Synthesis and RNA-selective hybridization of α-L-ribo- and β-D-lyxo-configured oligonucleotides

Tetrahedron 62 (2006) 2278–2294

Synthesis and RNA-selective hybridization of a-L-ribo- and

b-^D-lyxo-conﬁgured oligonucleotides

Gilles Gaubert,^aB. Ravindra Babu,^aStefan Vogel,^aTorsten Bryld,^aBirte Vester^b

and Jesper Wengel^a,

*

^aDepartment of Chemistry, Nucleic Acid Center, University of Southern Denmark, DK-5230 Odense M, Denmark

^bDepartment of Biochemistry and Molecular Biology, University of Southern Denmark, DK-5230 Odense M, Denmark

Received 26 October 2005; revised 28 November 2005; accepted 2 December 2005

Available online 27 December 2005

Abstract—Three a-L-ribofuranosyl analogues of RNA nucleotides (a-L-RNA analogues) have been synthesized and incorporated into

oligonucleotides using the phosphoramide approach on an automated DNA synthesizer. The 4⁰-C-hydroxymethyl-a-L-ribofuranosyl thymine

monomer was furthermore synthesized. Relative to the unmodiﬁed duplexes, incorporation of a single a-^L-RNA monomer into a DNA strand

leads to reduced thermal stability of duplexes with DNA complements but unchanged thermal stability of duplexes with RNA complements,

whereas incorporation of more than one a-^L-RNA monomer lead to moderately decreased thermal stability also of duplexes with RNA

complements. Efﬁcient hybridization with an RNA complement and no melting transition with a DNA complement were observed with

stereoregular chimeric oligonucleotides composed of a mixture of a-^L-RNA and afﬁnity enhancing a-L-LNA monomers (a-L-ribo-conﬁgured

locked nucleic acid). Furthermore, duplexes formed between oligodeoxynucleotides containing an a-^L-RNA monomer and complementary

RNA were good substrates for Escherichia coli RNase H. RNA-selective hybridization was also achieved by the incorporation of 1-(4-C-

hydroxymethyl-b-^D-lyxofuranosyl)thymine monomers into a DNA strand, whereas stable duplexes were formed with both complementary

DNA and RNA when these monomers were incorporated into an RNA strand.

1. Introduction

Conformational restriction of the single-stranded AON has

the potential to favour duplex formation entropically by

diminishing the loss of conformational freedom upon

duplex formation. a-^L-LNA (a-L-ribo-conﬁgured locked

nucleic acid),^2,3containing a 2⁰-O,4⁰-C-methylene linked

furanose ring,^†with three out of four chirality centers

inverted relative to RNA, forms duplexes with complemen-

tary RNA and DNA with highly increased thermal stability

and generally improved selectivity. a-^L-LNA can be most

adequately described as a DNA mimic as NMR spectro-

scopic studies of a-^L-LNA$RNA duplexes and molecular

dynamics simulation of fully modiﬁed a-^L-LNA$RNA

duplexes have shown the overall duplex geometry to be

very similar to the corresponding unmodiﬁed DNA$RNA

hybrid.³Fully modiﬁed and mix-meric a-L-LNA (consisting

The utilization of modiﬁed oligonucleotides (ONs) in the

antisense approach requires the formation of duplexes with

mRNA in order to speciﬁcally inhibit their translation into

proteins involved in various pathologic disorders. Essential

properties of successful antisense oligonucleotides (AON)

are good aqueous solubility, resistance against enzymatic

degradation, high binding afﬁnity and speciﬁcity for the

target RNA strand.¹It is furthermore desirable if they have

the ability to recruit the endogenous enzyme RNase H. An

AON basically has two possible modes of action, which

both involve hybridization to the RNA target. One is steric

blocking of the mRNA, and the other is recognition of the

RNA$AON duplex as a substrate for the enzyme RNase H,

which subsequently cleaves the RNA strand of the duplex.

In the latter scenario, one AON is able to pacify multiple

mRNA strands. A high binding afﬁnity towards RNA is

crucial, especially for the steric blocking approach.

^†a-L-LNA (a-L-ribo conﬁgured diastereoisomer of LNA; deﬁned as an

oligonucleotide containing one or more 2⁰-O,4⁰-C-methylene-a-L-

ribofuranosyl nucleotide monomers) has shown appealing hybridization

properties despite its unnatural conﬁguration. The furanose conformation

of an a-^L-LNA monomer is of N-type (C3⁰-endo, ³E). For further

information about the conformations of the nucleotides, see Eur. J.

Biochem. 1983, 131, 9 (abbreviations and symbols for the description of

conformations of polynucleotide chains, IUPAC-IUB Joint Commission

on Biochemical Nomenclature). A similar, but more ﬂexible, furanose

conformation is likely for an a-^L-RNA monomer.⁵

Keywords: a-L-ribo-Conﬁgured nucleic acid (a-L-RNA); b-D-Lyxofura-

nosyl nucleotide; a-^L-LNA; Preferential RNA hybridization; S-type

furanose conformation; Thermal denaturation studies.

Corresponding author. Tel.: C45 65502510; fax: C45 66158780;

e-mail: jwe@chem.sdu.dk

*

doi:10.1016/j.tet.2005.12.007

G. Gaubert et al. / Tetrahedron 62 (2006) 2278–2294

2279

of a mixture of a-L-LNA and unmodiﬁed DNA nucleotides)

supported in vitro Escherichia coli RNase H-mediated

cleavage of the RNA target, albeit at a very reduced rate and

at high enzyme concentration.^3cRNase H has been reported to

bind to the minor groove of substrate RNA$DNA hetero-

duplexes adopting a duplex form intermediate between the A-

and B-form, with a minor groove width also intermediate

between that of the A- and B-forms.⁴The furanose

conformations of the nucleotides in the RNA strand are of

theN-type, whereashybridizationofa DNAstrandtotheRNA

strand causes the furanose conformation₀of the DNA strand to

change from the typical S-type (C2 -endo) into E-type

conformations (O4⁰-endo range).⁴Thus, the activation of

RNase H proposedly ₀requires AONs with furanose rings able

to adopt E-type (O4 -endo), or perhaps S-type (C2⁰-endo),

conformations. The locked furanose conformations of a-^L-

LNA might therefore explain the limited ability of a-^L-

LNA$RNA duplexes to act as substrates for RNase H (high

enzyme concentration and extended reaction time) despite the

global DNA-mimicking nature of a-^L-LNA in a-L-LNA:RNA

duplexes.^3c

obtain efﬁcient RNA binding.^9,10The results obtained with

thymine a-^L-RNA/a-L-LNA chimeras,⁶that is, high binding

afﬁnity, RNA-selective hybridization and serum stability

motivated us to further investigate this class of RNA

stereoisomers.

Base

O

OH

O^–P O

^–O P O

^αLT^L: Base = T

^αLA^L: Base = A

DNA

RNA

^–O P O

–

O P O

T

Base

T

HO

O

HO

O

HO

O

HO

O

K

L

^αLT: Base = T⁶

^αLU: Base = U

^αLC: Base = C

^αLA: Base = A

a-L-RNA (a-L-ribo-conﬁgured RNA) has structural resem-

blance to a-^L-LNA. The furanose conformation of an a-L-

3

LNA monomer is of the N-type (C3⁰-endo, E),^†and a

Figure 1. Structures of DNA, RNA, a-L-RNA (^aLT, ^aLU, ^aLC and ^aLA), a-

L-LNA (^aLT^Land ^aLA^L),^2,34⁰-C-hydroxymethyl-a-L-RNA (K) and 4⁰-C-

hydroxymethyl-b-^D-lyxofuranosyl (L) monomers. The short notations

shown are used in Tables 1 and 2. TZthymin-1-yl, UZuracil-1-yl, CZ

cytosin-1-yl, AZadenin-9-yl.

similar furanose conformation, although more ﬂexible, is

likely to be preferred for an a-^L-RNA monomer as indicated

by calculations.⁵We had previously in a preliminary form

reported the synthesis and binding properties of the a-^L-

RNA monomer bearing a thymine unit as nucleobase (^aLT,

Fig. 1).⁶The a-L-RNA thymine monomer when incorpor-

ated into an ON impairs a higher tendency towards

hybridization with an RNA complement than with a DNA

complement. A single incorporation of an a-^L-RNA

nucleotide in a 9-mer mixed-base sequence (ON3) leads

to unchanged thermal stability towards RNA and reduced

thermal stability towards DNA (DT_mZK4 8C) when

compared to the DNA reference ON1 (Table 1).⁶When

three a-^L-RNA monomers were incorporated (ON4), the

destabilization against the RNA target was limited to

K16 8C, whereas no co-operative transition above 5 8C

could be detected against DNA.⁶10-mer ONs composed of

a mixture of a-^L-RNA monomers and afﬁnity enhancing a-

L-LNA^2,3monomers (ON13 and ON14) displayed efﬁcient

hybridization to the corresponding RNA complement

(DT_mZC10 and C8 8C, respectively), whereas no hybrid-

ization towards the corresponding DNA complement could

be detected under the applied conditions (Table 2).⁶

Moreover, the stability of a-L-RNA/a-L-LNA chimera

(ON13 and ON14) towards 3⁰-exonucleolytic degradation

in vitro (snake venom phosphodiesterase) is signiﬁcantly

improved relative to the unmodiﬁed DNA reference.⁶If the

pronounced RNA selectivity obtained for ON14 turns out to

be a general feature of a-^L-RNA/a-L-LNA chimeras, one

may envision improved speciﬁcity compared to the current

antisense molecules, which are known also to hybridize

towards DNA targets. A similar pronounced RNA selectiv-

ity has been reported for a few other ON analogues, for

example, b-^L-DNA,⁷arabinonucleic acids,⁸2⁰-O,3⁰-C

linked bicyclic oligonucleotides,⁹and a-D-LNA.¹⁰How-

ever, their usefulness as antisense molecules is hampered

either by comparatively low binding afﬁnity toward RNA^7,8

or the necessity of using fully modiﬁed oligomers in order to

Table 1. Thermal denaturation experiments^a

DNA

RNA

T_m(DT_m)

(8C)

T_m(DT_m)

(8C)

5⁰-d(GTGATATGC)

ON1

30^b/28^c

(Ref)

28^b/26^c/31^d

(Ref)

5⁰-r(GUGAUAUGC)

5⁰-d(GTGA(^aLT)ATGC)

5⁰-d(G(^aLT)GA(^aLT)A(^aLT)GC)

5⁰-d(GTGAKATGC)

5⁰-d(GKGAKAKGC)

5⁰-d(GTGALATGC)

5⁰-d(GLGALALGC)

5⁰-r(GUGALAUGC)

5⁰-r(GLGALALGC)

ON2

ON3

ON4

ON5

ON6

ON7

ON8

ON9

ON10

26 (Ref)

26 (K4)^b

nt^b

36 (Ref)

28 (G0)^b

12 (K16)^b

26 (G0)^c

13 (K18)^d

26 (G0)^c

21 (K5)^c

36 (G0)^c

34 (K2)^c

23 (K5)^c

nt^d

24 (K4)^c

nt^c

25 (K1)^c

20 (K6)^c

^aMelting temperatures (T_mvalues) were obtained from the maxima of the

ﬁrst derivatives of the melting curves (A₂₆₀vs temperature) recorded in

either medium salt buffer (10 mM sodium phosphate, 100 mM sodium

chloride, 0.1 mM EDTA, pH 7.0)^b,cor in high salt buffer (10 mM sodium

phosphate, 700 mM sodium chloride, 0.1 mM EDTA, pH 7.0)^dusing 1.5^b/

1.0^cmM concentrations of the two complementary strands (assuming

identical extinction coefﬁcients for all modiﬁed and unmodiﬁed

nucleotides); DT_mvalues are changes in the T_mvalue relative to the

unmodiﬁed reference duplex (Ref); AZadenin-9-yl monomer, CZ

cytosin-1-yl monomer, GZguanin-9-yl monomer, TZthymin-1-yl

monomer, UZuracil-1-yl monomer; see Figure 1 for the structures of

a-^L-RNA nucleotide monomer (^aLT), 4⁰-C-hydroxymethyl-a-L-RNA

thymine monomer K and 4⁰-C-hydroxymethyl-b-D-lyxofuranosyl thy-

mine monomer L; ‘nt’-No co-operative melting transition; DNA

sequences are shown as d(sequence) and RNA sequences are shown as

r(sequence).

^bRef. 6.

This report is focused on the synthesis of the a-^L-RNA

monomers of three of the naturally occurring RNA monomers

(U^aL, C^aLand A^aL) (Fig. 1), their incorporation into

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G. Gaubert et al. / Tetrahedron 62 (2006) 2278–2294

Table 2. Thermal denaturation experiments^a

DNA

RNA

T

_m(DT_m)

T_m(DT_m)

(8C)

ON11

ON12

ON13

ON14

ON15

ON16

ON17

ON18

ON19

ON20

ON21

ON22

ON23

ON24

20 (Ref)^b

30 (Ref)^c

nt

19 (Ref)^b

28 (Ref)^c

29 (C10)^b

27 (C8)^b

11 (K17)^c

49 (Ref)^c

47 (K2)^c

24 (Ref)^c

29 (C5)^c

5⁰-T₁₀

5⁰-T₁₄

5⁰-(^aLT)₄(^aLT^L)₄(^aLT)T

5⁰-[(^aLT)(^aLT)(^L)]₄(^aLT)T

5⁰-T₅(^aLT)₄T₅

5⁰-GTCTCTATGGACCT

5⁰-GTCTCTA(^aLU)GGACCT

5⁰-GTC(^aLU)CTATGGACCT

5⁰-G(^aLU)CTCTATGGACCT

5⁰-ATTATTATAAATTA

5⁰-^aL(A^LT^LUA^LUT^LA^LUA^LA^LAT^LT^L)A

5⁰-TATTTACTTTC

nt

45 (Ref)^c

41 (K4)^c

36 (K9)^c

40 (K5)^c

32 (Ref)^c

nt

23 (Ref)^c,d26 (Ref)^c,d

nt

16 (K10)^c,d

23 (K5)^c

5⁰-^aL(UA^LUT^LUA^LCT^LUT^L)C

5⁰-T₇LT₆

21 (K9)^c

a

Melting temperatures (T_mvalues) were obtained from the maxima of the ﬁrst

derivatives of the melting curves (A₂₆₀vs temperature) recorded in either

medium salt buffer^b,cor in high salt buffer (10 mM sodium phosphate, 1 M

sodium chloride, 0.1 mM EDTA, pH 7.0)^dusing 1.5^b/1.0^cmM concentrations of

the two complementary strands; see below Table 1 for other details; see Figure 1

for the structures of a-^L-RNA nucleotide monomers (^aLT, ^aLU, ^aLC and ^aLA),

Scheme 2. Reagents and conditions (and yields): (i) Ac₂O, pyridine, rt

(83%); (ii) (a) Lawesson’s reagent, 1,2-dichloroethane, reﬂux, (b) saturated

methanolic NH₃, 100 8C (74%); (iii) (a) TMSCl, pyridine, rt, (b) BzCl, rt,

(c) aq NH₃, rt (77%); (iv) DMTCl, pyridine, rt (95%); (v) TBDMSCl,

imidazole, pyridine, rt (49%); (vi) NC(CH₂)₂OP(Cl)N(i-Pr)₂, EtN(i-Pr)₂,

CH₂Cl₂, rt (52%); (vii) DNA synthesizer.

aL

a-^L-LNA monomers (^aLT^Land A^L) and 4⁰-C-hydroxymethyl-b-D-lyxofur-

d

anosyl thymine monomer L; DNA target [5⁰-d(AAAGTAAATA)] and RNA

target [5⁰-r(AAAGUAAAUA)] containing a sequence complementary to the

ﬁrst ten monomers of ON22 and ON23 were used.

