2.5%), to give 34 (160 mg, 98%) as a white glass:

FAB-LRMS m/z 390 (MH^þ); FAB-HRMS calcd for

1

524.1952, found 524.1963; H NMR (DMSO-d₆) d: 8.35 (s,

1H), 8.15 (s, 0.5H), 8.09 (s, 0.5H), 7.66e7.41 (m, 10H),

7.10 and 6.89 (each br s, 2H), 6.47 (dd, 0.5H, J¼4.0 and

22.3 Hz), 6.17 (dd, 0.5H, J¼4.0 and 13.7 Hz), 5.92e5.88

(m, 1H), 5.57e5.46 (dt, 0.5H, J¼4.0 and 49.8 Hz), 5.19e

5.07 (dt, 1H, J¼4.0 and 49.8 Hz), 4.57e4.53 (m, 0.5H),

4.36e4.34 (m, 0.5H), 4.14e3.95 (m, 2H), 3.80e3.76 (m,

0.5H), 3.59e3.57 (m, 0.5H), 1.00 (s, 9H); ¹³C NMR

(CDCl₃) d: 155.7, 155.6, 153.1, 152.9, 149.8, 149.7, 141.4,

139.4, 135.7, 135.6, 135.5, 132.7, 132.6, 132.6, 130.1,

130.1, 130.0, 127.9, 127.9, 127.9, 127.9, 119.9, 118.8, 96.8

(d, J¼191.6 Hz), 93.8 (d, J¼193.6 Hz), 75.2 (d,

J¼16.2 Hz), 73.0 (d, J¼16.2 Hz), 64.8, 64.2, 57.9 (d,

J¼29.5 Hz), 56.4 (d, J¼17.1 Hz), 52.8, 51.4, 26.9, 26.8,

19.3, 19.3.

1

C₁₇H₁₇FN₅O₃S (MH^þ) 390.1036, found 390.1032; H NMR

(DMSO-d₆) d: 11.2 (br s, 1H), 8.82 (s, 1H), 8.77 (s, 1H),

8.03 (d, 2H, J¼7.4 Hz), 7.64 (t, 1H, J¼7.4 Hz), 7.54 (t, 2H,

J¼7.4 Hz), 6.29 (dd, 1H, J¼4.5 and 13.1 Hz), 5.90 (d, 1H,

J¼5.7 Hz), 5.56e5.31 (dt, 1H, J¼4.5 and 50.4 Hz), 5.32 (t,

1H, J¼5.7 Hz), 4.51e4.47 (m, 1H), 3.89e3.84 (m, 1H,

J¼5.7 and 11.4 Hz), 3.74e3.69 (m, 1H, J¼5.7 and 11.4 Hz),

3.45e3.43 (m, 1H); ¹³C NMR (DMSO-d₆) d: 165.6, 152.1,

151.8, 150.5, 143.2, 133.2, 132.5, 128.5, 125.8, 96.0 (d,

J¼191 Hz), 71.5 (d, J¼16.2 Hz), 62.0, 59.0 (d, J¼27.7 Hz),

52.3.

3.24. N⁶-Benzoyl-9-[2-deoxy-2-ﬂuoro-5-O-(4,4⁰-

3.22. N⁶-Benzoyl-9-[5-O-(tert-butyldiphenylsilyl)-2-deoxy-2-

dimethoxytrityl)-4-thio-b-^D-furanosyl]adenine (35)

ﬂuoro-4-thio-b-^D-ribofuranosyl]adenine (33)

Compound 35 (250 mg, 95%) was obtained as a yellow

foam from 34 (150 mg, 0.38 mmol) as described above for

the synthesis of 5, after puriﬁcation by silica gel column chro-

matography, eluted with MeOH in CHCl₃(0e1.5%): FAB-

LRMS m/z 692 (MH^þ); FAB-HRMS calcd for C₃₈H₃₅FN₅O₅S

To a solution of 32 (1.5 g, 2.8 mmol) in dry pyridine

(30 mL) was added BzCl (1.7 mL, 14 mmol) at 0 ^ꢀC and the

mixture was stirred for 1 h at room temperature. The reaction

was quenched by the addition of saturated aqueous NaHCO₃.

