Synthesis and properties of 2′-O,4′-C-ethylene-bridged nucleic acids (ENA) as effective antisense oligonucleotides

Bioorganic & Medicinal Chemistry 11 (2003) 2211–2226

Synthesis and Properties of 2⁰-O,4⁰-C-Ethylene-Bridged Nucleic

Acids (ENA) as Eﬀective Antisense Oligonucleotides

Koji Morita,^aMiho Takagi,^aChikako Hasegawa,^aMasakatsu Kaneko,^a

Shinya Tsutsumi,^bJunko Sone,^bTomio Ishikawa,^bTakeshi Imanishi^c

and Makoto Koizumi^a,*

^aExploratory Chemistry Research Laboratories, Sankyo Co., Ltd., Tokyo 140-8710, Japan

^bBiomedical Research Laboratories, Sankyo Co., Ltd., Tokyo 140-8710, Japan

^cGraduate School of Pharmaceutical Sciences, Osaka University, Osaka 565-0871, Japan

Received 25 October 2002; accepted 11 February 2003

Abstract—Novel bicyclo nucleosides, 2⁰-O,4⁰-C-ethylene nucleosides and 2⁰-O,4⁰-C-propylene nucleosides, were synthesized as

building blocks for antisense oligonucleotides to further optimize the 2⁰-O,4⁰-C-methylene-linkage of bridged nucleic acids

(2⁰,4⁰-BNA) or locked nucleic acids (LNA). Both the 2⁰-O,4⁰-C-ethylene- and propylene-linkage within these nucleosides restrict the

sugar puckering to the N-conformation of RNA as do 2⁰,4⁰-BNA/LNA. Furthermore, ethylene-bridged nucleic acids (ENA) having

2⁰-O,4⁰-C-ethylene nucleosides had considerably increased the aﬃnity to complementary RNA, and were as high as that of

2⁰,4⁰-BNA/LNA (ÁT_m=+3ꢀ5 ^ꢁC per modiﬁcation). On the other hand, addition of 2⁰-O,4⁰-C-propylene modiﬁcations in oligo-

nucleotides led to a decrease in the aﬃnity to complementary RNA. As for the stability against nucleases, incorporation of one

2⁰-O,4⁰-C-ethylene or one 2⁰-O,4⁰-C-propylene nucleoside into oligonucleotides considerably increased their resistance against exo-

nucleases to an extent greater than 2⁰,4⁰-BNA/LNA. These results indicate that ENA is more suitable as an antisense oligonucleo-

tide and is expected to have better antisense activity than 2⁰,4⁰-BNA/LNA.

Introduction

the complement cascade and severe hypotension in

vivo.^3,4PS modiﬁcation involves a dilemma where using

PS ODN of longer size to oﬀset their low aﬃnity to

RNA inevitably leads to an increase in unspeciﬁc bind-

ing and toxic eﬀects. To overcome their limitations,

considerable eﬀort has been focused on the development

of other types of modiﬁed oligonucleotides or oligonu-

cleotide analogues as second-generation oligonucleo-

tides that combine high nuclease resistance with

superior RNA binding aﬃnity and decrease the like-

lihood of eliciting unspeciﬁc biological responses that

are not related to target mRNA binding.⁵Among

numerous modiﬁed nucleic acids, several types of

nucleic acid analogues are now under clinical develop-

ment as antisense drugs. 2⁰-O-Alkyl modiﬁcations are

known to be of value in enhancing the binding aﬃnity

to target RNA and nuclease resistance.^5ꢂ8With 2⁰-O-

modiﬁcations, nuclease resistance increases due to the

bulkiness of 2⁰-O-chain.⁶In particular, a 2⁰-O-(2-meth-

oxy)ethyl modiﬁcation showed high RNA aﬃnity

(average +2 ^ꢁC per modiﬁcation) with extraordinary

nuclease stability.^7,8Other backbone-modiﬁed nucleic

Antisense oligonucleotides are now in increasing

demand for their use as a gene-target validation tool in

genomic-based drug discovery^1,2and their potential to

be developed as a new class of drugs for the treatment

of inveterate diseases such as cancer, inﬂammation, and

viral diseases.³Phosphorothioate oligodeoxynucleotides

(PS ODN), currently the standard choice for most anti-

sense designs, have favorable properties such as high

nuclease resistance, and are able to be recognized by

RNase H, which leads to the degradative inactivation of

the target mRNA. However, they also have limitations

in their use, such as low aﬃnity to RNA derived from

mixtures of 2ⁿ(n=chain length ꢂ1) diﬀerent diaster-

eoisomers and nonsequence-speciﬁc protein binding,

which can be the cause of signiﬁcant side eﬀects, such as

inhibition of the blood clotting cascade, activation of

*Corresponding author. Tel.: +81-3-3492-3131; fax: +81-3-5436-

8570; e-mail: koizum@shina.sankyo.co.jp

doi:10.1016/S0968-0896(03)00115-9

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K. Morita et al. / Bioorg. Med. Chem. 11 (2003) 2211–2226

acids such as PNA,⁹morpholino¹⁰and phosphor-

amidate (3⁰-NP DNA)^11,12are capable of hybridizing to

mRNA with increased thermal stabilities (PNA:

+1ꢀ2 ^ꢁC per modiﬁcation, morpholino: not more than

1 ^ꢁC per modiﬁcation and 3⁰-NP DNA: 2.2–2.6 ^ꢁC per

modiﬁcation) due to the elimination of anionic repul-

sion between the phosphodiesters, and they also exhibit

complete nuclease resistance. They have a completely

diﬀerent backbone from endogenous DNA and work as

non-RNase H-mediated antisense molecules.

LNA and exhibit much more nuclease-resistance than

2⁰,4⁰-BNA/LNA.²²

Here, we report the synthesis of 2⁰-O,4⁰-C-ethylene

nucleosides and 2⁰-O,4⁰-C-propylene thymidine (f in Fig.

1) which is one carbon longer than ethylene-bridged

nucleosides, and evaluated the basic properties of their

oligonucleotides by circular dichroism (CD), UV melt-

ing analysis and nuclease resistance assay, to determine

whether oligonucleotides with ENA residues were more

suitable than those with propylene nucleic acid (PrNA)

residues for use as antisense oligonucleotides.

Recently, our group and Wengel’s group have indepen-

dently reported the synthesis of novel 2⁰-O,4⁰-C-methyl-

ene nucleosides whose sugar puckering is ﬁxed in the

N-conformation as in RNA, and that oligonucleotides

containing these bridged nucleosides (2⁰,4⁰-BNA/LNA)

showed an unprecedented level of aﬃnity (+4ꢀ8 ^ꢁC

per modiﬁcation) toward their complementary RNA,

higher than any other modiﬁed oligonucleotide (b in

Fig. 1).^13ꢂ15We have also carried out several studies on

2⁰,4⁰-BNA/LNA derivatives such as 3⁰,4⁰-BNA and

3⁰-amino-2⁰,4⁰-BNA.^16,17Wengel’s group reported the

synthesis of 2⁰,4⁰-BNA/LNA derivatives such as 2⁰-thio-

LNA,¹⁸2⁰-amino-LNA¹⁸and an a-l-ribo isomer of

LNA (a-l-LNA),¹⁹which all have a ﬁve-membered ring

with 2⁰-O,4⁰-C-methylene linkage. Some of these LNA

derivatives hybridize favorably to their target mRNA

but to a lesser extent than 2⁰,4⁰-BNA/LNA. Wang et al.

have reported the synthesis of 2⁰,4⁰-C-bridged 2⁰-deoxy-

nucleosides and their corresponding oligonucleotides (c

in Fig. 1). These show a moderate aﬃnity to RNA

(+1.9ꢀ3.3 ^ꢁC per modiﬁcation), compared with 2⁰,4⁰-

BNA/LNA.^20,21Other bridged nucleosides, in which the

oxygen at the 2⁰-position is replaced with a methylene

group, have been introduced as less eﬀective analogues

(d in Fig. 1).⁵Recently, we have reported the synthesis

of novel 2⁰-O,4⁰-C-ethylene thymidine, which has a less

strai₀ned six-membered ring than the ﬁve-membered ring

of 2 -O,4⁰-C-methylene thymidine (e in Fig. 1).²²The

corresponding oligonucleotides with 2⁰-O,4⁰-C-ethylene

thymidine retain a binding aﬃnity as high as 2⁰,4⁰-BNA/

Results and Discussion

Synthesis of 2⁰-O,4⁰-C-ethylene nucleosides and 2⁰-O,4⁰-

C-propylene thymidine

Novel nucleosides containing all possible natural

nucleobases (adenine, guanine, thymine, cytosine, uracil

and 5-methylcytosine) were synthesized by a procedure

shown in Scheme 1. The hydroxymethyl group at the

4-position of the known pentofuranose derivative 1²³

was converted to a hydroxyethyl group via Swern oxi-

dation, Wittig reaction and hydroboration, followed by

oxidation to give compound 4. Tosylation and acetolysis

of 4 aﬀorded compound 6, which is a useful common

intermediate for coupling reactions with a variety of

silylated nucleobases. Stereoselective coupling with 6-N-

benzoyladenine, 2-N-isobutyrylguanine, thymine, 4-N-

benzoylcytosine, uracil or 4-N-benzoyl-5-methylcytosine

by Vorbruggen’s method²⁴and the following base-

induced ring closure by the treatment with 1 M NaOH/

pyridine–H₂O

aﬀorded

2⁰-O,4⁰-C-ethylene-bridged

nucleosides 8a–f. Unfortunately, the yield of 7b was quite

low most likely due to a side reaction of debenzylation at

the 3⁰- or 5⁰-position and the following ring-closure

reaction of the 4⁰-p-toluenesulfonyloxyethyl group with

the debenzylated 3⁰- or 5⁰-hydroxyl group. Furthermore,

an undesired N-7 regioisomer that should have been

separated by silica gel chromatography after the ring-

closure reaction, was obtained. The stereochemistry of

each isomer was determined based on the UV spectra of

unprotected 2⁰-O,4⁰-C-ethylene-bridged guanosine 9h

which were analyzed under acidic, neutral and basic

conditions. The deprotected bicyclonucleosides 9a–f

were obtained by debenzylation of 8b, 8c and 8e using

10% Pd(OH)₂/C and of 8a, 8d and 8f using BCl₃. By

subsequent 5⁰-O-DMTr-protection and phosphitylation,

the 2⁰-O,4⁰-C-ethylene-bridged nucleoside phosphor-

amidites 11a–f were synthesized.

The synthesis of 2⁰-O,4⁰-C-propylene thymidine and its

corresponding phosphoramidite was performed as

shown in Scheme 2. Swern oxidation and chain exten-

sion by Horner–Wadsworth–Emmons oleﬁnation of the

known 4⁰-C-hydroxymethyl pentofuranose derivative 12²⁵

furnished the (E)-enoate 14. Pd(OH)₂-catalyzed reduction

for 6 h at 3.5 psi aﬀorded oleﬁn-selective hydrogenation of

14, and the following reduction using l-selectride led to

4-hydroxypropyl furanose derivative 16. Tosylation and

acetolysis of 16 aﬀorded 18, the intermediate for the

Figure 1. Structures of DNA and 2⁰,4⁰-bridged nucleic acid derivatives.

2⁰,4⁰-BNA/LNA: 2⁰-O,4⁰-C-methylene nucleic acids. ENA: 2⁰-O,4⁰-C-

ethylene nucleic acids. PrNA:2⁰-O,4⁰-C-propylene nucleic acids.

K. Morita et al. / Bioorg. Med. Chem. 11 (2003) 2211–2226

2213

Scheme 1. Synthesis of 2⁰-O,4⁰-C-ethylene nucleosides-3⁰-O-phosphoramidite and oligonucleotides. Reagents and conditions: (i) (COCl)₂, DMSO,

Et₃N, CH₂Cl₂, ꢂ78 ^ꢁC, 91%; (ii) Ph₃P⁺CH₃Br^ꢂ, NaH, DMSO, 80%; (iii) 9-BBN, THF then H₂O₂, NaOH, 93%; (iv) TsCl, Et₃N, CH₂Cl₂94%; (v)

Ac₂O, H₂SO₄, AcOH, 89% (a/b=1:5); (vi) silylated nucleobase, TMSOTf, ClC₂H₄Cl, reﬂux; (vii) 1 M NaOH, pyridine–H₂O; (viii) H₂, Pd(OH)₂,

MeOH or BCl₃, CH₂Cl₂, ꢂ78 ^ꢁC; (ix) DMTrCl, pyridine, CH₂Cl₂; (x) ((iPr)₂N)₂P(OC₂H₄CN), N,N-diisopropylammonium tetrazolide or (iPr)₂-

NP(Cl)(OC₂H₄CN), diisopropylethylamine; (xi) DNA/RNA synthesizer; (xii) succinic anhydride, DMAP, CH₂Cl₂; (xiii) 2,3,4,5,6-penta-

chlorophenol, DCC, DMF; long-chain alkylamino-CPG, triethylamine, DMF.

Scheme 2. Synthesis of 2⁰-O,4⁰-C-propylene thymidine-3⁰-O-phosphoramidite and oligonucleotides. Reagents and conditions: (i) (COCl)₂, DMSO,

Et₃N, CH₂Cl₂, ꢂ78 ^ꢁC, 99% (ii) (EtO)₂P(O)CH₂COOEt, NaH, THF, 86%; (iii) Pd(OH)₂, H₂(3.5 psi), MeOH, 71%; (iv) l-selectride, THF, 85%;

(v) TsCl, Et₃N, CH₂Cl₂77%; (vi) Ac₂O, H₂SO₄, AcOH, 79% (mainly b-anomer); (vii) silylated thymine, TMSOTf, ClC₂H₄Cl, reﬂux, 88%;

(viii) 1 M NaOH, pyridine–H₂O; (ix) TBAF, THF, 82% (two steps); (x) H₂, Pd(OH)₂, MeOH, 59%; (xi) DMTrCl, pyridine, CH₂Cl₂, quant;

(xii) ((iPr)₂N)₂P(OC₂H₄CN), N,N-diisopropylammonium tetrazolide, quant; (xiii) DNA/RNA synthesizer.

coupling reaction with silylated nucleobases. The reaction

of 18 with O,O⁰-bis(trimethylsilyl)thymine under Vor-

bruggen’s conditions²⁴aﬀorded the b-anomer of thymi-

dine derivative 19, which was converted to the desired

2⁰-O,4⁰-C-propylene nucleoside 20 by treatment with 1 M

NaOH/pyridine–H₂O. The debenzylation of 20 and the

following 5⁰-O-DMTr-protection and phosphitylation

gave 2⁰-O,4⁰-C-propylene thymidine phosphoramidite 23,

a building block for oligonucleotide synthesis.

