Synthesis and Conformational Properties of Oligonucleotides Incorporating 2′-O-Phosphorylated Ribonucleotides as Structural Motifs of Pre-tRNA Splicing Intermediates

J . Org. Chem. 2000, 65, 7479-7494

7479

Syn th esis a n d Con for m a tion a l P r op er ties of Oligon u cleotid es

In cor p or a tin g 2′-O-P h osp h or yla ted Ribon u cleotid es a s Str u ctu r a l

Motifs of P r e-tRNA Sp licin g In ter m ed ia tes

Hiroyuki Tsuruoka, Koh-ichiroh Shohda, Takeshi Wada, and Mitsuo Sekine*

Department of Life Science, Tokyo Institute of Technology, Nagatsuta, Midoriku,

Yokohama 226-8501, J apan

msekine@bio.titech.ac.jp

Received J uly 8, 1999

To synthesize oligonucleotides containing 2′-O-phosphate groups, four kinds of ribonucleoside 3′-

phosphoramidite building blocks 6a -d having the bis(2-cyano-1,1-dimethylethoxy)thiophosphoryl

(BCMETP) group were prepared according to our previous phosphorylation procedure. These

phosphoramidite units 6a -d were not contaminated with 3′-regioisomers and were successfully

applied to solid-phase synthesis to give oligodeoxyuridylates 15, 16 and oligouridylates 21, 22.

Self-complementary Drew-Dickerson DNA 12mers 24-28 replaced by a 2′-O-phosphorylated

ribonucleotide at various positions were similarly synthesized. In these syntheses, it turned out

that KI₃was the most effective reagent for oxidative desulfurization of the initially generated

thiophosphate group to the phosphate group on polymer supports. Without using this conversion

step, a tridecadeoxyuridylate 17 incorporating a 2′-O-thiophosphorylated uridine derivative was

also synthesized. To investigate the effect of the 2′-phosphate group on the thermal stability and

3D-structure of DNA(RNA) duplexes, T_mmeasurement of the self-complementary oligonucleotides

obtained and MD simulation of heptamer duplexes 33-36 were carried out. According to these

analyses, it was suggested that the nucleoside ribose moiety phosphorylated at the 2′-hydroxyl

function predominantly preferred C2′-endo to C3′-endo conformation in DNA duplexes so that it

did not significantly affect the stability of the DNA duplex. On the other hand, the 2′-modified

ribose moiety was expelled to give a C3′-endo conformation in RNA duplexes so that the RNA

duplexes were extremely destabilized.

In tr od u ction

mechanism of this tRNA splicing is quite different from

those of Group I and II self-splicing and eukaryotic

mRNA splicing.^3,6Therefore, this unique processing was

considered to be developed from a diverse origin. More-

over, Phizicky et al. reported that an NAD-dependent 2′-

phosphotransferase playing a critical role at the final step

of tRNA splicing transferred the 2′-phosphoryl function

from 2′-O-phosphorylated tRNA to NAD to give an ADP-

ribose 1′-2′ cyclic phosphate.⁷

In 1981, Konarska first discovered that when the

circular RNA fragments of tobacco mosaic virus RNAΩ73

were treated with wheat germ, an unprecedented species,

phosphorylated at the 2′-hydroxyl function, was formed

as the ligated product.^1,2This unique nucleotide has both

phosphomonoester and phosphodiester linkages on the

2′,3′-cis-diol.

After the discovery of such a species, Abelson and co-

workers also reported that pre-tRNA^Leu, transcribed from

DNA, underwent splicing in tRNA synthesis with elimi-

nation of its intron part to generate a spliced tRNA

product 2′-O-phosphorylated at the ribonucleoside at the

junction point near the 3′-side of the anticodon region.³

Since it is considered that the mature function of tRNA

would not be completely acquired unless the 2′-phosphate

group was removed,⁴the influence of the 2′-phosphate

group on the structure of tRNA was expected to be

connected with the recognition of aminoacyl-tRNA syn-

thetases (ARS). To date, a number of pre-tRNAs having

an intron have been found in yeast pre-tRNAs.⁵The

With respect to another biochemical property of 2′-

phosphorylated RNA species, Szostak and co-workers

reported that when the in vitro selection from a random

RNA pool and mutagenic PCR was performed to obtain

evolved ribozymes with polynucleotide 5′-O-thiophospho-

rylation activity, some of the products selected were

eventually phosphorylated at the internal 2′-position.⁸

This result strongly suggested the possibility that RNAs

involving a 2′-phosphorylated ribonucleoside exist in the

process of molecular evolution of nucleic acids. Interest-

ingly, they also disclosed that reverse transcription using

an RNA oligomer having a 2′-O-thiophosphoryl group as

(1) Konarska, M.; Filipowicz, W.; Domdey, H.; Gross, H. J . Nature

1981, 293, 112.

(2) Konarska, M.; Filipowicz; Gross, H. J . Proc. Natl. Acad. Sci.

U.S.A. 1982, 79, 1474.

(3) Greer, C. L.; Peebles, C. L.; Gegenheimer, P.; Abelson, J . Cell

1983, 32, 537.

(4) Zillmann, M.; Gorovsky, M. A.; Phizicky, E. M. J . Biol. Chem.

1992, 267, 10289-10294.

(5) Culbertson, M. R.; Winey, M. Yeast 1989, 5, 405-427.

(6) (a) Cech, T. R. Cell 1983, 34, 713-716. (b) Filipowicz, W.;

Shatkin, A. J . Cell 1983, 32, 547-557. (c) Mattoccia, E.; Baldi, I. M.;

Gandini-Attardi, D.; Ciafre, S.; Tocchini-Valentini, G. M. Cell 1988,

55, 731-738. (d) Rauhut, R.; Green, P. R.; Abelson, J . J . Biol. Chem.

1990, 30, 18180-18184.

(7) Culver, G. M.; McCraith, S. M.; Zillmann, M.; Kierzek, R.;

Michaud, M.; LaReau, R. D.; Turner, D. H.; Phizicky, E. M. Science

1993, 261, 206.

(8) Lorsch, J . R.; Szostak, J . W. Nature 1994, 371, 31-36.

Published on Web 10/07/2000

7480 J . Org. Chem., Vol. 65, No. 22, 2000

Tsuruoka et al.

Sch em e 1

Sch em e 2

the templates caused a pause in AMV and MMLV H^-

reverse trascriptases, while these ultimately read through

this point.⁹

nucleoside building blocks and their application to the

synthesis of oligonucleotides containing a 2′-O-phospho-

rylated ribonucleotide. Finally, to study the structural

effect of only one 2′-O-phosphate group on recognition of

ARSs, thermodynamic and conformational properties of

these modified oligomers were examined.

In consideration of these facts found in tRNA splicing

and reverse transcription, studies on the 3D-structure

of oligonucleotides containing a 2′-phosphorylated ribo-

nucleoside could provide valuable information for molec-

ular recognition of ARSs and HIV-reverse transcriptases

and structure-activity relationships. In our recent stud-

ies, an effective method for the polymer-supported syn-

thesis of 2′-O-phosphorylated RNAs has been established

to give oligouridylates (2-6mers) phosphorylated at the

2′-positions of all the uridines except the 3′-terminal one

by use of the phosphoramidite approach.¹⁰However, this

polymer support synthesis, which involved removal of the

2-cyano-1,1-dimethylethyl group used as a protecting

group for the thiophosphate group and the successive

conversion of the unprotected thiophosphate group into

a phosphate group, was accompanied with unexpected

side reactions and therefore limited to short oligonucle-

otides. Later, this polymer-supported synthesis was

improved by use of KI₃as the reagent for the S-O

conversion to give oligodeoxynucleotides incorporating a

2′-phosphorylated uridine as briefly reported in our

previous communication.¹¹Our recent NMR and CD

studies revealed that the 5′-upstream uridine ribose

residue of U(2′-p)pU has a C2′-endo conformation similar

to that of B-DNA rather than A-DNA.¹¹It was also shown

by T_mexperiments and MD simulation that, when

incorporated into a tridecadeoxyuridylate, the 2′-O-

phosphorylated uridine behaved as a rigid C2′-endo

nucleoside.

Resu lts a n d Discu ssion

Syn th esis of F ou r Kin d s of Ribon u cleosid e 3′-

P h osph or am idites Havin g th e 2′-O-BCMETP Gr ou p.

Previously, we first reported the 2′-O-phosphorylation of

ribonucleosides by the use of the bis(tert -butoxy)phos-

phoryl (BTBP) group masked by sterically hindered

protecting groups.¹²

However, removal of the two tert-butyl groups from the

BTBP group required rather drastic conditions (20%

trifluoroacetic acid in acetic acid at room temperature

for 3-5 h) which were apparently unsuitable for the

synthesis of longer oligonucleotides. To overcome this

problem, we considered the use of a bis(2-cyano-1,1-

dimethylethoxy)phosphoryl (BCMEP) group protected by

the 2-cyano-1,1-dimethylethyl (CME) groups (Scheme 2),

which not only was sterically hindered but also could be

simultaneously eliminated by treatment with a combined

reagent of DBU and bis(trimethylsilyl)acetamide (BSA).^10,13

(9) Lorsch, J . R.; Bartel, D. P.; Szostak, J . W. Nucleic Acids Res.

1995, 23, 15.

(10) Sekine, M.; Tsuruoka, H.; Iimura, S.; Kusuoku, H.; Wada, T.;

Furusawa, K. J . Org. Chem. 1996, 61, 4087-4100.

(11) Tsuruoka, H.; Shohda, K.; Wada, T.; Sekine, M. Tetrahedron

Lett. 1996, 37, 6741-6744.

(12) Sekine, M.; Iimura, S.; Furusawa, K. J . Org. Chem. 1993, 58,

3204-3208.

(13) Sekine, M.; Tsuruoka, H.; Iimura, S.; Wada, T. Nat. Prod. Lett.

1994, 5, 41-46.

In this paper, we report a general procedure for the

synthesis of four kinds of 2′-O-phosphorylated ribo-

Oligonucleotides with 2′-O-Phosphorylated Ribonucleotides

Sch em e 3

J . Org. Chem., Vol. 65, No. 22, 2000 7481

However, when the di-tert-butylsilanediyl (DTBS) group

was removed from the 3′,5′-positions after introduction

of the BCMEP group into the 2′-hydroxyl group of a

uridine derivative, 2′-3′ phosphoryl migration slightly

took place to give a mixture of the desired 2′-O-phospho-

rylated product and its 3′-regioisomer. It seemed that

such isomerization occurred because of the electron-

withdrawing effect of the two cyano groups involved in

the CME groups, although four covalent bonds exist

between the phosphorus and cyano groups. This result

led us to examine the bis(2-cyano-1,1-dimethylethoxy)-

thiophosphoryl (BCMETP) group, having the lesser elec-

tronegative sulfur atom instead of oxygen in the BCMEP

group. Consequently, we found that introduction of the

BCMETP group resulted in complete suppression of the

2′-3′ phosphoryl migration.

diethylaminophosphine (CMEAP)¹⁰was required to ob-

tain a satisfactory result in the 2′-O-phosphitylation in

CH₂Cl₂. The successive in situ sulfurization of the phos-

phitylated product in pyridine-CS₂for 30 min gave the

2′-thiophosphorylated derivative 3e quantitatively ac-

cording to the TLC analysis. Desilylation of the DTBS

group was successively carried out by treatment with

pyridinium hydrogenfluoride to produce a 3′,5′ unpro-

tected 2′-O-thiophosphorylated cytidine derivative 4e in

77% yield from 2e. The detailed analysis of ¹H, ¹³C, and

³¹P NMR of this product revealed that it was obtained

free from the 3′-regioisomer as reported in the case of

the 2′-O-thiophosphorylated uridine derivative 4a . After

the usual 5′-dimethoxytritylation of 4e, the 3′-hydroxyl

group of the resulting product 5e could subsequently be

phosphitylated cleanly. However, the corresponding phos-

phoramidite 6e was considerably unstable during silica

gel column chromatography. Attempts of other purifica-

tion methods, such as aluminum oxide and gel-filtration

column chromatography, and precipitation by use of

various solvent systems, failed, and consequently 6e

could not be obtained in sufficiently pure form. Therefore,

the phosphoromorpholidite 6f was prepared as a more

stable derivative¹⁵instead of 6e. Compound 6f could be

synthesized purely by use of O(CH₂CH₂)₂NP(OCE)Cl in

the presence of Et₃N without the 2′-3′ phosphoryl

migration. The condensation using 6f was attempted with

the 5′-hydroxyl of a uridine derivative bound to amino-

propyl-CPG 12b. However, the average coupling yield did

In the present study, our preliminary method of 2′-O-

phosphorylation using uridine derivatives was applied

to preparation of three other kinds of nucleoside 3′-

phosphoramidites building blocks having the 2′-O-BC-

METP group.