Ref. 6.

b

ONs containing 4⁰-C-hydroxymethyl nucleotide monomers

hybridize with both complementary DNA and RNA with

virtually identical or slightly improved binding afﬁnity

compared to the unmodiﬁed duplexes.^13,14The additional

C-alkyl branch faces the minor groove for b-^D-ribo-

conﬁgured derivatives allowing attachment of molecular

entities,^14,15for example, intercalators, lipophilic groups,

positive charged amines or a third strand to an ON.

Furthermore, ONs containing C4⁰-substituted nucleotides

have shown increased resistance towards enzymatic

degradation.^13,16,17In order to investigate the inﬂuence of

the 4⁰-C-hydroxymethyl moiety of a-L-ribo-conﬁgured

monomer K on the hybridization towards DNA and RNA

complements, we synthesized phosphoramidite 34 and

incorporated it into ONs (Scheme 4). The structural

resemblance of the ﬂexible monocyclic monomer K to the

bicyclic a-^L-LNA thymine monomer ^aLT^Ladded to its

interest (Fig. 1).

oligonucleotides and the study of the stability of duplexes

formed between these oligonucleotides and their complemen-

tary RNA and DNA strands. The enantiomeric a-^D-

ribonucleosides derived from uracil, cytosine and adenine

havebeendescribedpreviously^11,12butthelowyieldsreported

and the difﬁcult separation of the anomeric mixture in the case

of the adenine derivative make these strategies generally

unsuitable for the preparation of the enantiomeric a-^L-

ribonucleosides. Moreover, the utilization of ^D-ribose as a

starting material in these published strategies stimulated us,

becauseofthehighcostof^L-ribose, to reconsiderthe strategies

for the preparation of the phosphoramidite derivatives

(Schemes 1–3).

During the recent years a plethora of sugar-modiﬁed

nucleoside analogues has been chemically synthesized

with the aim of improving nucleic acid recognition.

However, lyxofuranosyl nucleosides, containing all the

three hydroxyls in the ‘up’ position, have received rather

limited attention. Thus, there are only a few reports in the

literature regarding their synthesis,^12,18biological evalu-

ation¹⁹and conformational investigation.²⁰And, to the best

of our knowledge, no attempts have been made to

incorporate a lyxofuranosyl nucleotide monomer into an

ON with the aim of evaluating its hybridization properties.

To promote such investigation we describe here the

synthesis of phosphoramidite 43 (Scheme 5), starting from

the common intermediate 26, required for the incorporation

of 4⁰-C-hydroxymethyl-b-D-lyxofuranosyl thymine mono-

mer L into ONs (Fig. 1).

Scheme 1. Reagents and conditions (and yields): (i) NH₂CN, K₂CO₃, DMF,

90 8C (82%); (ii) methyl propiolate, EtOH, reﬂux (81%); (iii) aq HCl

(0.2 N), reﬂux (77%); (iv) DMTCl, pyridine, rt; (v) TBDMSCl, imidazole,

pyridine, rt (42% from 3); (vi) NC(CH₂)₂OP(Cl)N(i-Pr)₂, EtN(i-Pr)₂,

CH₂Cl₂, rt (70%); (vii) DNA synthesizer.

G. Gaubert et al. / Tetrahedron 62 (2006) 2278–2294

2281

Scheme 3. Reagents and conditions (and yields): (i) two steps (Ref. 24); (ii) BnBr, NaH (60% in mineral oil), DMF, 0 8C to rt (86%); (iii) TBAF, THF, rt

(80%); (iv) BzCl, pyridine, rt (74%); (v) (a) aq AcOH (80%), cat. H₂SO₄, rt, (b) Ac₂O, pyridine, rt (73%); (vi) 6-N-benzoyladenine, SnCl₄, CH₃CN, rt (88%);

(vii) half-saturated methanolic NH₃, 0 8C (92%); (viii) (a) Tf₂O, pyridine, CH₂Cl₂, K30 8C, (b) KOAc, 18-crown-6 ether, toluene, CH₂Cl₂, reﬂux (79%); (ix)

aq NaOH (1 M), EtOH, pyridine, 0 8C; (x) Pd/C, HCO₂NH₄, abs EtOH, reﬂux (54% from 20); (xi) DMTCl, pyridine, rt (92%); (xii) TBDMSCl, imidazole,

pyridine, rt (48%); (xiii) NC(CH₂)₂OP(Cl)N(i-Pr)₂, EtN(i-Pr)₂, CH₂Cl₂, rt (73%); (xiv) DNA synthesizer.

Scheme 4. Reagents and conditions (and yields): (i) saturated methanolic NH₃, rt (96%); (ii) DMTCl, pyridine, rt, (97%); (iii) MsCl, DMAP, Et₃N, CH₂Cl₂, rt;

(iv) aq NaOH (2 M), EtOH, H₂O, reﬂux (65% from 28); (v) TBDMSCl, imidazole, pyridine, rt (32: 58% and 33: 23%); (vi) NC(CH₂)₂OP(Cl)N(i-Pr)₂, EtN(i-

Pr)₂, CH₂Cl₂, rt (71%); (vii) DNA synthesizer.

Scheme 5. Reagents and conditions (and yields): (i) TBDMSCl, imidazole, DMAP, DMF, 36 8C; (ii) saturated methanolic NH₃, MeOH, rt (80% from 35);

(iii) MsCl, Et₃N, DMAP, CH₂Cl₂, rt; (iv) DBU, CH₃CN, rt (83% from 37); (v) aq NaOH (2 M), EtOH–H₂O (1/1), reﬂux (74%); (vi) TBDMSCl, imidazole,

pyridine, rt (41: 36% and 42: 53%); (vii) NC(CH₂)₂OP(Cl)N(i-Pr)₂, EtN(i-Pr)₂, CH₂Cl₂, rt (66%); (viii) DNA synthesizer.

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G. Gaubert et al. / Tetrahedron 62 (2006) 2278–2294

2. Results and discussion

spectroscopic data of nucleoside 22 were consistent with

the literature data for its ^D-enantiomer.^12b

2.1. Synthesis of a-^L-ribonucleosides 3, 9 and 22

2.2. Synthesis of the a-L-RNA phosphoramidites 6, 12

and 25

a-^L-Uridine was synthesized following a slightly modiﬁed

procedure reported for the synthesis of the corresponding

D-enantiomer.¹¹In our hands, the synthesis of the oxazoline

1 was more efﬁcient with potassium bicarbonate in DMF

than with aqueous ammonia as previously described.¹¹

Oxazoline 1 was obtained in 82% yield from L-ribose.

Reaction of 1 with methyl propiolate afforded the anhydro

derivative 2 in 81% yield. Subsequent opening of this ring

with acid under aqueous conditions afforded the desired a-^L-

uridine 3 in 77% yield. The ¹H NMR data of 3 were found to

be consistent with the literature data for the corresponding

D-enantiomer (Scheme 1).¹²

Compounds 3, 9 or 22 were O5⁰-dimethoxytritylated using

standard conditions in satisfactory yields (95, 95 and 92%,

respectively). Silylation of nucleosides 4, 10 or 23 with tert-

butyldimethylsilyl chloride (TBDMSCl) in the presence of

imidazole and pyridine produced a mixture of a byproduct

(fast eluting; assigned as the 2⁰-O-TBDMS isomers) and 3⁰-

O-TBDMS (slow eluting) derivatives, which were separated

by silica gel column chromatography. The ¹H NMR spectral

data obtained for compounds 5, 11 or 24 were found to be

consistent with the literature data for their ^D-enantiomers.¹²

Nucleosides 5, 11 or 24 were dissolved in anhydrous

dichloromethane and phosphitylated using 2-cyanoethyl

N,N-diisopropylphosphoramidochloridite in the presence of

N,N-diisopropylethylamine to give the corresponding

phosphoramidites 6, 12 and 25 in yields of 70, 52 and

73%, respectively.

Compound 3 was per-acetylated with acetic anhydride in

pyridine to give 7 in 83% yield. Synthesis of a-^D-cytidine

has been reported^12bin a moderate 48% yield applying the

Sung methodology²¹to furnish the D-enantiomer of 8.

Nevertheless, reaction of 7 with the Lawesson’s reagent²²

followed by treatment with saturated methanolic ammonia

afforded a-^L-cytidine 8 in 74% yield. Using the transient

protection method,²³a-L-cytidine was N-benzoylated to

2.3. Synthesis of the 1-(4-C-hydroxymethyl-a-^L-ribo-

furanosyl)thymine phosphoramidite 34

1

afford nucleoside 9 in 77% yield. The H NMR spectro-

Complete deacylation of 1-[2-O-acetyl-3,5-(di-O-tert-butyl-

diphenylsilyl)-4-C-benzoyloxymethyl-b-^D-xylofurano-

syl]thymine (26)²⁸with saturated methanolic ammonia,

followed by regioselective dimethoxytritilation afforded

nucleoside 28. Activation of the 2⁰-OH in nucleoside 28 by

reaction with MsCl afforded the desired nucleoside 29 along

with the 2,2⁰-anhydro nucleoside 30. This crude mixture was

reﬂuxed under alkaline conditions, which also resulted in

complete desilylation, affording triol 31. Silylation of the triol

31 with 4 equiv of TBDMSCl afforded the desired 2⁰-O-

TBDMS isomer 32 in 58% yield along with the 3⁰-O-isomer

33 (23% yield). Phosphitylation of 32 by the standard protocol

afforded phosphoroamidite 34 (71% yield) that was used to

incorporate monomer K into ONs (Scheme 4). To ascertain

that no silyl migration occurred under the basic conditions

applied during phosphitylation, the isomers ₀of 34 were

separated and characterized. The signals of H3 appeared as

a double doublet with a large ²J_H,Pcoupling constant (major

isomer: 13.7 Hz; minor isomer: 13.0 Hz) conﬁrming that no

silyl migration occurred during the course of the reaction.²⁹

scopic data for compounds 8 and 9 were found to be

consistent with the literature data for their ^D-enantiomers

(Scheme 2).^12b

Using inexpensive L-arabinose as starting material, furanose

13 was prepared in a two step procedure developed by

Dahlman et al.²⁴The 3-hydroxy group of 13 was benzylated

using benzyl bromide and sodium hydride in DMF to give

furanose 14 in 86% yield. Cleavage of the silyl protecting

group of 14 using TBAF gave derivative 15 (80% yield) that

1

was benzoylated to give furanose 16 in 74% yield. The H

NMR spectroscopic data of furanoses 14 and 15 were

consistent with the literature data for their ^D-enantiomer.²⁵

Finally, the glycosyl donor 17 was obtained by a standard

two step procedure of isopropylidene group cleavage and

acetylation of the 1- and 2-hydroxy functions in 73% overall

yield. Furanose 17 was condensed with N-6-benzoyladenine

under the conditions initially reported by Saneyoshi and

Satoh²⁶where the base is directly reacted with the

O-acetylated sugar in the presence of stannic chloride.

Coupling proceeded in 88% yield to afford nucleoside 18. It

can be noticed that the participation of the 2⁰-O-acetyl group

provided only the desired a-anomer. Compound 18 was

selectively deprotected at the 2⁰-position by the action of

half-saturated methanolic ammonia to give nucleoside 19 in

good yield (92%). Inversion of the conﬁguration at C2⁰

proceeded in two steps. Firstly, the 2⁰-hydroxy group was

activated by reaction with triﬂic anhydride followed by the

reaction of the intermediate with potassium acetate to give

the expected inverted nucleoside 20 in 79% yield. Nucleo-

side 20 was selectively deacylated at the 2⁰- and 5⁰-positions

with aqueous sodium hydroxide in an ethanol–pyridine

mixture following a previously described procedure²⁷to

give nucleoside 21 in 77% yield. Finally, debenzylation of

compound 21 by treatment with ammonium formate and

palladium on carbon produced N-6-benzoyl-a-^L-adenosine

22 in a yield of 70% (Scheme 3). The ¹H NMR

2.4. Synthesis of the 1-(4-C-hydroxymethyl-b-D-lyxofura-

nosyl)thymine phosphoramidite 43

Selective removal of the primary silyl protection in the

commonintermediate26proveddifﬁcult, probablyduetosilyl

migration from the 3⁰-position. Therefore, complete desilyla-

tion of nucleoside 26 and subsequent O5⁰-tritylation followed

by O3⁰-silylation furnished nucleoside 36 in 66% overall yield

(from 26 via 35). Deacetylation afforded nucleoside 37, which

upon mesylation yielded a mixture of nucleosides 38 and 39

(w3:1, as judged from analytical TLC). Concomitant

treatment with aq NaOH furnished a complex mixture,

presumably via desilylalation of 38, followed by epoxide

formation and then opening of the epoxide under alkaline

conditions. The desired lyxo-conﬁgured nucleoside 40 was

obtained by complete conversion of the crude mixture (38C

G. Gaubert et al. / Tetrahedron 62 (2006) 2278–2294

2283

39) into O2⁰,C2-anhydronucleoside 39, followed by treatment

with aq NaOH in ethanol, affording nucleoside 40 in 61%

overall yield (from 37). Silylation of the triol 40 by reaction

with 4 equiv of TBDMSCl afforded the desired 2⁰-O-TBDMS

isomer 41 in 36% yield along with the O3⁰-isomer 42 (52%).

O3⁰-phosphitylation of nucleoside 41 afforded phosphoro-

amidite 43 (66% yield) that was used to incorporate monomer

L into ONs (Scheme 5). The signal of H3⁰in 43 appeared as a

(DT_mZC10, C8 and C5 8C, respectively), whereas no

hybridization towards the DNA target was detected. It should

be noted that ON15, having four consecutive a-^L-RNA T

monomers and no a-^L-LNA monomer, displayed signiﬁcantly

decreased afﬁnity towards the RNA complement. Similar

RNA-selective hybridization was seen with the a-^L-RNA/a-L-

LNA chimera ON23 consisting of alternate a-^L-RNA and a-L-

LNA monomers. Thus, although no co-operative transition

could be detected at medium salt conditions, a melting

temperature was observed against the RNA target under high

salt conditions.

2

double doublet with a large J_H,Pcoupling constant (major

isomer: 13.6 Hz) indicating that no silyl migration occurred

during phosphitylation.

A single incorporation of 1-(4-C-hydroxymethyl-a-^L-ribofur-

anosyl)thymine monomer K in the middle of a 9-mer mixed-

base sequence induced similar hybridization properties as

incorporation of the a-^L-RNA thymine monomer ^aLT (ON5

compared to ON3), that is, a decrease in T_mvalue (K5 8C)

against the DNA complement and no change against the RNA

complement. The partly modiﬁed 9-mer containing three

incorporations of monomer K induced a similar destabilizing

effect (DT_mZK6 8C/modiﬁcation, ON6 relative to ON1) on

the duplex formed with complementary RNA as seen above

with ON4 containing three ^aLT monomers (DT_mZK5.3 8C/

modiﬁcation, ON4 relative to ON1), indicating no unfavor-

able steric hindrance due to the additional 4⁰-C-alkyl chain.

3. Synthesis of ONs and thermal denaturation studies

All oligomers ON5–ON10, ON15, ON17–ON19, ON21,

ON23 and ON24 (Tables 1 and 2) were prepared in

0.2 mmol scale using the phosphoramidite approach (see the

Section 7 for details). The composition of the oligomers was

veriﬁed by MALDI-MS analysis (see the Section 7) and

their purity (O80%) by capillary gel electrophoresis.