Conformational

analysis

of

2⁰-O,4⁰-C-ethylene

nucleosides and 2⁰-O,4⁰-C-propylene nucleosides

The conformational analysis of the bridged nucleosides

obtained was carried out by means of ¹H NMR. In all of

the bicyclic nucleoside analogues (8–11, 20–23), the cou-

pling constant (J_{H1 ꢂH2}) was 0 Hz, which was identical to

that of 2⁰-O,4⁰-C-methylene nucleosides.¹³These results

indicate that the furanose pucker of 2⁰-O,4⁰-C-ethylene

0

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K. Morita et al. / Bioorg. Med. Chem. 11 (2003) 2211–2226

nucleosides and of 2⁰-O,4⁰-C-propylene thymidine

were all ﬁxed in the N-conformation, and C3⁰-endo

conformation. Similar results were obtained by X-ray

crystal structure analysis of the 2⁰-O,4⁰-C-ethylene-2-N-

isobutyrylguanosine (9b), 2⁰-O,4⁰-C-ethylene adenosine

(9g), 2⁰-O,4⁰-C-methylene adenosine and 2⁰-O,4⁰-C-

methylene thymidine (the cell unit of 2⁰-O,4⁰-C-methyl-

ene thymidine had two conformers), which had the

pseudorotation phase angle P of 12.1, 15.1, 20.2, 16.8

and 14.7^ꢁ, respectively (Fig. 2). These P values corre-

sponded to typical C3⁰-endo conformations.²⁶On the

b₀asis of these data and the previously reported data of

2 -O,4⁰-C-methylene uridine (P angle=17.4^ꢁ),¹³the

sugar puckering of 2⁰-O,4⁰-C-methylene nucleosides was

synthesized using the standard phosphoramidite

approach on a DNA/RNA synthesizer. Coupling yields

of 2⁰-O,4⁰-C-ethylene nucleoside units were more than

95%. However, the 2⁰-O,4⁰-C-propylene thymidine-3⁰-

O-phosphoramidite unit did not give good yield

(approximately 50–60%), even with the use of

5-ethylthio-1H-tetrazole,²⁷which is well-known as an

eﬀective activator. This is probably due to steric hin-

drance of the propylene bridge of the 2⁰-O,4⁰-C-pro-

pylene thymidine unit. Two 2⁰-O,4⁰-C-propylene

thymidine units were able to be incorporated into the

oligonucleotide chain in moderate overall yield. On

the other hand, to prepare oligonucleotides with

2⁰-O,4⁰-C-ethylene thymidine at their 3⁰end, 2⁰-O,4⁰-C-

ethylene thymidine-bound CPG 25c was synthesized

from 10c by standard methods (Scheme 1). The oligo-

nucleotides modiﬁed with ENA or PrNA residues were

puriﬁed by reverse-phase HPLC. The structures of oli-

gonucleotides were conﬁrmed by negative ion ESI mass

spectroscopy.

3

found to adopt an envelope or near-envelope form, E,

and that of 2⁰-O,4⁰-C-ethylene nucleosides, an unsym-

3

metrical form, T₂.²⁶These diﬀerences appear clearly in

the torsion angle d of C5⁰–C4⁰–C3⁰–O3⁰(see Fig. 1e)

which is one of the deﬁning angles of ribose conforma-

tion.²⁶The d angles of nucleosides described above were

78.9, 75.7, 62.5, 68.5 and 66.9^ꢁ, respectively. Further-

more the mean d angles of 2⁰-O,4⁰-C-ethylene nucleo-

sides and 2⁰-O,4⁰-C-methylene nucleosides were 77 and

66^ꢁ, respectively, and the mean d angle of 2⁰-O,4⁰-C-eth-

ylene nucleosides was approximately 11^ꢁlarger than that

of 2⁰-O,4⁰-C-methylene nucleosides. From the view of oli-

gonucleotide structure, this diﬀerence in each nucleoside

unit might result in a large diﬀerence overall which might,

in turn, inﬂuence the duplex formation of oligonucleotides

with complementary DNA and RNA (see section of CD

spectra). Compared with 2⁰-C,4⁰-C-bridged nucleosides

with the six-membered ring (c in Fig. 1), whose wide range

in p values indicates ﬂexibility (11.1–19.3^ꢁ),²¹the range in

p values of 2⁰-O,4⁰-C-ethylene nucleosides is relatively

narrow. The torsion angles of C3⁰–C2⁰–O2⁰–C7⁰of 2⁰-

O,4⁰-C-ethylene nucleosides, 9b and 9g, were 62.3 and

61.6^ꢁ, respectively, and those of C3⁰–C4⁰–C6⁰–C7⁰of the

same nucleosides were 60.7 and 59.9^ꢁ, respectively. These

results show that the six-membered rings of 2⁰-O,4⁰-C-

ethylene nucleosides have a typical chair conformation.

The torsion angles w of the glycoside bonds were almost

identical (from 157 to 169^ꢁ) in all nucleosides, which sug-

gests that these nucleosides adopt an anti orientation.

Circular dichroism (CD) analysis of the duplex of

2⁰-O,4⁰-C-bridged nucleic acids with complementary

DNA or RNA

To investigate the properties of oligonucleotides con-

taining novel 2⁰-O,4⁰-C-bridged nucleosides, the CD

spectra of duplexes of oligonucleotides containing ENA

or PrNA residues and their complementary DNA and

RNA were evaluated. Cognate duplexes containing

2⁰-O,4⁰-C-methylene nucleosides show A-like conforma-

tion.²⁸In Figure 3A, the spectrum of a duplex of an

oligonucleotide containing six ENA residues and com-

plementary DNA showed a positive cotton eﬀect at

276 nm, and an A-like conformation similar to that in

the spectrum of the duplex containing 2⁰-O,4⁰-C-meth-

ylene nucleosides. However, the intensity of the positive

band of the duplex of the oligonucleotide containing

ENA residues and complementary DNA was ca.

1.7-fold greater than that of the duplex with 2⁰-O,4⁰-C-

methylene nucleosides and was similar to that of a

duplex with six uridine residues (Fig. 3A). The results

indicate that a duplex with some ENA residues form an

A-like conformation. Their structures were closer to a

natural DNA/RNA duplex than structures of duplexes

with 2⁰,4⁰-BNA/LNA, maybe due to the diﬀerence of

the d angles between ENA and 2⁰,4⁰-BNA/LNA. In the

case of complementary RNA, no remarkable diﬀerence

between the CD spectra of a duplex with 2⁰-O,4⁰-C-

methylene nucleosides and that with 2⁰-O,4⁰-C-ethylene

nucleosides was observed (Fig. 3B). These data suggest

that the oligonucleotide containing ENA residues can

form a duplex with the complementary RNA strand and

be cleaved by RNase H. This is also the case for the

duplex of 2⁰,4⁰-BNA/LNA-DNA chimeric oligonucleo-

tide with the complementary RNA.²⁹Furthermore, the

CD spectrum of a duplex of an oligonucleotide con-

taining two PrNA residues with the complementary

DNA was almost identical to that of an oligonucleotide

containing ENA residues, even when bound to com-

plementary RNA at room temperature (Fig. 3C and

D). To investigate the diﬀerence in the properties of the

two types of duplexes containing either ENA or PrNA

Synthesis of oligonucleotides containing 2⁰-O,4⁰-C-

ethylene nucleosides or 2⁰-O,4⁰-C-propylene nucleosides

Oligonucleotides including the bridged nucleosides,

2⁰-O,4⁰-C-ethylene nucleosides, were successfully

Figure 2. X-ray crystal structures of 2⁰-O,4⁰-C-methylene adenosine

(left) and 2⁰-O,4⁰-C-ethylene adenosine (9g, right).

K. Morita et al. / Bioorg. Med. Chem. 11 (2003) 2211–2226

2215

Figure 3. CD spectra of duplexes of 2⁰-O,4⁰-C-bridged nucleic acids and their complementary DNA or RNA: (A) duplex of modiﬁed oligonucleo-

tides, 5⁰-d(GCGXXXXXXGCT)-3⁰and DNA, 5⁰-d(AGCAAAAAACGC)-3⁰; (B) duplex of modiﬁed oligonucleotides, 5⁰-d(GCGXXXXXXGCT)-3⁰

and RNA 5⁰-r(AGCAAAAAACGC)-3⁰; (C) duplex of modiﬁed oligonucleotides, 5⁰-d(GCGTTXTTXGCT)-3⁰and DNA, 5⁰-

d(AGCAAAAAACGC)-3⁰. (D) duplex of modiﬁed oligonucleotides, 5⁰-d(GCGTTXTTXGCT)-3⁰and RNA 5⁰-r(AGCAAAAAACGC)-3⁰. Blue:

X=thymidine, green: X=2⁰-O,4⁰-C-methylene thymidine, red: X=2⁰-O,4⁰-C-ethylene thymidine, purple: 2⁰-O,4⁰-C-propylene thymidine, sky blue:

duplex with 5⁰-r(GCGUUUUUUGCU)-3⁰, (U=uridine). Duplex concentration: 4 mM. Buﬀer: 100 mM NaCl, 10 mM sodium phosphate buﬀer

(pH 7.2).

Nuclease resistance of oligonucleotides modiﬁed with

2⁰-O,4⁰-C-bridged nucleosides

residues, the thermodynamic stability of these duplexes

was evaluated by UV melting temperature analysis.

We have shown that oligonucleotides modiﬁed with an

ENA residue have greater stability against exo- and

endonucleases than those modiﬁed with a 2⁰,4⁰-BNA/

LNA residue.²²To compare the stability of oligonu-

cleotides modiﬁed with 2⁰-O,4⁰-C-bridged nucleosides,

such as 2⁰,4⁰-BNA/LNA, ENA and PrNA, or a PS

linkage, a well known method to increase nuclease-

resistance, a relatively high concentration of 3⁰-exonu-

clease, snake venom phosphodiesterase (SVPD, 7.14 mg/

mL), was used, since it was previously found that

UV melting temperature analysis

It has been reported that the duplexes of the oligonu-

cleotide containing six ENA residues and com-

plementary RNA or DNA have a higher UV melting

temperature (T_m) than a natural DNA/DNA and DNA/

RNA duplex, which is also the case for duplexes con-

taining 2⁰,4⁰-BNA/LNA residues.²²We measured the T_m

of the duplexes in a buﬀer of 100 mM NaCl and 10 mM

sodium phosphate (pH 7.2). The T_mvalues of the

duplex of the oligonucleotide containing two ENA resi-

dues with complementary RNA or DNA were increased

by ca. 3.5 ^ꢁC or 0.5 ^ꢁC per modiﬁcation, respectively

(Table 1). These data conﬁrmed our previous result that

the incorporation of ENA residues into oligonucleotides

increases the stability of the cognate duplexes.²²On the

other hand, a decrease in the T_mvalues of the duplexes

of the oligonucleotide containing two PrNA residues

(ꢂ0.5 or ꢂ2.0 ^ꢁC per modiﬁcation) was observed, and it

was found that propylene-bridging of the nucleosides

between the 2⁰- and the 4⁰-position was not suitable for

the stabilization of the duplex and that bridging by a

methylene or ethylene linkage between the 2⁰- and the

4⁰-position gave the best results in stabilizing the duplex

formation.

Table 1. T_mvalues (^ꢁC) of the modiﬁed oligonucleotides towards

complementary RNA and DNA

Oligonucleotide

5⁰-d(GCGTTX-

TTXGCT)-3⁰

Complementary RNA

5⁰-r(AGCAAAAAAC

GC)-3⁰

Complementary DNA

5⁰-d(AGCAAAAAAC

GC)-3⁰

T_m

(^ꢁC)

ÁT_m(^ꢁC)/

modiﬁcation

T_m

(^ꢁC)

ÁT_m(^ꢁC)/

modiﬁcation

DNA (X=T)

ENA (X=eT)

PrNA (X=prT)

48

55

47

51

52

47

3.5

ꢂ0.5

0.5

ꢂ2.0

Duplex concentration: 4 mM. Buﬀer: 100 mM NaCl, 10 mM sodium

phosphate buﬀer (pH 7.2). eT: 2⁰-O,4⁰-C-ethylene thymidine; prT: 2⁰-

O,4⁰-C-propylene thymidine.

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K. Morita et al. / Bioorg. Med. Chem. 11 (2003) 2211–2226

hydrolysis of the oligonucleotide with the ENA residue

at 0.3 mg/mL of SVPD was very slow.²²Under these

conditions, natural DNA (oligo T₁₂) and an oligonu-

with a PrNA residue (Fig. 4). Although it has been

reported that 2⁰,4⁰-BNA/LNA oligonucleotides are

more stable than natural DNA,^22,29,31oligonucleotides

with the ENA or the PrNA residues were much more

resistant against exonucleases than 2⁰,4⁰-BNA/LNA

residues. As well, we found that ENA oligonucleotides

were stable in plasma, suggesting a potential for their

use as antisense therapeutics in vivo (data not shown).

cleotide containing

a

2⁰,4⁰-BNA/LNA residue

(5⁰-TTTTTTTTTTXT-3⁰: X=2⁰-O,4⁰-C-methylene thy-

midine) were degraded rapidly (Fig. 4). On the other

hand, the stability of an oligonucleotide modiﬁed with

the ENA residue (5⁰-TTTTTTTTTTXT-3⁰: X=2⁰-O,4⁰-

C-ethylene thymidine) was identical to that of an oligo-

nucleotide with a PS Rp diastereomer. Later, we

designed and synthesized an oligonucleotide containing

Conclusion

two

ENA

residues

(5⁰-TTTTTTTTTTXX-3⁰:

We synthesized 2⁰-O,4⁰-C-bridged nucleosides, such as

2⁰-O,4⁰-C-methylene, -ethylene and -propylene deriva-

tives, and their corresponding oligonucleotides, such as

2⁰,4⁰-BNA/LNA, ENA and PrNA. As well, we eval-

uated the basic properties and the possibility of these

oligonucleotides to be used as antisense drugs. Regard-

ing the coupling yields of the synthesized oligonucleo-

tides and their binding aﬃnity to complementary RNA,

2⁰,4⁰-BNA/LNA and ENA were superior to PrNA,

owing to diﬀerences in their steric hindrance. Another

important factor to consider in creating antisense oli-

gonucleotides as therapeutic agents is nuclease resis-

tance. Oligonucleotides lending more steric hindrance,

such as ENA and PrNA, are more eﬀective in increasing

nuclease resistance than 2⁰,4⁰-BNA/LNA. Moreover,

oligonucleotides with contiguous ENA modiﬁcation or

dual-modiﬁcation by incorporation of an ENA residue

and PS bond lead to the formation of much more stable

duplexes than those with PS ODN. ENA modiﬁcation

permits designing antisense oligonucleotides with or

without a PS bond. From these results, we consider that

the ENA modiﬁcation is most suitable for 2⁰-O,4⁰-C-

alkylene-bridged nucleosides and that oligonucleotides

with ENA residues have a potential to be used as antisense

therapeutics. Application of ENA oligonucleotides as

antisense oligonucleotides will be reported elsewhere in

the near future.

X=2⁰-O,4⁰-C-ethylene thymidine) to evaluate the total

resistance of contiguous ENA residues, since we have

previously reported that incorporating an ENA residue

in oligonucleotides increased not only the nuclease

resistance of the phosphodiester bond of the 3⁰-side of

the ENA residue but also that of the 5⁰-side.²²This oli-

gonucleotide was more stable than an oligonucleotide

with a PS Sp diastereomer which is known to be a stable

isomer.³⁰These data indicate that oligonucleotides

composed of contiguous ENA residues without PS

modiﬁcation could show more nuclease stability than

PS ODN. Surprisingly, an oligonucleotide with a PrNA

residue was the most stable in all tested compounds and

ca. 70% of which remained even after incubation for

90 min. These data suggest that the incorporation of a

longer alkylene linkage in the nucleoside between the 2⁰-

and the 4⁰-position gives greater nuclease resistance,

probably due to the blockage of the phosphodiester

bond of the 3⁰-side of the nucleosides against nuclease

attack, by the additional linkage on the sugar. In addi-

tion, dual-modiﬁcation of incorporating an ENA resi-

due and a PS bond into an oligonucleotide was

successfully carried out. This oligonucleotide showed a

high stability identical to that of the oligonucleotide

Experimental

General methods

¹H and ¹³C NMR were recorded on Varian Mercury

400 (400 MHz) and JEOL JNM-ECP500 (125 MHz),

respectively. IR spectra were recorded on a JASCO FT/

IR-610 spectrometer. All reactions were monitored by

thin-layer chromatography performed on Merck silica

gel plates (Merck Art 5715 silica gel 60 F₂₅₄plate) with

UV detection. For column chromatography, Merck Art

9385 silica gel 60 (230–400 mesh) or Kanto Chemicals

silica gel 60, spherical particles (40–100 mm) were used.