4-N-Benzoylated cytidine (1e) was silylated by treat-

ment with di(tert-butyl)dichlorosilane in the presence of

silver nitrate (Scheme 3). However, silylation of 1e was

slightly complicated due to the remaining reactivity of

the 3-nitrogen atom despite introduction of the benzoyl

group into the nucleobase. Furusawa reported that when

di(tert-butyl)silyl bis(trifluoromethanesulfonate) was em-

ployed in the presence of silver nitrate that could favor-

ably coordinate to the 3-nitrogen, the desired product was

obtained quantitatively.¹⁴In fact, 2e was synthesized in

85% yield. A characteristic feature of 2e was its low

solubility in various solvents such as CH₂Cl₂, THF, and

CH₃CN. Because of this inherent problem, an excess

amount (2.5 equiv) of bis(2-cyano-1,1-dimethylethoxy)-

(14) (a) Furusawa, K.; Ueno, K.; Katsura, T.; Chem. Lett. 1990, 97.

(b) Furusawa, K.; Katsura, T. Tetradedron Lett. 1985, 26, 887. (c)

Furusawa, K. Chem. Lett. 1989, 509 (d) Frusawa, K. Chem. Express

1991, 6, 763.

(15) Sinha, N. D.; Biernat, J .; McManut, J .; Ko¨ster, H. Nucleic Acids

Res. 1984, 12, 4539.

7482 J . Org. Chem., Vol. 65, No. 22, 2000

Tsuruoka et al.

not exceed 85% even when a slightly higher concentration

(0.15 M) of 6f was employed. It is desirable that conden-

sation in the solid-phase synthesis is performed in more

than 95% yield. Since the 2′-thiophosphorylated uridine

derivative (6a ) reported previously was stable enough to

isolate and difference between the 3′-phosphoramidites

6a and 6e was only in the structure of the base moiety,

the benzoyl group as a 4-N-protecting group of cytidine

was replaced by the isobutyryl group with the hope that

this more sterically hindered aliphatic protecting group

might stabilize the 3′-phosphoramidite function. Silyla-

tion of 4-N-isobutyrylcytidine (1c) followed by the sub-

sequent 2′-O-thiophosphorylation, desilylation, and 5′-

O-dimethoxytritylation gave the 5′-masked product 5c

in good yield. Introduction of the phosphoramidite func-

tion into the 3′-hydroxyl of 5c using iPr₂NP(OCE)Cl and

Et₃N gave the building unit 6c. It turned out that 6c did

not decompose during silica gel column chromatography.

It was, however, disclosed that this phosphitylation

accompanied the 2′-3′ migration of the 2′-thiophosphoryl

group so that a desired product was contaminated with

the 3′-thiophosphorylated cytidine 2′-O-phosphoramidite

7c, which could not be separated by any purification

methods as shown in Figure 1-A₁. It was likely that this

migration was ascribed to the basicity of Et₃N (pK_a; 10.7).

Therefore, 2,4,6-collidine (pK_a; 7.4) as a weaker base,

which Usman et al. used in the synthesis of 2′-O-silylated

ribonucleoside 3′-phosphoramidites,¹⁶was used to avoid

such a base-induced phosphoryl migration. Consequently,

migration was completely suppressed to give a sole

product 6c in 72% yield as evidenced by Figure 1-A₂.

Since the nucleobase structure of ribonucleosides con-

siderably affected the stability of the P-N bond at the

3′-position as well as 2′-3′ phosphoryl migration, these

major effects might be explained in terms of the electron-

withdrawing effect of the base moiety. To investigate the

inductive effect of cytosine derivatives, partial charges

of 1-methyl-carbon in 4-N-benzoyl-N¹-methylcytosine and

4-N-isobutyryl-N¹-methylcytosine as model compounds

were estimated by ab initio calculation using the HF/6-

31G* basis set. However, there was almost no difference

between them (data not shown). Accordingly, the induc-

tive effect would not be an essential factor.

Next, to prepare 2′-thiophosphorylated adenosine and

guanosine 3′-phosphoramidite 6b and 6d , 6-N-benzoyl-

adenosine 1b and 6-O-cyanoethyl-2-N-phenylacetylgua-

nosine 1d were treated with tBu₂SiCl₂and AgNO₃to give

the 3′,5′-O-protected derivatives 2b and 2d , respectively,

in good yields. Moreover, 2b and 2d were converted to

the corresponding 5′-O-dimethoxytritylated compounds

5b and 5d in 78 and 83% yields, respectively, via a four-

step reaction. No 2′-3′ phosphoryl migration could be

observed during these reactions. However, when the

phosphoramidite function was introduced into the 3′-

hydroxyl of 5b and 5d using iPr₂NP(OCE)Cl and Et₃N,

2′-3′ phosphoryl migration of 2′-thiophosphoryl group

was slightly caused like that of the N-isobutyrylated

cytidine derivative to simultaneously give the phosphi-

tylated regioisomers 7b and 7d . The use of 2,4,6-collidine

as the acid-scavenger allowed the complete suppression

of the phosphoryl migration during the conversion to the

3′-phosphoramidite. Therefore, this base is the most

appropriate reagent in the present approach.

Sta bility of th e 2′-O-P h osp h or yla ted Ribon u cleo-

sid e Der iva tives 4a -e in Va r iou s Solven ts. To evalu-

ate the utility of the 2′-BCMETP group, the stability of

four nucleotides 4a -e were systematically examined.

Compounds 4a -e were kept in pyridine, 80% acetic

acid, and methanol at 25 °C for 50 days and distribution

of the unchanged starting material, and the decomposi-

tion products in these solutions were estimated by ³¹P

NMR to monitor the phosphoryl migration and degrada-

tion. These results are shown in Figure 2. Only when the

3′-resioisomers could be spectroscopically assigned, their

amount was measured

As shown in Figure 2-A, it was found that all of the

nucleoside derivatives 4a -e in pyridine underwent deg-

radation of the 2′-BCMETP group rather than the 2′-3′

phosphoryl migration. The guanosine derivative 4d was

exceptionally the most stable compared with the other

derivatives 4a -c and 4e. The usual conditions (rt, 4 h

in pyridine) prescribed for the 5′-O-dimethoxytritylation

gave the corresponding 3′-regioisomers to a degree of less

than 0.1% in all cases. Interestingly, the 4-N-isobutyryl-

cytidine derivative 4c was apparently more stable and

more resistant to migration than the 4-N-benzoylcytidine

derivative 4e. This result was in agreement with those

observed in conversion of 5c and 5e to 6c and 6e,

respectively. In methanol as a protic solvent, most of

4a -e remained unchanged even after a few days as

shown in Figure 2-B. In contrast to the above result,

however, the guanosine derivative 4d was less stable

than 4a -c and 4e upon standing for prolonged periods.

In 80% acetic acid (Figure 2-C), the uridine and adenosine

derivatives 4a and 4b were more resistant to migration

and degradation than the other nucleoside derivatives.

In 4a and 4b, 2′-3′ thiophosphoryl migration mainly

occurred so that the 2′- and 3′-O-thiophosphorylated

isomers remained to a degree of 90% even after 50 days.

The guanosine derivative 4d and the cytidine derivative

4c and 4e were relatively unstable, and only less than

20% of these compounds remained after 50 days.

In consideration of these stabilities and actual reaction

conditions such as above, the present results suggested

that 4a -d were useful intermediates to meet the stability

requirements during the synthesis of the building blocks

6a -d and that protecting groups of the base moieties

considerably affect the stability of the 2′-O-thiophos-

phoryl group.

Im p r oved P olym er -Su p p or ted Syn th esis of Oligo-

n u cleotides In cor por atin g 2′-O-P h osph or ylated an d

2′-O-Th iop h osp h or yla ted Ribon u cleosid es. It was

previously reported by us that oligouridylates (2-6mers),

phosphorylated at all internal 2′-positions, were prepared

on polymer supports.¹⁰In the present study, we first

attempted to apply this previous original method to

incorporation of a 2′-O-phosphorylated uridine into oligo-

deoxyuridylates 10mer and 13mer only at one position

(Scheme 4).

With respect to polymer-supported synthesis of the

desired oligomers, the classical conditions used in the

phosphoramidite method were basically used, but in the

case of condensation using 6a the reaction should be

performed at a slightly high concentration (0.15 M) of

the phosphoramidite reagent. If not, the average coupling

yield was insufficient (ca. 85%). To synthesize deoxy-

uridylate 10mer dU₄U(2′-p)dU₅13, after the chain elon-

gation, the 2-cyano-1,1-dimethylethyl (CME) and 2-cyano-

ethyl (CE) groups were removed from the fully protected

(16) Scaringe, S. A.; Francklyn, C.; Usman, N. Nucleic Acids Res.

1990, 18, 5433-5441.

F igu r e 1. 2′-3′ Thiophosphoryl migration of the BCMETP group of 5c and 5d upon 3′-phosphitylation. A₁: The ³¹P NMR spectrum of the product obtained by phosphitylation of

5c in the presence of triethylamine. A₂: The ³¹P NMR spectrum of the product obtained in phosphitylation of 5c in the presence of 2,4,6-collidine. B₁: The ³¹P NMR spectrum of the

product obtained in phosphitylation of 5d in the presence of triethylamine. B₂: The ³¹P NMR spectrum of the product obtained in phosphitylation of 5d in the presence of 2,4,6-

collidine.

7484 J . Org. Chem., Vol. 65, No. 22, 2000

Tsuruoka et al.

F igu r e 2. Stability of 2′-thiophosphorylated ribonucleoside derivatives 4a -e in various solvents at 25 °C: solid dot, 4a ; open

dot, 4b; open square, 4c; solid triangle, 4d ; solid squere, 4e; solid line, the remaining amount of the starting material 4a -d ;

dotted line, the amount of 3′-isomerized product; A: Each sample (0.06 mmol) was dissolved in Py-Py-d₅(9:1, v/v, 0.6 mL). B:

Each sample (0.06 mmol) was dissolved in CD₃OD (0.6 mL). C: Each sample (0.06 mmol) was dissolved in acetic acid-D₂O (4:1,

v/v, 0.6 mL).

Sch em e 4

oligomer bound to the polymer support by use of DBU in

the presence of N,O-bis(trimethylsilyl)acetamide (BSA)¹⁰

without release of the oligomer from the CPG. Oxidative

conversion of the unprotected 2′-O-phosphorothioate

monoester function to the corresponding phosphate was

successively carried out by treatment with iodine, which

was employed in the synthesis of U(2′-p)pU in the

previous paper.¹⁰Finally, the succinate linker was cleaved

by treatment with concentrated NH₃, and the crude

material was analyzed by ion exchange HPLC.

carefully checked. As a consequence, it was disclosed that

the random 5-iodination of the uracil base with iodine

occurred. About 30% of 2′-deoxyuridine was iodinated at

the 5-position by 10 equiv of iodine after 48 h. Therefore,

in the general polymer-supported synthesis, iodine has

to be used carefully, especially if its reaction time is

prolonged. In our previous paper¹⁰with regard to the

synthesis of U(2′-p)pU, however, such an electrophilic

substitution on the pyrimidine residue was not observed

at all. It was assumed that this previous better result

could be explained in terms of the electron-withdrawing

effect by the N³-protecting group which suppressed the

side reaction. Accordingly, the N³-benzoylated 2′-deoxy-

uridine phosphoramidite 11 was separately prepared,

As seen in Figure 3-A₁, a large broad peak at 23-33

min appeared, suggesting a cluster of many compounds

having similar structures were formed. To clarify what

happened during a series of treatments, each step was

Oligonucleotides with 2′-O-Phosphorylated Ribonucleotides

J . Org. Chem., Vol. 65, No. 22, 2000 7485

F igu r e 3. The ion-exchange HPLC profiles of uridylate 13mer containing a 2′-phosphoryl group. A₁: A crude material of 13

obtained after iodine oxidation followed by concd NH₃treatment. A₂: A crude material of 16 obtained after KI₃-oxidation followed

by concd NH₃treatment. A₃: A purified material of 16. A₄: A product obtained in dephosphorylation of 16 by treatment with

alkaline phosphatase. (B₁) A purified material of 17. (B₂) A product obtained in thermolysis of 17 at pH 7.0, 90 °C for 20 min.

Sch em e 5

and the synthesis of oligonucleotide 16 was performed

in the same manner. However, the HPLC profile taken

after treatment with concentrated NH₃was as compli-

cated as before in spite of the use of 11. Since the

oxidative S-O conversion by iodine not only took long

(t_comp) 48 h) but also was accompanied 5-iodination,

improvement of this reaction would require the use of

an alternative to iodine.