Results from hybridization experiments (T_mvalues) towards

single-stranded DNA and RNA complements are shown in

Tables 1 and 2. A single replacement of a DNA thymine

monomer in a 9-mer mixed-base sequence by its a-^L-RNA

counterpart ^aLT resulted in destabilization of the duplex by

4 8C when hybridized to complementary DNA (Table 1, ON3

relative to ON1), while no change in the duplex stability was

seenwhenhybridizedtotheRNAcomplement.⁶Incorporation

ofafew isolated^aLTmonomersintoa DNAstrandreducedthe

afﬁnitytowardstheRNA target(DT_mZK16 8C, ON4relative

to ON1), but the effect was more pronounced towards the

DNA target (no co-operative transition above 5 8C could be

detected).⁶A single incorporation of an a-L-RNA U monomer

in a 14-mer mixed-base sequence ON18 leads to a decrease in

duplex stability against the DNA target (DT_mZK9 8C) when

compared to the DNA reference ON16; the effect is less

detrimental when the substitution is either ₀in the centre

(ON17, DT_mZK4 8C) or towards the 5 -end (ON19,

DT_mZK5 8C). However, against the RNA complement the

stability (T_mZ47 8C, DT_mZK2 8C) was comparable with

that of the DNA$RNA reference duplex. The fact that

incorporation of a single a-^L-RNA monomer is tolerated in a

duplex with complementary RNA is likely explained by

conformational adaptation that is impossible for the relatively

short duplexes following incorporation of more than one a-^L-

RNA monomer. The stereoregular (almost) fully modiﬁed

a-^L-RNA/a-L-LNA chimera ON13,⁶ON14⁶and ON21

consisting of a mixture of a-^L-RNA and a-L-LNA monomers

displayed very efﬁcient hybridization towards the RNA target

The 4⁰-C-hydroxymethyl-b-D-lyxofuranosyl thymine mono-

mer L, containing all three hydroxyls in ‘up’ position,

displayed some interesting hybridization properties. A

single incorporation of monomer L in a 9-mer mixed-base

sequence ON7 showed preference for binding to its RNA

complement (T_munchanged) relative to its DNA comp-

lement (DT_mZK4 8C). However, the duplex stability

decreased when the modiﬁcation was placed in the centre

of a 14-mer homopyrimidine sequence (ON24, DT_m-

ZK9 8C against the DNA target and DT_mZK5 8C against

the RNA target). A T_mvalue of 21 8C was observed with the

RNA complement hybridized to the partly modiﬁed stereo-

irregular 9-mer mixed-base sequence ON8, but no co-

operative transition above 5 8C was observed with the DNA

complement. In contrast, efﬁcient recognition of both DNA

and RNA targets was achieved when monomer L was

incorporated into an RNA strand, and satisfactory binding

afﬁnity towards DNA and RNA complements was observed

with one (ON9) and three (ON10) incorporations.

4. Molecular modeling

Molecular modeling (see the Section 7 for details) was used

to rationalize the thermal stability results for monomers

Figure 2. ON:DNA duplexes: (a) the tilted base is indicated for the overlaid structures of standard DNA thymine monomer (yellow) and ^aLT monomer;

(b) overlaid structures of DNA (T) and K monomers; (c) overlaid structures of DNA (T) and L monomers; (d) the S-type furanose conformation is shown for

standard DNA (yellow) and ^aLT, K, and L monomers (all monomer structures have been cut out of the corresponding duplex helix structure and overlaid with

the sugar ring system kept ﬁxed).

2284

G. Gaubert et al. / Tetrahedron 62 (2006) 2278–2294

Figure 3. ON:RNA duplexes. See legend to Figure 2 for further details.

^aLT, K and L. All monomers showed S-type furanose

conformations^†in both ON$DNA (Fig. 2) and ON$RNA

(Fig. 3) hybrids. The most pronounced change was the

tilting of the nucleobase in all the three modiﬁed monomers

when compared to the reference DNA (T) monomer

(Table 3).

6–7 and 7–8. The cleavage pattern for ON19 modiﬁed at

position 2 is very similar to the pattern seen with the

reference ON16. The RNA complement can thus be

cleaved both to the 3⁰and 5⁰site of a-L-RNA residues in

the corresponding oligonucleotide, showing that RNase H

cleavage can take place in close proximity to an a-^L-

RNA monomer. These results show that properly

designed a-^L-RNA/DNA mixmers can be attractive

molecules for antisense applications.

Table 3. Torsion angle c (O4⁰–C1⁰–N1–C2)

Modiﬁcation

Torsion angle c (8)

[ON:RNA]

[ON:DNA]

DNA-T (Ref)

K96

K168

K169

K151

K135

K156

K152

K97

^aLT

K

L

The calculated structures for monomers ^aLT, K, and L are in

agreement with the observed thermal stabilities. Single

incorporations of the monomers ^aLT, K, or L in a 9-mer ON

leads to reduced afﬁnity towards the complementary DNA

target, which can be attributed to signiﬁcant change in the

torsion angle c in the modiﬁed monomers (Table 3). The

base displacement causes reduced stacking within the strand

and a loss of hydrogen bonding towards the complementary

base. However, in duplexes with the RNA complement the

change in the torsion angle c in monomers ^aLT, K, and L

(compared to DNA-T monomer) is more limited, which

offers an explanation for the RNA-selective hybridization

induced by the incorporation of these monomers.

Figure 4. Autoradiogram showing gel electrophoresis of RNase H cleavage

of labelled RNA hybridized to complementary ON16–ON19. C are

hybridized samples incubated in the absence of RNase H.

6. Conclusion

Oligonucleotides containing a-^L-RNA monomers display in

general decreased duplex stability relative to the unmodiﬁed

reference duplexes, but at the same time preferential binding

towards the RNA complement. Despite the unnatural

conﬁguration of the a-^L-RNA monomer, DNA ONs contain-

ing a single incorporation of an a-^L-RNA monomer retain the

ability to elicit RNase H activity. Moreover, increased

binding afﬁnity and RNA-selective hybridization was

induced by combining the a-^L-RNA monomers with afﬁnity

enhancing a-^L-LNA monomers. As furthermore the a-L-

RNA/a-^L-LNA chimeras displayed signiﬁcant stabilization

towards 3⁰-exonucleolytic degradation,⁶these classes of

molecules are excellent candidates for use within the

antisense technology. The presence of a 4⁰-C-alkyl group

in 4⁰-hydroxymethyl-a-L-RNA monomer K had no inﬂuence

on the duplex stability when compared to the a-^L-RNA

monomer, and could therefore function as a handle for the

attachment of amino functionalities to improve the binding

afﬁnity or the pharmacokinetic properties of ONs containing

a-^L-RNA monomers. RNA-selective hybridization was also

achieved by the incorporation of 1-(4-C-hydroxymethyl-b-

D-lyxofuranosyl)thymine monomer L into a DNA strand,

5. RNase H cleavage

Stimulated by the satisfactory RNA binding character-

istics of the a-^L-RNA modiﬁed ONs, we studied RNase

H degradation of [³²P] labelled RNA that was comp-

lementary to ON16–ON19. Hybridized samples were

digested for different time intervals and the RNA was

electrophoresed on an acryamide gel and visualized by

autoradiography. Basic hydrolysis of RNA (Fig. 4) was

used to identify the cleaved positions. As can be seen in

Figure 4, the unmodiﬁed reference ON16 mainly

supports RNase H cleavage at phosphodiester bonds

opposite positions 4–5, 6–7 and 7–8. ON17 that is

modiﬁed at position 8 is less efﬁciently cleaved than

ON16 showing no 4–5 cleavage band, a weak 5–6 band

and a strong 7–8 band. This indicates that the

modiﬁcation interferes moderately with initial binding

of RNase H but also shows that the enzyme can cleave

opposite 5⁰to the modiﬁcation. ON18 modiﬁed at

position 4 provides an even better cleavage than the

reference ON16 with cleavage mainly opposite positions

G. Gaubert et al. / Tetrahedron 62 (2006) 2278–2294

2285

whereas stable duplexes towards both complementary DNA

and RNA were formed upon incorporation of monomer L

into an RNA strand.

12.0 Hz, H5⁰a), 3.57 (1H, m, H4⁰), 3.46 (1H, m, H5⁰b); d_C

(DMSO-d₆) 171.0, 160.7, 136.8, 108.8, 88.6, 81.4, 80.7,

69.8, 59.5; MALDI-MS: m/z 249 ([MCNa]^C, C₉H₁₀N₂O₅-

Na^Ccalcd 249).

7. Experimental

7.1. General

7.1.3. 1-(a-^L-Ribofuranosyl)uracil (3). A solution of

nucleoside 2 (2.17 g, 9.6 mmol) in aqueous hydrochloric

acid (0.2 N, 10 mL) was reﬂuxed for 1 h. After cooling to

room temperature, the solution was neutralized using

Amberlyst IRA 410 [OH^K]. The resin was ﬁltered off and

washed with lukewarm H₂O. The combined ﬁltrate was

evaporated to dryness under reduced pressure. The residue

was puriﬁed on a silica gel column, [15% (v/v) MeOH in

EtOAc] affording 1.80 g (77%) of nucleoside 3 as a white

solid material. d_C(DMSO-d₆) 163.5, 150.7, 142.8, 99.8,

85.1, 84.0, 70.4, 70.3, 61.2; MALDI-MS: m/z 267 ([MC

Reactions were conducted under an atmosphere of nitrogen

when anhydrous solvents were used. All reactions were

monitored by thin-layer chromatography (TLC) using silica

plates with ﬂuorescence indicator (SiO₂-60, F-254) visua-

lizing under UV light and by revelation with 5% concd

sulfuric acid in ethanol (v/v) followed by heating. Silica gel

60 (particle size 0.040–0.063 mm, Merck) was used for

ﬂash column chromatography. Light petroleum of the

distillation range 60–80 8C was used. After column

chromatography fractions containing product were pooled,

evaporated to dryness under reduced pressure and dried for

12 h under vacuum to give the product unless otherwise

1

Na]^C, C₉H₁₂N₂O₆Na^Ccalcd 267). The H NMR data of

were found to be consistent with the literature data for the

corresponding ^D-enantiomer.^12b

7.1.4. 1-(5-O-(4,4⁰₀-Dimethoxytrityl)-a-L-ribofuranosyl)-

uracil (4). 4,4 -Dimethoxytrityl chloride (0.43 g,

1.3 mmol) was added to a solution of nucleoside 3

(0.26 g, 1.07 mmol) in anhydrous pyridine (5 mL). The

reaction mixture was stirred at room temperature for 12 h

whereupon methanol (2 mL) was added. After stirring for

additional 10 min, the mixture was poured into saturated aq

NaHCO₃(25 mL). Extraction was performed with CHCl₃

(3!20 mL), and the combined organic phase was dried

(Na₂SO₄), ﬁltered and evaporated to dryness under reduced

pressure. The residue was puriﬁed by column chromato-

graphy [5–8% MeOH in CHCl₃containing 0.5% Et₃N (v/v/

v)] to give nucleoside 4 (550 mg) as a white foam. NMR

spectroscopic data revealed the compound to be contami-

nated with traces of Et₃N. d_C(CDCl₃) 164.3, 158.4, 150.9,

144.6, 142.7, 135.8, 135.7, 130.0, 129.9, 128.1, 127.8,

126.8, 113.2, 100.4, 86.6, 86.3, 84.3, 72.0, 71.2, 63.3, 55.2;

MALDI-MS: m/z 569 ([MCNa]^C, C₃₀H₃₀N₂O₈Na^Ccalcd

569). The ¹H NMR data were found to be consistent with the

literature data for the corresponding ^D-enantiomer.^12b

speciﬁed. ¹H NMR spectra were recorded at 300 MHz, ¹³

C

NMR spectra at 75.5 MHz, and ³¹P NMR spectra at

121.5 MHz. Chemical shifts are reported in ppm relative

to either tetramethylsilane or the deuterated solvent as

internal standard for ¹H and ¹³C NMR, and relative to 85%

H₃PO₄as external standard for ³¹P NMR. Assignments of

NMR spectra, when given, are based on 2D spectra and

follow the standard carbohy₀drate/nucleoside nomenclature

(the carbon atom of the C-4 -subsituent is numbered C5⁰⁰).

The assignments of methylene protons, when given, may be

interchanged. Coupling constants (J values) are given in

Hertz. MALDI-HRMS were recorded in positive ion mode

on an IonSpec Fourier Transform mass spectrometer.

7.1.1. 2-Amino-a-^L-ribofurano[1⁰,2⁰:4,5]-2-oxazoline (1).

A mixture of ^L-ribose (2.00 g, 13.3 mmol), cyanamide

(0.67 g, 16.0 mmol) and powdered potassium bicarbonate

(0.07 g, 0.05 mmol) was stirred at 90 8C for 1 h in

anhydrous DMF (15 mL). After cooling to room tempera-

ture, the mixture was evaporated under reduced pressure to

half volume and the resulting solution was stored for 20 h at

5 8C. The precipitate obtained was ﬁltered off and

recrystallized from 96% aq EtOH to give 1.90 g of

oxazoline 1 (82%) as a white solid material. d_H(DMSO-

d₆) 6.26 (2H, br s, NH₂), 5.58 (1H, d, JZ4.8 Hz, H1⁰), 5.17

(1H, br s, OH), 4.59–4.56 (2H, m, H2⁰and OH), 3.74–3.63

(2H, m, H3⁰and H4⁰), 3.42–3.25 (2H, m, H5⁰); d_C(DMSO-

d₆) 163.8, 98.3, 80.8, 77.8, 71.2, 60.4; MALDI-MS: m/z 197

([MCNa]^C, C₆H₁₀N₂O₄Na^Ccalcd 197).

7.1.5.

1-(2-O-tert-Butyldimethylsilyl-5-O-(4,4⁰-di-

methoxytrityl)-a-^L-ribofuranosyl)uracil (5). Nucleoside

4 (3.20 g) and imidazole (1.04 g, 15.2 mmol) were

dissolved in anhydrous pyridine (60 mL). TBDMSCl

(1.15 g, 7.6 mmol) was added and the solution was stirred

at room temperature for 24 h. The reaction mixture was then

poured into saturated aq NaHCO₃(120 mL) and extraction

was performed with CHCl₃(3!80 mL). The combined

organic phase was dried (Na₂SO₄), ﬁltered and evaporated

to dryness under reduced pressure. The residue was puriﬁed

by column chromatography [5–7% (v/v) acetone in CH₂Cl₂]

yielding the 2⁰-O-tert-butyldimethylsilyl isomer 5 (2.90 g,

42% from 3) as a clear oil and [9–10% (v/v) acetone in

CH₂Cl₂] a byproduct tentatively assigned as the 3⁰-O-tert-

butyldimethylsilyl isomer (yield not determined). d_C

(CDCl₃) 163.3, 158.7, 150.6, 149.9, 144.5, 142.0, 135.7,

135.4, 130.0, 128.1, 128.0, 127.1, 123.8, 113.4, 113.3,

101.0, 87.0, 85.9, 84.5, 72.8, 72.7, 64.2, 55.3, 25.7, 18.1,

K5.2, K5.3; ESI-HRMS: m/z 683.2723 ([MCNa]^C,

C₃₆H₄₄N₂O₈SiNa^Ccalcd 683.2759). The ¹H NMR data

were found to be consistent with the literature data for the

corresponding D-enantiomer.^12a

7.1.2. 2,2⁰-Anhydro-1-(a-L-ribofuranosyl)uracil (2). A

mixture of oxazoline 1 (1.00 g, 5.75 mmol) in 96% aq

EtOH (10 mL) and methyl propiolate (1.69 g, 20.1 mmol)

was heated under reﬂux for 2 h. After cooling to room

temperature, the reaction mixture was evaporated to dryness

under reduced pressure and then coevaporated several times

with 96% aq EtOH to give 1.05 g of nucleoside 2 as a white

solid material (81%) after recrystallization from EtOH. d_H

(DMSO-d₆) d 7.85 (1H, d, JZ7.4 Hz, H6), 6.20 (1H, d, JZ

5.2 Hz, H1⁰), 5.88 (1H, d, JZ7.4 Hz, H5), 5.74 (1H, d, JZ

6.9 Hz, 3⁰-OH), 5.23 (1H, t, JZ5.2 Hz, H2⁰), 4.86 (1H, t,

JZ5.0 Hz, 5⁰-OH), 4.05 (1H, m, H3⁰), 3.70 (1H, dd, JZ5.0,

2286

G. Gaubert et al. / Tetrahedron 62 (2006) 2278–2294

7.1.6. 1-(2-O-tert-Butyldimethylsilyl-3-O-[2-cyanoethox-

y(diisopropylamino)phosphino]-5-O-(4,4⁰-dimethoxy-

trityl)-a-^L-ribofuranosyl)uracil (6). To a stirred solution of

nucleoside 5 (0.26 g, 0.40 mmol) in CH₂Cl₂(10 mL) at room

temperature was added N,N-diisopropylethylamine (0.69 mL,

3.95 mmol). After dropwise addition of 2-cyanoethyl N,N⁰-

diisopropylphosphoramidochloridite (0.38 mL, 1.98 mmol),

the reaction mixture was stirred for another 15 h. CH₂Cl₂

(20 mL)wasaddedandthe mixturewaswashedwithsaturated

aq NaHCO₃(25 mL). The organic phase was dried (Na₂SO₄),

ﬁltered and evaporated todryness under reduced pressure. The

residue was puriﬁed by column chromatography [45–50%

EtOAc in n-hexane, containing 0.5% Et₃N (v/v/v)] to yield

phosphoramidite 6 as a white foam (240 mg, 70%). d_P

(DMSO-d₆) 151.0, 149.9; ESI-HRMS: m/z 883.3838 ([MC

Na]^C, C₄₅H₆₁N₄O₉PSiNa^Ccalcd 883.3843).