Figure 4. Stability of oligonucleotides with a phosphorothioate link-

age, ENA or LNA, against snake venom phosphodiesterase. Oligo

concentration: 26 mg/mL, enzyme concentration: 7.14 mg/mL; buﬀer:

50 mM Tris–HCl (pH 8.0), 10 mM MgCl₂, at 37 ^ꢁC. Open squares

(green): 5⁰-TTT TTT TTT TXT-3⁰(X=2⁰-O,4⁰-C-propylene thymi-

dine), closed circles (purple): 5⁰-TTT TTT TTT TXT-3⁰(X=2⁰-O,4⁰-C-

ethylene thymidine-3⁰-phosphorothioate), closed triangles (orange): 5⁰-

TTT TTT TTT TXX-3⁰(X=2⁰-O,4⁰-C-ethylene thymidine), open dia-

monds (blue): 5⁰-TTT TTT TTT TXT-3⁰(X=thymidine-3⁰-phosphor-

othioate (Sp)), closed circles (sky blue): 5⁰-TTT TTT TTT TXT-3⁰

[X=thymidine-3⁰-phosphorothioate (Rp)], open squares (red): 5⁰-TTT

TTT TTT TXT-3⁰(X=2⁰-O,4⁰-C-ethylene thymidine), closed squares

(black): 5⁰-TTT TTT TTT TXT-3⁰(X=2⁰-O,4⁰-C-methylene thymidine).

3,5-Di-O-benzyl-4-C-formyl-1,2-O-isopropylidene-ꢀ-^D-

erythropentofuranose (2). Oxalyl chloride (6.02 mL,

69.0 mmol) was added to CH₂Cl₂(200 mL) cooled at

ꢂ78 ^ꢁC. A solution of DMSO (7.87 mL, 110 mmol) in

anhydrous CH₂Cl₂(100 mL) was added dropwise to this

solution. After stirring for 20 min a solution of 1²³

(9.21 g, 23.02 mmol) in anhydrous CH₂Cl₂(100 mL) was

added dropwise to this mixture and the mixture

was stirred for 30 min. Triethylamine (28 mL, 200 mmol)

K. Morita et al. / Bioorg. Med. Chem. 11 (2003) 2211–2226

2217

was added to this reaction mixture and the mixture was

slowly warmed to room temperature. The reaction mix-

ture was partitioned between CH₂Cl₂and H₂O. The

organic layer was washed with H₂O and brine, dried

over MgSO₄and then concentrated in vacuo. The resi-

due was puriﬁed by chromatography on silica gel (hex-

ane/EtOAc=5:1) to give 2 as a colorless oil (8.31 g,

20.88 mmol, 91%). ¹³C NMR (125 MHz, CDCl₃) d

26.09, 26.51, 69.18, 72.80, 73.78, 178.36, 79.69, 89.76,

104, 86, 114.14, 127.70, 127.80, 127.96, 128.11, 128.40,

128.48, 137.04, 137.57, 199.94. ¹H NMR (400 MHz,

CDCl₃) d 1.35 (3H, s), 1.60 (3H, s), 3.61 (1H, d, 11 Hz),

3.68 (1H, d, 11 Hz), 4.37 (1H, d, 4.4 Hz), 4.46 (1H, d,

12 Hz), 4.52 (1H, d, 12 Hz), 4.59 (1H, d, 12 Hz), 4.59

(1H, dd, 3.4, 4.4 Hz), 4.71 (1H, d, 12 Hz), 5.84 (1H, d,

3.4 Hz), 7.3 (10H, m), 9.91 (1H, s). IR (ﬁlm) n_max3064,

3032, 2987, 2939, 2865, 1731, 1101, 1022 cm^ꢂ1. FAB-

MS (mNBA): 397 (MꢂH)⁺, 421 (M+Na)⁺. ESI-

HRMS (positive): calcd for C₂₃H₂₆O₆Na [M+Na]⁺

421.1627, found 421.1631. [a]_D+27.4^ꢁ(0.51, MeOH).

30% aqueous hydrogen peroxide solution was added

while keeping the temperature between 30 and 50 ^ꢁC.

This mixture was stirred for 30 min and partitioned

between H₂O and EtOAc. The organic layer was

washed with neutral phosphate buﬀer solution and

brine, dried over MgSO₄and concentrated in vacuo.

The residue was puriﬁed by chromatography on silica

gel (hexane/EtOAc=2:1) to give 4 as a colorless oil

(5.37 g, 12.97 mmol, 93%). ¹³C NMR (125 MHz,

CDCl₃) d 26.03, 26.38, 33.87, 58.77, 72.43, 73.23, 73.55,

78.52, 79.12, 87.21, 104.23, 113.60, 127.63, 127.71,

127.83, 127.88, 128.38, 137.73, 137.83. ¹H NMR

(400 MHz, CDCl₃) d 1.33 (3H, s), 1.66 (3H, s), 1.78 (1H,

ddd, 4.0, 8.5, 15 Hz), 2.51 (1H, ddd, 3.4, 6.4, 15 Hz),

3.31 (1H, d, 10 Hz), 3.54 (1H, d, 10 Hz), 3.80 (2H, m),

4.13 (1H, d, 5.3 Hz), 4.43 (1H, d, 12 Hz), 4.52 (1H, d,

12 Hz), 4.55 (1H, d, 12 Hz), 4.65 (1H, dd, 4.0, 5.3 Hz),

4.77 (1H, d, 12 Hz), 5.77 (1H, d, 4.0 Hz), 7.3 (10H, m).

IR (ﬁlm) n_max3507, 2937, 2870, 1104, 1026, cm^ꢂ1. FAB-

MS (mNBA): 415 (M+H)⁺. FAB-HRMS (positive):

calcd for C₂₄H₃₀O₆Na [M+Na]⁺437.1940, found

437.1945. [a]_D+57.4^ꢁ(0.91, MeOH).

3,5-Di-O-benzyl-4-C-vinyl-1,2-O-isopropylidene-ꢀ-^D-ery-

thropentofuranose (3). Sodium hydride (60% in mineral,

3.7 g, ca. 93.6 mmol) was added to DMSO (35 mL)

under a nitrogen atmosphere and the mixture was stir-

red for 45 min at 80 ^ꢁC. After the mixture was cooled

down to room temperature, a solution of methyl-

triphenylphosphonium bromide (33.4 g, 93.6 mmol) in

anhydrous DMSO (72 mL) was added dropwise to the

mixture at 0 ^ꢁC and the mixture was stirred for 20 min at

room temperature. A solution of 2 (28.7 g, 71.98 mmol)

in anhydrous DMSO (50 mL) was added dropwise and

the reaction mixture was stirred for 1 h at room tem-

perature. The reaction mixture was poured into H₂O in

an ice-bath and extracted with EtOAc. The organic

layer was washed with brine, dried over MgSO₄and

then evaporated in vacuo. The residue was puriﬁed by

chromatography on silica gel (hexane/EtOAc=18:1) to

give 3 as a colorless oil (22.7 g, 57.3 mmol, 80%). ¹³C

NMR (125 MHz, CDCl₃) d 25.63, 26.10, 72.49, 72.76,

73.44, 76.79, 77.34, 78.34, 86.46, 103.91, 113.36, 116.30,

127.57, 127.58, 127.86, 127.93, 128.08, 128.13, 128.31,

128.36, 128.48, 135.49, 137.89, 138.10. ¹H NMR

(400 MHz, CDCl₃) d 1.28 (3H, s), 1.52 (3H, s), 3.31 (1H,

d, 11 Hz), 3.34 (1H, d, 11 Hz), 4.25 (1H, d, 4.9 Hz), 4.40

(1H, d, 12 Hz), 4.52 (1H, d, 12 Hz), 4.57 (1H, dd, 3.9,

4.9 Hz), 4.59 (1H, d, 12 Hz), 4.76 (1H, d, 12 Hz), 5.25

(1H, dd, 1.8, 11 Hz), 5.52 (1H, dd, 1.8, 18 Hz), 5.76 (1H,

d, 3.9 Hz), 6.20 (1H, dd, 11, 18 Hz), 7.3 (10H, m). IR

(ﬁlm) n_max3357, 3065, 3032, 2985, 2939, 2865, 1725,

1104, 1027 cm^ꢂ1. FAB-MS (mNBA): 419 (M+Na)⁺.

ESI-HRMS (positive): calcd for C₂₄H₂₈O₅Na

[M+Na]⁺419.1834, found 419.1829.

3,5-Di-O-benzyl-4-C-(p-toluenesulfonyloxyethyl)-1,2-O-

isopropylidene-ꢀ-^D-erythropentofuranose (5). Triethyl-

amine (1.8 mL, 13 mmol), N,N-dimethylaminopyridine

(DMAP, 30 mg, 0.25 mmol), and p-toluenesulfonyl

chloride (858 mg, 4.5 mmol) were added to a solution of

4 (1.03 g, 2.5 mmol) in anhydrous CH₂Cl₂(35 mL)

under a nitrogen atmosphere at 0 ^ꢁC and the mixture

was stirred at room temperature overnight. The reaction

mixture was partitioned between the CH₂Cl₂and satu-

rated NaHCO₃solution. The organic layer was washed

with saturated NaHCO₃solution and brine, dried over

MgSO₄, and then concentrated in vacuo. The residue

was puriﬁed by chromatography on silica gel (hexane/

EtOAc=3:1) to give 5 as a colorless oil (1.34 g,

2.6 mmol, 94%). ¹³C NMR (125 MHz, CDCl₃) d 21.60,

25.93, 26.43, 31.26, 67.55, 72.35, 73.30, 73.52, 78.50,

78.80, 85.34, 104.26, 113.09, 127.61, 127.67, 127.75,

127.86, 127.88, 128. 38, 129.74, 133.24, 137.70, 137. 84,

144.47. ¹H NMR (400 MHz, CDCl₃) d 1.33 (3H, s), 1.49

(3H, s), 1.99 (1H, dt, 7.6 and 15 Hz), 2.47 (3H, s), 2.60

(1H, ddd, 5.7, 7.6, 15 Hz), 3.28 (1H, d, 10 Hz), 3.45 (1H,

d, 10 Hz), 4.11 (1H, d, 5.3 Hz), 4.32 (2H, m), 4.42 (1H,

d, 12 Hz), 4.50 (1H, d, 12 Hz), 4.54 (1H, d, 12 Hz), 4.62

(1H, dd, 4.0, 5.2 Hz), 4.76 (1H, d, 12 Hz), 5.74 (1H, d,

4.0 Hz), 7.3 (12H, m), 7.78 (2H, d, 8.3 Hz). IR (ﬁlm)

n_max2940, 2868, 1359, 1176, 1099, 1024, 958, cm^ꢂ1

.

FAB-MS (mNBA): 569 (M+H)⁺. ESI-HRMS (posi-

tive): calcd for C₃₁H₃₆O₈SNa [M+Na]⁺591.2029,

found 591.2050.

3,5-Di-O-benzyl-4-C-(p-toluenesulfonyloxyethyl)-1,2-di-

O-acetyl-^D-erythropentofuranose (6). Acetic anhydride

(1.88 mL, 20 mmol) and concentrated H₂SO₄(0.01 mL)

were added to a solution of 5 (1.34 g, 2.36 mmol) in

acetic acid (15 mL) and the mixture was stirred at room

temperature for 1 h. The reaction mixture was poured

into H₂O (60 mL) in an ice-bath, stirred for 30 min and

then partitioned between brine and EtOAc. The organic

layer was washed with neutral phosphate buﬀer solu-

tion, saturated NaHCO₃solution, brine and dried over

3,5-Di-O-benzyl-4-C-hydroxyethyl-1,2-O-isopropylidene-

ꢀ-^D-erythropentofuranose (4). A 0.5 M THF solution of

9-BBN (9-borabicyclo[3.3.1]nonane) (80 mL, 40 mmol)

was added dropwise to a solution of 3 (5.50 g,

13.89 mmol) in anhydrous THF (200 mL) under a

nitrogen atmosphere and the mixture was stirred at

room temperature overnight. H₂O was added to the

reaction mixture until evolution of gas ceased, 3 N

NaOH solution (30 mL) was added and then slowly

2218

K. Morita et al. / Bioorg. Med. Chem. 11 (2003) 2211–2226

MgSO₄and then concentrated. The residue was puriﬁed

by chromatography on silica gel (hexane/EtOAc=2:1)

to give 6 as a colorless oil (1.29 g, 2.11 mmol, 89%,

a:b=1:5). ¹³C NMR (125 MHz, CDCl₃) d 20.65, 20.90,

21.59, 32.24, 67.24, 73.37, 74.16, 74.27, 78.96, 85.64,

97.50, 127.61, 127.74, 127.85, 127.94, 128.38, 128.42,

129.77, 133.27, 137.37, 137.74, 144.54, 169.05, 169.49.

¹H NMR (400 MHz, CDCl₃) d (b derivative) 1.86 (3H,

s), 2.05 (3H, s), 2.08 (1H, m), 2.18 (1H, m), 2.42 (3H, s),

3.30 (1H, d, 10 Hz), 3.33 (1H, d, 10 Hz), 4.23 (1H, d,

5.1 Hz), 4.24 (2H, m), 4.42 (2H, s), 4.45 (1H, d, 12 Hz),

4.55 (1H, d, 12 Hz), 5.28 (1H, d, 5.1 Hz), 6.01 (1H, s),

7.3 (12H, m), 7.73 (2H, d, 8.3 Hz). IR (ﬁlm) n_max3032,

2868, 1749, 1361, 1218, 1099, 952, cm^ꢂ1. FAB-MS

(mNBA): 613 (M+H)⁺. ESI-HRMS (positive): calcd

for C₃₂H₃₆O₁₀SNa [M+Na]⁺635.1927, found

635.1938.

9.00 (1H, brs). FAB-MS (mNBA): 578 (M+H)⁺. ESI-

HRMS (positive): calcd for C₃₃H₃₂N₇O₇[M+H]⁺

578.2404, found 578.2407.