Conversion of 30 as a model compound to 32 by other

oxidants was examined (Scheme 5, Table 1). After

removal of the two CME groups from the thymidine

derivative 29 thiophosphorylated at the 3′-position and

successive hydrolysis of the silyl ester, the oxidant was

added in situ, and the time-dependent product distribu-

tion was pursued by ³¹P NMR (see Supporting Informa-

tion). Most of the reagents were found to promote

formation of a byproduct 31 (δp: ca. 15 ppm) having the

disulfide bond rather than desulfurization, but only

iodine and KI₃could gradually convert the once-formed

31 to the desired product 32. (See Supporting Informa-

tion) Although the desulfurization by mCPBA was too

rapid to observe any intermediates, a small amount of

more complicated. Among them, the triiodide reagent KI₃,

which was reported by Nussbaum et al.¹⁸in activation

of the P-SEt bond of phosphorothioate derivatives, gave

exclusively the desired product 32 during a relatively

short period (t_comp) 6 h), and more importantly it was

pyrophosphate derivative (δ_P: ca. -10 ppm) was formed;

therefore, this reaction is unsuitable for application to

solid-phase synthesis. Reagents tBuOOH, H₂O₂, and K₃-

(17) Eckstein, F. J . Am. Chem. Soc. 1966, 88, 4292.

(18) Poonian, M. S.; Nowoswiat, E. F.; Nussbaum, A. L. J . Am.

17

Fe(CN)₆readily accumulated a large amount of a

disulfide derivative, and the reaction mixture became

Chem. Soc. 1972, 94, 3992-3997.

7486 J . Org. Chem., Vol. 65, No. 22, 2000

Tsuruoka et al.

Ta ble 1. Oxid a tive Desu lfu r iza tion of 30 to 32

(2′-p)dU₃U(2′-p)dU₃U(2′-p)dU₂18 and [U(2′-p)]₁₂U 19

bearing a higher number of 2′-O-phosphorylated nucleo-

sides. However, HPLC analysis suggested that these

reactions were complicated by intra- or intermolecular

formation of disulfide bonds to give dimerization-, trim-

erization-, and larger products (see Supporting Informa-

tion). These S-S bonds in relatively long oligonucleotides

were difficult to convert to the corresponding phosphate

because of steric hindrance.

oxidant

(10 equiv)

a

t_comp

l₂

Kl₃

mCPBA

tBuOOH

H₂O₂

48 h

6 h

1 h^b

c

-

c

K₃Fe(CN)₆

-

a

t_comprefers to the time when the reaction has completely

Furthermore, a tridecaeoxyuridylate 17 bearing a 2′-

O-thiophosphorylated uridine was also synthesized by a

modified procedure without the KI₃treatment and char-

acterized by the facile 2′-dethiophosphorylation using

thermolysis at pH 7.0 and 90 °C, which was recently

reported by us,¹⁹to give dU₆UdU₆in nearly quantitative

yield without appreciable internucleotidic bond cleavage.

Interestingly, this 2′-O-thiophosphate group was elimi-

nated in only 20 min, and the first-order rate constant

of this hydrolysis was 1.9 × 10^-3s^-1, which was greater

than any other values such as that of U(2′-ps)pU (1.4 ×

10^-3s^-1, t_comp) 1 h).

The synthetic method described above was applied to

incorporation of a 2′-O-phosphorylated nucleoside into

tridecauridylates. These modified oligoribonucleotides are

a simple motif of the ligation site of tRNA splicing

intermediates. To prepare the 13mers 21 and 22, com-

pound 6a and the normal uridine phosphoramidite, in

which the 2′-hydroxyl was masked by the tert-butyldi-

methylsilyl group, were employed for chain elongation.

In these syntheses, an additional treatment with TBAF

at the last step was carried out. Finally, the crude

material was purified by HPLC to give the oligomers 21

and 22, having a 2′-O-phosphorylated uridine at the same

positions as 15 and 16, respectively.

b

finished. A small amount of pyrophosphate was formed. ^cA

considerable amount of S-S bond was formed.

Ta ble 2. Isola ted Yield s a n d T_mVa lu es of Oligom er s

Con ta in in g th e 2′-O-P h osp h or yl Gr ou p s

sequence

yield (%) T_m(°C) ∆T_m(°C)

d(UUUUUUUUUU) (13)

d(UUUUUUUUUUUUU) (14)

d(UUUUUUUUUUUUU) (15)

d(UUUUUUUUUUUUU) (16)

d(UUUUUUUUUUUUU (17)

d(UUUUUUUUUUUUU) (18)

UUUUUUUUUUUUU (19)

UUUUUUUUUUUUU (20)

UUUUUUUUUUUUU (21)

UUUUUUUUUUUUU (22)

d(CGCGAAUUCGCG) (23)

d(CGCGAAUUCGCG) (24)

d(CGCGAAUUCGCG) (25)

d(CGCGAAUUCGCG) (26)

d(CGCGAAUUCGCG) (27)

d(CGCGAAUUCGCG) (28)

-

20

28

19

-

30.9^a

28.8^a

26.2^a

25.5^a

-

-2.1

-4.7

-5.4

-

7

6

35.1^a

27.8^a

23.6^a

55.7^b

53.3^b

55.7^b

41.2^b

40.5^b

51.3^b

-7.3

-11.5

-

16

29

22

20

27

-2.4

0

-14.5

-15.2

-4.4

a

In the T_mmeasurement of oligomers 14-22, dA₁₃or A₁₃was

used as a complementary strand. Conditions: 10 mM sodium

b

phosphate, 0.1 mM EDTA, 1

M

NaCl, pH 7.0. In the T_m

measurement of self-complementary oligomers 23-28. Conditions:

10 mM sodium phosphate, 0.1 mM EDTA, 150 mM NaCl, pH 7.0.

Bold and underlined letters in sequences refer to the 2′-phospho-

rylated and 2′-thiophosphorylated ribonucleotide, respectively. U

refers to 2′-O-thiophosphorylated uridine.

As the results, we were able to establish the solid-

phase synthesis for DNA and RNA oligomers with a 2′-

O-phosphoryl group using the phosphoramidites 6a -d .

found to be inert against the electrophilic substitution

on the uracil moiety. Therefore, we chose to apply this

KI₃-oxidation procedure to the preparation of oligonucleo-

tides involving a 2′-O-phosphorylated ribonucleoside.

To obtain the 13mer 16 incorporating a 2′-O-phos-

phorylated uridine having no base-protecting group, the

usual protocol involving chain elongation, removal of the

CME groups, and oxidation by use of KI₃was performed.

As shown in the HPLC profile after treatment with

concentrated NH₃(Figure 3-A₂), a main peak correspond-

ing to dU₆U(2′-p)dU₆appeared without the 5-iodination.

After purification by HPLC, this main product was

isolated and characterized by treatment with alkaline

phosphatase. As expected, this enzymatic treatment gave

the 2′-dephosphorylated product dU₆UdU₆(Figure 3-A₃

and 3-A₄), which was further characterized by enzymatic

assay using snake venom phosphodiesterase. In a similar

way, a tridecadeoxyuridylates 15 incorporating a 2′-O-

phosphorylated uridine at another site was synthesized.

Furthermore, self-complementary dodecadeoxynucleo-

tides 24-28 with the Drew-Dickerson sequence contain-

ing the four common nucleobases could also be prepared

in good yields using phosphoramidites 6a -d (Table 2).

Therefore, compounds 6a -d have proved to be generally

useful building blocks capable of incorporating a 2′-O-

phosphorylated ribonucleoside (The HPLC profiles of the

crude materials obtained after cleavage of the succinate

linker are summarized in Supporting Information). More-

over, we attempted to synthesize oligonucleotides dU₂U-

Du p lex Sta bility of Oligon u cleotid es Con ta in in g

2′-O-P h osp h or yla ted Ribon u cleosid e. The UV-melt-

ing curves for duplexes between these oligomers and

complementary strands were measured to study the

effect of the 2′-O-phosphoryl group on the duplex stabil-

ity.

These T_mmeasurements of the duplexes formed by

oligouridylates and oligoadenylates were carried out by

mixing them in a 1:1 ratio. As shown in the figure of

Supporting Information, the melting curves of both

modified and unmodified nucleotide duplexes clearly

suggested two states in all cases. Therefore, we concluded

that these oligonucleotides formed duplexes under the

condition described in the present paper. The T_mvalues

obtained are summarized in Table 2.

In the case of DNAs hybridized with dA₁₃as comple-

mentary strand, the T_mvalue of the duplex 15/dA₁₃(28.8

°C) was lower by 2.1 °C than that of unmodified dU₁₃

14/dA₁₃(30.9 °C). This difference was rather smaller than

expected since the electrostatic repulsion due to negative

charge of the 2′-O-phosphomonoester was estimated to

be more detrimental for duplex formation. We previously

reported the MD simulation of the duplex of dA₇/dU₃U-

(2′-p)dU₃, suggesting that 2′-O-phosphorylated uridine

U(2′-p) of dU₃U(2′-p)dU₃has a rigid C2′-endo conforma-

(19) Tsuruoka, H.; Shohda, K.; Wada, T.; Sekine, M. J . Org. Chem.

1997, 62, 2813-2822.

Oligonucleotides with 2′-O-Phosphorylated Ribonucleotides

J . Org. Chem., Vol. 65, No. 22, 2000 7487

The duplexes formed by the self-complementary DNAs

24-28 involved a 2′-O-phosphorylated nucleoside in each

strand. Therefore, it was anticipated that the stability

of these duplexes should be influenced more considerably

than that of oligomers 15-17/dA₁₃. Indeed, most of the

T_mvalues of these modified self-complimentary duplexes

decreased more significantly than that of the unmodified

duplex 23/23. Particularly, the duplex 27/27 was ex-

tremely destabilized by 15.2 °C. To our surprise, the T_m

value of the duplex 25/25 having a 2′-O-phosphorylated

guanosine was the same (55.7 °C) as that of the control

23/23. In nucleic acid chemistry, the phosphate group has

been generally thought as an electrostatically disadvan-

tageous function as far as stability of duplexes is con-

cerned. The present study, however, indicates the pos-

sibility that this is not always the case. In 28/28, the

duplex was not destabilized only by 4.4 °C compared with

that of the unmodified duplex.

Con for m a tion a l An a lysis by Con str a in ed Molec-

u la r Dyn a m ics Sim u la tion . The 2′-O-phosphate group

inhibits various tRNA-processing enzymes from recogniz-

ing tRNA intermediates.⁴We speculated that consider-

able destabilization of RNA duplexes containing a 2′-O-

phosphorylated nucleoside as described above was ascribed

to distortion caused by unfitting of the 2′-O-phosphoryl-

ated nucleoside for the A-type duplex. First, the CD

spectra and native 20% PAGE of single- and double-

stranded oligonucleotides containing a 2′-O-phosphoryl-

ated nucleoside were analyzed. However, they did not

give any helpful information with respect to the 3D-

structure of the modified duplexes (see the Supporting

Information).

To study the structural effects of the 2′-O-phosphoryl-

ated nucleoside on the duplex in more detail, computa-

tional molecular dynamics (MD) simulations were carried

out for 1 ns by using the duplexes containing a 2′-O-

phosphorylated nucleoside or not (Figure 4).

Indeed, dU₃U(2′-p)dU₃/dA₇34 and dU₇/dA₇33 as DNA

duplexes and U₃U(2′-p)U₃/A₇36 and U₇/A₇35 as RNA

duplexes were simulated. Figure 5 shows the MD trajec-

tory of distinctive torsion angles (the ꢀ and ú angles

defined in nucleic acid chemistry²⁰) and phase angle (P),

of the ribose in the fourth uridine residue which is or is

not phosphorylated at the 2′-position.

With respect to the DNA duplex 34, the structural

parameters of this duplex did not change significantly

after simulation. In detail, the phase angle (P) of the

unmodified fourth deoxyuridine in 33 was relatively

flexible and frequently changed from C2′-endo (137° < P

< 194°) to C3′-endo conformer (-1° < P < 34°) and vice

versa. On the contrary, the C2′-O-phosphorylated sugar

in 34 moved sluggishly only within the range of the C2′-

endo conformer during the 1 ns-MD simulation. (Scheme

6)

This result was in agreement with the fact that the

ribose residue of 2′-O-phosphorylated dinucleotides was

found to have a rigid C2′-endo conformation as evidenced

by NMR analysis.¹¹Therefore, the 2′-O-phosphorylated

nucleoside could entropically stabilize the B-DNA duplex.

As a unique structure, it was found that the ꢀ angle of

the 2′-O-phosphorylated uridine in 34 was sometimes

transferred to g^-conformation although that of the

unmodified fourth uridine has trans conformation during

F igu r e 4. Snapshot of DNA duplex having a 2′-phosphoryl-

ated ribonucleoside for 1 ns-MD simulation and sequences of

DNA and RNA duplex used in this simulation. Bold letters

refer to 2′-phosphorylated ribonucleoside.

tion.¹¹The present result also suggests that conformation

of the 2′-O-phosphorylated uridine was more favorable

for the B-DNA duplex than normal deoxyribonucleosides.