trimethylchlorosilane (3.13 mL, 24.7 mmol). The reaction

mixture was stirred at room temperature for 1 h whereupon

benzoyl chloride (2.38 mL, 20.6 mmol) was added. After

stirring for another 5 h the resulting mixture was cooled in

an ice bath, H₂O (10 mL) was added and stirring was

continued for additional 5 min. Aqueous ammonia (10 mL,

29%, w/w) was added, and the resulting mixture was stirred

at room temperature for 15 min and evaporated to dryness

under reduced pressure. The residue was coevaporated with

toluene (2!5 mL) and then puriﬁed by column chromato-

graphy [5–8% (v/v) MeOH in EtOAc] affording nucleoside

9, which was crystallized from absolute ethanol as colour-

less crystals (1.1 g, 77%). d_C(DMSO-d₆) 167.2, 162.9,

154.6, 147.0, 133.2, 132.7, 128.4, 128.2, 95.0, 86.7, 83.7,

70.6, 70.0, 60.9; MALDI-HRMS: m/z 370.1009 ([MC

Na]^C, C₁₆H₁₇N₃O₆Na^Ccalcd 370.1015). The ¹H NMR data

were found to be consistent with the literature data for the

corresponding ^D-enantiomer.^12b

7.1.7. 1-(2,3,5-Tri-O-acetyl-a-^L-ribofuranosyl)uracil (7).

Acetic anhydride (2.32 mL, 24.5 mmol) was added to a

solution of nucleoside 3 (1.71 g, 7.0 mmol) in anhydrous

pyridine (10 mL). The reaction mixture was stirred at room

temperature for 12 h. MeOH (5 mL) was added and the

reaction mixture was stirred for another 10 min and then

concentrated to dryness under reduced pressure. The residue

was dissolved in EtOAc (50 mL) and washing was

performed ﬁrst with saturated aq NaHCO₃(25 mL) and

then brine (25 mL). The separated organic phase was dried

(Na₂SO₄), ﬁltered and evaporated to dryness under reduced

pressure. The residue was puriﬁed by column chromatog-

raphy [3–5% (v/v) MeOH in CHCl₃] to afford nucleoside 7

(2.16 g, 83%) as a white foam. d_H(CDCl₃) 9.47 (1H, br s,

NH), 7.47 (1H, d, JZ8.2 Hz, H6), 6.39 (1H, d, JZ4.7 Hz,

H1⁰₀), 5.77 (1H, d, JZ8.1 Hz, H5), 5.71 (1H, t, JZ4.9 Hz,

H2 ), 5.42 (1H, t, JZ5.4 Hz, H3⁰), 4.55 (1H, m, H4⁰), 4.36

(1H, dd, JZ3.2, 12.3 Hz, H5⁰a), 4.18 (1H, dd, JZ4.2,

12.2 Hz, H5⁰b), 2.15, 2.07 and 2.03 (3H each, 3s, 3!

COCH₃); d_C(CDCl₃) 170.5, 169.3, 168.7, 163.3, 150.2,

140.2, 101.6, 84.3, 79.9, 70.9, 70.3, 63.1, 20.9, 20.5, 20.4;

MALDI-HRMS: m/z 393.0885 ([MCNa]^C, C₁₅H₁₈N₂O₉-

Na^Ccalcd 393.0905).

7.1.10. 4-N-Benzoyl-1-[5-O-(4,4⁰-dimethoxytrityl)-a-L-

ribofuranosyl]cytosine (10). 4,4⁰-Dimethoxytrityl chloride

(0.33 g, 1.0 mmol) was added to a solution of nucleoside 9

(0.12 g, 0.49 mmol) in anhydrous pyridine (5 mL) and the

resulting mixture was stirred at room temperature for 12 h.

MeOH (2 mL) was added, stirring was continued for

another 10 min whereupon the reaction mixture was poured

into saturated aq NaHCO₃(25 mL). Extraction was

performed with CHCl₃(3!20 mL), and the combined

organic phase was dried (Na₂SO₄), ﬁltered and evaporated

to dryness under reduced pressure. The residue obtained was

puriﬁed by column chromatography [6–8% (v/v) MeOH in

CHCl₃] to furnish nucleoside 10 (180 mg, 95%) as a white

foam. d_C(CDCl₃) 166.7, 162.5, 158.6, 156.2, 146.5, 144.7,

136.0, 135.8, 133.2, 133.0, 130.2, 129.1, 128.2, 128.0,

127.8, 127.0, 113.3, 96.3, 88.9, 86.5, 84.5, 72.1, 71.3, 63.8,

55.3; MALDI-MS: m/z 649 ([MCNa]^C, C₃₇H₃₅N₃O₈Na^C

1

calcd 649). The H NMR data were found to be consistent

with the literature data for the corresponding

D-enantiomer.^12b

7.1.8. 1-(a-^L-Ribofuranosyl)cytosine (8). The Lawesson’s

reagent (1.80 g, 4.45 mmol) was added to a stirred solution

of nucleoside 7 (2.06 g, 5.57 mmol) in anhydrous 1,2-

dichloroethane (50 mL). The reaction mixture was heated

under reﬂux for 4 h and then cooled to room temperature.

Methanol (20 mL) was added and the reaction mixture

concentrated to dryness under reduced pressure. The residue

was immediately dissolved in a saturated solution of

ammonia in methanol (100 mL) and heated at 100 8C for

3 h in an autoclave. After cooling to room temperature, the

reaction mixture was evaporated to dryness under reduced

pressure. The residue was puriﬁed by column chromatog-

raphy [5–10% (v/v) MeOH in EtOAc] to give nucleoside 8

(1.0 g, 74%) as a white powder. d_C(DMSO-d₆) 165.5,

155.2, 143.1, 92.2, 85.6, 83.1, 70.6, 70.1, 61.1; MALDI-MS:

7.1.11. 4-N-Benzoyl-1-(2-O-tert-butyldimethylsilyl-5-O-

(4,4⁰-dimethoxytrityl)-a-L-ribofuranosyl)cytosine (11).

tert-Butyldimethylsilyl chloride (0.19 g, 1.05 mmol) was

added to a solution of nucleoside 10 (0.44 g, 0.81 mmol)

and imidazole (0.14 g, 2.10 mmol) in anhydrous pyridine

(10 mL) and stirring was continued at room temperature for

24 h. The reaction mixture was poured into a saturated aq

NaHCO₃(25 mL) and extraction was performed with

CHCl₃(3!20 mL). The combined organic phase was

dried (Na₂SO₄), ﬁltered and evaporated to dryness under

reduced pressure. The residue obtained was puriﬁed by

column chromatography [5–8% (v/v) acetone in CH₂Cl₂] to

give the 2⁰-O-tert-butyldimethylsilyl isomer 11 as a white

foam (0.30 g, 49%). Further elution [8–10% (v/v) acetone

in CH₂Cl₂] yielded a byproduct tentatively assigned as the

3⁰-O-tert-butyldimethylsilyl isomer (yield not determined).

d_C(CDCl₃) 162.1, 158.7, 146.5, 144.6, 135.8, 135.6, 133.3,

130.1, 129.2, 128.2, 128.1, 127.7, 127.1, 113.4, 95.5, 87.3,

86.9, 84.4, 73.0, 72.7, 64.0, 55.4, 25.9, 18.2, K5.1, K5.3;

ESI-MS: m/z 786 ([MCNa]^C, C₄₃H₄₉N₃O₈SiNa^Ccalcd

786). The ¹H NMR data were found to be consistent with the

literature data for the corresponding D-enantiomer.^12b

1

m/z 266 ([MCNa]^C, C₉H₁₃N₃O₅Na^Ccalcd 266). The H

NMR data were found to be consistent with the literature

data for the corresponding ^D-enantiomer.^12b

7.1.9. 4-N-Benzoyl-1-(a-L-ribofuranosyl)cytosine (9). To

a stirred solution of nucleoside 8 (1.0 g, 4.11 mmol) in

anhydrous pyridine (20 mL) at 0 8C was added

G. Gaubert et al. / Tetrahedron 62 (2006) 2278–2294

2287

7.1.12. 4-N-Benzoyl-1-(2-O-tert-butyldimethylsilyl-3-O-

[2-cyanoethoxy(diisopropylamino)phosphino]-5-O-(4,4⁰-

dimethoxytrityl)-a-^L-ribofuranosyl)cytosine (12). To a

stirred solution of nucleoside 11 (70 mg, 0.09 mmol) and N,N-

diisopropylethylamine (0.16 mL, 0.92 mmol) in CH₂Cl₂

(4 mL) at room temperature was added 2-cyanoethyl N,N⁰-

diisopropylphosphoramidochloridite (0.09 mL, 0.46 mmol)

and stirring was continued for 15 h. CH₂Cl₂(10 mL) was

added and the resulting mixture was washed with saturated aq

NaHCO₃(10 mL). The organic phase was dried (Na₂SO₄),

ﬁltered and concentrated to dryness under reduced pressure.

The residue obtained was puriﬁed by column chromatography

[45–50% EtOAc in n-hexane, containing 0.5% Et₃N (v/v/v)]

affording phosphoramidite 12 (50 mg, 52%) as a white foam.

d_P(DMSO-d₆) 151.4, 150.9.

reduced pressure, the residue obtained was dissolved in

ethyl acetate (100 mL), and washing was performed ﬁrst

with saturated aq NaHCO₃(100 mL) and then with brine

(100 mL). The separated organic phase was dried (Na₂SO₄),

ﬁltered, evaporated to dryness under reduced pressure and

then coevaporated with toluene (2!5 mL). The residue

obtained was puriﬁed by column chromatography [15–20%

(v/v) EtOAc in light petroleum] to give furanose 16 (2.60 g,

74%) as a clear oil. d_H(CDCl₃) 8.00 (2H, d, JZ8.0 Hz),

7.59–7.25 (8H, m), 5.95 (1H, d, JZ3.6 Hz, H1), 4.70 (1H, d,

JZ3.6 Hz, H2), 4.66 (1H, d, JZ11.8 Hz, CH₂Ph), 4.58 (1H,

d, JZ11.9 Hz, CH₂Ph), 4.50–4.40 (3H, m, H4 and H5), 4.08

(1H, d, JZ2.8 Hz, H3), 1.55 and 1.35 [3H each, 2s,

CH₃(isopropylidene)]; d_C(CDCl₃) 166.1, 136.9, 133.1,

129.7, 128.5, 128.4, 128.3, 128.0, 127.9, 127.8, 127.7,

113.1, 105.8, 84.8, 82.7, 82.2, 71.8, 64.3, 27.1, 26.3;

MALDI-HRMS: m/z 407.1475 ([MCNa]^C, C₂₂H₂₄O₆Na^C

calcd 407.1471).

7.1.13. 3-O-Benzyl-5-O-tert-butyldiphenylsilyl-1,2-O-iso-

propylidene-b-^L-arabinofuranose (14). To a solution of

5-O-tert-butyldiphenylsilyl-1,2-O-isopropylidene-b-^L-ara-

binofuranose 13²⁴(7.00 g, 16.4 mmol) in anhydrous DMF

(50 mL) at 0 8C was added NaH (1.31 g, 60% suspension in

mineral oil, 32.7 mmol) and benzyl bromide (3.9 mL,

32.7 mmol). The reaction mixture was stirred at room

temperature for 5 h and then concentrated to dryness under

reduced pressure. The residue obtained was dissolved in

diethyl ether (100 mL) and washing was performed

successively with saturated aq NaHCO₃(100 mL) and

brine (100 mL). The separated organic phase was dried

(Na₂SO₄), ﬁltered and evaporated to dryness under reduced

pressure. The residue obtained was puriﬁed by column

chromatography [5–10% (v/v) EtOAc in light petroleum] to

afford nucleoside 14 as a clear oil (7.20 g, 86%). d_C(CDCl₃)

137.5, 135.6, 135.5, 133.1, 129.7, 128.5, 127.8, 127.7,

127.68, 127.64, 112.4, 105.7, 85.2, 85.1, 82.8, 71.6, 63.4,

26.9, 26.8, 26.1, 19.2; MALDI-HRMS: m/z 541.2382 ([MC

Na]^C, C₃₁H₃₈O₅SiNa^Ccalcd 541.2386). The ¹H NMR data

were found to be consistent with the literature data for the

corresponding ^D-enantiomer.^25b

7.1.16. 1,2-Di-O-acetyl-5-O-benzoyl-3-O-benzyl-a,b-^L-

arabinofuranose (17). Concentrated sulfuric acid

(0.02 mL) was added to a solution of furanose 16 (2.21 g,

5.76 mmol) in 80% aq acetic acid (20 mL) and stirring was

continued for 1 h at 50 8C. The reaction mixture was

allowed to cool to room temperature and then concentrated

under reduced pressure to approximately half of the original

volume. Pyridine (30 mL) and acetic anhydride (1.63 mL,

17.3 mmol) were added and the resulting mixture was

stirred at 50 8C for 6 h and then concentrated to dryness

under reduced pressure. The residue obtained was dissolved

in ethyl acetate (50 mL) and then washed ﬁrst with saturated

aq NaHCO₃(50 mL) followed by brine (50 mL). The

separated organic phase was dried (Na₂SO₄), ﬁltered,

evaporated to dryness under reduced pressure and then

coevaporated with toluene (2!5 mL). The residue obtained

was puriﬁed by column chromatography [15–20% (v/v)

EtOAc in light petroleum] yielding a 1:1 mixture of

anomers 17 as a clear oil (1.80 g, 73%). d_H(CDCl₃) 8.06–

8.01 (4H, m), 7.60–7.55 (2H, m), 7.46–7.40 (4H, m), 7.30–

7.26 (10H, m), 6.39 (1H, d, JZ4.6 Hz), 6.23 (1H, s), 5.31

(1H, m), 5.27 (1H, s), 4.77 (1H, d, JZ12.0 Hz), 4.70–4.34

(9H, m), 4.01–3.99 (2H, m), 2.12 (3H, s), 2.06 (3H, s), 2.05

(3H, s), 1.95 (3H, s), d_C(CDCl₃) 169.6, 169.5, 169.2, 166.1,

166.0, 137.2, 137.1, 133.2, 133.1, 129.8, 129.7, 129.6,

129.5, 128.5, 128.4, 128.3, 128.0, 127.9, 127.8, 127.7,

127.6, 99.9, 93.9, 83.1, 82.9, 80.6, 79.6, 79.5, 77.0, 72.7,

72.4, 64.3, 63.4, 21.1, 20.9, 20.7, 20.4; MALDI-HRMS: m/z

451.1375 ([MCNa]^C, C₂₃H₂₄O₈Na^Ccalcd 451.1369).