2⁰-O,4⁰-C-Ethylene-6-N-benzoyladenosine (9a). A 1 M

boron trichloride solution (1.5 mL, 1.5 mmol) in CH₂Cl₂

was slowly added dropwise to a solution of 8a (116 mg,

0.20 mmol) in anhydrous CH₂Cl₂(5 mL) at ꢂ78 ^ꢁC and

the mixture was stirred at ꢂ78 ^ꢁC for 3 h. To the reac-

tion mixture was added a 1 M boron trichloride solution

(1.5 mL, 1.5 mmol) in CH₂Cl₂and the mixture was stir-

red for 2 h. The mixture was slowly warmed to room

temperature and then quickly cooled to ꢂ78 ^ꢁC. Then,

MeOH (5 mL) was added to the mixture. The reaction

mixture was slowly warmed to room temperature and

concentrated in vacuo. The residue was puriﬁed by

chromatography on a silica gel column (CH₂Cl₂/

MeOH=9:1) to aﬀord 9a as a white powder (49 mg,

3⁰,5⁰-Di-O-benzyl-4⁰-C-(p-toluenesulfonyloxyethyl)-2⁰-O-

acetyl-6-N-benzoyladenosine (7a). Trimethylsilylated

6-N-benzoyladenosine (500 mg, about 2.0 mmol), which

was prepared according to Vorbruggen’s method,²⁴was

added to a solution of 6 (600 mg, 0.98 mmol) in anhy-

drous 1,2-dichloroethane (15 mL) at room temperature

under a nitrogen atmosphere. After dropwise addition

of trimethylsilyl triﬂuoromethanesulfonate (TMSOTf,

0.36 mL, 2 mmol) to the mixture, the mixture was stirred

at 50 ^ꢁC for 4 h. Saturated NaHCO₃solution and

CH₂Cl₂were added to the reaction mixture and the

mixture was partitioned between these two layers. The

organic layer was washed with saturated NaHCO₃

solution and brine, dried over MgSO₄and then con-

centrated in vacuo. The residue was puriﬁed by chro-

matography on silica gel (CH₂Cl₂/MeOH=50:1) to give

7a as a colorless amorphous solid (405 mg, 0.51 mmol,

0.17 mmol, 84%). H NMR (400 MHz, CD₃OD) d 1.45

1

(1H, dd, 4.3 and 13 Hz), 2.12 (1H, m), 3.72 (1H, d,

12 Hz), 3.79 (1H, d, 12 Hz), 4.04 (1H, dd, 7.3 and

12 Hz), 4.15 (1H, dt, 4.3 and 9.4 Hz), 4.36 (1H, d,

3.2 Hz), 4.43 (1H, d, 3.2 Hz), 6.57 (1H, s), 7.57 (2H, m),

7.66 (1H, m), 8.09 (2H, d, 8.0 Hz), 8.72 (1H, s), 8.85

(1H, s). FAB-MS (mNBA): 398 (M+H)⁺. ESI-HRMS

(positive): calcd for C₁₉H₂₀N₅O₅[M+H]⁺398.1464,

found 398.1460.

2⁰-O,4⁰-C-Ethyleneadenosine (9g). A solution of 9a

(14 mg, 0.035 mmol) in MeOH saturated with ammonia

(1 mL) was left standing overnight. The mixture was

concentrated and the residue was puriﬁed by chroma-

tography on a silica gel column (CH₂Cl₂/MeOH=10:1)

to aﬀord 9g as a white powder (10 mg, 0.034 mmol,

1

98%). H NMR (400 MHz, CD₃OD) d 1.32 (1H, dd, 4

1

52%). H NMR (400 MHz, CDCl₃) d 2.0 (1H, m), 2.06

and 13 Hz), 2.04 (1H, dt, 7.4 and 12 Hz), 3.53 (1H, dd, 5

and 12 Hz), 3.61 (1H, dd, 5.2 and 12 Hz), 3.90 (1H, dd,

7.4 and 12 Hz), 3.97 (1H, dt, 4 and 12 Hz), 4.15 (1H, d,

3.1 Hz), 4.21 (1H, d, 3.1 Hz), 5.27 (1H, t, 5.2 Hz), 5.39

(1H, d, 3.1 Hz), 6.33 (1H, s), 7.29 (2H, s), 7.66 (1H, m),

8.14 (1H, s), 8.42 (1H, s). FAB-MS (mNBA): 294

(3H, s), 2.32 (1H, dt, 6.0 and 15 Hz), 2.40 (3H, s), 3.36

(1H, d, 10 Hz), 3.58 (1H, d, 10 Hz), 4.22 (2H, m), 4.39

(1H, d, 12 Hz), 4.45 (1H, d, 12 Hz), 4.47 (1H, d, 12 Hz),

4.59 (1H, d, 12 Hz), 4.62 (1H, d, 5.6 Hz), 5.94 (1H, dd,

4.5 and 5.6 Hz), 6.21 (1H, d, 4.5 Hz), 7.2-7.3 (12H, m),

7.54 (2H, m), 7.62 (1H, dt, 1.2 and 6.2 Hz), 7.72 (2H, d,

8.3 Hz), 8.02 (2H, m), 8.21 (1H, s), 8.75 (1H, s), 8.97

(1H, brs). FAB-MS (mNBA): 792 (M+H)⁺.

(M+H)⁺

.

ESI-HRMS (positive): calcd for

C₁₂H₁₆N₅O₄[M+H]⁺294.1202, found 294.1196. UV

(l_max): 260 (pH 7), 260 (pH 1), 258 (pH 13).

3⁰,5⁰-Di-O-benzyl-2⁰-O,4⁰-C-ethylene-6-N-benzoyladeno-

sine (8a). Of a 2 N NaOH mixture solution, comprising

pyridine/MeOH/H₂O=65:30:5, 5 mL was added to 7a

(238 mg, 0.30 mmol) in pyridine/MeOH/H₂O=65:30:5

(5 mL) at 0 ^ꢁC and the mixture was stirred at room

temperature for 15 min. The reaction mixture was neu-

tralized with 1 N HCl solution and extracted with

EtOAc. The organic layer was washed with saturated

NaHCO₃solution and brine, dried over MgSO₄and

then concentrated in vacuo. The residue was puriﬁed by

chromatography on a silica gel column (CH₂Cl₂/

MeOH=50:1) to aﬀord 8a as a colorless amorphous

5⁰-O-Dimethoxytrityl-2⁰-O,4⁰-C-ethylene-6-N-benzoylade-

nosine (10a). To a solution of 9a (14 mg, 0.035 mmol) in

anhydrous pyridine (1 mL) 4,4⁰-dimethoxytritylchloride

(18 mg, 0.053 mmol) was added and the mixture was

stirred at 40 ^ꢁC for 5 h. A small amount of MeOH was

added to the reaction mixture and then the solvent was

evaporated in vacuo. The residue was partitioned

between H₂O and CHCl₃. The organic layer was washed

with saturated NaHCO₃solution and brine and con-

centrated in vacuo. The residue was puriﬁed by chro-

matography on

MeOH=100:5) to aﬀord 10a (18 mg, 0.026 mmol, 73%)

a

silica gel column (CH₂Cl₂/

1

solid (133 mg, 0.23 mmol, 78%). H NMR (400 MHz,

as a colorless amorphous solid. H NMR (400 MHz,

CDCl₃) d 1.44 (1H, d, 13 Hz), 2.31 (1H, dd, 13 and

19 Hz), 3.56 (1H, d, 11 Hz), 3.70 (1H, d, 11 Hz), 4.10

(2H, m), 4.24 (1H, s), 4.45 (1H, d, 12 Hz), 4.53–4.67

(4H, m), 6.52 (1H, s), 7.3 (10H, m), 7.53 (2H, m), 7.62

(1H, m), 8.03 (2H, d, 7.6 Hz), 8.66 (1H, s), 8.78 (1H, s),

CDCl₃) d 1.63 (1H, m), 2.14 (1H, 7.5, 12, and 13 Hz),

3.37 (1H, d, 11 Hz), 3.41 (1H, d, 11 Hz), 3.79 (6H, s),

4.10 (2H, m), 4.48 (1H, d, 3.3 Hz), 4.59 (1H, d, 3.3 Hz),

6.54 (1H, s), 6.85 (4H, m), 7.2-7.6 (12H, m), 8.02 (2H,

m), 8.45 (1H, s), 8.82 (1H, s), 9.02 (1H, brs). FAB-MS

K. Morita et al. / Bioorg. Med. Chem. 11 (2003) 2211–2226

2219

(mNBA): 700 (M+H)⁺. ESI-HRMS (positive): calcd

for C₄₀H₃₈N₅O₇[M+H]⁺700.2771, found 700.2747.

(20 mg). The mixture was stirred under a hydrogen

atmosphere at atmospheric pressure for 5 h. The reac-

tion mixture was ﬁltered in order to remove the catalyst

and the ﬁltrate was concentrated in vacuo. The residue

was puriﬁed by chromatography on a silica gel column

(CH₂Cl₂/MeOH=10:2) to aﬀord 9b as a colorless oil

(5 mg, 0.013 mmol, 72%). ¹H NMR (400 MHz,

CD₃OD) d 1.21 (3H, s), 1.22 (3H, s), 1.41 (1H, dd, 4 and

13 Hz), 2.18 (1H, m), 2.69 (1H, qui, 6.9 Hz), 3.69 (1H, d,

12 Hz), 3.76 (1H, d, 12 Hz), 4.0 (2H, m), 4.26 (1H, d,

3.2 Hz), 4.30 (1H, d, 3.2 Hz), 6.30 (1H, s), 8.40 (1H, s).

FAB-MS (mNBA): 380 (M+H)⁺. ESI-HRMS (posi-

tive): calcd for C₁₆H₂₂N₅O₆[M+H]⁺380.1570, found

380.1555.

5⁰-O-(4,4⁰-Dimethoxytrityl-2⁰-O,4⁰-C-ethylene-6-N-ben-

zoyladenosine-3⁰-O-(2-cyanoethyl N,N-diisopropyl)phos-

phoramidite (11a). To a solution of 10a (16 mg,

0.023 mmol) in anhydrous CH₂Cl₂(0.5 mL), N,N-diiso-

propylammonium tetrazolide (10 mg) was added and

then

2-cyanoethyl

N,N,N⁰,N⁰-tetraisopropylphos-

phoramidite (20 mL) was added dropwise in an ice bath.

The mixture was stirred at room temperature overnight.

The reaction mixture was washed with saturated

NaHCO₃solution and brine and concentrated in vacuo.

The residue was puriﬁed by chromatography on a silica

gel column (CH₂Cl₂/EtOAc=2:1) to aﬀord 11a (20 mg,

0.022 mmol, 97%) as a white solid. ¹H NMR (400 MHz,

CDCl₃) d 1.0–1.2 (12H, m), 1.54 (1H, m), 2.15 (1H, m),

2.33 (2H, m), 3.3–3.6 (6H, m), 3.80 (6H, s), 4.08 (2H,

m), 4.65 (1H, m), 4.75 (1H, m), 6.53 (1H, s), 6.84 (4H, m),

7.2–7.6 (12H, m), 8.01 (2H, m), 8.53 (1H, s), 8.83 (1H, s),

9.01 (1H, brs). FAB-MS (mNBA): 900 (M+H)⁺. ESI-

HRMS (positive): calcd for C₄₉H₅₅N₇O₈P [M+H]⁺

900.3850, found 900.3860.

2⁰-O,4⁰-C-Ethyleneguanosine (9h). A solution of 9b

(0.5 mg) in MeOH saturated with ammonia (0.5 mL)

was allowed to stand at 60 ^ꢁC for 5 h. The mixture was

concentrated to aﬀord 9h as a white powder (0.4 mg).

¹H NMR (400 MHz, DMSO-d₆) d 1.24 (1H, m), 2.01

(1H, m), 3.50 (1H, dd, 5.1 and 12 Hz), 3.58 (1H, dd, 5.1

and 12 Hz), 3.87 (2H, m), 4.08 (2H, m), 5.17 (1H, t,

5.1 Hz), 5.32 (1H, d, 5.1 Hz), 6.08 (1H, s), 6.55 (2H, brs),

7.14 (1H, brs), 8.01 (1H, s). FAB-MS (mNBA): 310

(M+H)⁺. ESI-HRMS (positive): calcd for C₁₂H₁₆N₅O₅

[M+H]⁺310.1152, found 310.1160. UV (l_max): 255

(pH 7), 256 (pH 1), 258–266 (pH 13).

3⁰,5⁰-Di-O-benzyl-4⁰-C-(p-toluenesulfonyloxyethyl)-2⁰-O-

acetyl-2-N-isobutyrylguanosine (7b). Trimethylsilylated

2-N-isobutyrylguanosine (650 mg, about 1.5 mmol),

which was prepared according to Vorbruggen’s

method,²⁴was added to a solution of 6 (400 mg,

0.65 mmol) in anhydrous 1,2-dichloroethane (10 mL) at

room temperature under a nitrogen atmosphere. After

addition of TMSOTf (0.2 mL, 1.2 mmol) to the mixture,

the mixture was stirred at 50 ^ꢁC for 4 h. Saturated

NaHCO₃solution was added to the reaction mixture

and the organic layer was washed with saturated

NaHCO₃solution and brine, dried over MgSO₄and then

concentrated in vacuo to give a product 7b which was

used in the next reaction without further puriﬁcation.

5⁰-O-(4,4⁰-Dimethoxytrityl)-2⁰-O,4⁰-C-ethylene-2-N-iso-

butyrylguanosine (10b). The preparation of 10b was car-

ried out according to the same procedure used for 10a

to give 10b (94%) as a colorless solid. ¹H NMR

(400 MHz, CDCl₃): 1.26 (3H, d, 1.4 Hz), 1.28 (3H, d,

1.4 Hz), 1.66 (1H, m), 2.15 (1H, m), 2.59(1H, qui,

6.9 Hz), 3.65 (1H, m), 3.78 (1H, m), 4.06 (2H, m), 4.35

(1H, m), 4.38 (1H, d, 3.2 Hz), 6.23 (1H, s), 6.8 (4H, m),

7.2–7.5 (9H, m), 8.01 (1H, s), 8.19 (1H, brs). FAB-MS

(mNBA): 682 (M+H)⁺. ESI-HRMS (positive): calcd

for C₃₇H₄₀N₅O₈[M+H]⁺682.2877, found 682.2891.

3⁰,5⁰-Di-O-benzyl-2⁰-O,4⁰-C-ethylene-2-N-isobutyrylgua-

nosine (8b). An aqueous solution of 1 N NaOH (2 mL)

was added to a solution of 7b (about 200 mg) in pyridine

(2 mL) and the mixture was stirred at room temperature

for 15 min. The reaction mixture was neutralized with

1 N HCl solution and extracted with EtOAc. The

organic layer was washed with saturated NaHCO₃solu-

tion and brine, dried over MgSO₄and then concentrated

in vacuo. The residue was puriﬁed by chromatography

on a silica gel column (CH₂Cl₂:MeOH=50:1) to aﬀord

8b as a colorless amorphous solid (20 mg, 0.036 mmol,

6%, 2 steps). ¹H NMR (400 MHz, CDCl₃) d 1.27 (3H, s),

1.29 (3H, s), 1.43 (1H, dd, 3 and 13 Hz), 2.28 (1H, m),

2.59 (1H, qui, 6.9 Hz), 3.54 (1H, d, 11Hz), 3.68 (1H, d,

11Hz), 4.03 (2H, m), 4.15 (1H, d, 3.0 Hz), 4.31 (1H, d,

3.0 Hz), 4.45 (1H, d, 12 Hz), 4.56 (1H, d, 12 Hz), 4.61 (1H,

d, 12Hz), 4.63 (1H, d, 12Hz), 6.18 (1H, s), 7.2–7.4 (10H,

m), 8.19 (1H, s), 11.93 (1H, brs). FAB-MS (mNBA): 560

(M+H)⁺. ESI-HRMS (positive): calcd for C₃₀H₃₄N₅O₆

[M+H]⁺560.2509, found 560.2503.

5⁰-O-(4,4⁰-Dimethoxytrityl)-2⁰-O,4⁰-C-ethylene-2-N-iso-

butyrylguanosine-3⁰-O-(2-cyanoethyl N,N-diisopropyl)-

phosphoramidite (11b). The preparation of 11b was

carried out according to the same procedure used for

11a to give 11b (73%) as a white solid. ¹H NMR

(400 MHz, CDCl₃) d 1.1–1.4 (19H, m), 2.1(1H, m), 2.4

(2H, m), 2.6 (1H, m), 3.3–3.6 (6H, m), 3.8 (6H, s), 4.0–

4.6 (4H, m), 6.2 (1H, s), 6.8 (4H, m), 7.2–7.5 (9H, m),

8.1 (1H, s). FAB-MS (mNBA): 882(M+H)⁺. ESI-

HRMS (positive): calcd for C₄₆H₅₇N₇O₉P [M+H]⁺

882.3955, found 882.3948.