On the other hand, the T_mexperiment of 16/dA₁₃(26.2

°C) suggested that the duplex stability decreased less

significantly when a 2′-O-phosphoryl group was intro-

duced at a position closer to the center of the strand. The

duplex of 17/dA₁₃was destabilized more significantly by

5.4 °C than that of 14. This may be explained in terms

of more unfavorable steric disruption by the bigger-sized

sulfur atom as well as stronger electrostatic repulsion

due to the 2′-O-thiophosphate dianion species. It is known

that the pK_avalues of monoalkyl esters of thiophosphoric

acids are generally 0.5 unit lower than those of the

corresponding monoalkyl esters of phosphoric acids so

that the electrostatic charge distribution of the 2′-O-

thiophosphate group is greater than that of the 2′-

phosphate at pH 7.0.

On the contrary, the T_mvalues of RNA duplexes 21/

A₁₃and 22/A₁₃were markedly decreased by the existence

of the 2′-O-phosphate group compared with those of DNA

duplexes 15/dA₁₃and 16/dA₁₃with identical sequences.

The reason for this destabilization might be ascribed to

the essential incompatibility between the A-type RNA

backbone structure and the B-type favored 2′-O-phos-

phorylated uridine as well as electrostatic repulsion.

Accordingly, incorporation of 2′-O-phosphorylated uridine

caused conformational change of the A-type duplex

structure to a more disordered one.

(20) Saenger, W. Principles of Nucleic Acid Structure; Springer-

Verlag Inc.: New York, 1984.

F igu r e 5. The trajectory of phase angles (P) and tortion angles (ꢀ and ú) of the fourth uridines, which were phosphorylated at the 2′-position or not, in the duplexes 33, 34, 35, and

36 in molecular dynamics simulation at 300 K for 1 ns.

Oligonucleotides with 2′-O-Phosphorylated Ribonucleotides

J . Org. Chem., Vol. 65, No. 22, 2000 7489

Sch em e 6

nantly the C3′-endo conformation and stabilize the A-type

double helix when incorporated into DNA strands and

hybridized with the target mRNA.²¹

It is known that the population of the C3′-endo

conformer is in proportion to the strength of electro-

negativity of a 2′-substituent.²⁰Chattopadhyaya and co-

workers have recently reported that the electronegativity

of a 3′-substituent has a close connection with the ∆H

value obtained by the equilibrium between the C2′-endo

and C3′-endo conformers, and concluded that this es-

sential factor is explained in terms of the gauche effect

between the 3′-substituent and the oxygen of the sugar

ring.²²A similar discussion was deduced in the case of

2′-substituted nucleosides.

this simulation like the ordinary case.²⁰However, the ú

angle of the 2′-O-phosphorylated uridine simultaneously

transferred from normal g^-conformation to trans so as

to cancel the distortion generated at the ꢀ angle. In

consideration of these results, since the ribose residue

of the 2′-O-phosphorylated uridine incorporated into 34

exists rigidly in a C2′-endo conformation, it seemed that

the total stability of 34 did not decrease despite the

electrostatic repulsion and change in the ꢀ and the ú

angle.

We previously showed that 2′-O-phosphorylated nucleo-

sides interestingly prefer the C2′-endo conformer to the

C3′-endo though the monoester-type phosphate group is

generally regarded as a weak electron-withdrawing

group. In pre-tRNA splicing products, the 2′-O-phos-

phorylated nucleosides are mostly located at the border

between the tRNA anticodon stem and loop, which

involves the typical A-type single strand. Therefore, it is

interesting to estimate how the 2′-phosphate group

affects the local conformation in this region. In the

present study we could show that, if a 2′-phosphate group

exists in an RNA duplex, the duplex stability is signifi-

cantly destabilized due to incompatibility between the

unique DNA-like property of the 2′-O-phosphorylated

nucleoside and the conservative A-type RNA strand.

These results apparently indicate that a 2′-O-phos-

phorylated nucleoside of the pre-tRNA splicing product

causes the distortion of the strictly arranged A-type

strand in the anticodon loop region.

During simulation of RNA duplexes U₇/A₇35 and U₃U-

(2′-p)U₃/A₇36, having the same sequence described

above, the fourth 2′-O-phosphorylated uridine first seemed

to have a rigid C2′-endo conformation similar to that of

the typical DNA duplex. However, this ribonucleoside

was unexpectedly found to behave as a C3′-endo con-

former. Its ꢀ and ú angles also gave the same conforma-

tion as those of the normal ribonucleosides in the A-type

RNA duplex, which has trans and g^-conformations,

respectively. In this 1 ns-MD simulation, it was irrelevant

whether the used initial structure of the fourth 2′-O-

phosphorylated nucleoside was C2′- or C3′-endo. Since

A-RNA duplexes are structurally rigid, tight, and con-

servative, as is well-known,²⁰probably the 2′-O-phos-

phorylated uridine could not come to a C2′-endo confor-

mation because of stronger pressure of the neighbor

A-type circumstance regardless of its propensity to the

C2′-endo conformation so that the duplex was extremely

destabilized, especially at the modified nucleoside point.

Consequently, the DNA duplex containing a 2′-O-phos-

phorylated nucleoside is stable due to rigid C2′-endo

puckering despite the existence of difference in the two

torsional angles compared with the normal ribonucleo-

side, while the RNA duplex is entropically destabilized

by the expelled structural force prone to the C3′-endo

form. Unexpectedly, the total structure of the RNA

duplex 36 containing a 2′-O-phosphorylated nucleoside

did not change significantly in the present simulation.

In a tRNA intermediate during splicing, however, a 2′-

O-phosphorylated nucleoside was located at the border

between the tRNA anticodon stem and loop like an A-type

helical structure. Therefore, it is necessary to examine a

partial structure having an actual 2′-O-phosphorylated

tRNA sequence, if we want to confirm the structural

effect of tRNA intermediates on biological activities.

An increasing number of MD simulation studies in

nucleic acid chemistry have recently been reported.^23-27

MD simulation is, indeed, a powerful tool to estimate the

thermodynamic behavior of a modified nucleoside incor-

porated into a DNA or RNA strand. In this study, we

also showed that the 2′-O-phosphorylated uridine in

dU₃U(2′-p)dU₃/dA₇preserves its favored C2′-endo con-

formation and fits the B-type helix through a simulta-

neous change in the two torsion angles. This result is a

typical case which shows the very flexibility of the DNA

backbone.

Furthermore, we have recently found that the 2′-O-

phosphate group is eventually hydrolyzed more rapidly

than expected due to the neighboring participation of the

proximal 3′,5′-phosphodiester moiety, and suggested a

novel possibility as a catalytic function in RNA.¹⁹Actu-

ally, Chattopadhyaya reported a new finding that 2′-O-

phosphorylated cyclic RNA species has a catalytic activity

(21) Crooke S. T. Handbook of Experimental Pharmaceutics: Anti-

sense Research and Application; Springer-Verlag Inc.: New York, 1998;

Vol. 131.

(22) (a) Plavec, J .; Tong, W.; Chattopadhyaya, J . J . Am. Chem. Soc.

1993, 115, 9734-9746. (b) Thibaudeau, C.; Plavec, J .; Garg, N.;

Papchikhin, A.; Chattopadhyaya, J . J . Am. Chem. Soc. 1994, 116,

4038-4043.

Con clu sion

(23) Herzyk, P.; Neidle, S.; Goddfellow, J . M. J . Biomol. Struct.

Dynam. 1992, 10, 97-139.

In recent studies on new antisense oligonucleotides,

many 2′-modified ribonucleoside derivatives, such as 2′-

halogenated and 2′-O-alkylated nucleosides, have been

synthesized and their conformational properties have

been investigated in great detail. These studies disclosed

that ribonucleoside derivatives, substituted by electron-

withdrawing groups at the 2′-position, have predomi-

(24) Sekharudu, C. Y.; Yathindra, N.; Sundralingam, M. J . Biomol.

Struct. Dynam. 1993, 11, 225-245.

(25) Cho, Y.; Zhu, F. C.; Luxon, B. A.; Gorenstein, D. G. J . Biomol.

Struct. Dynam. 1993, 11, 685-702.

(26) Shibata, M.; Zielinski, T. J .; Rein, R. Biopolymers 1991, 31,

211-232.

(27) Fedoroff, O. Y.; Salazar, M.; Reid, B. R. J . Mol. Biol. 1993, 233,

509-523.

7490 J . Org. Chem., Vol. 65, No. 22, 2000

Tsuruoka et al.

of 2′-O-phosphodiester.²⁸Two vicinal phosphate groups,

such as branched RNA and 2′-phosphorylated RNA,

might have more unique chemical properties.

Through our continuous studies, we have realized that

oligonucleotides incorporating a 2′-O-phosphorylated

nucleoside at the specific position of the sequence having

the four common nucleobases could be effectively syn-

thesized. Compounds of this kind would be promising

tools to elucidate the mechanism of tRNA processing in

molecular biology.

suspended in acetonitrile (220 mL). Hexamethyldisilazane

(26.4 mL, 125 mmol) was added, and the mixture was refluxed

for 1 h. The resulting clear solution was evaporated under

reduced pressure, and the last traces of pyridine were removed

by coevaporation with ethanol. The residue was dissolved in

dry CH₂Cl₂(220 mL). To the solution were added successively

(dimethylamino)pyridine (153 mg, 1.25 mmol), triethylamine

(13.9 mL, 100 mmol), and mesitylenesulfonyl chloride (6.56 g,

30 mmol). After being stirred at room temperature for 1 h,

the mixture was cooled to 0 °C. To the solution was added

pyrolidine (26.0 mL, 250 mmol). After the mixture was stirred

at 0 °C for 40 min, 2-cyanoethanol (17.1 mL, 250 mmol) and

DBU (11.2 mL, 75 mmol) were added, and stirring was

continued for 30 min. CH₂Cl₂-MeOH and phosphate buffer

(pH 6.0) were added. The organic layer was collected, dried

over Na₂SO₄, filtered, and evaporated under reduced pressure.

The residue was dissolved in a 1:1 mixture of CH₂Cl₂-MeOH

500 mL), and trifluoroacetic acid (5 mL) was added. After being

stirred for 30 min, the mixture was evaporated under reduced

pressure. The residue was chromatographed on a column of

silica gel with CH₂Cl₂-MeOH to give 1d (4.5 g, 40%): ¹H NMR

(270 MHz, TMS) δ 2.96 (2H, t, J ) 6.6 Hz), 3.76 (1H, dd, J )

2.6, 12.5 Hz), 3.84 (2H, s), 3.88 (1H, dd, J ) 3.0, 12.5 Hz),

4.23 (1H, m), 4.47 (1H, dd, J ) 3.0, 5.0 Hz), 4.63 (1H, dd, J )

5.0, 5.6 Hz), 4.71 (2H, t, J ) 6.6 Hz), 5.91 (1H, d, J ) 5.6 Hz),

7.32-7.41 (5H, m), 8.14 (1H, s); ¹³C NMR (67.8 MHz, R-proton

of pyridine-d₅) δ 18.37, 44.48, 62.43, 72.12, 76.27, 79.74, 87.43,

90.09, 118.52, 118.66, 127.34, 129.05, 130.23, 136.27, 136.31,

142.07, 152.84, 152.87, 153.75. 160.01, 170.84. Anal. Calcd for

Exp er im en ta l Section

Gen er a l Meth od s. ¹H NMR spectra were measured at 270

and 400 MHz with TMS (for CDCl₃) or DDS (for D₂O) as

internal standard. ¹³C NMR spectra were obtained at 67.8 and

100.6 MHz, respectively, with CDCl₃(for CDCl₃) as internal

standard or DDS (for D₂O) as external standard. ³¹P NMR

spectra were recorded at 109 MHz with the external reference

of 85% H₃PO₄. UV spectra were taken on a U-2000 spectro-

photometer. Reversed phase HPLC was performed on a Waters

LC module 1 with a µBondasphere 5 µm C18 100 Å (3.9 × 150

mm) column using a linear gradient of acetonitrile (0-30%)

in 0.1 M ammonium acetate buffer (pH 7.0) for 30 min at flow

rate of 1 mL/min at 50 °C. Ion exchange HPLC was carried

out at a flow rate of 1 mL/min for 30 min at 50 °C on a

Whatman Partisil 10 SAX WCS analytical column (4.6 × 250

mm) using a linear gradient of 0-100% solution A (20% CH₃-

CN in 0.5 M KH₂PO₄) in solution B (20% CH₃CN in 0.005 M

KH₂PO₄) Paper chromatography was carried out by use of a

descending technique with Whatman 3MM Chr papers using

iPrOH-concd NH₃-H₂O (6:1:3, v/v/v). Thin-layer chromatog-

raphy was performed using Merck Kieselgel 60F-254 (0.25

mm) with developing solvent of CH₂Cl₂-CH₃OH (9:1, v/v) or

iPrOH-concd NH₃-H₂O (7:1:2, v/v/v). Column chromatography

was performed with silica gel C-200 purchased from Wako Co.