7.1.14. 3-O-Benzyl-1,2-O-isopropylidene-b-L-arabino-

furanose (15). To a solution of furanose 14 (9.84 g,

19.0 mmol) in THF (150 mL) was added TBAF (38.0 mL,

1 M in THF, 38.0 mmol) and stirring was continued at room

temperature for 12 h. The reaction mixture was concen-

trated to dryness under reduced pressure and the residue

dissolved in ethyl acetate (200 mL) whereupon washing was

performed with brine (2!100 mL). The separated organic

phase was dried (Na₂SO₄), ﬁltered and evaporated to

dryness under reduced pressure. The residue obtained was

puriﬁed by column chromatography [3–5% (v/v) MeOH in

CH₂Cl₂] to give furanose 15 (4.20 g, 80%) as a clear oil. d_C

(CDCl₃) 137.1, 128.5, 128.4, 128.0, 127.9, 127.7, 112.9,

105.5, 85.5, 85.2, 82.7, 71.8, 62.7, 27.1, 26.3; MALDI-MS:

m/z 303.1207 ([MCNa]^C, C₁₅H₂₀O₅Na^Ccalcd 303.1208).

7.1.17. 9-(2-O-Acetyl-5-O-benzoyl-3-O-benzyl-a-^L-

arabinofuranosyl)-6-N-benzoyladenine (18). To a suspen-

sion of anomers 17 (0.70 g, 1.64 mmol) and 6-N-benzoy-

ladenine (0.59 g, 2.45 mmol) in anhydrous acetonitrile

(6 mL) was added SnCl₄(0.4 mL, 3.3 mmol) and the

resulting mixture was stirred at room temperature for 4 h.

Saturated aq NaHCO₃was added until the evolution of

carbon dioxide ceased whereupon the mixture was ﬁltered

through a layer of Celite 545, that was subsequently ﬂushed

with CHCl₃(2!50 mL). The combined ﬁltrate was washed

successively with saturated aq NaHCO₃(3!100 mL) and

brine (2!100 mL), dried (Na₂SO₄), ﬁltered and evaporated

to dryness under reduced pressure. The residue obtained was

puriﬁed by column chromatography [4–5% (v/v) MeOH in

1

The H NMR data were found to be consistent with the

literature data for the corresponding ^D-enantiomer.²⁵

7.1.15. 5-O-Benzoyl-3-O-benzyl-1,2-O-isopropylidene-

b-^L-arabinofuranose (16). Benzoyl chloride (1.6 mL,

13.9 mmol) was added dropwise to a solution of furanose

15 (2.60 g, 9.29 mmol) in anhydrous pyridine (10 mL) and

the resulting mixture was stirred at room temperature for

2 h. The mixture was then concentrated to dryness under

2288

G. Gaubert et al. / Tetrahedron 62 (2006) 2278–2294

CHCl₃] affording nucleoside 18 (870 mg, 88%) as a

white solid material. d_H(CDCl₃) 9.16 (1H, br s, NH), 8.81

(1H, s, H8), 8.39 (1H, s, H2), 8.04–8.00 (4H,₀m), 7.62–7.42

(6H, m), 7.28–7.23 (5H, m), 6.48 (1H, s, H1 ), 5.81 (1H, t,

JZ1.4 Hz, H2⁰), 4.83 (1H, m, H4⁰), 4.73 (1H, d, JZ

11.9 Hz, CH₂Ph), 4.65 (1H, d, JZ12.0 Hz, CH₂Ph), 4.51

(1H, d, JZ5.4 Hz, H5⁰a), 4.50 ₀(1H, d, JZ5.6 Hz, H5⁰b),

4.24 (1H, dd, JZ1.3, 3.3 Hz, H3 ), 2.11 (3H, s, COCH₃); d_C

(CDCl₃) 169.7, 166.2, 164.7, 153.0, 151.8, 149.7, 141.6,

136.4, 133.7, 133.5, 132.9, 129.9, 129.5, 129.0, 128.7,

128.6, 128.4, 128.04, 128.01, 123.1, 88.5, 84.3, 82.7, 80.5,

72.7, 63.5, 20.9; MALDI-HRMS: m/z 630.1966 ([MC

Na]^C, C₃₃H₂₉N₅O₇Na^Ccalcd 630.1965).

169.3, 166.2, 164.9, 152.8, 149.8, 149.6, 142.8, 136.5,

133.6, 132.9, 129.8, 129.4, 129.1, 128.9, 128.8, 128.7,

128.6, 128.3, 128.2, 128.1, 125.4, 123.9, 82.7, 81.0, 76.8,

73.8, 70.9, 63.5, 20.6; MALDI-HRMS: m/z 630.1960 ([MC

Na]^C, C₃₃H₂₉N₅O₇Na^Ccalcd 630.1965).

7.1.20. 6-N-Benzoyl-9-(a-^L-ribofuranosyl)adenine (22).

Aqueous sodium hydroxide (1, 2.7 mL) was added to an ice-

cold solution of nucleoside 20 (0.36 g, 0.59 mmol) in a

mixture of ethanol (1.8 mL) and pyridine (3.5 mL). The

reaction mixture stirred at 0 8C for 30 min and then

neutralized with Dowex 50WX2(H^C). Dowex was ﬁltered

off and the ﬁltrate was concentrated to dryness under

reduced pressure. The oily residue was dissolved in EtOAc

(100 mL) whereupon washing was performed using brine

(2!75 mL). The separated organic phase was dried

(Na₂SO₄), ﬁltrated and concentrated to dryness under

reduced pressure. The residue obtained was coevaporated

with toluene and puriﬁed by column chromatography [3–5%

MeOH in EtOAc] to give nucleoside 21 (tentatively

assigned) as a white solid material (0.21 g). Ammonium

formate (200 mg) and Pd/C (100 mg) were added to a

solution of nucleoside 21 (0.21 g) in EtOH (5 mL). The

resulting mixture was heated under reﬂux for 2 h, cooled to

room temperature, ﬁltered across a pad of Celite and then

concentrated to dryness. The crude product was recrystalli-

zed from EtOH to give nucleoside 22 as white needles

(0.12 g, 54% from 20). d_C(DMSO-d₆) 165.5, 152.6, 151.4,

149.9, 144.9, 133.4, 132.4, 128.5, 125.0, 85.3, 83.6, 70.7,

70.6, 61.4; MALDI-HRMS: m/z 394.1120 ([MCNa]^C,

C₁₇H₁₇N₅O₅Na^Ccalcd 394.1127). The ¹H NMR data were

found to be consistent with the literature data for the

corresponding ^D-enantiomer.^12b

7.1.18. 6-N-Benzoyl-9-(5-O-benzoyl-3-O-benzyl-a-^L-ara-

binofuranosyl)adenine (19). To a solution of nucleoside 18

(0.64 g, 1.06 mmol) in MeOH (16 mL) was added saturated

methanolic ammonia (16 mL) and the mixture was stirred at

0 8C for 1.5 h. The reaction mixture was concentrated to

dryness under reduced pressure and the residue was

coevaporated with toluene (5!10 mL). The residue

obtained was puriﬁed by column chromatography [5–6%

(v/v) MeOH in CHCl₃] affording nucleoside 19 (550 mg,

92%) as a white solid material. d_H(CDCl₃) 9.07 (1H, br s,

NH), 8.71 (1H, s, H8), 8.21 (1H, s, H2), 8.05–8.01 (4H, m),

7.62–7.43 (6H, m), 7.30–7.25 (5H, m), 6.12 (1H, d, JZ

4.0 Hz, H1⁰), 4.97 (1H, t, JZ4.7 Hz, H2⁰), 4.80 (1H, d, JZ

11.9 Hz, CH₂Ph), 4.75 (1H, m, H4⁰), 4.71 (1H, d, JZ

12.0 Hz, CH₂Ph), 4.63 (1H, dd, JZ3.7, 12.4 Hz, H5⁰a), 4.51

(1H, dd, JZ5.0, 12.3 Hz, H5⁰b), 4.33 (1H, t, JZ5.3 Hz,

H3⁰); d_C(CDCl₃) 166.4, 164.7, 152.5, 151.1, 149.6, 141.4,

137.2, 133.6, 133.5, 133.1, 129.9, 129.8, 129.7, 129.1,

128.7, 128.6, 128.5, 128.3, 128.1, 128.0, 123.2, 91.4, 82.3,

80.6, 72.7, 63.9; MALDI-HRMS: m/z 588.1863 ([MC

Na]^C, C₃₁H₂₇N₅O₆Na^Ccalcd 588.1859).

7.1.21. 6-N-Benzoyl-9-[5-O-(4,4⁰-dimethoxytrityl)-a-L-

ribofuranosyl]adenine (23). 4,4⁰-Dimethoxytrityl chloride

(0.33 g, 1.0 mmol) was added to a solution of nucleoside 22

(0.13 g, 0.35 mmol) in anhydrous pyridine (7 mL) and

stirring was continued at room temperature for 12 h. MeOH

(5 mL) was added and after stirring for another 10 min the

reaction mixture was poured into saturated aq NaHCO₃

(25 mL). Extraction was performed with CHCl₃(3!

20 mL) and the combined organic phase was dried

(Na₂SO₄), ﬁltered and evaporated to dryness under reduced

pressure. The residue obtained was puriﬁed by column

chromatography [6–8% (v/v) MeOH in CHCl₃] affording

nucleoside 23 (220 mg, 92%) as a white foam. d_C(CDCl₃)

158.7, 152.0, 151.1, 149.4, 144.7, 143.9, 143.8, 135.9,

135.7, 133.7, 133.0, 130.2, 130.0, 129.0, 128.2, 128.1,

128.0, 127.9, 127.8, 127.1, 122.9, 113.4, 113.2, 87.2, 86.9,

86.0, 72.8, 72.2, 64.4, 55.4.; ESI-HRMS: m/z 696.2429

([MCNa]^C, C₃₈H₃₅N₅O₇Na^Ccalcd 696.2434). The ¹H

NMR data were found to be consistent with the literature

data for the corresponding ^D-enantiomer.^12b

7.1.19. 9-(2-O-Acetyl-5-O-benzoyl-3-O-benzyl-a-^L-ribo-

furanosyl)-6-N-benzoyladenine (20). Nucleoside 19

(0.45 g, 0.80 mmol) was dissolved in a mixture of

anhydrous CH₂Cl₂(20 mL) and anhydrous pyridine

(4 mL). The stirred solution was cooled to K30 8C and

triﬂuoromethanesulfonic anhydride (0.35 mL, 2.15 mmol)

was added. After 1.5 h, the reaction mixture was allowed to

warm to 0 8C and saturated aq NaHCO₃(10 mL) and

CH₂Cl₂(60 mL) were added. The organic phase was

separated, washed with saturated aq NaHCO₃(3!70 mL),

dried (Na₂SO₄), ﬁltered and concentrated to dryness under

reduced pressure. The residue was dissolved in a mixture of

anhydrous toluene (24 mL) and anhydrous CH₂Cl₂(24 mL)

whereupon KOAc (0.39 g, 3.98 mmol) and 18-crown-6

(0.74 g, 2.79 mmol) were added at room temperature under

stirring. The temperature was raised to 50 8C and stirring

was continued for another 16 h. After cooling to room

temperature, CH₂Cl₂(100 mL) was added, and the reaction

mixture was washed with saturated aq NaHCO₃(3!

50 mL), dried (Na₂SO₄), ﬁltered and evaporated to dryness

under reduced pressure. The residue obtained was puriﬁed

by column chromatography [3–5% (v/v) MeOH in CHCl₃]

to yield nucleoside 20 (380 mg, 79%) as a white solid

material. d_H(CDCl₃) 9.17 (1H, br s, NH), 8.80 (1H, s, H8),

8.35 (1H, s, H2), 8.04–7.43 (15H, m), 6.47 (1H, d, JZ

5.2₀Hz, H1⁰), 5.87 (1H, m, H2⁰), 4.72–4.25 (6H, m, H3⁰,

H4 , H5⁰and CH₂Ph), 2.08 (3H, s, COCH₃); d_C(CDCl₃)

7.1.22. 6-N-Benzoyl-9-[2-O-tert-butyldimethylsilyl-5-O-

(4,4⁰-dimethoxytrityl)-a-L-ribofuranosyl]adenine (24).

tert-Butyldimethylsilyl chloride (0.06 g, 0.43 mmol) was

added to a solution of nucleoside 23 (0.22 g, 0.33 mmol)

and imidazole (0.06 g, 0.85 mmol) in anhydrous pyridine

(5 mL) and stirring was continued at room temperature for

15 h. The reaction mixture was poured into saturated aq

NaHCO₃(25 mL) and extraction was performed with

G. Gaubert et al. / Tetrahedron 62 (2006) 2278–2294

2289

CHCl₃(3!20 mL). The combined organic phase was dried

(Na₂SO₄), ﬁltered and evaporated to dryness under reduced

pressure. The residue obtained was puriﬁed by column

chromatography [4–5% (v/v) acetone in CH₂Cl₂] to give the

required 2⁰-O-tert-butyldimethylsilyl isomer 24 as a white

foam (0.12 g, 48%). Further elution [5–7% (v/v) acetone i₀n

CH₂Cl₂] yielded a byproduct tentatively assigned as the 3 -

O-tert-butyldimethylsilyl isomer (yield not determined). d_C

(CDCl₃) 164.6, 158.73, 158.71, 152.5, 151.8, 149.5, 144.6,

143.6, 135.7, 135.4, 133.9, 132.8, 130.1, 130.0, 128.9,

128.1, 127.9, 127.1, 122.6, 113.4, 113.37, 86.9, 85.8, 85.4,

73.2, 72.9, 64.4, 55.3, 25.4, 17.8, K5.2, K5.3; ESI-HRMS:

m/z 810.3286 ([MCNa]^C, C₄₄H₄₉N₅O₇SiNa^Ccalcd

stirring the mixture 12 h at room temperature, toluene

(20 mL) was added and the solution was concentrated to

approximately one-fourth the original volume under

reduced pressure. CH₂Cl₂(100 mL) was added whereupon

washing was performed with saturated aq NaHCO₃(2!

50 mL). The separated organic phase was dried (Na₂SO₄),

ﬁltered and concentrated to dryness under reduced pressure.

The residue was coevaporated with toluene (2!10 mL) and

then puriﬁed by column hromatography [40–50% EtOAc in

light petroleum, containing 0.5% Et₃N (v/v/v)] to afford

nucleoside 28 as a white solid (5.68 g, 97%). R_f0.33

(MeOH/CH₂Cl₂5:95, v/v); d_H(CDCl₃) 8.89 (1H, s, NH),

7.64–7.61 (2H, m), 7.58–7.55 (2H, m), 7.48 (1H, d, JZ

1.2 Hz, H6), 7.44–7.40 (7H, m), 7.36–7.30 (9H, m), 7.28–

7.12 (9H, m), 6.73 (4H, d, JZ8.7 Hz), 5.82 (1H, d, JZ

3.7₀Hz, H1⁰), 4.35 (1H, d, JZ3.2 Hz, H3⁰), 4.07 (1H, m,

H2 ), 4.03 (1H, d, JZ11.7 Hz, H5⁰a), 3.92 (1H, d, JZ

11.8 Hz, H5⁰b), 3.77 and 3.76 (3H each, 2s, 2!OCH₃), 3.57

(1H, d, JZ9.6 Hz, H5⁰⁰a), 3.18 (1H, d, JZ9.5 Hz, H5⁰⁰b),

2.89 (1H, d, JZ5.4 Hz, 2⁰-OH), 1.65 (3H, s, 5-CH₃), 1.00

and 0.83 (9H each, 2s, 2!C(CH₃)₃); d_C(CDCl₃) 163.9

(C4), 158.5, 158.4, 150.8 (C2), 144.4, 136.0, 135.8, 135.7,

135.6, 135.5, 133.2, 133.0, 132.9, 132.0, 130.4, 130.2,

130.1, 129.9, 129.8, 128.4, ₀127.9, 127.8, 126.8, 113.2, 110.5

(C5), 91.4 (C1⁰), 89.9 (C4 ), 87.0 (CAr₃), 83.2 (C2⁰), 80.0

(C3⁰), 65.0 and 63.6 (C5⁰and C5⁰⁰), 55.3 (2!OCH₃), 27.0

and 26.9 (2!C(CH₃)₃), 19.5 and 19.2 (2!SiC(CH₃)₃), 12.4

(5-CH₃); MALDI-HRMS: m/z 1089.4576 ([MCNa]^C,

C₆₄H₇₀N₂O₉Si₂Na^Ccalcd 1089.4512).