3⁰,5⁰-Di-O-benzyl-4⁰-C-(p-toluenesulfonyloxyethyl)- 2⁰-O-

acetyl-5-methyluridine (7c). Trimethylsilylated thymine

(500 mg, about 2 mmol), which was prepared according

to Vorbruggen’s method was added to a solution of 6

(650 mg, 1.06 mmol) in anhydrous 1,2-dichloroethane

(15 mL) at room temperature under nitrogen atmo-

sphere. TMSOTf (0.36 mL, 2 mmol) was added drop-

wise to the mixture and the mixture was stirred at 50 ^ꢁC

for 1 h. Saturated NaHCO₃solution was added to the

reaction mixture and the mixture was ﬁltered through

Celite. CH₂Cl₂was added to the ﬁltrate. The organic

2⁰-O,4⁰-C-Ethylene-2-N-isobutyrylguanosine (9b). To a

solution of 8b (10 mg, 0.018 mmol) in MeOH (2 mL)

was added 20% palladium hydroxide on carbon

2220

K. Morita et al. / Bioorg. Med. Chem. 11 (2003) 2211–2226

layer was washed with saturated NaHCO₃solution and

brine, dried over MgSO₄and then concentrated in

vacuo. The residue was puriﬁed by chromatography on

silica gel (using hexane/EtOAc=1.2:1) to give 7c as a

colorless amorphous solid (432 mg, 0.64 mmol, 60%).

¹H NMR (400 MHz, CDCl₃) d 1.52 (3H, d, 0.9 Hz), 1.94

(1H, dt, 7.5 and 15 Hz), 2.06 (3H, s), 2.23 (1H, dt, 6.0

and 15 Hz), 2.42 (3H, s), 3.38 (1H, d, 10 Hz), 3.67 (1H,

d, 10 Hz), 4.17 (2H, m), 4.36 (1H, d, 6.0 Hz), 4.41 (1H,

d, 12 Hz), 4.44 (1H, d, 12 Hz), 4.48 (1H, d, 12 Hz), 4.58

(1H, d, 12 Hz), 5.39 (1H, dd, 5.1 and 6.0 Hz), 6.04 (1H,

d, 5.1 Hz), 7.3 (12H, m), 7.73 (2H, dt, 1.8 and 8.3 Hz),

8.18 (1H, s). FAB-MS (mNBA): 679 (M+H)⁺.

(mNBA): 587 (M+H)⁺. ESI-HRMS (positive): calcd

for C₃₃H₃₄N₂O₈Na [M+Na]⁺609.2213, found

609.2213.

5⁰-O-(4,4⁰-Dimethoxytrityl)-2⁰-O,4⁰-C-ethylene-5-methy-

luridine-3⁰-O-(2-cyanoethyl N,N-diisopropyl)phosphora-

midite (11c). The preparation of 11c was carried out

according to the same procedure used for 11a to give

1

11c (89%) as a colorless amorphous solid. H NMR

(400 MHz, CDCl₃) d 1.1–1.2 (15H, m), 1.4 (1H, m), 2.08

(1H, m), 2.4 (2H, m), 3.2–4.0 (14H, m), 4.38 (2H, m),

4.47 (1H, m), 6.06 (1H, s), 6.8–6.9 (4H, m), 7.2–7.5 (9H,

m), 7.91 (1H, m). FAB-MS (mNBA): 787 (M+H)⁺.

ESI-HRMS (positive): calcd for C₄₂H₅₁N₄O₉PNa

[M+Na]⁺809.3291, found 809.3275.

3⁰,5⁰-Di-O-benzyl-2⁰-O,4⁰-C-ethylene-5-methyluridine

(8c). Of 2 N NaOH mixture solution, comprising pyri-

dine/MeOH/H₂O=65:30:5, 5 mL was added to 7c

(418 mg, 0.62 mmol) in pyridine/MeOH/H₂O=65:30:5

(5 mL) at 0 ^ꢁC and the mixture was stirred at room

temperature for 15 min. The reaction mixture was

neutralized with 1 N HCl solution and extracted with

EtOAc. The organic layer was washed with saturated

NaHCO₃solution and brine, dried over MgSO₄and

then concentrated in vacuo. The residue was puriﬁed

by chromatography on a silica gel column (hexane/

EtOAc=1:1) to aﬀord 8c as a colorless amorphous

3⁰,5⁰-Di-O-benzyl-4⁰-C-(p-toluenesulfonyloxyethyl)-2⁰-O-

acetyl-4-N-benzoylcytidine (7d). Trimethylsilylated 4-N-

benzoylcytosine (300 mg, about 1.0 mmol), which was

prepared according to Vorbruggen’s method,²⁴was

added to a solution of 6 (383 mg, 0.626 mmol) in anhy-

drous 1,2-dichloroethane (4 mL). TMSOTf (0.18 mL,

0.995 mmol) at 0 ^ꢁC was added to the mixture and the

mixture was stirred at 50 ^ꢁC for 1 h. Saturated NaHCO₃

solution and CH₂Cl₂was added to the mixture and the

mixture was stirred. The resulting white precipitates

were removed by ﬁltrating through Celite. The organic

layer of the ﬁltrate was washed with brine, dried over

MgSO₄and then concentrated in vacuo to give 7d as a

colorless amorphous solid (397 mg, 83%). ¹H NMR

(400 MHz, CDCl₃) d 8.70 (1H, br), 8.18 (1H, d, 7.4 Hz),

7.87 (2H, d, 7.5 Hz), 7.72 (2H, d, 8.3 Hz), 7.61–7.57 (1H,

m), 7.51–7.48 (2H, m), 7.43–7.21 (13H,m), 6.02 (1H, d,

2.9 Hz), 5.40 (1H, dd, 5.8, 2.9 Hz), 4.57 (1H, d, 11 Hz),

4.39 (1H, d, 11 Hz), 4.32–4.28 (3H, m), 4.19–4.16 (2H,

m), 3.69 (1H, d, 11 Hz), 3.31 (1H, d, 11 Hz), 2.40 (3H,

s), 2.30–2.23 (1H, m), 2.06 (3H, s), 1.95–1.89 (1H, m).

FAB-MS (mNBA): 768 (M+H)⁺.

1

solid (228 mg, 0.49 mmol, 79%). H NMR (400 MHz,

CDCl₃) d 1.35 (1H, d, 13 Hz), 1.41 (3H, s), 2.28 (1H,

dt, 9.4 and 13 Hz), 3.60 (1H, d, 11 Hz), 3.76 (1H, d,

11 Hz), 3.94 (1H, d, 3.0 Hz), 4.10 (1H, d, 7.0 Hz), 4.14

(1H, d, 7.0 Hz), 4.31 (1H, d, 3.0 Hz), 4.51 (1H, d,

12 Hz), 4.54 (1H, d, 12 Hz), 4.58 (1H, d, 12 Hz), 4.75

(1H, d, 12 Hz), 6.06 (1H, s), 7.3 (10H, m), 7.91 (1H,

s), 8.42 (1H, brs). FAB-MS (mNBA): 465(M+H)⁺.

ESI-HRMS (positive): calcd for C₂₆H₂₈N₂O₆[M+H]⁺

465.2025, found 465.2018.

2⁰-O,4⁰-C-Ethylene-5-methyluridine (9c). To a solution

of 8c (195 mg, 0.42 mmol) in MeOH (10 mL) was added

20% palladium hydroxide on carbon (100 mg). The

mixture was stirred under hydrogen atmosphere at

atmospheric pressure for 5 h. The reaction mixture was

ﬁltered in order to remove the catalyst and the ﬁltrate

was concentrated in vacuo. The residue was puriﬁed by

chromatography on a silica gel column (CH₂Cl₂/

MeOH=10:1) to aﬀord 9c as a colorless powder (76 mg,

0.268 mmol, 64%). ¹H NMR (400 MHz, CD₃OD) d 1.33

(1H, dd, 3.8 and 13 Hz), 1.86 (3H, d, 0.9 Hz), 1.94 (1H,

ddd, 7.5, 11.7 and 13 Hz), 3.68 (1H, d, 12 Hz), 3.75 (1H,

d, 12 Hz), 3.9–4.0 (2H, m), 4.05 (1H, d, 3.2 Hz), 4.09

(1H, d, 3.2 Hz), 6.00 (1H, s), 8.28 (1H, d, 1.1 Hz). FAB-

MS (mNBA): 285 (M+H)⁺. ESI-HRMS (positive):

calcd for C₁₂H₁₇N₂O₆[M+H]⁺285.1086, found

285.1074.

3⁰,5⁰-Di-O-benzyl-2⁰-O,4⁰-C-ethylene-4-N-benzoylcytidine

(8d). Of a 2 N NaOH solution, 68 mL was added to a

solution of 7d (6.80 g, 8.86 mmol) in pyridine (136 mL)

at 0 ^ꢁC and the mixture was stirred at room temperature

for 1 h. The reaction mixture was neutralized by drop-

wise addition of aqueous 20% acetic acid and extracted

with chloroform. The organic layer was washed with

brine and concentrated in vacuo. The residue was pur-

iﬁed by chromatography on a silica gel column (CH₂Cl₂/

MeOH=100:3) to aﬀord 8d (3.3 g, 6.02 mmol, 68%). ¹H

NMR (400 MHz, CDCl₃) d 8.64 (2H, brs), 7.89 (2H, d,

7.6 Hz), 7.64–7.60 (1H, m), 7.54–7.51 (2H, m), 7.48–7.37

(3H, m), 7.36–7.26 (8H, m), 6.18 (1H,s), 4.70 (1H, d,

11 Hz), 4.60 (1H, d, 11 Hz), 4.55 (1H, d, 11 Hz), 4.46 (1H,

d, 2.9 Hz), 4.42 (1H, d, 11 Hz), 4.10–4.02 (2H,m), 3.89

(1H, d, 2.9 Hz), 3.75 (1H, d, 11 Hz), 3.62 (1H, d, 11 Hz),

2.34–2.26 (1H, m), 1.39–1.36 (1H, m). FAB-MS

(mNBA): 554 (M+H)⁺. ESI-HRMS (positive): calcd

for C₃₂H₃₂N₃O₆[M+H]⁺554.2291, found 554.2305.

5⁰-O-(4,4⁰-Dimethoxytrityl)-2⁰-O,4⁰-C-ethylene-5-methyl-

uridine (10c). The preparation of 10c was carried out

according to the same procedure used for 10a to give

1

10c (81%) as a colorless amorphous solid. H NMR

(270 MHz, DMSO-d₆) d 11.36 (1H, s), 7.68 (1H, s),

6.90–7.44 (13H, m), 5.89 (1H, s), 5.55 (1H, d), 4.09 (1H,

m), 4.04 (1H, d), 3.82 (2H, m), 3.74 (6H, s), 3.19 (2H,

m), 1.99 (1H, m), 1.36 (1H, m), 1.17 (3H, s). FAB-MS

2⁰-O,4⁰-C-Ethylene-4-N-benzoylcytidine (9d). A solution

(31.7 mL) of 1.0 M trichloroborane in CH₂Cl₂was

added dropwise to a solution of 8d (2.06 g, 3.72 mmol)

in anhydrous CH₂Cl₂(317 mL) at ꢂ78 ^ꢁC and the mix-

K. Morita et al. / Bioorg. Med. Chem. 11 (2003) 2211–2226

2221

ture was stirred at ꢂ78 ^ꢁC for 1 h. The reaction mixture

was slowly warmed to ꢂ20 ^ꢁC and stirred at between

20 ^ꢁC and ꢂ10 ^ꢁC for 2 h. MeOH (12 mL) was slowly

added to the mixture and the mixture was stirred for

10 min. The pH of the reaction mixture was adjusted to

7–8 by dropwise addition of saturated NaHCO₃solu-

tion. The mixture was warmed to room temperature and

concentrated in vacuo. The residue was puriﬁed by

chromatography on a silica gel column (CH₂Cl₂/

MeOH=100:5) to aﬀord 9d (1.21 g, 3.24 mmol, 87%) as

0.8 mmol) to the mixture, the mixture was stirred at

70 ^ꢁC for 1 h. Saturated NaHCO₃solution was added to

the reaction mixture. Then, the mixture was ﬁltered

through Celite and CH₂Cl₂was added to the ﬁltrate.

The organic layer was washed with saturated NaHCO₃

solution and brine, dried over MgSO₄and then con-

centrated in vacuo. The residue was puriﬁed by chro-

matography on silica gel (CH₂Cl₂/MeOH=100:2) to

give 7e as a colorless oil (199 mg, 0.299 mmol, 92%). ¹H

NMR (400 MHz, CDCl₃) d 1.94 (1H, dt, 7.4 and 15 Hz),

2.07 (3H, s), 2.23 (1H,dt,5.9 and 15 Hz), 2.43 (3H, s),

3.36 (1H, d, 10 Hz), 3.65 (1H, d, 10 Hz), 4.17 (2H, dd, 6

and 7 Hz), 4.31 (1H, d, 5.9 Hz), 4.38 (1H, d, 11 Hz), 4.39

(1H, d, 11 Hz), 4.40 (1H, d, 11 Hz), 4.58 (1H, d, 11 Hz),

5.29 (1H, dd, 2.4 and 8.2 Hz), 5.33 (1H, dd, 4.5 and

6 Hz), 6.00 (1H, d, 4.5 Hz), 7.2–7.4 (12H, m), 7.61 (1H,

d, 8.2 Hz), 7.74 (1H, d, 8.3 Hz), 8.14 (1H, brs). FAB-MS

(mNBA): 665 (M+H)⁺.

1

a white solid. H NMR (500 MHz, DMSO-d₆) d 11.23

(1H, brs), 8.70 (1H, d, 7.2 Hz), 8.00 (2H, d, 7.5 Hz), 7.3-

6 (4H, m), 5.97 (1H, s), 5.35 (1H, dd, 5 and 10 Hz), 4.10

(1H, dd, 5 and 10 Hz), 4.03 (1H, d, 3.2 Hz), 3.95–3.85

(2H, m) 3.83 (1H, d, 3.2 Hz), 3.65–3.51 (2H, m), 2.06–

1.98 (1H, m), 1.26 (1H, m). FAB-MS (mNBA): 374

(M+H)⁺. ESI-HRMS (positive): calcd for C₁₈H₂₀N₃O₆

[M+H]⁺374.1352, found 374.1341.

2⁰-O,4⁰-C-Ethylenecytidine (9i). A solution of 9d (0.1 g,

0.268 mmol) in MeOH saturated with ammonia (12 mL)

was allowed to stand overnight. The mixture was con-

centrated in vacuo to aﬀord 9i (54 mg, 75%) as a white

3⁰,5⁰-Di-O-benzyl-2⁰-O,4⁰-C-ethyleneuridine (8e). Of 1 N

NaOH solution, 2 mL was added to a solution of 7e

(194 mg, 0.292 mmol) in pyridine (3 mL) at 0 ^ꢁC and the

mixture was stirred at room temperature for 30 min.

The reaction mixture was neutralized with 1 N HCl

solution and extracted with EtOAc. The organic layer

was washed with saturated NaHCO₃solution and brine,

dried over MgSO₄and then concentrated in vacuo. The

residue was puriﬁed by chromatography on a silica gel

column (CH₂Cl₂/MeOH=100:3) to aﬀord 8e as a col-

orless oil (105 mg, 0.233 mmol, 80%). ¹H NMR

(400 MHz, CDCl₃) d 1.36 (1H, m), 2.29 (1H, m), 3.63

(1H, d, 11 Hz), 3.74 (1H, d, 11 Hz), 3.87 (1H, d, 2.9 Hz),

4.03 (2H, m), 4.29 (1H, d, 2.9 Hz), 4.49 (1H, d, 12 Hz),

4.50 (1H, d, 11 Hz), 4.53 (1H, d, 11 Hz), 4.73 (1H, d,

12 Hz), 5.20 (1H, dd, 2 and 8 Hz), 6.04 (1H, s), 7.2–7.4

(10H, m), 8.13 (1H, d, 8.2 Hz), 8.57 (1H, brs). FAB-MS

(mNBA): 451 (M+H)⁺. ESI-HRMS (positive): calcd

for C₂₅H₂₆N₂O₆Na [M+Na]⁺473.1688, found

473.1695.