Ltd., and a minipump for a goldfish bowl was conveniently

used to attain sufficient pressure for rapid chromatographic

separation. Elemental analyses were performed by the Mi-

croanalytical Laboratory, Tokyo Institute of Technology, at

Nagatsuta.

3′,5′-O-(Di-t er t -b u t ylsila n e d iyl)-4-N -isob u t yr ylcyt i-

d in e (2c). 4-N-Isobutyrylcytidine (1c) (313 mg, 1 mmol) was

rendered anhydrous by repeated coevaporation with dry

dimethylformamide (3 mL × 2) and dissolved with dimethyl-

formamide (3 mL). After addition of silver nitrate (170 mg, 1

mmol) and di-tert-butylsilyl bis(trifluoromethanesulfonate)

(360 µL, 1.1 mmol), the solution was stirred vigorously at room

temperature for 30 min. The mixture was added with triethy-

lamine (1.25 mL, 9 mmol), and the solvent was removed under

reduced pressure. The residue was dissolved with CH₂Cl₂(50

mL) and washed with saturated NaHCO₃and water. The

organic layer was collected, dried over Na₂SO₄, filtered, and

concentrated under reduced pressure. The crude material was

chromatographed on a column of silica gel eluted with CH₂-

Cl₂-MeOH to give 7: ¹H NMR (270 MHz, TMS) δ 1.01-1.13

(18H), 1.23 (6H, d, J ) 6.9 Hz, 6H), 2.61 (1H, sept), 3.91 (1H,

dd, J ) 4.6, 9.6 Hz), 4.05 (1H, dd, J ) 8.9 Hz, 10.2 Hz), 4.24-

4.29 (2H, m), 4.55 (1H, dd, J ) 5.0 Hz, 8.9 Hz), 5.72 (1H, s),

7.52 (1H, d, J ) 7.5 Hz), 7.76 (1H, d, J ) 7.5 Hz); ¹³C NMR

(67.8 MHz, CDCl₃) δ 18.64, 18.71, 20.20, 22.52, 26.85, 27.08,

36.34, 67.26, 73.55, 74.66, 75.58, 93.82, 97.13, 143.34, 155.65,

162.93, 177.93. Anal. Calcd for C₂₁H₃₅N₃O₆Si‚2H₂O: C, 51.51;

H, 7.21; N, 8.58. Found: C, 51.65; H, 7.35; N, 8.27.

C

₂₁H₂₂N₆O₆‚1.2 H₂O: C, 52.98; H, 5.21; N, 17.65. Found: C,

53.29; H, 4.76; N, 16.94.

3′,5′-O-(Di-ter t-bu tylsila n ed iyl)-2-N-p h en yla cetyl-6-O-

(2-cya n oet h yl)gu a n osin e (2d ). 2-N-Phenylacetyl-6-O-(2-

cyanoethyl)guanosine (1d ) (181 mg, 0.4 mmol) was coevapo-

rated with dimethylformamide (2 mL × 2) and dissolved in

dimethylformamide (2 mL). To the solution were added silver

nitrate (190 mg, 1.12 mmol) and di-tert-butyldichlorosilane

(110 µL, 0.52 mmol). After being stirred vigorously at 0 °C for

15 min, the mixture was diluted with CH₂Cl₂-pyridine (2:1,

v/v, 10 mL). The solvent was removed under reduced pressure,

and the residue was partitioned between CH₂Cl₂and water.

The organic phase was collected, dried over Na₂SO₄, filtered,

and concentrated under reduced pressure. The residue was

chromatographed on a column of silica gel eluted with CH₂-

Cl₂-MeOH to give 2d (220 mg, 92%): ¹H NMR (270 MHz,-

CDCl₃, TMS) δ 1.04 (9H, s), 1.09 (9H, s), 2.92 (2H, t, J ) 6.4

Hz), 3.43 (1H, s), 3.95-4.13 (4H, m), 4.46 (1H, dd, J ) 3.5 Hz,

J ) 7.9 Hz), 4.54 (1H, dd, J ) 7.9 Hz, J ) 5.0 Hz), 4.70 (1H,

d, J ) 5.0 Hz), 5.97 (1H, d), 7.27-7.38 (5H, m), 7.89 (1H, s),

8.27 (1H, s); ¹³C NMR (67.8 MHz, CDCl₃) δ 17.65, 20.06, 22.36,

26.88, 27.05, 43.87, 53.30, 61.44, 67.05, 73.23, 74.45, 75.72,

90.57, 116.93, 117.84, 126.90, 138.41, 129.18, 134.02, 140.61,

151.41, 151.99, 159.28, 169.36. Anal. Calcd for C₂₉H₃₈N₆O₆Si:

C, 58.57; H, 6.44; N, 14.13. Found: C, 58.33; H, 6.37; N, 13.91.

2′-O-Bis(2-cya n o-1,1-d im eth yleth oxy)th iop h osp h or yl-

6-N-ben zoyla d en osin e (4b). 3′,5′-O-(di-tert-butylsilanediyl)-

6-N-benzoyladenosine (2b) (205 mg, 0.4 mmol) and 1H-

tetrazole (63 mg, 0.9 mmol) was rendered anhydrous by

repeated coevaporation with dry pyridine (2 mL × 2) and dry

toluene (2 mL × 1). To the mixture was added a 0.25 M

solution of bis(2-cyano-1,1-dimethylethyl) N,N-diethylphos-

phoramidite (1.2 mmol) in CH₂Cl₂. After the mixture was

stirred at room temperature for 2 h, a 1 M solution of S₈in

CS₂-pyridine (4 mL, 20 mmol) was added. The solution was

stirred at room temperature for 2 h and then evaporated under

reduced pressure. The residue was dissolved with CH₂Cl₂(50

mL). The precipitate was removed by filtration. The filtrate

was washed twice with 5% NaHCO₃and with water, dried over

Na₂SO₄, filtered, and evaporated under reduced pressure. The

residue was dissolved in THF (2 mL), and a mixture of (HF)‚Py

(231 µL)/Py (1.2 mL) was added. The mixture was stirred at

room temperature for 10 min, and then an excess amount of

pyridine (3 mL) was added. The mixture was partitioned

between CH₂Cl₂and 5% NaHCO₃. The organic phase was

collected, washed with 5% NaHCO₃and water, dried over Na₂-

6-O-(2-Cya n oeth yl)-2-N-p h en yla cetylgu a n osin e (1d ).

2-N-Phenylacetylguanosine²⁹(10.3 g, 25 mmol) was rendered

anhydrous by repeated coevaparation with dry pyridine and

(28) (a) Sund, C.; Agback, P.; Chattopadhyaya, J . Tetrahedron 1993,

49, 649. (b) Agback, P.; Glemarec, C.; Yin, L.; Sandstro¨m, A.; Plavec,

J .; Sund, C.; Yamakage, S.-I.; Viswanadham, G.; Rousse, B.; Puri, N.;

Chattopadhyaya, J . Tetrahedron Lett. 1993, 34, 3929.

(29) Chaix, C.; Duplaa, A. M.; Molko, D.; Teoule, R. Nucleic Acids

Res. 1989, 17, 7381-7393.

Oligonucleotides with 2′-O-Phosphorylated Ribonucleotides

J . Org. Chem., Vol. 65, No. 22, 2000 7491

SO₄, filtered, and evaporated under reduced pressure. The

residue was chromatographed on a column of silica gel with

CH₂Cl₂-MeOH containing 0.5% pyridine to give 4b (172 mg,

82%): ¹H NMR (270 MHz,CDCl₃, TMS) δ 1.42-1.59 (12H, m),

2.65-2.87 (4H, m,), 3.70-3.76 (1H, m), 3.91 (1H, dd, J ) 1.6

Hz, J ) 13.2 Hz), 4.32 (1H, s), 4.71 (1H, d, J ) 4.6 Hz), 5.72

(1H, ddd, J ) 7.2 Hz, J ) 4.6 Hz, J ) 11.9 Hz), 6.11 (1H, d, J

) 7.2 Hz), 7.42-7.57 (3H, m), 8.14 (1H, s), 8.94 (2H, d), 8.73

(1H, s), 9.14 (1H, s); ¹³C NMR (67.8 MHz, CDCl₃) δ 26.92,

26.97, 27.24, 27.31, 31.48, 62.28, 70.71, 78.40, 82.75, 82.86,

87.24, 87.69, 87.84, 116.55, 116.80, 123.76, 127.89, 128.05,

128.66, 132.79, 133.23, 142.98, 150.04, 150.84, 152.09, 165.00;

³¹P NMR (109 MHz, CDCl₃, 85% H₃PO₄) δ 48.90. Anal. Calcd

for C₂₇H₃₂N₇O₇PS: C, 51.5; H, 5.12; N, 15.57. Found: C, 51.65;

H, 5.25; N, 15.41.

2′-O-Bis(2-cya n o-1,1-d im eth yleth oxy)th iop h osp h or yl-

4-N-isobu tyr ylcytid in e (4c). 3′,5′-O-(Di-tert-butylsilanediyl)-

4-N-isobutyrylcytidine (2c) (2.4 g, 5 mmol) was converted to

4c (2.45 g, 86%) using the same method as described in 4b.

4c: ¹H NMR (270 MHz, DSS) δ 1.22 (6H, d, J ) 6.9 Hz), 1.69-

1.74 (12H, m), 2.62 (1H, sept, J ) 6.9 Hz), 2.84-3.17 (4H, m),

3.94 (2H, m), 4.21 (1H, m), 4.64 (1H, m), 5.23 (1H, ddd, J )

4.3 Hz, J ) 4.6 Hz, J ) 11.2 Hz), 6.12 (1H, d, J ) 4.3 Hz),

7.47 (1H, d, J ) 7.6 Hz), 8.33 (1H, d, J ) 7.6 Hz), 8.51 (1H,

brs); ¹³C NMR (67.8 MHz, CDCl₃) δ 18.65, 26.60, 26.74, 26.79,

26.85, 26.90, 26.96, 27.01, 27.14, 27.19, 31.09, 31.16, 35.94,

36.03, 60.16, 68.07, 68.14, 79.68, 82.43, 82.55, 82.59, 82.71,

84.55, 89.49, 89.52, 96.98, 116.87, 123.81, 148.63, 155.29,

162.68, 177.43; ³¹P NMR (109 MHz, CDCl₃, 85% H₃PO4) δ

49.725. Anal. Calcd for C₂₃H₃₄N₅O₈PS‚3/2H₂O: C, 46.14; H,

5.73; N, 11.70. Found: C, 46.43; H, 5.91; N, 11.42.

2′-O-Bis(2-cya n o-1,1-d im eth yleth oxy)th iop h osp h or yl-

2-N-p h en yla cetyl-6-O-(2-cya n oeth yl)gu a n osin e (4d ). 3′,5′-

O-(Di-tert-butylsilanediyl)-2-N-phenylacetyl-6-O-(2-cyanoethyl)-

guanosine 2d (214 mg, 0.36 mmol) was converted to 4d (217

mg, 84%) using the same method as described in 4b. 4d : ¹H

NMR (270 MHz,CDCl₃, TMS) δ 1.48-1.63 (12H, m), 2.77-2.99

(6H, m), 3.81 (1H, dd, J ) 2.0 Hz, J ) 12.9 Hz), 3.87 (2H, s)

3.94 (1H, dd, J ) 2.0 Hz, J ) 12.9 Hz), 4.31-4.33 (1H, m),

4.73 (2H, t, J ) 6.2 Hz), 4.87 (1H, dd, J ) 4.8 Hz, J ) 1.7 Hz),

5.68 (1H, ddd, J ) 6.9 Hz, J ) 4.8 Hz, J ) 9.8 Hz), 6.09 (1H,

d, J ) 6.9 Hz), 7.32-7.42 (5H, m), 8.02 (1H, s), 8.18 (1H, s);

¹³C NMR (67.8 MHz, CDCl₃) δ 17.95, 21.37, 26.94, 27.05, 27.10,

27.46, 27.53, 31.43, 31.50, 44.55, 61.74, 62.32, 70.55, 70.60,

78.44, 78.51, 82.84, 82.93, 82.97, 87.39, 87.53, 116.55, 116.77,

116.87, 119.07, 125.21, 127.42, 128.14, 128.95, 129.42, 133.89,

142.32, 151.34, 152.45, 159.82, 161.71, 169.16; ³¹P NMR (109

MHz, CDCl₃, 85% H3PO₄) δ 48.60. Anal. Calcd for C₃₁H₃₇N₈O₈-

PS‚1/2H₂O: C, 51.59; H, 5.31; N, 15.52; S, 4.44. Found: C,

51.87; H, 5.27; N, 15.12; S, 4.93.