1

810.3299). The H NMR data were found to be consistent

with the literature data for the corresponding

D-enantiomer.^12b

7.1.23. 6-N-Benzoyl-9-[2-O-tert-butyldimethylsilyl-3-O-

(2-cyanoethoxy(diisopropylamino)phosphino)-5-O-(4,4⁰-

dimethoxytrityl)-a-^L-ribofuranosyl]adenine (25). To a

stirred solution of nucleoside 24 (70 mg, 0.09 mmol) and

N,N-diisopropylethylamine (0.16 mL, 0.92 mmol) in

CH₂Cl₂(2 mL) at₀room temperature was added 2-cya-

noethyl

N,N -diisopropylphosphoramidochloridite

(0.09 mL, 0.46 mmol) and stirring was continued for 15 h.

CH₂Cl₂(10 mL) was added and the resulting mixture was

washed with saturated aq NaHCO₃(10 mL). The organic

phase was dried (Na₂SO₄), ﬁltered and concentrated to

dryness under reduced pressure. The residue obtained was

puriﬁed by column chromatography [50–55% EtOAc in

n-hexane, containing 0.5% Et₃N (v/v/v)] furnishing phos-

phoramidite 25 (65 mg, 73%) as a white foam. d_P(DMSO-

d₆) 151.8, 150.4; ESI-MS m/z 988.4 [MCH]^C, 1010.5

([MCNa]^C, C₅₃H₆₆N₇O₈PSiNa^Ccalcd 1010.5).

7.1.26. 1-[3,5-Di-O-(tert-butyldiphenylsilyl)-4-C-(4,4⁰-

dimethoxytrityloxymethyl)-2-O-methanesulfonyl-b-^D-

xylofuranosyl]thymine (29) and 2,2⁰-anhydro-1-[3,5-di-

O-(tert-butyldiphenylsilyl)-4-C-(4,4⁰-dimethoxytrityloxy-

methyl)-b-^D-lyxofuranosyl]thymine (30). Nucleoside 28

(3.2 g, 3.0 mmol) was dissolved in a 1:1 mixture of

anhydrous CH₂Cl₂–Et₃N (10 mL). DMAP (440 mg,

3.6 mmol) was added followed by methanesulfonyl chloride

(413 mg, 3.6 mmol) and the resulting mixture was stirred at

room temperature for 12 h. Analytical TLC showed the

formation of two products. The reaction mixture was diluted

with CH₂Cl₂(100 mL) and washing was performed with

saturated aq NaHCO₃(2!50 mL). The separated organic

phase was dried (Na₂SO₄), ﬁltered and concentrated to

dryness under reduced pressure. An analytical sample was

puriﬁed by column chromatography [40–45% (v/v) EtOAc

in light petroleum, containing 0.5% Et₃N (v/v/v)] to give as

the major product nucleoside 29, R_f0.38 (MeOH/CH₂Cl₂

5:95, v/v), and [70–75% (v/v) EtOAc in light petroleum,

containing 0.5% Et₃N (v/v/v)] as the minor product the

anhydro nucleoside 30, R_f0.28 (MeOH/CH₂Cl₂5:95, v/v),

(both as white solid materials). Data for compound 29: d_H

(CDCl₃) 8.70 (1H, s, NH), 7.70–7.68 (4H, m), 7.54–7.52

(2H, m), 7.47–7.25 (16H, m), 7.23–7.14 (8H, m), 6.76–6.73

(4H, m), 6.03 (1H, d, JZ4.7 Hz, H1⁰), 5.17 (1H, dd, JZ4.3,

4.7 Hz, H2⁰), 4.67 (1H, d, JZ4.1 Hz, H3⁰), 4.1₀7 (1H, d, JZ

11.2 Hz, H5⁰a), 4.12 (1H, d, JZ11.2 Hz, H5 b), 3.77 and

3.76 (3H each, 2s, 2!OCH₃), 3.39 (1H, d, JZ9.4 Hz,

H5⁰⁰a), 3.09 (1H, d, JZ9.1 Hz, H5⁰⁰b), 2.50 (3H, s,

SO₂CH₃), 1.44 (3H, s, 5-CH₃), 1.03 and 0.87 (9H each,

2s, 2!C(CH₃)₃); d_C(CDCl₃) 163.5 (C4), 158.5, 150.4 (C2),

144.7, 136.0, 135.9, 135.8, 135.6, 135.4, 133.5, 132.9,

132.3, 131.1, 130.5, 130.3, 130.2, 129.9, 128.3, 128.1,

7.1.24. 1-[3,5-Di-O-(tert-butyldiphenylsilyl)-4-C-hydro-

xymethyl-b-^D-xylofuranosyl]thymine (27). A solution of

nucleoside 26²⁸(5.50 g, 6.04 mmol) in saturated methanolic

ammonia (100 mL) was stirred for 48 h at room tempera-

ture. After evaporation to dryness under reduced pressure,

the resulting residue was coevaporated with toluene (2!

5 mL) and puriﬁed by column chromatography [60–66% (v/

v) EtOAc in light petroleum] to afford nucleoside 27

(4.45 g, 96%) as a white solid material. R_f0.24 (MeOH/

CH₂Cl₂15:85, v/v); d_H(CDCl₃) 9.50 (1H, s), 7.66 (2H, dd,

JZ1.3, 7.9 Hz), 7.59 (2H, dd, JZ1.3, 7.9 Hz), 7.50–7.31

(13H, m), 7.27–7₀.23 (4H, m), 5.86 (1H, d, JZ2.9 Hz, H1⁰),

4.43 (1H, br s, 2 -OH), 4.41 (1H, d, JZ2.7 Hz, H2⁰), 4.22

(1H, br s, H3⁰), 4.02 (1₀H₀, d, JZ11.6 Hz, H5⁰a), 3.86 (1H,

dd, JZ4.5, 11.9 Hz, H5 a), 3.71 (1H, d, JZ11.9 Hz, H5⁰b),

3.48 (1H, dd, JZ6.0, 11.7 Hz, H5⁰⁰b), 2.76 (1H, m, 5⁰-OH),

1.66 (3H, d, JZ1.0 Hz, 5-CH₃), 1.06 and 0.91 (2!

SiC(CH₃)₃); d_C(CDCl₃) 164.1, 151.0, 136.0, 135.9, 135.8,

135.7, 132.9, 132.8, 132.6, 132.2, 130.3, 130.2, 130.0,

128.0, 127.9, 127.8, 110.8, 92.0, 90.7, 83.2, 79.6, 65.2, 63.6,

27.0, 26.9, 19.4, 19.2, 12.4; MALDI-MS: m/z 787 ([MC

Na]^C, C₄₃H₅₂N₂O₇Si₂Na^Ccalcd 787).

7.1.25. 1-[3,5-Di-O-(tert-butyldiphenylsilyl)-4-C-(4,4⁰-

dimethoxytrityloxymethyl)-b-^D-xylofuranosyl]thymine

(28). 4,4⁰-Dimethoxytrityl chloride (2.05 g, 6.05 mmol) was

added in one portion to a stirred solution of nucleoside 27

(4.2 g, 5.49 mmol) in anhydrous pyridine (20 mL). After

2290

G. Gaubert et al. / Tetrahedron 62 (2006) 2278–2294

128.0, 127.9, 12₀6.8, 113.2, ₀111.8 (C5), 88.3 (C4⁰₀), 86.7

(CAr₃), 85.6 (C1 ), 85.2 (C2 ), 77.0 (C3⁰), 64.4 (C5 ), 62.5

(C5⁰⁰), 55.3 (2!OCH₃), 38.3 (SO₂CH₃), 27.2 and 27.0 (2!

C(CH₃)₃), 19.6 and 19.3 (2!SiC(CH₃)₃), 11.9 (5-CH₃);

data for compound 30: d_H(CDCl₃) 7.57–7.54 (2H, m), 7.41–

7.19 (22H, m), 7.16–7₀.07 (6H, m), 6.77–6.73 (4H, m), 6.07

(1H, d, JZ6.2 Hz, H1 ), 4.82 ₀(1H, dd, JZ6.0, 6.3 Hz, H2⁰),

4.65 (1H, d, JZ5.9 Hz, H3 ), 3.87 (1H, d, JZ10.5 Hz,

H5⁰a), 3.79 (6H, s, 2!OCH₃), 3.58 (1H, d, JZ12.2 Hz,

H5⁰⁰a), 3.15₀(1H, d, JZ12.2 Hz, H5⁰⁰b), 2.75 (1H, d, JZ

10.3 Hz, H5 b), 1.99 (3H, s, 5-CH₃), 0.88 and 0.81 (9H each,

2s, 2!C(CH₃)₃); d_C(CDCl₃) 172.4 (C4), 159.9 (C2), 158.6,

144.5, 136.1, 135.8, 135.6, 135.5, 135.4, 133.1, 132.7,

132.5, 132.3, 130.3, 130.2, 130.1, 129.9, 129.8, 129.7,

128.1, 127.9, 127.8, 127.7, 127.6, 127.0, 119.0 (C5), 113.₀2,

90.8 (C4⁰), 89.3 (C1⁰), 86.9 (CAr₃), 81.2 (C2⁰), 73.3 (C3 ),

64.9 and 64.4 (C5⁰and C5⁰⁰), 55.3 (2!OCH₃), 26.8 and 26.7

(2!C(CH₃)₃), 19.4 and 19.2 (2!SiC(CH₃)₃), 14.2 (5-

CH₃); MALDI-HRMS: m/z 1071.4466 ([MCNa]^C, C₆₄-

H₆₈N₂O₈Si₂Na^Ccalcd 1071.4406).

residue was coevaporated with toluene (2!5.0 mL) and

puriﬁed by column chromatography [30–40% (v/v) EtOAc

in light petroleum] to give nucleoside 32 (955 mg, 58%) and

[45–50% (v/v) EtOAc in light petroleum] nucleoside 33

(384 mg, 23%) (both as white solid materials). R_f0.26, 0.32

(MeOH/CH₂Cl₂5:95, v/v); data for 32: d_H(CDCl₃) 8.74

(1H, s, NH), 7.43–7.39 (3H, m), 7.31–7.19 (7₀H, m), 6.82

(4H, d, JZ8.7 Hz), 6.48 (1H, d, JZ5.9 Hz, H1 ₀), 4.77 (1H,

dd, JZ5.3, 5.5 Hz, H2⁰₀), 4.04–4.00 (2H, m, H3 and H5⁰a),

3.77–3.74 (7H, m, H5 b and 2!OCH₃), 3.33 (1H, d, JZ

9.8 Hz, H5⁰⁰a), 3.23 (1H, d, JZ10.1 Hz, H5⁰⁰b), 2.89 (1H, d,

JZ1.6 Hz, 3⁰-OH), 1.91 (3H, s, 5-CH₃), 0.80 and 0.76 (9H

each, 2s, 2!C(CH₃)₃), 0.04, K0.02, K0.04 and K0.06 (3H

each, 4s, 4!SiCH₃); d_c(CDCl₃) 163.8 (C4), 158.6, 150.7

(C2), 144.5, 138.2, 135.8, 135.5, 130.1, 130.0, 128.1, 128.0,

127.0, 113.4, 113.3, 109.2 (C5), 87.2, 86.8 and 85.2 (C1⁰,

C4⁰and CAr₃), 73.0 and 72.7 (C2⁰and C3⁰), 66.1 and 63.5

(C5⁰and C5⁰), 55.3 (2!OCH₃), 25.8 and 25.6 (2!

C(CH₃)₃), 18.2 and 18.0 (2!C(CH₃)₃), 12.6 (5-CH₃),

K5.2, K5.3, K5.4 and K5.5 (4!SiCH₃); MALDI-

HRMS: m/z 841.3900 ([MCNa]^C, C₄₄H₆₂N₂O₉Si₂Na^C

calcd 841.3886); data for 33: d_H(CDCl₃) 8.81 (1H, s, NH),

7.60 (1H, d, JZ1.0 Hz, H6), 7.42 (2H, d, JZ7.2 Hz), 7.33–

7.21 (7H, m), 6.83 (4H, d, JZ9.0 Hz), 6.10 (1H, d, JZ

2.7 Hz, H1⁰), 4.67 (1H, d, JZ10.2 Hz, 2⁰-OH), 4.53 (1H, ₀d,

JZ4.8 Hz, H3⁰), 4.12 (1H, ddd, JZ2.7, 5.1, 10.2 Hz, H2 ),

3.79 (6H, s, 2!OCH₃), 3.74 (1H, d, JZ10.6 Hz, H5⁰a),

3.59 (1H, d, JZ10.8 Hz, H5⁰b), 3.15 (1H, d, JZ9.9 Hz,

H5⁰⁰a), 3.04 (1H, d, JZ9.9 Hz, H5⁰⁰b), 1.94 (3H, s, 5-CH₃),

0.95 and 0.81 (9H each, 2s, 2!C(CH₃)₃), 0.16, 0.14, 0.02

and K0.05 (3H each, 4s, 4!SiCH₃); d_C(CDCl₃) 163.9,

158.6, 150.4, 144.4, 137.5, 135.6, 135.5, 130.1, 130.0,

128.1, 128.0, 127.0, 113.3, 108.8, 86.9, 86.6, 85.0, 74.4,

71.2, 65.3, 63.5, 55.3, 26.0, 25.8, 18.3, 18.1, 12.6, K4.7,

K5.1, K5.4 and K5.5; MALDI-HRMS: m/z 841.3908

([MCNa]^C, C₄₄H₆₂N₂O₉Si₂Na^Ccalcd 841.3886).

7.1.27. 1-[5-O-(4,4⁰-Dimethoxytrityloxymethyl)-4-C-

hydroxymethyl-a-^L-ribofuranosyl]thymine (31). The

crude mixture (3.35 g) obtained from the mesylation of 28

was dissolved in ethanol (50 mL), H₂O (45 mL) and aq

NaOH (2 M solution, 5.0 mL) were added and the resulting

solution was heated under reﬂux for 16 h. Toluene (100 mL)

was added and the resulting mixture was concentrated to

approximately one-third of the original volume. After

partitioning between EtOAc (200 mL) and saturated aq

NaHCO₃(200 mL), the aqueous phase was separated and

extracted with EtOAc (100 mL). The combined organic

phase was washed with brine (200 mL), dried (Na₂SO₄),

ﬁltered and concentrated to dryness under reduced pressure.

The residue obtained was puriﬁed by column chromatog-

raphy [4–6% MeOH in CH₂Cl₂, containing 0.5% Et₃N (v/v/

v)] to afford nucleoside 31 as a white solid material (1.15 g,

65% from 28). R_f0.12 (MeOH/CH₂Cl₂5:95, v/v); d_H

(CDCl₃) 10.30 (1H, s, NH), 7.81 (1H, s, H6), 7.40 (2H, d,

JZ7.3 Hz), 7.31–7.15 (7H, m), 6.81 (4H, d, JZ8.5 Hz),

6.09 (1H, d, JZ3.8 Hz, H1⁰), 4.66₀(1H, dd, JZ4.3, 4.6 Hz,

H2⁰), 4.38 (1H, d, JZ5.1 Hz, H3 ), 3.90 (2H, br s, H5⁰⁰),

3.74 (6H, s, 2!OCH₃), 3.25 (1H, d, JZ9.9 Hz, H5⁰a), 3.17

(1H, d, JZ10.1 Hz, H5⁰b), 1.81 (3H, s, 5-CH₃); d_C(CDCl₃)

165.4, 158.6, 151.0, 144.5, 138.5, 135.6, 135.5, 130.2,

130.1, 128.1, 128.0, 127.0, 113.3, 113.2, 108.9, 87.3, 86.8,

86.2, 73.9, 70.9, 66.2, 63.3, 55.3, 12.5; MALDI-HRMS: m/z

613.2180 ([MCNa]^C, C₃₂H₃₄N₂O₉Na^Ccalcd 613.2157).