1

solid. H NMR (500 MHz, DMSO-d₆) d 8.18 (1H, d,

7.4 Hz), 7.10 (2H, br), 5.84 (1H, s), 5.69 (1H, d, 7.6 Hz),

5.27–5.24 (2H, m), 3.86 (1H, d, 3.2 Hz), 3.90–3.78 (2H,

m), 3.76 (1H, d, 3.2 Hz), 3.56 (1H, dd, 5.5 and 12 Hz),

3.49 (1H, dd, 5.5 and 12 Hz), 2.01–1.93 (1H,dt, 7.5 and

12 Hz), 1.22 (1H, dd, 3.6 and 13 Hz). FAB-MS

(mNBA): 270 (M+H)⁺. ESI-HRMS (positive): calcd

for C₁₁H₁₆N₃O₆[M+H]⁺270.1090, found 270.1092.

5⁰-O-(4,4⁰-Dimethoxytrityl)-2⁰-O,4⁰-C-ethylene-4-N-benz-

oylcytidine (10d). The preparation of 10d was carried

out according to the same procedure used for 10a to

give 10d (90%) as a colorless amorphous solid. ¹H

NMR (270 MHz, DMSO-d₆) d 11.27 (1H, brs), 8.59

(1H, m), 6.92–8.01 (19H, m), 6.03 (1H, s), 5.56 (1H, m),

4.17 (1H, m), 4.08 (1H, m), 3.86 (2H, m), 3.77 (6H, s),

3.24 (2H, m), 1.98 (1H, m), 1.24 (1H, m). FAB-MS

(mNBA): 676 (M+H)⁺. ESI-HRMS (positive): calcd

for C₃₉H₃₈N₃O₈[M+H]⁺676.2659, found 676.2648.

2⁰-O,4⁰-C-Ethyleneuridine (9e). To a solution of 8e

(100 mg, 0.222 mmol) in MeOH (4 mL) was added 20%

palladium hydroxide on carbon (90 mg). The mixture

was stirred under a hydrogen atmosphere at atmo-

spheric pressure for 5 h. The reaction mixture was ﬁl-

tered in order to remove the catalyst and the ﬁltrate was

concentrated in vacuo. The residue was puriﬁed by

chromatography on a silica gel column (CH₂Cl₂/

MeOH=10:1) to aﬀord 9e as a colorless oil (45 mg,

0.167 mmol, 75%). ¹H NMR (400 MHz, CD₃OD) d 1.35

(1H, dd, 4 and 13 Hz), 2.13 (1H, ddd, 7, 11 and 13 Hz),

3.66 (1H, d, 12 Hz), 3.73 (1H, d, 12 Hz), 3.91–4.08

(2H,m), 4.01 (1H, d, 3.2 Hz), 4.12 (1H, d, 3.2 Hz), 5.66

(1H, d, 8.2 Hz), 6.00 (1H, s), 8.37 (1H, d, 8.2 Hz). FAB-

MS (mNBA): 271 (M+H)⁺. FAB-HRMS (positive):

calcd for C₁₁H₁₅N₂O₆[M+H]⁺271.0930, found

271.0924.

5⁰-O-(4,4⁰-Dimethoxytrityl-2⁰-O,4⁰-C-ethylene-4-N-benz-

oylcytidine-3⁰-O-(2-cyanoethyl

N,N-diisopropyl)phos-

phoramidite (11d). The preparation of 11b was carried

out according to the same procedure used for 11a to

1

give 11d (84%) as a colorless compound. H NMR

(400 MHz, CDCl₃) d 1.1–1.2 (12H, m), 1.35 (1H,m),

2.11 (1H, m), 2.3 (2H, m), 3.35–3.7 (6H, m), 3.8 (6H,

m), 3.9–4.1 (2H, m), 4.33 (1H, m), 4.45 (1H, m), 6.23

(1H, s), 6.9 (4H, m), 7.3–7.9 (15H, m), 8.7–8.8 (1H, m).

FAB-MS (mNBA): 876 (M+H)⁺. ESI-HRMS (posi-

tive): calcd for C₄₈H₅₅N₅O₉P [M+H]⁺876.3738, found

876.3744.

3⁰,5⁰-Di-O-benzyl-2⁰-O-acetyl-4⁰-C-(p-toluenesulfonylox-

yethyl)uridine (7e). Trimethylsilylated uracil (200 mg,

about 0.8 mmol), which was prepared according to

Vorbruggen’s method, was added to a solution of 6

(200 mg, 0.327 mmol) in anhydrous 1,2-dichloroethane

(8 mL) at room temperature under a nitrogen atmo-

sphere. After dropwise addition of TMSOTf (0.145 mL,

5⁰-O-(4,4⁰-Dimethoxytrityl)-2⁰-O,4⁰-C-ethyleneuridine

(10e). The preparation of 10e was carried out accord-

ing to the same procedure used for 10a to give 10e

1

(42%) as a colorless oil. H NMR (400 MHz, CD₃OD)

d 1.35 (1H, dd, 3 and 14 Hz), 2.03 (1H, ddd, 8, 11 and

2222

K. Morita et al. / Bioorg. Med. Chem. 11 (2003) 2211–2226

14 Hz), 2.46 (1H, d, 8>Hz), 3.36 (1H, d, 11 Hz), 3.41

(1H, d, 11 Hz), 3.80 (3H, s), 3.81 (3H, s), 3.97 (2H, m),

4.21 (1H, d, 3.2 Hz), 4.33 (1H, brm), 5.31 (1H, m), 6.10

(1H, s), 6.86 (4H, m), 7.2–7.5 (9H, m), 8.27 (1H, d,

8.2 Hz), 8.43 (1H, brs). FAB-MS (mNBA): 573

2⁰-O,4⁰-C-Ethylene-4-N-benzoyl-5-methylcytidine (9f). A

1.0 M boron trichloride solution (1.6 mL) in CH₂Cl₂

was added dropwise to a solution of 8f (120 mg,

0.211 mmol) in anhydrous CH₂Cl₂(5 mL) at ꢂ78 ^ꢁC and

the mixture was stirred at ꢂ78 ^ꢁC for 4 h. MeOH (1 mL)

was slowly added dropwise to the mixture and the mix-

ture was stirred for 10 min. The pH of the reaction

mixture was adjusted to 7–8 by dropwise addition of

saturated NaHCO₃solution. The reaction mixture was

warmed to room temperature and concentrated in

vacuo. The residue was puriﬁed by chromatography on

a silica gel column (CH₂Cl₂/MeOH=100:6) to aﬀord 9f

(M+H)⁺.

ESI-HRMS

(positive):

calcd

for

C₃₂H₃₂N₂O₈Na [M+Na]⁺595.2056, found 595.2039.

5⁰-O-(4,4⁰-Dimethoxytrityl)-2⁰-O,4⁰-C-ethyleneuridine-3⁰-

O-(2-cyanoethyl N,N-diisopropyl)phosphoramidite (11e).

The preparation of 11e was carried out according to

the same procedure used for 11a to give 11e (quant.) as

1

a white solid. H NMR (400 MHz, CDCl₃) d 1.1–1.2

(29 mg, 0.075 mmol, 36%) as a white solid. H NMR

(13H, m), 2.09 (1H, m), 2.4 (2H, m), 3.3–3.6 (6H, m),

3.81 (6H, m), 3.94 (2H, m), 4.35 (1H, m), 4.47 (1H, m),

5.18 (1H, d, 8.2 Hz), 6.08 (1H, s), 6.86 (4H, m), 7.2–7.4

(9H, m), 8.31 (1H, d, 8.2 Hz). FAB-MS (mNBA): 773

(400 MHz, DMSO-d₆) d 1.24 (1H, m), 2.01 (3H, s), 2.0

(1H, m), 3.54 (1H, dd, 5.4 and 12 Hz), 3.64 (1H, dd, 5.4

and 12 Hz), 3.88 (3H, m), 4.10 (1H, m), 5.36 (1H, d,

5.4 Hz), 5.49 (1H, t, 5.0 Hz), 5.95 (1H, s), 7.4–7.6 (3H,

m), 8.21 (2H, m), 8.49 (1H, s), 13.17 (1H, brs). FAB-MS

(mNBA): 388 (M+H)⁺. ESI-HRMS (positive): calcd

for C₁₉H₂₁N₃O₆Na [M+Na]⁺410.1328, found

410.1336.

(M+H)⁺.

ESI-HRMS

(positive):

calcd

for

C₄₁H₄₉N₄O₉PNa [M+Na]⁺795.3135, found 795.3167.

2⁰-O-Acetyl-3⁰,5⁰-di-O-benzyl-4⁰-C-(p-toluenesulfonyloxy-

ethyl)-4-N-benzoyl-5-methylcytidine (7f). Trimethylsily-

lated N-4-benzoyl 5-methylcytosine (400 mg, about

1.2 mmol), which was prepared according to Vor-

bruggen’s method²⁴was added to a solution of 6 (400 mg,

0.653 mmol) in anhydrous 1,2-dichloroethane (6 mL).

After addition of TMSOTf (0.180 mL, 1.0 mmol) to the

mixture at 0 ^ꢁC, the mixture was stirred at 50 ^ꢁC for 1h.

The reaction mixture was warmed to room temperature.

Saturated NaHCO₃solution and CH₂Cl₂were added to

the reaction mixture and the mixture was stirred. The

mixture was ﬁltered through Celite in order to remove the

white precipitate. The organic layer of the ﬁltrate was

washed with brine, dried over MgSO₄and then con-

centrated in vacuo to give 7f as a colorless amorphous solid

(320 mg, 0.409 mmol, 63%). ¹H NMR (400MHz, CDCl₃)

d 1.68 (3H, s), 1.95 (1H, dt, 7.3 and 15 Hz), 2.07 (3H, s),

2.25 (1H, dt, 6 and 15 Hz), 2.43 (3H, s), 3.40 (1H, d, 10 Hz),

3.71(1H, d, 10 Hz), 4.18 (2H, m), 4.37 (1H, d, 5.8 Hz), 4.42

(1H, d, 12Hz), 4.46 (1H, d, 12Hz), 4.51 (1H, d, 12Hz),

4.61 (1H, d, 12 Hz), 5.42 (1H, dd, 4.9 and 5.8 Hz), 6.07 (1H,

d, 4.9 Hz), 7.2–7.6 (17H, m), 7.74 (2H, d, 8.3 Hz), 8.28 (2H,

d, 7.0 Hz). FAB-MS (mNBA): 782 (M+H)⁺.

2⁰-O,4⁰-C-Ethylene-5-methylcytidine (9j). A solution of

9f (11.6 mg, 0.030 mmol) in MeOH saturated with

ammonia (2 mL) was allowed to stand overnight. The

mixture was concentrated to aﬀord 9j as a white solid

(8.5 mg, 0.030 mmol, quant.). ¹H NMR (400 MHz,

DMSO-d₆) d 1.20 (1H, m), 1.82 (3H, s), 1.97 (1H, m),

3.49 (1H, dd, 5 and 12 Hz), 3.58 (1H, dd, 5 and 12 Hz),

3.85 (2H, m), 5.23 (1H, d, 5 Hz), 5.32 (1H, t, 5 Hz), 5.84

(1H, s), 6.7 (1H, brs), 7.2 (1H, brs), 8.08 (1H, s). FAB-

MS (mNBA): 284 (M+H)⁺. ESI-HRMS (positive):

calcd for C₁₂H₁₈N₃O₅[M+H]⁺284.1246, found

284.1238. UV (l_max): 279 (pH7), 289 (pH1), 279 (pH13).

5⁰-O-(4,4⁰-Dimethoxytrityl)-2⁰-O,4⁰-C-ethylene-4-N-ben-

zoyl-5-methylcytidine (10f). The preparation of 10f was

carried out according to the same procedure used for

10a to give 10f (93%) as a colorless oil. ¹H NMR

(400 MHz, CDCl₃) d 1.46 (1H, m), 1.49 (3H, s), 2.06 (1H,

m), 2.59 (1H, d, 8.6 Hz), 3.36 (1H, d, 11 Hz), 3.39 (1H, d,

11 Hz), 3.80 (3H, s), 3.81 (3H, s), 3.99 (2H, m), 4.30 (1H,

d, 3.3 Hz), 4.39 (1H, m), 6.12 (1H, s), 6.85 (4H, m), 7.2–

7.5 (12H, m), 8.03 (1H, s), 8.28 (2H, m). FAB-MS

(mNBA): 690 (M+H)⁺. FAB-HRMS (positive): calcd

for C₄₀H₄₁N₃O₈[M+H]⁺690.2815, found 690.2812.

3⁰,5⁰-Di-O-benzyl-2⁰-O,4⁰-C-ethylene-4-N-benzoyl-5-me-

thylcytidine (8f). Of 1 N NaOH solution, 5 mL was

added to a solution of 7f (310 mg, 0.396 mmol) in pyri-

dine (5 mL) at 0 ^ꢁC and the mixture was stirred at room

temperature for 20 min. The reaction mixture was neu-

tralized by dropwise addition of aqueous 20% acetic

acid and extracted with CH₂Cl₂. The organic layer was

washed with brine and concentrated in vacuo. The resi-

due was puriﬁed by chromatography on a silica gel col-

umn (CH₂Cl₂/MeOH=100:2) to aﬀord 8f (190 mg,

5⁰-O-(4,4⁰-Dimethoxytrityl)-2⁰-O,4⁰-C-ethylene-4-N-ben-

zoyl-5-methylcytidine-3⁰-O-(2-cyanoethyl N,N-diisopro-

pyl)phosphoramidite (11f). The preparation of 11b was

carried out according to the same procedure used for

11a to give 11f (89%) as a white solid. ¹H NMR

(400 MHz, CDCl₃) d 1.1–1.2 (12H, m), 1.36 (3H, s), 1.37

(1H, m), 2.10 (1H, m), 2.36 (2H, m), 3.3–3.6 (6H, m),

3.81 (6H, m), 3.98 (2H, m), 4.42 (1H, m), 4.49 (1H, m),

6.11 (1H, s), 6.88 (4H, m), 7.2–7.5 (12H, m), 8.14 (1H,

s), 8.28 (2H, m). FAB-MS (mNBA): 890 (M+H)⁺.

ESI-HRMS (positive): calcd for C₄₉H₅₇N₅O₉P

[M+H]⁺890.3894, found 890.3884.

1

0.334 mmol, 84%). H NMR (400 MHz, CDCl₃) d 1.37

(1H, m), 1.58 (3H, s), 2.30 (1H, dt, 10 and 13 Hz), 3.64

(1H, d, 11 Hz), 3.79 (1H, d, 11 Hz), 3.95 (1H, d, 3.0 Hz),

4.04 (2H, dd, 2.3 and 10 Hz), 4.37 (1H, d, 3.0 Hz), 4.50

(1H, d, 12 Hz), 4.56 (1H, d, 11 Hz), 4.61 (1H, d, 11 Hz),

4.76 (1H, d, 12 Hz), 6.11 (1H, s), 7.2–7.5 (13H, m), 8.09

(1H, s), 8.29 (2H, m). FAB-MS (mNBA): 568

(M+H)⁺. ESI-HRMS (positive): calcd for C₃₃H₃₄N₃O₆

[M+H]⁺568.2448, found 568.2455.