2′-O-Bis(2-cya n o-1,1-d im eth yleth oxy)th iop h osp h or yl-

4-N-ben zoylcytid in e (4e). A mixture of 3′,5′-O-(di-tert-bu-

tylsilanediyl)-4-N-benzoylcytidine (2e) (3.45 g, 7.07 mmol) and

1H-tetrazole (1.88 g, 26.9 mmol) was rendered anhydrous by

repeated coevaporation with dry pyridine (2 mL × 2) and dry

toluene (2 mL × 1). To the mixture was added a 0.25 M

solution of bis(2-cyano-1,1-dimethylethyl) N,N-diethylphos-

phoramidite (17.7 mmol) in CH₂Cl₂. After the mixture was

stirred at room temperature for 19 h, the solvent was removed

in vacuo, and the residue was dissolved in 1 M solution of S₈

(18 g, 71 mmol) in CS₂-pyridine (1:1, v/v, 80 mL). The mixture

was stirred at room temperature for 2 h and then evaporated

under reduced pressure. After the residue was dissolved in

CH₂Cl₂(200 mL), the precipitate was removed by filtration.

The filtrate was washed with 5% NaHCO₃and water, dried

over Na₂SO₄, and evaporated under reduced pressure. The

residue was dissolved in THF (25 mL), and a mixture of

pyridinium hydrogenfluoride (4.1 mL)/Py (18.3 mL) was added.

After the mixture was stirred for 10 min, a large excess of

pyridine (20 mL) was added. The mixture was partitioned

between CH₂Cl₂and 5% NaHCO₃, and the organic layer was

collected, dried over Na₂SO₄, and evaporated under reduced

pressure. The crude material was chromatographed on a

column of silica gel with CH₂Cl₂-MeOH containing 0.5%

pyridine to give 4e (3.29 g, 77%): ¹H NMR (270 MHz,CDCl₃,

TMS) δ 1.69 (6H, s), 1.73 (6H, s), 2.80-3.18 (4H, m), 3.90 (1H,

dd, J ) 1.7 Hz, J ) 12.4 Hz), 4.00 (1H, dd, J ) 1.7 Hz, J )

12.4 Hz), 4.24 (1H, dd, J ) 4.3 Hz, J ) 1.7 Hz), 4.63 (1H, t, J

) 4.3 Hz, J ) 4.3 Hz), 5.23 (1H, dt, J ) 4.3 Hz, J ) 4.3 Hz, J

) 11.2 Hz), 6.19 (1H, d, J ) 4.3 Hz), 7.15-7.63 (4H, m), 7.90

(2H, d), 8.40 (1H, d, J ) 7.6 Hz); ¹³C NMR (67.8 MHz, CDCl₃)

δ 27.05, 27.10, 27.17, 27.35, 27.41, 27.55, 27.60, 31.25, 31.34,

31.94, 60.56, 68.64, 80.00, 80.06, 82.62, 82.75, 82.98, 83.09,

84.85, 89.60, 97.38, 117.05, 117.11, 123.79, 127.69, 128.84,

132.72, 133.14, 136.23, 146.33, 149.38, 162.73; ³¹P NMR (109

MHz, CDCl₃, 85% H₃PO₄) δ 49.68. Anal. Calcd for C₂₆H₃₂N₅O₈-

PS‚1/2H₂O: C, 50.81; H, 5.41; N, 11.39; S, 5.22. Found: C,

50.56; H, 5.65; N, 11.31; S,5.47.

2′-O-Bis(2-cya n o-1,1-d im eth yleth oxy)th iop h osp h or yl-

5′-O-(4,4′-d im et h oxyt r it yl)-6-N-b en zoyla d en osin e (5b ).

Compound 4b (1.3 g, 2.06 mmol) was rendered anhydrous by

repeated coevaporation with dry pyridine (20 mL × 2) and

finally dissolved in dry pyridine (20 mL). To the mixture was

added 4,4′-dimethoxytrityl chloride (1.3 g, 3.75 mmol), and the

solution was kept at room temperature for 4 h. Extraction was

performed with CH₂Cl₂and 5% NaHCO₃. The usual workup

followed by silica gel column chromatography eluted with CH₂-

Cl₂-MeOH containing 0.5% pyridine gave 5b (1.82 g, 95%):

¹H NMR (270 MHz,CDCl₃, TMS)δ 1.43-1.70 (12H, m), 2.62-

3.14 (4H, m,), 3.41 (1H, d, J ) 8.6 Hz), 3.50 (1H, d, J ) 8.6

Hz), 3.77 (6H, s,), 4.33 (1H, s), 4.88 (1H, d, J ) 6.3 Hz), 5.79

(1H, ddd, J ) 6.3 Hz, J ) 6.3 Hz, J ) 10.9 Hz), 6.45 (1H, d, J

) 6.3 Hz), 6.82 (4H, d), 7.14-7.58 (12H, m), 8.0 (2H, d), 8.33

(1H, s), 8.73 (1H, s), 9.23 (1H, s); ¹³C NMR (67.8 MHz, CDCl₃)

δ 26.56, 26.99, 27.03, 27.10, 27.35, 27.41, 31.29, 31.36, 31.57,

31.65, 55.08, 63.07, 770.35, 79.35, 79.43, 82.80, 82.91, 83.04,

84.39, 85.14, 86.70, 112.92, 113.14, 116.46, 116.71, 122.79,

126.83, 127.64, 127.71, 127.87, 127.96, 128.70, 129.94, 132.69,

133.41, 135.27, 135.35, 141.76, 144.24, 149.45, 151.73, 152.65,

158.38, 164.53; ³¹P NMR (109 MHz, CDCl₃, 85% H₃PO₄) δ

49.91. Anal. Calcd for C₄₈H₅₀N₇O₉PS: C, 61.86; H, 5.41; N,

10.50. Found: C, 61.62; H, 5.54; N, 10.03.

2′-O-Bis(2-cya n o-1,1-d im eth yleth oxy)th iop h osp h or yl-

5′-O-(4,4′-d im et h oxyt r it yl)-4-N-isob u t yr ylcyt id in e (5c).

Compound 4c (1.7 g, 3.0 mmol) was converted to 5c (2.5 g,

96%) using the same method as described in 5b. 5c: ¹H NMR

(270 MHz, DSS) δ 1.21 (6H, 2d, J ) 6.9 Hz), 1.67-1.75 (12H,

m), 2.55 (1H, sept, J ) 6.9 Hz), 2.88-3.10 (4H, m), 3.53 (2H,

m), 3.81 (6H, s), 4.20 (1H, m), 4.65 (1H, m), 5.15 (1H, ddd, J

) 3.0 Hz, J ) 3.9 Hz, J ) 11.5 Hz), 6.24 (1H, d, J ) 3.0 Hz),

6.81-6.88 (4H, m), 7.08 (1H, d, J ) 7.6 Hz), 7.15-7.42 (9H,

m), 8.14 (1H, brs), 8.36 (1H, d, J ) 7.6 Hz); ¹³C NMR (67.8

MHz, CDCl₃) δ 18.82, 18.89, 27.01, 27.05, 27.12, 27.17, 27.28,

27.33, 31.20, 31.27, 31.47, 31.54, 36.21, 55.02, 61.73, 68.91,

80.59, 80.67, 82.71, 82.84, 83.16, 83.29, 87.05, 87.94, 88.07,

96.80, 113.21, 116.78, 116.91, 126.97, 127.89, 128.05, 129.83,

129.96, 134.97, 135.33, 143.74, 144.80, 154.97, 158.49, 162.61,

176.87; ³¹P NMR (109 MHz, CDCl₃, 85% H₃PO₄) δ 50.339.

Anal. Calcd for C₄₄H₅₂N₅O₁₀PS‚5/2H₂O: C, 57.51; H, 5.70; N,

7.62. Found: C, 57.79; H, 5.90; N, 7.35.

2′-O-Bis(2-cya n o-1,1-d im eth yleth oxy)th iop h osp h or yl-

5′-O-(4,4′-d im eth oxytr ityl)-2-N-p h en yla cetyl-6-O-(2-cya -

n oeth yl)gu a n osin e (5d ). Compound 4d (2.08 g, 2.92 mmol)

was converted to 5d (2.9 g, 99%) using the same method as

described in 5b. 5d : ¹H NMR (270 MHz, DSS) δ 1.40-1.69

(12H, m), 2.62-3.14 (6H, m), 3.30-3.48 (4H, m), 3.76 (6H, s),

4.28 (1H, s), 4.74 (2H, t, J ) 6.4 Hz), 4.89 (1H, s), 5.29 (1H,

m), 6.21 (1H, d, J ) 6.6 Hz), 6.77 (2H, s), 6.80 (2H, s), 7.15-

7.50 (14H, m), 8.07 (1H, s), 8.60-8.63 (1H, br); ¹³C NMR (67.8

MHz, CDCl₃) δ 17.95, 26.63, 26.70, 26.74, 26.79, 26.99, 27.05,

27.51, 31.38, 31.43, 31.57, 31.63, 43.85, 55.13, 61.64, 63.29,

70.08, 82.79, 82.86, 82.89, 82.97, 84.49, 86.54, 113.19, 116.53,

116.80, 116.87, 118.19, 123.72, 126.92, 127.15, 127.94, 128.05,

128.12, 128.68, 128.93, 129.38, 130.01, 133.91, 135.51, 135.65,

141.51, 141.56, 144.55, 151.52, 153.03, 158.49, 159.66, 169.02;

³¹P NMR (109 MHz, CDCl₃, 85% H₃PO₄) δ 49.58. Anal. Calcd

for C₅₂H₅₅N₈O₁₀PS: C, 61.53; H, 5.46; N, 11.04; S, 3.16.

Found: C, 61.53; H, 5.47; N, 10.75; S, 2.84.

2′-O-Bis(2-cya n o-1,1-d im eth yleth oxy)th iop h osp h or yl-

5′-O-(4,4′-d im eth oxytr ityl)-4-N-ben zoylcytidin e (5e). Com-

7492 J . Org. Chem., Vol. 65, No. 22, 2000

Tsuruoka et al.

pound 4e (2.5 g, 4.1 mmol) was converted to 5e (3.4 g, 91%) in

the same manner of 5b. 5e: ¹H NMR (270 MHz, DSS) δ 1.62

(6H, s), 1.76 (6H, s), 2.86-3.06 (4H, m), 3.55 (2H, d, J ) 1.7

Hz), 3.77 (6H, s), 4.22 (1H, dd, J ) 1.7 Hz, J ) 6.3 Hz), 4.69

(1H, dd, J ) 3.3 Hz, J ) 6.3 Hz), 5.18 (1H, dt, J ) 3.3 Hz, J

) 3.3 Hz, J ) 11.6 Hz), 6.27 (1H, d, J ) 3.3 Hz), 6.87 (2H, s),

6.90 (2H, s), 7.16-7.65 (13H, m), 7.88 (2H, d), 8.44 (1H, d, J

) 7.3 Hz); ¹³C NMR (67.8 MHz, CDCl₃) d 27.15, 27.28, 27.42,

31.36, 31.43, 31.61, 31.70, 55.15, 61.64, 68.97, 80.63, 80.67,

80.70, 82.88, 82.98, 83.27, 83.47, 83.60, 87.24, 88.00, 88.07,

113.33, 113.46, 116.78, 117.02, 123.74, 125.19, 127.12, 127.51,

128.05, 128.12, 128.93, 129.06, 129.99, 130.06, 132.94, 132.94,

132.97, 133.10, 135.00, 135.35, 143.90, 144.87, 144.91, 144.98,

145.01, 158.62, 162.28; ³¹P NMR (109 MHz, CDCl₃, 85%

H3PO4) δ 50.27. Anal. Calcd for C₄₇H₅₀N₅O₁₀PS‚H₂O: C, 60.96;

H, 5.66; N, 7.56. Found: C, 60.83; H, 5.43; N, 7.19.