The reaction mixture was concentrated in vacuo, and the resi-

due was partitioned between AcOEt and H₂O. The separated

organic layer was washed with saturated aqueous NaHCO₃

followed by brine. The organic layer was dried (Na₂SO₄)

and concentrated in vacuo. The residue was dissolved in

MeOH (30 mL). To this solution, 1 N aqueous NaOH

(10 mL) was added and the mixture was stirred for 30 min

at room temperature. The reaction mixture was neutralized

with 1 N aqueous HCl. The mixture was partitioned between

AcOEt and H₂O. The separated organic layer was washed

with saturated aqueous NaHCO₃followed by brine. The or-

ganic layer was dried (Na₂SO₄) and concentrated in vacuo.

The residue was puriﬁed by a ﬂush silica gel column, eluted

with hexane/AcOEt (1:1 to 0:1), to give 33 (740 mg, 42%)

as a white foam: FAB-LRMS m/z 628 (MH^þ); FAB-HRMS

calcd for C₃₃H₃₅FN₅O₃SSi (MH^þ) 628.2214, found

1

(MH^þ) 692.2343, found 692.2343; H NMR (CDCl₃) d: 9.16

(br s, 1H), 8.71 (s, 1H), 8.26 (s, 1H), 7.98 (d, 2H,

J¼7.4 Hz), 7.60e7.21 (m, 12H), 6.84 (d, 4H, J¼9.1 Hz),

6.23 (dd, 1H, J¼3.4 and 13.7 Hz), 5.47e5.35 (dt, 1H,

J¼3.4 and 53.2 Hz), 4.51e4.47 (m, 1H), 3.75e3.71 (m, 1H),

3.63e3.53 (m, 2H), 3.39 (br s, 1H); ¹³C NMR (CDCl₃) d:

164.6, 158.6, 152.8, 151.6, 149.6, 144.2, 141.9, 135.4,

133.4, 132.8, 130.0, 128.8, 128.1, 128.0, 127.9, 127.1,

123.5. 113.3, 96.3 (d, J¼191 Hz), 87.0, 74.3 (d, J¼17.9 Hz),

64.2, 60.1 (d, J¼29.8 Hz), 55.2, 49.3.

3.25. X-ray crystallography

Crystal data for 8: C₉H₁₂FN₃O₃S$2H₂O, M¼297.30, ortho-

˚

rhombic, a¼6.5004 (1), b¼6.9843 (1), c¼27.1636 (6) A,

3

˚

V¼1233.28 (4) A , T¼100 K, space group P2₁2₁2₁(no. 19),

1

628.2220; H NMR (CDCl₃) d: 9.14 (br s, 1H), 8.73 (s, 1H),

Z¼4, m(Cu Ka)¼27.02 cm^ꢁ1, 14,437 reﬂections measured,

2266 unique (R_int¼0.034), which were used in the last least-

squares reﬁnement. The ﬁnal R was 0.025 for 2234 reﬂections

with I>2s(I).

8.30 (s, 1H), 7.98 (d, 2H, J¼7.7 Hz), 7.70e7.38 (m, 13H),

6.25 (dd, 1H, J¼3.4 and 13.1 Hz), 5.42 (dt, 1H, J¼3.4

and 49.8 Hz), 4.60e4.57 (m, 1H), 4.07e3.95 (m, 2H),

4324

M. Takahashi et al. / Tetrahedron 64 (2008) 4313e4324

10. Tobin, K. A. Insight 2006, 31, 11.

Acknowledgements

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This work was supported in part by Grant-in-Aid for

Scientiﬁc Research from the Japan Society for Promotion of

Science (No. 18109001). We would like to thank Ms. H.

Matsumoto and Ms. A. Maeda (Center for Instrumental

Analysis, Hokkaido University) for elemental analysis. We

also would like to thank Ms. S. Oka (Center for Instrumental

Analysis, Hokkaido University) for measurement of mass

spectra.

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Article Doi

Synthesis and crystal structure of 2′-deoxy-2′-fluoro-4′-thioribonucleosides: substrates for the synthesis of novel modified RNAs

DOI: 10.1016/j.tet.2008.02.071

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