5-O-(tert-Butyldiphenylsilyl)-4-C-formyl-3-O-benzyl-1,2-

O-isopropylidene-ꢀ-^D-erythropentofuranose (13). Oxalyl

chloride (8.3 mL, 95 mmol) was added to anhydrous

K. Morita et al. / Bioorg. Med. Chem. 11 (2003) 2211–2226

2223

CH₂Cl₂(200 mL) at ꢂ78 ^ꢁC. A solution of DMSO

(11.4 mL, 160 mmol) in anhydrous CH₂Cl₂(100 mL)

was added dropwise to this solution. After stirring for

20 min, a solution of 12 (26.3 g, 47.9 mmol) in anhy-

drous CH₂Cl₂(100 mL) was added dropwise to this

mixture and the mixture was stirred for 30 min. Tri-

ethylamine (42 mL, 300 mmol) was added to this reac-

tion mixture and this was slowly warmed to room

temperature. The reaction was quenched with H₂O. The

organic layer was washed with H₂O and brine, dried

over MgSO₄and then evaporated in vacuo. The residue

was puriﬁed by chromatography on silica gel (hexane/

EtOAc=7:1) to give 13 as a colorless oil (26.1 g,

47.8 mmol, 99%). ¹³C NMR (125 MHz, CDCl₃) d 19.21,

26.13, 26.66, 26.76, 63.05, 72.73, 78.57, 79.08, 90.62,

104.91, 114.15, 127.76, 127.79, 127.83, 128.07, 128.51,

129.81, 129.86, 132.51, 132.82, 135.49, 135.57, 136.99,

200.17. ¹H NMR (400 MHz, CDCl₃) d 0.98 (9H, s), 1.37

(3H, s), 1.62 (3H, s), 3.79 (1H, d, 11 Hz), 3.88 (1H, d,

11 Hz), 4.54 (1H, d, 4.4 Hz), 4.62 (1H, d, 12 Hz), 4.65

(1H, dd, 3.7 and 4.4 Hz), 4.74 (1H, d, 12 Hz), 5.87 (1H,

d, 3.7 Hz), 7.3–7.6 (15H), 9.89 (1H, s). IR (ﬁlm) n_max

2933, 2858, 1732, 1113, 1022, 702 cm^ꢂ1. ESI-HRMS

(positive): calcd for C₃₂H₃₈O₆SiNa [M+Na]⁺569.2335,

found 569.2354.

puriﬁed by chromatography on silica gel (hexane/

EtOAc=7:1) to give 15 as a colorless oil (18.5 g,

29.9 mmol, 71%) and the debenzylated compound

(6.0 g, 11.34 mmol, 27%). ¹³C NMR (125 MHz, CDCl₃)

d 14.16, 19.20, 26.19, 26.59, 26.80, 27.00, 29.02, 60.12,

66.29, 72.35, 77.97, 79.16, 86.88, 104.17, 113.27, 127.67,

127.73, 127.76, 128.40, 129.68, 129.76, 132.89, 133.17,

135.53, 135.61, 137.92, 173.88. ¹H NMR (400 MHz,

CDCl₃) d 0.98 (9H, s), 1.18 (3H, t, 7.0 Hz), 1.35 (3H, s),

1.64 (3H, s), 1.82 (1H, m), 2.16 (1H, m), 2.52 (2H, m),

3.40 (1H, d, 11 Hz), 3.61 (1H, d, 11 Hz), 4.04 (2H, dd,

7.0 and 14 Hz), 4.30 (1H, d, 5.1 Hz), 4.58 (1H, d, 12 Hz),

4.67 (1H, dd, 4.4 and 5.1 Hz), 4.80 (1H, d, 12 Hz), 5.78

(1H, d, 4.4 Hz), 7.3–7.7 (15H, m). IR (KBr) n_max2933,

2858, 1733, 1111, 1025, 703 cm^ꢂ1. FAB-MS (mNBA):

617 [M-H], 641 [M+Na]⁺. ESI-HRMS (positive): calcd

for C₃₆H₄₆O₇SiNa [M+Na]⁺641.2911, found

641.2910.

5-O-(tert-Butyldiphenylsilyl)-4-C-hydroxypropyl-3-O-

benzyl-1,2-O-isopropylidene-ꢀ-^D-erythropentofuranose

(16). To a solution of 15 (1.95 g, 3.15 mmol) in anhy-

drous THF (40 mL) was added 1.0 M of lithium tri-s-

butylborohydride in THF (l-selectride, 10 mL,

10 mmol) and the reaction mixture was stirred for

30 min at room temperature. The reaction was quen-

ched with aqueous 20% acetic acid and ﬁltered through

Celite. The ﬁltrate was extracted with EtOAc and the

organic layer was washed with saturated NaHCO₃

solution and brine, dried over MgSO₄and then evapo-

rated in vacuo. The residue was puriﬁed by chromato-

graphy on silica gel (hexane/EtOAc=4:1) to give 16 as a

colorless oil (1.55 g, 2.69 mmol, 85%). IR (ﬁlm) n_max

5-O-(tert-Butyldiphenylsilyl)-4-C-(2-ethoxycarbonyl-(E)-

vinyl)-3-O-benzyl-1,2-O-isopropylidene-ꢀ-^D-erythropen-

tofuranose (14). Sodium hydride (60% in mineral, 2.3 g,

about 57.4 mmol) was added to diethylphosphonoacetic

acid ethyl ester (11.5 mL, 57.4 mmol) in anhydrous THF

(200 mL) at 0 ^ꢁC. After stirring for 10 min, a solution of

13 (26.1 g, 47.8 mmol) in anhydrous THF (200 mL) was

added dropwise to this solution and the mixture was

stirred for 30 min at room temperature. The reaction

was quenched with H₂O and extracted with EtOAc. The

organic layer was washed with brine, dried over MgSO₄

and then evaporated in vacuo. The residue was puriﬁed

by chromatography on silica gel (hexane/EtOAc=7:1)

to give 14 as a colorless oil (25.4 g, 41.2 mmol, 86%).

¹³C NMR (125 MHz, CDCl₃) d 14.23, 19.25, 25.66,

26.07, 26.77, 60.21, 65.60, 72.53, 77.21, 78.08, 86.68,

103.99, 113.48, 122.66, 127.68, 127.73, 127.80, 127.96,

128.49, 129.73, 129.78, 132.71, 133.12, 135.49, 135.64,

3487, 2934, 2858, 1112, 1025, 703 cm^{ꢂ1 13}C NMR

.

(125 MHz, CDCl₃) d 19.21, 26.06, 26.58, 26.77, 26.80,

27.51, 62.57, 65.97, 72.42, 77.83, 79.15, 87.95, 104.13,

113.14, 127.68, 127.73, 127.82, 128.42, 129.70, 129.75,

132.92, 133.29, 135.53, 135.65, 137.95. ¹H NMR

(400 MHz, CDCl₃) d 0.98 (9H, s), 1.35 (3H, s), 1.63

(3H, s), 1.54–1.70 (2H, m), 1.97 (1H, t, 6.1 Hz), 2.17

(1H, m), 3.44 (1H, d, 11 Hz), 3.56 (2H, m), 3.74 (1H,

11 Hz), 4.35 (1H, d, 5.1 Hz), 4.59 (1H, d, 12 Hz), 4.68

(1H, dd, 4.4 and 5.1 Hz), 4.80 (1H, d, 12 Hz), 5.79 (1H,

d, 4.4 Hz), 7.3–7.7 (15H, m). FAB-HRMS (mNBA):

calcd for C₃₄H₄₄O₆SiNa [M+Na]⁺599.2805, found

599.2819.

1

137.56, 145.51, 166.21. H NMR (400 MHz, CDCl₃) d

1.04 (9H, s), 1.30 (3H, t, 7.3 Hz), 1.45 (3H, s), 1.62 (3H,

s), 3.54 (1H, d, 10 Hz), 3,58 (1H, d, 10 Hz), 4.2 (2H, m),

4.39 (1H, d, 6.3 Hz), 4.59 (1H, d, 10 Hz), 4.58 (1H, dd,

4.4 and 6.3 Hz), 4.77 (1H, d, 10 Hz), 5.90(1H, d, 4.4 Hz),

6.24 (1H, d, 16 Hz), 7.3–7.7 (15H, m). IR (KBr) n_max

5-O-(tert-Butyldiphenylsilyl)-4-C-(p-toluenesulfonyloxy-

propyl)-3-O-benzyl-1,2-O-isopropylidene-ꢀ-^D-erythropen-

tofuranose (17). Pyridine (0.24 mL, 3.0 mmol) and

p-toluenesulfonyl chloride (286 mg, 1.5 mmol) were

added to a solution of 16 (610 mg, 1.06 mmol) in anhy-

drous CH₂Cl₂(10 mL) under a nitrogen atmosphere at

0 ^ꢁC and the mixture was stirred at room temperature

overnight. The reaction mixture was partitioned

between CH₂Cl₂and saturated NaHCO₃. The organic

layer was washed with brine, dried over MgSO₄and

then evaporated in vacuo. The residue was puriﬁed by

chromatography on silica gel (hexane/EtOAc=7:1) to

give 17 as a colorless oil (600 mg, 0.82 mmol, 77%). IR

2929, 2857, 1717, 1302, 1139, 1106, 1034, 704 cm^ꢂ1

.

FAB-MS (mNBA): 639 [M+Na]⁺. ESI-HRMS (posi-

tive): calcd for C₃₆H₄₄O₇SiNa [M+Na]⁺639.2754,

found 639.2755.

5-O-(tert-Butyldiphenylsilyl)-4-C-(2-ethoxycarbonyle-

thyl)-3-O-benzyl-1,2-O-isopropylidene-ꢀ-^D-erythropento-

furanose (15). To a solution of 14 (26.0 g, 42.14 mmol) in

EtOAc (100 mL) was added 20% palladium hydroxide

on carbon (8.0 g). The mixture was stirred under a

hydrogen atmosphere at an atmospheric pressure of

3.5 psi for 10 h. The reaction mixture was ﬁltered and

the ﬁltrate was evaporated in vacuo. The residue was

(ﬁlm) n_max2933, 2858, 1360, 1176, 1112, 704 cm^{ꢂ1 13}C

.

NMR (125 MHz, CDCl₃) d 19.17, 21.56, 23.20, 26.17,

26.73, 26.77, 27.80, 66.33, 71.09, 72.37, 78.04, 79.25,

2224

K. Morita et al. / Bioorg. Med. Chem. 11 (2003) 2211–2226

87.20, 104.16, 113.13, 127.70, 127.76, 127.81, 127.87,

128.42, 129.70, 129.79, 132.85, 133.11, 133.13, 135.51,

135.58, 137.89, 144.44. H NMR (400 MHz, CDCl₃) d

0.96 (9H, s), 1.32 (3H, s), 1.50 (3H, s), 1.42–1.56 (2H,

m), 1.82 (1H, m), 2.05 (1H, m), 2.39 (3H, s), 3.34 (1H, d,

11 Hz), 3.56 (1H, d, 11 Hz), 3.90–4.00 (2H, m), 4.25 (1H,

d, 5.1 Hz), 4.54 (1H, d, 12 Hz), 4.63 (1H, dd, 3.7 and

5.1 Hz), 4.77 (1H, d, 12 Hz), 5.74 (1H, d, 3.7 Hz), 7.3–

7.8 (19H, m). ESI-HRMS: calcd for C₄₁H₅₀O₈SiSNa

[M+Na]⁺753.2893, found 753.2878.

3⁰-O-Benzyl-2⁰-O,4⁰-C-propylene-5-methyluridine (20).

Of 1.0 N NaOH solution, 3 mL was added to a solution

of 19 (475 mg, 0.56 mmol) in pyridine (5 mL) at 0 ^ꢁC and

the mixture was stirred at room temperature for 20 min.

The reaction mixture was neutralized by dropwise

addition of aqueous 20% acetic acid and extracted with

CH₂Cl₂. The organic layer was washed with neutral

phosphate buﬀer and brine and evaporated in vacuo.

The obtained crude compound was diluted with THF

(5 mL) and 1.0 M of tetrabutylammonium ﬂuoride in

THF solution was added to this solution. The reaction

mixture was stirred for 6 h at room temperature and

then partitioned between H₂O and CH₂Cl₂. The organic

layer was washed with brine, dried over MgSO₄and

then evaporated in vacuo. The residue was puriﬁed by

chromatography on silica gel (CH₂Cl₂/MeOH=40:1) to

give 20 as a colorless amorphous solid (180 mg,

1

5-O-(tert-Butyldiphenylsilyl)-4-C-(p-toluenesulfonyloxy-

propyl)-3-O-benzyl-1,2-di-O-acetyl-^D-erythropentofura-

nose (18). Acetic anhydride (1.0 mL, 10 mmol) and

concentrated H₂SO₄(0.01 mL) were added to a solution

of 17 (600 mg, 0.82 mmol) in acetic acid (5 mL) and the

mixture was stirred at room temperature for 30 min.

The reaction mixture was poured into H₂O (10 mL) in

an ice-bath, stirred for 30 min and then extracted with

EtOAc. The organic layer was washed with neutral

phosphate buﬀer, saturated NaHCO₃solution and

brine, dried over MgSO₄and then evaporated in vacuo.

The residue was puriﬁed by chromatography on silica

gel (hexane/EtOAc=5:1) to give mainly a b-anomer of

18 as a colorless oil (500 mg, 0.65 mmol, 79%). IR (ﬁlm)

1

0.46 mmol, 82%). H NMR (400 MHz, CDCl₃) d 1.50–

1.90 (4H, m), 1.87 (3H, s), 3.70 (1H, d, 12 Hz), 3.80 (1H,

d, 12 Hz), 4.05 (1H, m), 4.30 (1H, dt, 1.5 and 11 Hz),

4.37 (1H, d, 5.9 Hz), 4.42 (1H, d, 5.9 Hz), 4.56 (1H, d,

11 Hz), 4.82 (1H, d, 11 Hz), 6.02 (1H, s), 7.3–7.4 (5H,

m), 7.72 (1H, d, 1.5 Hz), 8.25 (1H, brs). IR (KBr) n_max

3393, 2956, 1468, 1118 cm^ꢂ1. FAB-MS (mNBA): 389

[M+H]⁺. ESI-HRMS (positive): calcd for C₂₀H₂₅N₂O₆

[M+H]⁺389.1713, found 389.1714.

n_max1748, 1362, 1220, 1176, 1112, 704 cm^{ꢂ1 13}C NMR

.

(125 MHz, CDCl₃) d 19.32, 20.75, 20.96, 21.58, 22.85,

26.90, 28.23, 67.30, 71.15, 73.50, 74.75, 79.07, 87.79,

97.70, 127.51, 127.77, 127.84, 128.43, 129.74, 129.82,

129.94, 132.71, 133.05, 133.24, 135.49, 135.59, 137.59,

2⁰-O,4⁰-C-Propylene-5-methyluridine (21). To a solution

of 20 (175 mg, 0.45 mmol) in MeOH (5 mL) was added

20% palladium hydroxide on carbon (105mg). The mix-

ture was stirred under a hydrogen atmosphere at atmo-

spheric pressure overnight. The reaction mixture was

ﬁltered and the ﬁltrate was evaporated in vacuo. The resi-

due was puriﬁed by chromatography on silica gel (CH₂Cl₂/

MeOH=10:1) to give 21 as a colorless amorphous solid

(81 mg, 0.27 mmol, 59%). ¹H NMR (400 MHz, CD₃OD) d

1.58 (1H, m), 1.71 (1H, m), 1.84 (2H, m), 1.85 (3H, s), 3.36

(1H, s), 3.66 (1H, d, 12 Hz), 3.74 (1H, d, 12Hz), 3.94 (1H,

dt, 3.7 and 11 Hz), 4.11 (1H, d, 5.9 Hz), 4.28 (1H, m), 4.50

(1H, 5.9 Hz), 5.95 (1H, s), 8.28 (1H, s). FAB-MS (mNBA):

299 [M+H]⁺. ESI-HRMS (positive): calcd for

C₁₃H₁₉N₂O₆[M+H]⁺299.1243, found 299.1237.