7.1.29. 1-[3-O-(2-Cyanoethoxy(N,N-diisopropylamino)-

phosphino)-2,5-di-O-(tert-butyldimethylsilyl)-4-C-(4,4⁰-

dimethoxytrityloxymethyl)-b-^D-lyxofuranosyl]thymine

(34). 2-Cyanoethyl N,N-diisopropylphosphoramidochlori-

dite (473 mg, 2.0 mmol) was added dropwise to a stirred

solution of the nucleoside 32 (819 mg, 1.0 mmol) and N,N-

diisopropylethylamine (1.0 mL) in anhydrous CH₂Cl₂

(10 mL). After stirring the resulting mixture for 12 h at

room temperature, the reaction mixture was diluted with

EtOAc (50 mL). Washing was performed with saturated aq

NaHCO₃(2!25 mL). The separated organic phase was

dried (Na₂SO₄), ﬁltered and concentrated to dryness under

reduced pressure. The residue obtained was puriﬁed by

column chromatography [33–40% EtOAc in n-hexane

containing 0.5% Et₃N (v/v/v)] to give amidite 34 as a

white solid material (723 mg, 71%). R_f0.31, 0.32 (MeOH/

CH₂Cl₂5:95, v/v); d_P153.9 and 151.6; d_H(CDCl₃, major

isomer) 8.13 (br s, NH), 7.52 (s, H6), 7.42 (d, JZ7.7 Hz),

7.32 (d, JZ8.8 Hz), 7.27–7.21 (m), 6.84 (d, JZ8.7 Hz),

6.35 (d, JZ5.7 Hz, H1⁰), 4.59 (dd, JZ5.1, 5.7 Hz, H2⁰),

4.45 (dd, JZ5.1, 13.7 Hz, H3⁰), 4.02 (d, JZ11.3 Hz, H5⁰a),

3.84 (d, JZ11.6 Hz, H5⁰b), 3.79 (s, 2!OCH₃), 3.75–3.56

(m, OCH₂and 2!CH(CH₃)₂), 3.44 (d, JZ9.9 Hz, H5⁰⁰a),

3.22 (d, JZ9.8 Hz, H5⁰⁰b), 2.34 (dd, JZ6.5, 11.4 Hz,

CH₂CN), 1.90 (s, 5-CH₃), 1.19 (d, JZ6.3 Hz, C(CH₃)₂),

1.18 (d, JZ6.4 Hz, C(CH₃)₂), 0.81 (s, 2!C(CH₃)₃), 0.06,

7.1.28. 1-[2,5-Di-O-(tert-butyldimethylsilyl)-4-C-(4,4⁰-

dimethoxytrityloxymethyl)-b-^D-lyxofuranosyl]thymine

(32) and 1-[3,5-di-O-(tert-butyldimethylsilyl)-4-C-(4,4⁰-

dimethoxytrityloxymethyl)-b-^D-lyxofuranosyl]thymine

(33). tert-Butyldimethylsilyl chloride (1.21 g, 8.0 mmol)

and imidazole (1.09 g, 16.0 mmol) were added to a stirred

solution of nucleoside 31 (1.18 g, 2.0 mmol) in anhydrous

pyridine (10 mL). The reaction mixture was stirred at room

temperature for 12 h and MeOH (1.0 mL) was then added.

After stirring for 30 min the reaction mixture was

concentrated to dryness under reduced pressure. The residue

was dissolved in EtOAc (100 mL) and washed with

saturated aq NaHCO₃(2!50 mL). The organic phase was

dried (Na₂SO₄), ﬁltered and concentrated to dryness. The

G. Gaubert et al. / Tetrahedron 62 (2006) 2278–2294

2291

K0.05, K0.06 and K0.10 (4s, 4!SiCH₃); MALDI-

HRMS: m/z 1041.4963 ([MCNa]^C, C₅₃H₇₉N₄O₁₀PSi₂Na^C

calcd 1041.4965).

(C2⁰), 78.2 (C3⁰), 63.3 and 63.1 (C5⁰and C5⁰⁰), 55.2 (2!

OCH₃), 25.4 (C(CH₃)₃), 17.7 (C(CH₃)₃), 12.5 (5-CH₃),

K5.1 (SiCH₃), K5.6 (SiCH₃); MALDI-HRMS: m/z

831.3273 ([MCNa]^C, C₄₅H₅₂N₂O₁₀SiNa^Ccalcd

831.3283).

7.1.30.

1-[2-O-Acetyl-5-O-benzoyl-3-O-(tert-butyl-

dimethylsilyl)-4-C-(4,4⁰-dimethoxytrityloxymethyl)-a-L-

arabinofuranosyl]thymine (36). tert-Butyldimethylsilyl

chloride (2.0 g, 13.3 mmol), imidazole (1.82 g,

26.7 mmol) and DMAP (100 mg, 0.82 mmol) were added

to a stirred solution of 1-[2-O-acetyl-5-O-benzoyl-4-C-

(4,4⁰-dimethoxytrityloxymethyl)-a-L-arabinofuranosyl]thy-

mine (35²⁸, 3.28 g, 4.45 mmol) dissolved in anhydrous

DMF (10 mL). The reaction mixture was allowed to stir at

36 8C for 12 h and was then partitioned between CH₂Cl₂

(100 mL) and saturated aq KHSO₄(100 mL). The separated

organic phase was washed with saturated aq NaHCO₃

(50 mL), then dried (Na₂SO₄), concentrated and coevapo-

rated with toluene (3!5.0 mL). The crude product was

puriﬁed by column chromatography [45–50% (v/v) EtOAc

in light petroleum] to give nucleoside 36 as white solid

material (3.33 g). R_f0.2 (MeOH/CH₂Cl₂5:95, v/v); d_H

(CDCl₃) 8.79 (1H, s, NH), 7.83 (2H, d, JZ7.8 Hz), 7.58

(1H, dd, JZ7.4, 7.6 Hz), 7.47–7.39 (6H, m), 7.34 (4H, d,

JZ8.5 Hz), 7.28–7.21 (2H, m), 6.78 (4H, d, JZ8.7 Hz),

6.30 (1H, d, JZ3.5 Hz, H1⁰), 5.26 (1H, dd, JZ3.3, 3.7 Hz,

H2⁰), 4.65 (1H, d, JZ10.8 Hz, H5⁰⁰a), 4.5₀6 (1H, d, JZ

10.9 Hz, H5⁰⁰b), 4.28 (1H, d, JZ3.2 Hz, H3 ), 3.76 (6H, s,

2!OCH₃), 3.64 (1H, d, JZ10.2 Hz, H5⁰a), 3.35 (1H, d, JZ

10.1 Hz, H5⁰b), 2.13 (3H, s, COCH₃), 1.57 (3H, s, 5-CH₃),

0.70 (9H, s, C(CH₃)₃), K0.01 (3H, s, SiCH₃), K0.19 (3H, s,

SiCH₃); d_C(CDCl₃) 169.7, 165.7, 163.6, 158.7, 158.6,

150.4, 144.3, 136.0, 135.7, 135.3, 133.4, 130.3, 130.2,

129.8, 129.4, 128.5, 128.4, 128.0, 127.1, 113.3, 111.1, 88.1,

87.4, 86.8, 82.0, 76.3, 63.1, 55.3, 25.6, 20.9, 17.8, 12.2,

K4.9, K5.5. NMR spectroscopic data revealed the

compound to be contaminated with traces of DMF;

MALDI-HRMS: m/z 873.3393 ([MCNa]^C, C₄₇H₅₄N₂O₁₁-

SiNa^Ccalcd 873.3389).

7.1.32. 1-[5-O-Benzoyl-3-O-(tert-butyldimethylsilyl)-4-C-

(4,4⁰-dimethoxytrityloxymethyl)-2-O-methanesulfonyl)-

a-^L-arabinofuranosyl]thymine (38). Nucleoside 37

(2.56 g, 3.16 mmol) was dissolved in a 3:1 mixture of

anhydrous CH₂Cl₂and Et₃N (20 mL), and DMAP (580 mg,

4.75 mmol) was added. Methanesulfonyl chloride (544 mg,

4.75 mmol) was added dropwise and the reaction mixture

was stirred at room temperature. After 4 h analytical TLC

showed the formation of two products. Saturated aq

NaHCO₃(50 mL) was added and the phases were separated.

The aqueous phase was extracted with CH₂Cl₂(2!50 mL)

and the combined organic phase was washed ﬁrst with aq

HCl (1 M, 2!50 mL) and then with saturated aq NaHCO₃

(50 mL). The organic phase was dried (Na₂SO₄), ﬁltered

and concentrated to dryness under reduced pressure. The

crude product was coevaporated with toluene affording a

white foam. An analytical sample was obtained by quick

fractionation through silica gel [50–60% (v/v) EtOAc in

light petroleum] affording the major product nucleoside 38

as a white solid material. R_f0.23 (MeOH/CH₂Cl₂5:95, v/v);

d_H(CDCl₃) 9.51 (1H, s, NH), 7.85–7.82 (2H, m), 7.58 (1H,

m), 7.45–7.38 (6H, m), 7.33–7.29 (4H, m), 7.24–7.22 (2H,

m), 6.78–6.74 (4H, m), 6.29 (1H, d, JZ3.1 Hz, H1⁰), 5.08

(1H, dd, JZ2.6, 2.8 Hz, H2⁰), 4.84₀(1H, d, JZ11.3 Hz,

H5⁰a), 4.49₀(1H, d, JZ2.8 Hz, H3 ), 4.41 (1H, d, JZ

10.9 Hz, H5 b), 3.75 and 3.74 (3H each, 2s, 2!OCH₃), 3.65

(1H, d, JZ10.4 Hz, H5⁰⁰a), 3.37 (1H, d, JZ10.7 Hz, H5⁰⁰b),

3.20 (3H, s, SO₂CH₃), 1.54 (3H, s, 5-CH₃), 0.69 (9H, s,

C(CH₃)₃), K0.02 (3H, s, SiCH₃), K0.13 (3H, s, SiCH₃); d_C

(CDCl₃) 165.5 (COPh), 163.7 (C4), 158.7, 158.6, 150.7

(C2), 144.1, 135.5, 135.4, 135.1, 133.4, 130.3, 130.2, 130.1,

129.8, 129.4, 128.5, 128.4,₀128.1, 127.1, 113.4, 113.3, 111.3

(C5), 89.3 (C4⁰), 88.1 (C1 ), 86.8 and 86.6 (C2⁰and CAr₃),

76.6 (C3⁰), 62.9 and 62.8 (C5⁰and C5⁰⁰), 55.3 (2!OCH₃),

39.0 (SO₂CH₃), 25.5 (C(CH₃)₃), 17.7 (C(CH₃)₃), 12.3 (5-

CH₃), K4.8 (SiCH₃), K5.6 (SiCH₃); MALDI-HRMS: m/z

909.3040 ([MCNa]^C, C₄₆H₅₄N₂O₁₂SSiNa^Ccalcd

909.3059).

7.1.31. 1-[5-O-Benzoyl-3-O-(tert-butyldimethylsilyl)-4-C-

(4,4⁰-dimethoxytrityloxymethyl)-a-L-arabinofuranosyl]-

thymine (37). To a stirred solution of nucleoside 36 (3.0 g,

3.53 mmol) in MeOH (50 mL) was added methanol

saturated with ammonia (10 mL) and the resulting mixture

was stirred at room temperature for 5 h. The reaction

mixture was concentrated to dryness under reduced pressure

and the residue obtained was coevaporated with toluene

(2!2 mL). The crude product was puriﬁed by column

chromatography [50–60% (v/v) EtOAc in light petroleum]

furnishing nucleoside 37 as a white solid material (2.6 g,

80% from 35). R_f0.13 (MeOH/CH₂Cl₂5:95, v/v); d_H

(CDCl₃) 10.50 (1H, s, NH), 7.82–7.80 (2H, m), 7.53–7.47

(5H, m), 7.45–7.31 (6H,₀m), 7.23–7.19 (2H, m), 6.75–6.71

(4H, m), 6.12 (1H, s, H1 ), 5.41 (1H, d, JZ3.5 Hz, 2⁰-OH),

4.92 (1H, d, JZ10.3 Hz, ₀H5⁰a), 4.74 (1H, d, JZ10.8 Hz,

H5⁰b), 4.32 (1H, br s, H3 ), 4.17 (1H, br s, H2⁰), 3.73 and

3.72 (3H each, 2s, 2!OCH₃), 3.63 (1H, d, JZ10.7 Hz,

H5⁰⁰a), 3.31 (1H, d, JZ10.8 Hz, H5⁰⁰b), 1.57 (3H, s, 5-CH₃),

0.66 (9H, s, C(CH₃)₃), K0.03 (3H, s, SiCH₃), K0.15 (3H, s,

SiCH₃); d_C(CDCl₃) 165.6 (COPh), 164.6 (C4), 158.6,

158.5, 151.0 (C2), 144.6, 136.5, 135.9, 135.5, 133.0, 130.2,

130.0, 129.9, 129.7, 128.3, 128.2, 128.0, 126.9, 113.3,

113.2, 110.1 (C5), 93.3 (C1⁰), 90.3 (C4⁰), 86.3 (CAr₃), 83.5

7.1.33. 2,2⁰-Anhydro-1-[5-O-benzoyl-3-O-(tert-butyl-

dimethylsilyl)-4-C-(4,4⁰-dimethoxytrityloxymethyl)-a-L-

ribofuranosyl]thymine (39). The crude product obtained

after mesylation of nucleoside 37 was coevaporated with

anhydrous CH₃CN (2!5 mL) and then dissolved in CH₃CN

(10 mL), and DBU (609 mg, 4 mmol) was added. The

resulting mixture was stirred 12 h at room temperature and

then evaporated to dryness under reduced pressure. CHCl₃

(50 mL) was added whereupon washing was performed with

saturated aq NaHCO₃(2!50 mL). The organic phase was

dried (Na₂SO₄), ﬁltered and concentrated to dryness under

reduced pressure. The residue was puriﬁed by column

chromatography [4% (v/v) MeOH in CH₂Cl₂] to give

nucleoside 39 as a white solid material (2.08 g, 83% from

37). R_f0.11 (MeOH/CH₂Cl₂5:95, v/v); d_H(CDCl₃) 7.91–

7.89 (2H, m), 7.60 (1H, dd, JZ7.3, 7.6 Hz), 7.44 (2H, dd,

JZ7.4, 7.9 Hz), 7.36–7.33 (2H, m), 7.27–7.12 (8H, m),

6.77–6.72 (4H, m), 6.15 (1H, d, JZ5.8 Hz), 5.23 (1H, dd,

2292

G. Gaubert et al. / Tetrahedron 62 (2006) 2278–2294

JZ5.9, 6.0 Hz), 4.80 (1H, d, JZ12.1 Hz), 4.49 (1H, d, JZ

6.2 Hz), 4.29 (1H, d, JZ11.8 Hz), 3.76 (3H, s), 3.75 (3H, s),

3.34 (1H, d, JZ11.3 Hz), 3.21 (1H, d, JZ11.3 Hz), 1.97

(3H, s), 0.67 (9H, s), 0.04 (3H, s), K0.15 (3H, s); d_C

(CDCl₃) 172.1, 165.9, 159.9, 158.6, 144.5, 135.7, 135.2,

133.6, 130.2, 130.0, 129.9, 129.7, 129.4, 128.7, 128.0,

127.9, 127.0, 118.9, 113.3, 113.2, 89.2, 88.9, 86.3, 81.5,

73.5, 64.5, 63.3, 55.3, 25.6, 17.9, 14.3, K4.6, K5.6;

MALDI-HRMS: m/z 813.3160 ([MCNa]^C, C₄₅H₅₀N₂O₉-

SiNa^Ccalcd 813.3178).