2′-O-Bis(2-cya n o-1,1-d im eth yleth oxy)th iop h osp h or yl-

5′-O-(4,4′-d im eth oxytr ityl)-4-N-ben zoylcytid in e-3′-(2-cya -

n oeth yl)-m or p h olin op h osp h or a m id ite (6f). Compound 5e

(2.1 g, 2.2 mmol) was rendered anhydrous by successive

coevaporations with dry pyridine (10 mL × 3) and dry toluene

(10 mL × 2) and finally dissolved in dry CH₂Cl₂(22 mL). To

the mixture were added triethylamine (1.22 mL, 8.8 mmol)

and chloro(2-cyanoethoxy)morpholinophosphine (0.69 mL, 4.4

mmol). After being stirred for 40 min, the reaction mixture

was diluted with CH₂Cl₂(20 mL). The CH₂Cl₂extract was

washed with 5% NaHCO₃and water. The usual workup

followed by silica gel column chromatography eluted with

hexanes-ethyl acetate containing 1% pyridine gave 6f (1.54

g, 61%): ¹H NMR (270 MHz, CDCl₃, TMS) δ 1.72-1.76 (12H,

m), 2.40-2.69 (2H, m), 2.81-3.20 (8H, m), 3.42-4.09 (8H, m),

3.82 (6H, s), 4.28-4.33 (1H, m), 4.74-4.78 (1H, m), 5.20-5.26

(1H, m), 6.32-6.38 (1H, m), 6.87 (2H, s), 6.90 (2H, s), 7.15-

7.63 (12H, m), 7.88 (2H, m), 8.44-8.48 (1H, m), 8.53-8.76 (1H,

brs); ¹³C NMR (67.8 MHz, CDCl₃) δ 20.07, 20.16, 20.22, 20.31,

21.35, 26.97, 27.03, 27.14, 27.26, 27.32, 27.39, 27.46, 27.49,

27.60, 27.64, 31.04, 31.07, 31.14, 31.54, 31.61, 42.98, 43.13,

43.24, 43.36, 53.39, 55.19, 58.22, 58.51, 58.98, 59.23, 61.31,

61.44, 67.62, 67.69, 67.78, 67.85, 78.56, 78.65, 78.73, 82.28,

82.41, 82.52, 82.57, 82.71, 83.11, 83.18, 83.24, 83.31, 87.35,

87.48, 87.66, 87.71, 113.32, 116.71, 116.77, 117.07, 117.25,

117.57, 125.19, 127.22, 127.33, 127.49, 128.01, 128.12, 128.30,

128.41, 128.91, 129.99, 130.06, 130.15, 130.21, 130.28, 133.08,

134.75, 134.90, 135.08, 137.74, 143.77, 144.94, 145.00, 158.63,

158.72, 158.78, 162.26; ³¹P NMR (109 MHz, CDCl₃, 85% H₃-

PO₄) δ 147.25, 146.83, 50.60, 50.50. Anal. Calcd for C₅₄H₆₁N₇O₁₂-

P2S‚1/2H₂O: C, 58.8; H, 5.66; N, 8.89. Found: C, 59.07; H,

5.77; N, 8.44.

2′-O-Bis(2-cya n o-1,1-d im eth yleth oxy)th iop h osp h or yl-

5′-O-(4,4′-d im et h oxyt r it yl)-4-N-isob u t yr ylcyt id in e-3′-(2-

cya n oeth yl)-N,N-d iisop r op ylp h osp h or a m id ite (6c). Com-

pound 5c (175 mg, 0.2 mmol) was rendered anhydrous by

successive coevaporations with dry pyridine (2 mL × 2) and

dry toluene (2 mL × 1) and finally dissolved in dry CH₂Cl₂(2

mL). To the mixture were added 2,4,6-collidine (158 mL, 1.2

mmol) and chloro(2-cyanoethoxy)diisopropylaminophosphine

(86 µL, 0.4 mmol). After being stirred over period of 1 h, the

mixture was diluted with CH₂Cl₂(10 mL) and washed with

5% NaHCO₃and water. The organic layer was collected and

concentrated in vacuo. The residue was dissolved in CH₂Cl₂

(1 mL), and the mixture was added dropwise to a solution of

hexanes-ether containing 1% pyridine (8:2, v/v, 100 mL) with

vigorous stirring. The precipitate was collected and washed

with hexanes-ether (8:2, v/v) containing 1% pyridine. The

usual workup followed by silica gel column chromatography

eluted with hexanes-ethyl acetate containing 1% pyridine

gave the title compound 6c (155 mg, 72%): ¹H NMR (270 MHz,

CDCl₃, TMS) δ 1.00-1.29 (18H, m), 1.66-1.80 (12H, m), 2.40-

2.70 (3H, m), 2.85-3.20 (4H, m), 3.40-3.98 (10H, m), 4.25-

4.35 (1H, m), 4.58-4.72 (3H, m), 5.10-5.40 (1H, m), 6.35, 6.45

(1H, 2d, J ) 5.3, J ) 4.3 Hz), 6.83-6.90 (4H, m), 6.97-7.21

(1H, m), 7.26-7.46 (9H, m), 8.15-8.20 (1H, br); ¹³C NMR (67.8

MHz, CDCl₃) δ 18.71, 19.01, 19.97, 20.22, 24.37, 24.46, 24.57,

24.78, 24.89, 26.99, 27.046, 27.19, 27.37, 27.66, 31.09, 31.16,

31.36, 36.43, 42.98, 43.16, 55.10, 58.83, 59.09, 62.43, 78.58,

82.19, 82.30, 82.41, 82.86, 82.98, 83.11, 83.22, 87.24, 87.30,

96.64, 96.71, 113.17, 113.23, 116.59, 116.82, 116.93, 117.02,

117.31, 117.79, 127.08, 127.91, 127.96, 128.12, 128.25, 130.06,

130.14, 134.72, 134.88, 135.02, 135.11, 143.65, 145.03, 145.14,

155.02, 158.56, 162.44, 176.68; ³¹P NMR (109 MHz, CDCl₃,

85% H₃PO₄) δ 152.41, 151.90, 50.70, 50.35. Anal. Calcd for

C₅₃H₆₉N₇O₁₁P₂S‚3/2H₂O: C, 57.81; H, 6.31; N, 8.90. Found: C,

57.92; H, 6.42; N, 8.52.

2′-O-Bis(2-cya n o-1,1-d im eth yleth oxy)th iop h osp h or yl-

5′-O-(4,4′-d im eth oxytr ityl)-2-N-p h en yla cetyl-6-O-(2-cya -

n oeth yl)gu an osin e-3′-(2-cyan oeth yl)-N,N-diisopr opylph os-

p h or a m id ite (6d ). Compound 5d (1.62 g, 1.6 mmol) was

rendered anhydrous by successive coevaporations with dry

pyridine (5 mL × 2) and dry toluene (5 mL × 1) and finally

dissolved in dry CH₂Cl₂(4.8 mL). To the mixture were added

2,4,6-collidine (1.27 mL, 9.6 mmol) and chloro(2-cyanoethoxy)-

diisopropylaminophosphine (0.7 mL, 3.2 mmol). After being

stirred for 1 h, the mixture was diluted with CH₂Cl₂(50 mL)

and washed with 5% NaHCO₃and water. The organic layer

was collected and concentrated in vacuo. The usual workup

followed by silica gel column chromatography eluted with

hexanes-ethyl acetate containing 1% pyridine gave the title

compound 6d (1.95 g, 74%): ¹H NMR (270 MHz, CDCl₃, TMS)

δ 0.97-1.70 (26H, m), 2.49-3.04 (6H, m), 3.22-4.03 (6H, m),

3.76 (6H, s), 4.30-4.40 (1H, m), 4.71-4.79 (3H, m), 6.02-6.12

(1H, m), 6.19-6.28 (1H, m), 6.79-6.84 (4H, m), 7.08-7.82

(14H, m), 8.10-8.12 (1H, m); ¹³C NMR (67.8 MHz, CDCl₃) δ

17.94, 20.34, 20.42, 21.33, 24.37, 24.46, 24.57, 24.67, 24.80,

26.67, 26.74, 26.90, 26.96, 27.21, 27.26, 31.23, 31.39, 31.47,

43.00, 43.18, 43.40, 43.63, 55.13, 59.23, 59.46, 61.65, 63.06,

63.25, 82.30, 82.34, 82.41, 82.45, 82.48, 82.59, 82.70, 84.64,

84.67, 84.96, 85.09, 85.39, 86.69, 86.78, 113.26, 116.32, 116.41,

116.62, 116.86, 117.23, 118.04, 118.17, 125.18, 126.97, 127.03,

128.03, 128.10, 128.16, 128.50, 128.55, 128.91, 129.38, 129.97,

130.12, 133.98, 135.38, 135.51, 135.72, 141.53, 144.46, 144.60,

151.55, 153.28, 153.33, 158.54, 159.62, 159.66, 168.86; ³¹P

NMR (109 MHz, CDCl₃, 85% H₃PO4) δ 153.01, 150.85, 50.24,

49.95. Anal. Calcd for C₆₁H₇₂N₁₀O₁₁P₂S: C, 60.29; H, 5.97; N,

11.53. Found: C, 59.94; H, 6.07; N, 11.11.

2′-O-Bis(2-cya n o-1,1-d im eth yleth oxy)th iop h osp h or yl-

5′-O-(4,4′-d im et h oxyt r it yl)-6-N-b en zoyla d en osin e-3′-(2-

cya n oeth yl)-N,N-d iisop r op ylp h osp h or a m id ite (6b). Com-

pound 5b (129 mg, 0.135 mmol) was rendered anhydrous by

successive coevaporations with dry pyridine (2 mL × 2) and

dry toluene (2 mL × 1) and finally dissolved in dry CH₂Cl₂

(1.5 mL). To the mixture were added 2,4,6-collidine (71 µL,

0.54 mmol) and chloro(2-cyanoethoxy)diisopropylaminophos-

phine (63 mg, 0.27 mmol). After being stirred for 1 h, the

mixture was diluted with CH₂Cl₂(5 mL) and washed with 5%

NaHCO₃and water. The organic layer was collected and

concentrated in vacuo. The usual workup followed by silica

gel column chromatography eluted with hexanes-ethyl acetate

containing 1% pyridine gave the title compound 6b (110 mg,

72%): ¹H NMR (270 MHz, CDCl₃, TMS) δ 1.06-1.78 (26H,

m), 2.36-3.04 (6H, m), 3.30-4.06 (4H, m), 3.76 (6H, s), 3.37-

4.44 (1H, m), 4.34-4.94 (3H, m), 5.77-5.97 (1H, m), 6.416.52

(1H, m), 6.79-6.84 (4H, m), 7.19-7.64 (12, m), 8.03-8.09 (2H,

m), 8.37-8.42 (1H, 2s), 8.76-8.79 (1H, 2s), 9.05-9.07 (1H,

2brs); ¹³C NMR (67.8 MHz, CDCl₃) δ 16.85, 16.97, 17.91, 17.94,

18.02, 18.29, 18.39, 20.71, 20.75, 20.80, 22.37, 22.45, 22.56,

22.69, 22.80, 24.65, 25.04, 25.11, 25.28, 25.62, 29.32, 29.43,

41.05, 41.23, 43.10, 43.19, 53.09, 55.64, 56.00, 57.12, 57.35,

60.87, 61.05, 69.55, 74.57, 75.05, 75.25, 75.52, 75.72, 80.48,

80.58, 80.67, 80.78, 82.26, 82.72, 82.87, 82.97, 84.77, 84.86,

111.15, 114.29, 114.38, 114.62, 115.28, 115.79, 120.76, 120.87,

124.84, 125.71, 125.87, 125.99, 126.08, 126.69, 126.87, 127.97,

130.61, 131.51, 1332.22, 133.27, 133.36, 140.06, 142.17, 142.24,

147.48, 149.91, 150.65, 156.45, 162.49; ³¹P NMR (109 MHz,

CDCl₃, 85% H₃PO₄) δ 152.84, 151.31, 50.78, 50.31. Anal. Calcd

for C₅₇H₆₇N₉O₁₀P₂S‚1/2H₂O: C, 59.50; H, 6.05; N, 10.95.

Found: C, 60.00; H, 6.59; N, 10.52.

3-N-Ben zoyl-2′-d eoxyu r id in e (8). Hexamethyldisilazane

(25 mL, 0.12 mol) was added to a solution of 2′-deoxyuridine

(6.85 g, 30 mmol) in CH₃CN (300 mL). After being refluxed

for 1 h, the solvent was removed under reduced pressure to

Oligonucleotides with 2′-O-Phosphorylated Ribonucleotides

J . Org. Chem., Vol. 65, No. 22, 2000 7493

give a colorless foam. To the residue dissolved in CH₂Cl₂(500

mL) - 0.2 M sodium carbonate solution (500 mL) were added

tetrabutylammonium bromide (0.8 g, 2.4 mmol) and benzoyl

chloride (5.2 mL, 45 mmol) with vigorous stirring. After 3 h,

the organic layer was extracted with water, collected, dried

over Na₂SO₄, and concentrated in vacuo. The resulting residue

was refluxed in CHCl₃(300 mL) for 1 h and evaporated under

reduced pressure. The residue was dissolved in methanol (300

mL) containing 6 mL of trifluoroacetic acid. After being stirred

for 20 min, the mixture was neutralized by addition of pyridine

(20 mL). Extraction was performed with CH₂Cl₂-pyridine (2:

1, v/v, 300 mL) and saturated NaHCO_3.The crude material

was chromatographed on a column of silica gel with CH₂Cl₂-

methanol to give 8 (9.2 g, 92%): ¹H NMR (270 MHz, C₅D₅N)

δ 2.52-2.69 (2H, m), 4.10 (1H, dd, J ) 2.6 Hz, J ) 12.9 Hz),

4.20 (1H, dd, J ) 3.0 Hz, J ) 12.9 Hz), 4.43 (1H, m), 4.97 (1H,

m), 5.93 (1H, d, J ) 7.9 Hz), 6.81 (1H, dd, J ) 6.3 Hz, J ) 6.3

Hz), 7.34-7.40 (2H, m), 7.49-7.53 (1H, m), 8.15-8.18 (2H,

m), 8.59 (1H, d, J ) 7.9 Hz); ¹³C NMR (67.8 MHz, C₅D₅N) δ

39.68, 59.80, 68.91, 84.06, 86.99, 99.89, 127.42, 128.68, 130.21,

133.15, 139.41, 160.41, 167.89. Anal. Calcd for C₁₆H₁₆N₂O₆: C,

57.83; H, 4.85; N, 8.43. Found: C, 57.33; H, 4.87; N, 8.38.