1

144.50, 169.20, 169.71. H NMR (400 MHz, CDCl₃) ꢀ

:

1.03 (9H, s), 1.60–1.90 (4H, m), 1.80 (3H, s), 2.06 (3H,

s), 2.40 (3H, s), 3.53 (2H, s), 3.98 (2H, m), 4.34 (1H, d,

5.2 Hz), 4.50 (1H, d, 11 Hz), 4.57 (1H, d, 11 Hz), 5.33

(1H, d, 5.2 Hz), 6.08 (1H, s), 7.3–7.8 (19H, m). ESI-

HRMS: calcd for C₄₂H₅₀O₁₀SiSNa [M+Na]⁺

797.2792, found 797.2798.

5⁰-O-(tert-Butyldiphenylsilyl)-4⁰-C-(p-toluenesulfonyloxy-

propyl)-3⁰-O-benzyl-2⁰-O-acetyl-5-methyluridine (19).

Trimethylsilylated thymine (500 mg, about 2 mmol),

which was prepared according to Vorbruggen’s

method,²⁴was added to a solution of 18 (490 mg,

0.64 mmol) in anhydrous 1,2-dichloroethane (15 mL) at

5⁰- O - (4,4⁰- Dimethoxytrityl) - 2⁰- O,4⁰- C - propylene - 5 -

methyluridine (22). The preparation of 22 was carried

out according to the same procedure used for 10a to

give 22 as a colorless amorphous solid (quant.). ¹³C

NMR (125 MHz, CDCl₃) d 11.83, 25.03, 31.46, 55.26,

66.10, 68.18, 70.64, 80.89, 86.94, 89.38, 91.37, 110.14,

113.36, 123.81, 127.18, 128.09, 128.13, 130.10, 135.17,

135.19, 135.37, 136.13, 144.22, 149.68, 149.99, 158.72,

room temperature under

a nitrogen atmosphere.

TMSOTf (0.2 mL, 1.1 mmol) was added dropwise to the

mixture and the mixture was stirred at 50 ^ꢁC for 3 h. The

reaction mixture was quenched with saturated NaHCO₃

solution and ﬁltered through Celite. The organic layer

was washed with saturated NaHCO₃solution and brine,

dried over MgSO₄and then evaporated in vacuo. The

residue was puriﬁed by chromatography on silica gel

(CH₂Cl₂/MeOH=100:3) to give 19 as a colorless amor-

phous solid (475 mg, 0.56 mmol, 88%). IR (KBr) n_max

1

158.74, 163.80. H NMR (400MHz, CDCl₃) d 1.32 (3H,

s), 1.67 (1H, m), 1.76 (1H, m), 1.83 (2H, m), 2.73 (1H, brs),

3.37 (2H, s), 3.79 (6H, s), 4.06 (1H, m), 4.20 (1H, m), 4.30

(1H, d, 6.6 Hz), 4.72 (1H, d, 6.6 Hz), 5.99 (1H, s), 6.84

(4H, m), 7.3–7.7 (9H, m), 8.44 (1H, s), 8.61 (1H, m). IR

(KBr) n_max3403, 2951, 1688, 1509, 1252cm^ꢂ1. FAB-MS

(mNBA): 601 [M+H]⁺. ESI-HRMS (positive): calcd for

C₃₄H₃₆N₂O₈Na [M+H]⁺623.2369, found 623.2365.

1

2931, 1748, 1693, 1361, 1228, 1176, 1111, 703 cm^ꢂ1. H

NMR (400 MHz, CDCl₃) d 1.10 (9H, s), 1.40–1.52 (2H,

m), 1.55 (3H, s), 1.72–1.82 (2H, m), 2.08 (3H, s), 2.40

(3H, s), 3.49 (1H, d, 12 Hz), 3.75 (1H, d, 12 Hz), 3.92

(2H, m), 4.37 (1H, d, 6.6 Hz), 4.02 (1H, d, 12 Hz), 4.57

(1H, d, 12 Hz), 5.36 (1H, t, 6.6 Hz), 6.12 (1H, d, 6.6 Hz),

7.3–7.7 (19H, m), 7.71 (1H, d, 2.2 Hz), 8.08 (1H, s). ESI-

HRMS: calcd for C₄₅H₅₂N₂O₁₀SiSNa [M+Na]⁺

863.3009, found 863.2991.

5⁰-O-(4,4⁰-Dimethoxytrityl)-2⁰-O,4⁰-C-propylene-5-methyl-

uridine-3⁰-O-(2-cyanoethyl

N,N-diisopropyl)phosphor-

amidite (23). The preparation of 23 was carried out

K. Morita et al. / Bioorg. Med. Chem. 11 (2003) 2211–2226

2225

according to the same procedure used for 11a to give 23

as a colorless amorphous solid (quant.). ¹H NMR

(400 MHz, CDCl₃) d 1.20 (3H, s), 1.22 (3H, s), 1.29 (3H,

s), 1.31 (3H, s), 1.78 (3H, s), 1.5-2.4 (6H, m), 2.6-2.8

(2H, m), 3.6–3.8 (4H, m), 3.80 (6H, s), 4.03 (1H, m),

4.13 (1H, m), 4.36 (1H, d, 6.6 Hz), 4.98 (1H, d, 6.6 Hz),

6.04 (1H, s), 6.8–6.9 (4H, m), 7.3-7.5 (9H), 7.91 (1H, d,

1.5 Hz), 8.25 (1H, brs). FAB-MS (mNBA): 801

mentary publication nos. CCDC 195962-195965. Copies

of the data can be obtained, free of charge, upon appli-

cation to CCDC, 12 Union Road, Cambridge, CB2

1EZ, UK, (fax: +44- (0)1223-336033 or e-mail: deposit

@ccdc.cam.ac.uk).

Synthesis of modiﬁed oligonucleotides containing 2⁰-O,4⁰-

C-ethylene nucleosides or 2⁰-O,4⁰-C-propylene nucleo-

sides. Modiﬁed oligonucleotides containing 2⁰-O,4⁰-C-

ethylene nucleosides were prepared by solid-phase

phosphoramidite chemistry using Applied Biosystems

DNA/RNA synthesizer 392. Reagent solutions were

purchased from Applied Biosystems. The coupling of

2⁰-O,4⁰-C-ethylene nucleoside-3⁰-phosphoramidite was

performed according to standard synthesis cycles except

for an elongation of the coupling time (15 min). After

synthesis, the CPGs were treated with concentrated

aqueous ammonia at 60 ^ꢁC for 5 h. The crude products

were puriﬁed by C18 silica gel column chromatography

with a gradient of CH₃CN (Cosmosil 75 C18-OPN,

Nacalai Tesque, Japan, 8 ꢃ 100 mm, 50 mM triethyl-

ammonium bicarbonate (pH 7.5)), treated with 80%

acetic acid in H₂O for 20 min, and then puriﬁed by

reverse phase HPLC with a gradient of CH₃CN

(Wakosil DNA, Wako Pure Chemical Industries, Ltd,

Japan, 10 ꢃ 250 mm, 0.1 M triethylammonium acetate

(pH 7.0)). The structures of modiﬁed oligonucleotides

were determined by negative ion ESI mass spectro-

scopy. 5⁰-d(GCGXXXXXXGCT)-3⁰, X=mT: 2⁰-O,4⁰-

C-methylene thymidine, calcd: 3801.47, found: 3801.04,

5⁰-d(GCGXXXXXXGCT)-3⁰, X=eT: 2⁰-O,4⁰-C-ethylene

thymidine, calcd: 3885.63, found: 3885.26, 5⁰-d(GCGTT

XTTXGCT)-3⁰, X=eT, calcd: 3717.45, found: 3717.02,

5⁰-d(GCGTTXTTXGCT)-3⁰, X=prT: X=2⁰-O,4⁰-C-

propylene thymidine, calcd: 3745.50, found: 3745.05, 5⁰-

d(TTTTTTTTTTXT)-3⁰, X=mT, calcd: 3616.40, found:

3616.04, 5⁰- d(TTTTTTTTTTXT) - 3⁰, X=eT, calcd:

3630.43, found: 3630.06, 5⁰- d(TTTTTTTTTTXT) - 3⁰,

X=prT, calcd: 3644.43, found: 3643.84, 5⁰-d(TTTTTT

TTTTXT)-3⁰, X=Ts(Sp): thymidine 3⁰-phosphorothioate

(Sp isomer), calcd: 3604.43, found: 3603.94, 5⁰-

d(TTTTTTTTTTXT)-3⁰, X=Ts(Rp), thymidine 3⁰-phos

phorothioate (Rp isomer), calcd: 3604.43, found:

3604.52, 5⁰-d(TTTTTTTTTTXT) - 3⁰, eTs: 2⁰- O,4⁰-C -

ethylene thymidine 3⁰-phosphorothioate, calcd: 3646.46,

found, 3646.05, 5⁰-d(TTTTTTTTTTXX)-3⁰, X=eT, calcd:

3672.43, found: 3672.28.

[M+H]⁺.

C₄₃H₅₄N₄O₉P [M+H]⁺801.3628, found 801.3648.

ESI-HRMS

(positive):

calcd

for

5⁰-O-(4,4⁰-Dimethoxytrityl)-3⁰-O-succinyl-2⁰-O,4⁰-C-eth-

ylene thymidine (24c). To a stirred solution of 10c

(580 mg, 0.988 mmol) and DMAP (217 mg, 1.78 mmol)

in 10 mL of pyridine was added succinic anhydride

(178 mg, 1.78 mmol). After stirring overnight, the reac-

tion mixture was concentrated in vacuo. The residue

was puriﬁed by silica gel column chromatography (8%

MeOH in CH₂Cl₂) to aﬀord 24c (580 mg, 85%). IR

(KBr) n_max3179, 3064, 2931, 1694, 1509, 1252,

1176 cm^ꢂ^{1 13}C NMR (125 MHz, CDCl₃) d 11.78, 28.36,

.

29.08, 29.11, 55.24, 60.48, 62.87, 66.89, 76.30, 83.42,

85.94, 86,81, 110.27, 113.31, 113.32, 124.11, 125.29,

127.19, 128.06, 128.21, 128.24, 129.02, 130.17, 134.87,

135.04, 135.45, 137.04, 137.84, 143.77, 148.51, 149.69,

158.72, 158.74, 165.15, 171.34, 175.81. ¹H NMR

(400 MHz, CDCl₃) d 1.23 (3H, s), 1.45 (1H, d, 12 Hz),

2.07 (1H, dd, 8.3 and 12 Hz), 2.54 (1H, m), 2.64 (1H,

m), 2.75 (2H, m), 3.17 (1H, d, 11 Hz), 3.43 (1H, d,

11 Hz), 3.79 (6H, s), 4.02 (2H, m), 4.55 (1H, d, 3.0 Hz),

5.40 (1H, d, 3.0 Hz), 6.11 (1H, s), 7.16 (4H, m), 7.1–7.4

(9H, m), 7.93 (1H, s), 8.61 (1H, brs). FAB-MS (mNBA):

686 [M–H]. ESI-HRMS (positive): calcd for

C₃₇H₃₈N₂O₁₁Na [M+Na]⁺709.2373, found 709.2372.

5⁰-O-(4,4⁰-Dimethoxytrityl)-2⁰-O,4⁰-C-ethylene thymi-

dine-bound controlled pore glass (CPG, 25c). To a solu-

tion of 24 (515 mg, 0.75 mmol) in 6 mL of DMF were

added 2,3,4,5,6-pentachlorophenol (330 mg, 1.12 mmol)

and 1,3-dicyclohexylcarbodiimide (DCC, 231 mg,

1.12 mmol). After stirring for 24 h, insoluble material

was removed by ﬁltration. The ﬁltrate was concentrated

in vacuo, and benzene was added to the residue. Any

insoluble material was removed by repeated-ﬁltration to

give the desired ester. Long-chain alkylamino CPG

(CPG Inc., 6.0 g, 170 mmol amino group/gram) was sus-

pended in 15 mL of DMF containing 280 mL (2 mmol)

of triethylamine, and to this was added penta-

chlorophenyl ester. After the mixture was left standing

for 8 h at 60 ^ꢁC and for 24 h at room temperature, the

CPG was ﬁltered, washed with CH₂Cl₂and MeOH, and

dried in vacuo to aﬀord CPG 25c. The loaded nucleo-

side amount, as determined by acid treatment and

measurement of the trityl cation (A₅₀₀; e=71,700), was

62 mmol/g. The residual amino groups were capped with

acetic anhydride in the presence of 1-methylimidazole in

pyridine-THF.

UV melting analysis

Each modiﬁed oligonucleotide and their corresponding

RNA or DNA were mixed (ﬁnal concentration, 4 mM)

and dissolved into a buﬀer containing 10 mM phosphate

(pH 7.2) and 100 mM NaCl, heated at 95 ^ꢁC for 10 min,

and then cooled down to room temperature. The melt-

ing temperature (T_m) of the complexes was measured

with UV spectrometer UV-3100PC (Shimadzu, Japan)

equipped with a temperature controller TCC-controller

(Shimadzu, Japan).

X-Ray crystallographic data

Crystallographic data (excluding structure factors) for

the structures in this paper have been deposited at the

Cambridge Crystallographic Data Centre as supple-

Measurement of CD spectra of duplexes. The CD spec-

tra of the complexes described above were measured

with a JASCO J-500C spectropolarimeter.

2226

K. Morita et al. / Bioorg. Med. Chem. 11 (2003) 2211–2226

R. G.; DeDionisio, L. A.; Ratmeyer, L.; Wilson, W. D. Proc.

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Nuclease stability of oligonucleotides

12. Tereshko, V.; Gryaznov, S.; Egi, M. J. Am. Chem. Soc.

1998, 120, 269.

To each solution of 40 mg of oligonucleotides in

1.425 mL of reaction buﬀer (50 mM Tris–HCl (pH8.0)

and 10 mM MgCl₂) was added 10.7 mg of snake venom

phosphodiesterase (Worthington) in 75 mL of H₂O at

37 ^ꢁC. Of the reaction mixture, 200 mL was taken at each

sampling time point and immediately heated at 90 ^ꢁC

for 4 min to inactivate the enzyme. The reaction mixture

was analyzed by reverse phase HPLC. The residual ratio

of oligonucleotides to the initial amount was determined

from their peak areas ratios.

13. Obika, S.; Nanbu, D.; Hari, Y.; Morio, K.; In, Y.; Ishida,

T.; Imanishi, T. Tetrahedron Lett. 1997, 38, 8735.

14. Obika, S.; Uneda, T.; Sugimoto, T.; Nanbu, D.; Minami,

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Acknowledgements

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The authors wish to thank Dr. Youji Furukawa for his

support in X-ray crystal structure analysis and Ms

Junko Kawakami for her support in 2⁰-O,4⁰-C-ethylene

guanosine synthesis.

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Article Doi

Synthesis and properties of 2′-O,4′-C-ethylene-bridged nucleic acids (ENA) as effective antisense oligonucleotides

DOI: 10.1016/S0968-0896(03)00115-9

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Article abstract of DOI:10.1016/S0968-0896(03)00115-9

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