5-CH₃), 0.91 and 0.78 (9H each, 2s, 2!C(CH₃)₃), 0.10 (3H,

s, SiCH₃), 0.07 (6H, s, 2!SiCH₃), K0.01 (3H, s, SiCH₃);

d_C(CDCl₃) 163.8 (C4), 158.7, 158.6, 150.5 (C2), 144.8,

138.3, 136.0, 135.7, 130.3, 130.2, 128.3, 128.0, ₀126.9,

113.2, 109.1 (C5), 88.4 (C4⁰), 86.4 (CAr₃), 85.6 (C1 ), 72.9

(C2⁰and C3⁰), 67.3 (C5⁰), 63.7 (C5⁰⁰), 55.3 (2!OCH₃), 26.1

and 25.6 (2!C(CH₃)₃), 18.4 and 18.0 (2!C(CH₃)₃), 12.6

(5-CH₃), K5.2, K5.3, K5.4 and K5.5 (4!SiCH₃);

MALDI-HRMS: m/z 841.3859 ([MCNa]^C, C₄₄H₆₂N₂O₉-

Si₂Na^Ccalcd 841.3886); data for 42: d_H(CDCl₃) 8.83 (1H,

s, NH), 7.54 (1H, s, H6), 7.46–7.44 (2H, m), 7.36–7.22 (7H,

m), 6.81 (4H, d, JZ9.0 Hz), 6.23 (1H, d, JZ4.1 Hz, H1⁰),

4.52 (1H, d, JZ5.4 Hz, H3⁰), 4.39 (1H, m, H2⁰), 3.78 (6H, s,

2!OCH₃), 3.73 (1H, d, JZ10.6 Hz, H5⁰a), 3.68 (1H, d, JZ

10.7 Hz, H5⁰b), 3.47 (1H, d, JZ10.4 Hz, H5⁰⁰a), 3.24 (1H, d,

JZ6.6 Hz, 2⁰-OH), 3.16 (1H, d, JZ10.5 Hz, H5⁰⁰b), 1.69

(3H, s, 5-CH₃), 0.87 and 0.76 (9H each, 2s, 2!C(CH₃)₃),

0.07, 0.03, 0.01 and K0.11 (3H each, 4s, 4!SiCH₃); d_C

(CDCl₃) 164.0 (C4), 158.7, 158.6, 150.7 (C2), 144.5, 138.0,

135.9, 135.6, 130.3, 130.2, 128.4, 128.0, 127.1, ₀113.3,

113.2, 109.1 (C5), 87.5 (C4⁰), 87.1 (CAr₃), 85.4 (C1 ), 73.4

(C3⁰), 71.7 (C2⁰), 64.7 and 64.1 (C5⁰and C5⁰⁰), 55.3 (2!

OCH₃), 25.9 and 25.7 (2!C(CH₃)₃), 18.3 and 18.2 (2!

C(CH₃)₃), 12.6 (5-CH₃), K5.0, K5.2, K5.3 and K5.4 (4!

SiCH₃); MALDI-HRMS: m/z 841.3891 ([MCNa]^C, C₄₄-

H₆₂N₂O₉Si₂Na^Ccalcd 841.3886).

7.1.34. 1-[5-O-(4,4⁰-Dimethoxytrityl)-4-C-hydroxy-

methyl-b-^D-lyxofuranosyl]thymine (40). To a suspension

of nucleoside 39 (1.7 g, 2.15 mmol) in a 1:1 mixture of

EtOH and H₂O (20 mL) was added 2 M aqueous sodium

hydroxide (1.5 mL), and the reaction mixture was heated

under reﬂux for 6 h, then cooled and evaporated to

approximately half of the original volume. The residue

was partitioned between EtOAc (100 mL) and NaHCO₃

(50 mL). The separated organic phase was dried (Na₂SO₄),

ﬁltered and concentrated to dryness under reduced pressure.

The crude product was puriﬁed by column chromatography

[6–7% (v/v) MeOH in CH₂Cl₂] to give nucleoside 40 as a

white solid material (935 mg, 74%). R_f0.21 (MeOH/

CH₂Cl₂10:90, v/v); d_H(CDCl₃) 10.30 (1H, s), 7.49 (1H,

s), 7.45 (2H, d, JZ7.3 Hz), 7.33 (4H, d, JZ7.7 Hz), 7.25–

7.15 (3H, m), 6.79 (4H, d, JZ8.3 Hz), 6.28 (1H, d, JZ

5.2 Hz), 5.17 (1H, d, JZ4.9 Hz), 4.75 (1H, m), 4.32 (1H,

dd, JZ4.3, 4.4 Hz), 3.79 (1H, m), 3.74–3.62 (7H, m), 3.58

(1H, d, JZ2.9 Hz), 3.53 (1H, d, JZ10.3 Hz), 3.38 (1H, d,

JZ10.4 Hz), 3.34 (1H, br s), 1.64 (3H, s); d_C(CDCl₃) 165.4,

158.6, 151.3, 144.6, 139.0, 135.7, 135.5, 130.2, 130.1,

128.2, 128.0, 127.0, 113.3, 109.0, 88.1, 86.8, 85.8, 72.5,

71.3, 65.5, 63.5, 55.3, 12.4.

7.1.36.

1-[3-O-(2-Cyanoethoxy(diisopropylamino)-

phosphino)-2,5-di-O-(tert-butyldimethylsilyl)-4-C-(4,4⁰-

dimethoxytrityloxymethyl)-a-^L-ribofuranosyl]thymine

(43). Cyanoethyl N,N⁰-diisopropylphosphoramidochloridite

(170 mg, 0.72 mmol) was added dropwise to a stirre₀d

solution of nucleoside 41 (295 mg, 0.36 mmol) and N,N -

diisopropylethylamine (0.5 mL) in anhydrous CH₂Cl₂

(5 mL). After stirring for 12 h at room temperature, the

reaction mixture was diluted with EtOAc (50 mL). Washing

was performed with saturated aq NaHCO₃(2!25 mL). The

separated organic phase was dried (Na₂SO₄), ﬁltered and

concentrated to dryness under reduced pressure. The residue

obtained was puriﬁed by column chromatography [30–35%

EtOAc in n-hexane containing 0.5% Et₃N (v/v/v)] to give

amidite 43 as a white solid material (242 mg, 66%). R_f0.32,

0.35 (MeOH/CH₂Cl₂5:95, v/v); d_P152.9 and 150.7; d_H

(CDCl₃, major isomer) 8.20 (br s, NH), 7.41 (d, JZ8.1 Hz),

7.40 (s, H6), 7.32–7.16 (m), 6.78 (d, JZ8.7 Hz), 6.20 (d,

JZ4.1 Hz, H1⁰), 4.69 (dd, JZ4.8, 13.6 Hz, H3⁰₀), 4.40 (dd,

JZ4.3, 4.5 Hz, H2⁰), 4.22 (d,₀₀JZ10.8 Hz, H5 a), 3.77 (s,

2!OCH₃), 3.74–3.68 (m, H5 a and OCH₂), 3.54 (d, JZ

10.8 Hz, H5⁰b), 3.44–3.41 (m, 2!CH(CH₃)₂), 3.17 (d, JZ

10.5 Hz, H5⁰⁰b), 2.56 (t, JZ6.4 Hz, CH₂CN), 1.78 (s, 5-

CH₃), 1.11 (d, JZ6.6 Hz, C(CH₃)₂), 0.93 (s, C(CH₃)₃), 0.87

(d, JZ6.7 Hz, C(CH₃)₂), 0.71 (s, C(CH₃)₃), 0.12, 0.11, 0.04

and K0.17 (4s, 4!SiCH₃); d_C(CDCl₃) 163.6, 158.5, 150.4,

145.1, 139.3, 136.2, 136.0, 130.2, 130.1, 128.2, 128.0,

126.8, 117.3, 113.3, 113.2, 108.9, 86.8 (d, JZ4.6 Hz), 85.9,

85.4, 73.5 (d, JZ16.9 Hz), 72.6 (d, JZ2.2 Hz), 64.8, 64.4,

58.3 (d, JZ21.8 Hz), 55.3, 43.2, 43.1, 26.1, 25.8, 24.8, 24.7,

24.6, 24.5, 20.4 (d, JZ8.0 Hz), 18.5, 17.9, 12.7, K4.7,

K4.8, K5.1, K5.2; MALDI-HRMS: m/z 1041.4926 ([MC

Na]^C, C₅₃H₇₉N₄O₁₀PSi₂Na^Ccalcd 1041.4965).

7.1.35. 1-[2,5-Di-O-(tert-butyldimethylsilyl)-4-C-(4,4⁰-

dimethoxytrityloxymethyl)-a-^L-ribofuranosyl]thymine

(41) and 1-[3,5-di-O-(tert-butyldimethylsilyl)-4-C-(4,4⁰-

dimethoxytrityloxymethyl)-a-^L-ribofuranosyl]thymine

(42). tert-Butyldimethylsilyl chloride (816 mg, 5.42 mmol)

and imidazole (740 mg, 10.9 mmol) were added to a stirred

solution of nucleoside 40 (800 mg, 1.35 mmol) dissolved in

anhydrous pyridine (8 mL). The reaction mixture was

stirred at room temperature for 12 h whereupon MeOH

(1.0 mL) was added. After stirring for 30 min the resulting

mixture was concentrated to dryness under reduced

pressure. The residue was dissolved in EtOAc (100 mL)

and washed with saturated aq NaHCO₃(2!50 mL). The

organic phase was dried (Na₂SO₄), ﬁltered, concentrated to

dryness under reduced pressure and coevaporated with

toluene (2!5 mL). The crude product was puriﬁed by

column chromatography [30–35% (v/v) EtOAc in light

petroleum] yielding nucleoside 41 (395 mg, 36%) and [35–

45% (v/v) EtOAc in light petroleum] nucleoside 42

(590 mg, 53%), both as white solid materials. R_f0.24,

0.34 (MeOH/CH₂Cl₂5:95, v/v); data for 41: d_H(CDCl₃)

8.45 (1H, s, NH), 7.47–7.44 (2H, m), 7.33 (4H, dd, JZ1.8,

8.9 Hz), 7.27–7.18 (4H, m)₀, 6.80 (4H, dd, JZ1.8, 8.9 Hz),

6.39 (1H, d, JZ6.2 Hz, H1 ), 4.76 (1H,₀dd, JZ5.4, 6.1 Hz,

H2⁰), 4.11 (1H, dd, JZ2.2, 5.5 Hz, H3 ), 3.87 (1H, d, JZ

10.7 Hz, H5⁰a), 3.76 (6H, s, 2!OCH₃), 3.70 (1H, d, JZ

10.8 Hz, H5⁰b), 3.49 (1H, d, JZ9.9 Hz, H5⁰⁰a), 3.33 (1H, d,

JZ10.1 Hz, H5⁰⁰b), 2.67 (1H, br s, 3⁰-OH), 1.80 (3H, s,

Synthesis and puriﬁcation of modiﬁed oligonucleotides.

The oligomers ON5–ON10, ON15, ON17–ON19, ON21,

G. Gaubert et al. / Tetrahedron 62 (2006) 2278–2294

2293

ON23 and ON24 (Table 1 and 2) were synthesized in

0.2 mmol scale on CPG solid support on an automated

DNA-synthesizer using the phosphoramidite approach.³⁰

The stepwise coupling yield for the a-L-RNA phosphor-

amidites (T-monomer,⁶6, 12 and 25) was above 90%

(1H-tetrazole as activator, 20 min coupling time), for the

phosphoramidite 34 approximately 88% (1H-tetrazole as

activator, 60 min pre-activation by mixing 34 and

activator, 120 min coupling time), and for phosphorami-

dite 43 approximately 95% (pyridinium chloride as

activator, 10–16 min coupling time). After detritylation

with 80% aq acetic acid, cleavage from the solid support

and deacylations were effected using 40% aqueous

methylamine (10 min, 55 8C). After cooling to K18 8C,

the solid support was removed (centrifugation), washed

[2!0.25 cm³; EtOH–CH₃CN–H₂O (3/1/1, v/v/v)], and

the combined liquid phase evaporated to dryness under

reduced pressure. Desilylation of the oligomers was

accomplished using a method described earlier³¹for 20 h

(at 55 8C) and precipitation was then performed from

t-BuOH. Standard conditions of the synthesizer were

used for incorporation of DNA monomers whereas the

incorporation of a-^L-LNA monomers followed procedures

described earlier.^2,3The composition of the ONs was

veriﬁed by MALDI-MS (negative ion mode) on a

Micromass Tof Spec E mass spectrometer using a matrix

of diammonium citrate and 2,6-dihydroxyacetophenone.

Analysis by capillary gel electrophoresis veriﬁed the

purity of the oligomers as being O80%. MALDI-MS of

selected ONs m/z ([MKH]^K, found/calcd): ON5, 2800/

2799; ON6, 2894/2891; ON7, 2800/2799; ON8, 2893/

2891; ON9, 2900/2899; ON10, 2992/2987; ON17, 4238/

4231; ON18, 4235/4231; ON19, 4237/4231; ON24,

4241/4242.

was incubated in the presence of a four-fold excess of

complementary ON16, ON17, ON18 or ON19 in

hybridization buffer (20 mM Tris–HCl, pH 7.5, 100 mM

KCl) at 65 8C for 2 min followed by slow cooling to

37 8C. The RNase H digest was performed in 20 mM

Tris–HCl, pH 7.5, 100 mM KCl, 10 mM MgCl₂, 1 mM

with 0.01 U of E. coli RNase H (Amersham) enzyme at

37 8C. Aliquots of 10 mL samples were withdrawn and

mixed with 5 mL formamide loading dye with 10 mM

EDTA on ice at the time points 2, 10 and 60 min after

RNase H addition. A basic hydrolysis of labelled RNA

was performed by heating to 90 8C for 15 min in

100 mM Na₂CO₃(pH 9.0, 2 mM EDTA) followed by

cooling on ice and addition of formamide dye. All

reaction products were analyzed by PAGE (20%

polyacrylamide containing 8.3 M urea). The radioactive

RNA bands were visualized by autoradiography of the

dried gels.

Acknowledgements

We thank The Danish National Research Foundation for

funding, Ms. B. M. Dahl, University of Copenhagen and

Ms. Kirsten Østergaard, University of Southern Denmark,

for oligonucleotide synthesis, and Dr. M. Meldgaard,

Exiqon A/S for MALDI-MS analysis and L. H. Hansen

for the RNase H assay.

Supplementary data

Supplementary data associated with this article can be

found, in the online version, at doi:10.1016/j.tet.2005.12.

007. H contains copies of ¹³C NMR spectra of compounds

1–3, 5, 7–11, 14–20, 22–24, 27–33, 36–42 and 43 (major

isomer) and ³¹P NMR spectra of compounds 6, 12, 25, 34

(major isomer), 34 (minor isomer), 43 (major isomer) and

43 (minor isomer).

Thermal denaturation studies. Melting temperatures (T_m

values, 8C) were determined by measuring the absor-

bance at 260 nm against increasing temperature (1.0 8C/

min) on equimolar mixtures (1.0 or 1.5 mM in each

strand) of modiﬁed ONs and their complementary DNA/

RNA strand in 10 mM phosphate buffers with different

NaCl concentration (see captions to Tables 2 and 3) and

were performed on a Perkin-Elmer UV–vis spectrometer

ﬁtted with a PTP-6 temperature programmer.

References and notes

Molecular modelling. A DNA duplex of sequence

5⁰-d(GTGATATGC) and a DNA:RNA hybrid NMR solution

structure³²were used as template and further modiﬁed within

the MacroModel V8.0 program suite.³³The modiﬁed residue

was ﬁrst partially optimized (MMFF94s force ﬁeld, 1000

cycles) and subsequently submitted to a 5 ns stochastic

dynamics (300 K, 2 fs timestep, 1000 structures were sampled

and minimized) using the SHAKE algorithm to keep X–H

bondlengths ﬁxedduringthe simulation. The non-bonded cut-

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Article Doi

Synthesis and RNA-selective hybridization of α-L-ribo- and β-D-lyxo-configured oligonucleotides

DOI: 10.1016/j.tet.2005.12.007

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