5′-O-(4,4′-Dim et h oxyt r it yl)-3-N-Ben zoyl-2′-d eoxyu r i-

d in e (9). N³-Benzoyl-2′-deoxyuridine 8 (5.0 g, 15 mmol) was

converted to 9 in the usual manner in 99% yield. 9: ¹H NMR

(270 MHz, CDCl₃) δ 2.23-2.43 (2H, m), 3.43 (1H, dd, J ) 3.0

Hz, J ) 11.2 Hz), 3.48 (1H, dd, J ) 3.3 Hz, J ) 11.2 Hz), 3.79

(6H, s), 3.98-4.01 (1H, m), 4.53-4.58 (1H, m), 5.46 (1H, d, J

) 8.3 Hz), 6.26 (1H, dd, J ) 5.9 Hz, J ) 6.3 Hz), 6.83-6.88

(4H, m), 7.15-7.63 (12H, m), 7.90-7.94 (3H, m); ¹³C NMR

(67.8 MHz, CDCl₃) δ 41.33, 55.22, 62.75, 71.21, 85.34, 86.09,

87.08, 101.98, 112.83, 113.10, 123.86, 127.17, 127.75, 127.85,

128.01, 128.05, 128.99, 129.11, 129.49, 130.03, 130.21, 130.24,

130.31, 130.46, 131.36, 135.06, 135.11, 135.22, 135.29, 136.24,

140.09, 144.17, 144.17, 149.15, 158.69, 162.16, 168.82. Anal.

Calcd for C₃₇H₃₄N₂O₈: C, 70.02; H, 5.34; N, 4.41. Found: C,

69.71; H, 5.35; N, 4.47.

168.77; ³¹P NMR (109 MHz, CDCl₃) δ 149.37, 149.76. Anal.

Calcd for C₄₆H₅₁N₄O₉P: C, 66.18; H, 6.16; N, 6.71. Found: C,

66.05; H, 6.12; N, 6.35.

Gen er a l Meth od for P olym er Su p p or ted Syn th esis of

Oligon u cleotid es. Syn th esis of d U_mU(2′-p )d Un (15-17).

A CPG gel having deoxyuridine (1 µmol, 25 µmol/g) as the 3′-

terminus was used. The synthetic unit 6a obtained above and

deoxyuridine phosphoramidite (or 11) were used for condensa-

tion. Chain extension was performed according to the protocol

described in our previous paper.¹⁸After all operations were

over (DMTr off), the CPG gel was evaporated with acetonitrile

and suspended in a solution of bis(trimethylsilyl)acetamide-

pyridine (0.4 mL, 1:1, v/v), and DBU (12 µL, 0.08 mmol) was

added. The mixture was slowly rotated in a vessel at room

temperature for 2 h. The CPG gel was then filtered and

washed with pyridine (1 mL × 3). The CPG gel was treated

with a 0.1 M solution of KI₃in pyridine-water (0.3 mL, 9:1,

v/v) at room temperature for 24 h. Filtration was performed,

and the CPG gel was washed with pyridine (1 mL × 7). The

CPG gel was collected and transferred to a round flask.

Concentrated ammonia-pyridine (20 mL, 9:1, v/v) was added,

and the suspension was slowly rotated by a rotary evaporator

for 18 h. The CPG gel was removed by filtration and washed

with water. The filtrate and washings were combined and

evaporated under reduced pressure. The residue was purified

by ion exchange and reversed phase HPLC to give the desired

products. (15: 18 A₂₆₀, 28%; 16: 13 A₂₆₀, 20%)

Syn th esis of d U₆U(2′-p s)d U₆(17). A CPG gel having

deoxyuridine (1 µmol, 25 µmol/g) as the 3′-terminus was used.

The same procedure to synthesize 15 was carried out without

the KI₃treatment. The crude material was purified by ion

exchange and reversed phase HPLC to give the desired

products 17 (13 A₂₆₀, 19%).

Syn th esis of U_mU(2′-p )Un (21, 22). A CPG gel having

uridine (1 µmol, 25 µmol/g) as the 3′-terminus was used. The

same procedure to synthesize 15 was carried out until treat-

ment with concentrated ammonia-pyridine (20 mL, 9:1, v/v)

and removal of CPG gel. After evaporation under reduced

pressure, the residue was dissolved in 1 M TBAF/THF (1 mL,

1 mmol). After being stirred for 15 h, the solution was directly

chromatographed by Sephadex G-15. The appropriate fraction

was collected and concentrated. The residue was purified by

ion exchange and reversed phase HPLC to give the desired

products (21: 3 A₂₆₀, 7%; 22: 4 A₂₆₀, 6%).

5′-O-(4,4′-Dim eth oxytr ityl)-3′-O-su ccin yl-3-N-Ben zoyl-

2′-d eoxyu r id in e (10). Compound 9 was converted to 10 in

the usual method in 98% yield. 10: ¹H NMR (270 MHz, CDCl₃)

δ 2.38-2.60 (6H, m), 3.44 (1H, dd, J ) 2.3 Hz, J ) 10.9 Hz),

3.54 (1H, dd, J ) 2.6 Hz, J ) 10.9 Hz), 3.79 (6H, s), 4.19-4.20

(1H, m), 5.41 (1H, d, J ) 8.2 Hz), 5.48-5.50 (1H, m), 6.32 (1H,

dd, J ) 5.9 Hz, J ) 7.9 Hz), 6.84-6.89 (4H, m), 7.15-7.66

(12H, m), 7.87-7.93 (3H, m); ¹³C NMR (67.8 MHz, CDCl₃) δ

29.85, 30.26, 38.42, 55.17, 63.29, 74.50, 84.21, 85.09, 87.21,

102.21, 113.21, 127.15, 128.01, 128.01, 128.12, 129.06, 130.01,

130.39, 131.36, 134.83, 135.04, 139.93, 144.04, 149.13, 158.64,

En zym a tic Assa y of Oligon u cleotid es (15-17, 21, 22).

Tr ea tm en t w ith Ca lf In testin a l Alk a lin e P h osp h a ta se.

An oligonucleotide (0.4 A₂₆₀) was dissolved in 50 mM Tris-HCl

buffer (pH 8.5, 200 µL) containing 0.1 mM EDTA. To the

mixture was added a solution (10 µL) of CIAP (4 units/µL).

The incubation was done at 50 °C for 1 h. After being heated

at 100 °C for 1 min, the mixture was analyzed by ion exchange

HPLC.

161.96, 168.71, 172.58, 176.28. Anal. Calcd for C₄₁H₃₈N₂O₁₁

3/4H₂O: C, 67.41; H, 6.12; N, 4.75. Found: C, 67.87; H, 5.97;

N,4.37.

‚

5′-O-(4,4′-Dim eth oxytr ityl)-3-N-Ben zoyl-2′-deoxyu r idin e-

3′-(2-cya n oeth yl)-N,N-d iisop r op ylp h osp h or a m id ite (11).

Compound 9 (4.44 g, 7 mmol) was rendered anhydrous by

coevaporations with dry pyridine (5 mL × 2) and dry toluene

(5 mL × 1) and finally dissolved in dry CH₂Cl₂(70 mL). To

the mixture were added diisopropylethylamine (2.44 mL, 14

mmol) and chloro(2-cyanoethoxy)diisopropylaminophosphine

(1.67 mL, 6.7 mmol). After being stirred for 1 h, the mixture

was diluted with CH₂Cl₂(100 mL) and washed with 5%

NaHCO₃and water. The organic layer was collected and

concentrated in vacuo. The usual workup followed by silica

gel column chromatography eluted with hexanes-ethyl acetate

containing 1% pyridine gave the title compound 11 (5.41 g,

93%): ¹H NMR (270 MHz, CDCl₃) δ 1.07-1.19 (12H, m), 2.34-

2.63 (4H, m), 3.43-3.84 (12H, m), 4.14-4.19 (1H, m), 4.71-

4.76 (1H, m), 5.44-5.45 (1H, 2d, J ) 8.3 Hz, J ) 8.3 Hz), 6.27-

6.34 (1H, m), 6.84-6.89 (4H, m), 7.18-7.66 (12H, m), 7.93-

8.01 (3H, m); ¹³C NMR (67.8 MHz, CDCl₃) δ 20.06, 20.18, 20.29,

24.30, 24.33, 24.44, 24.49, 40.20, 40.27, 40.40, 42.98, 43.02,

43.16, 43.22, 53.35, 55.11, 57.83, 58.10, 62.23, 62.41, 72.18,

72.42, 72.63, 72.89, 85.19, 85.28, 85.34, 85.54, 85.59, 56.88,

101.92, 101.96, 113.14, 117.32, 117.48, 127.04, 127.87, 128.01,

128.86, 128.99, 130.35, 131.32, 134.86, 134.93, 135.08, 135.11,

139.89, 139.95, 144.04, 144.08, 149.06, 149.11, 158.58, 161.98,

Gen er a l Meth od for T_mMea su r em en t. An appropriate

trideca(deoxy)uridylate (14-22) (0.603 A₂₆₀, 5 nmol) and

trideca(deoxy)adenylate (0.673 A₂₆₀, 5 nmol) were dissolved in

2.5 mL of buffer solution (10 mM sodium phosphate, 0.1 mM

EDTA, 1 M NaCl, pH 7.0). The solution was heated at 65 °C

for 5 min and annealed from 65 °C to 0 °C at a decreasing

rate of 1 °C/min. After being kept at 0 °C for 10 min, the

solution was heated to 65 °C at an increasing rate of 1 °C/

min. The UV-melting curve was measured at 254 nm as

temperature was increasing, and the T_mvalue was determined

by calculation of transition point. (In the case of a self-

complementary DNA, 2.0 A₂₆₀of oligonucleotide was used, and

the following conditions were used: 10 mM sodium phosphate

buffer, 0.1 mM EDTA, 150 mM NaCl (pH 7.0))

Gen er a l Meth od for Molecu la r Dyn a m ics Sim u la tion .

The present MD simulation was calculated on MacroModel 5.0/

Batchmin software using Indigo 2 workstation of Silicon

Graphics Inc.

MD Sim u la tion of U₇/d A₇(33) a n d d U₃U(2′-p )d U₃/d A₇

(34). To construct the duplex 34, a typical B-DNA duplex of

dU₇/dA₇was generated on MacroModel 5.0 and the R-side 2′-

hydrogen of the fourth deoxyuridine in the duplex was replaced

by a phosphate group in the monoanion form. (In the simula-

7494 J . Org. Chem., Vol. 65, No. 22, 2000

Tsuruoka et al.

tion of 33, a typical B-DNA duplex was used as an initial

structure.) The length of the hydrogen bonds of both 3′- and

5′-terminus base pairs was constrained on 1.9 Å. The energy

minimization of 34 was carried out using the united atom mode

on the Amber* force field and the GB/SA water model³⁰to give

a local minimized structure. It was used as the initial structure

in MD simulation. The first simulation was broadly performed

for 20 ps to equilibrate energy of the duplex. The detailed

simulation at the second step was carried out using the initial

structure obtained by the first simulation. The detailed

simulation condition was summarized as described below.

First Step. Mode: stochastic dynamics; time step: 1.5 fs;

shake on (H/Lp only); simulation temperature: 300 K; dielec-

tric constant: 1; solvation: GB/SA water model; simulation

time: 20 ps.

was generated on MacroModel 5.0, and the 2′-hydroxyl of the

fourth uridine in duplex was attached to a phosphate group

in the monoanion form. (In the simulation of 35, a typical

A-RNA duplex was used as an initial structure.) The other

simulation method and conditions were the same as described

in the case of 34.

Ack n ow led gm en t. This work was supported by

Toray Scientific Foundation, J apan Society for the

promotion of Science for Young Scientists, and a Grant-

in-Aid for Scientific Research on Priority Areas from the

Ministry of Education, Science, Sports and Culture,

J apan.

Second Step. Mode: stochastic dynamics, time step: 0.5 fs,

shake off, simulation temperature: 300 K, dielectric con-

stant: 1, sampling: 1000 conformer, solvation: GB/SA water

model; simulation time: 1000 ps.

MD Sim u la tion of U₇/A₇(35) a n d U₃U(2′-p )U₃/A₇(36).

To construct the duplex 36, a typical A-RNA duplex of U₇/A₇

Su p p or tin g In for m a tion Ava ila ble: Copies of ¹H and ¹³C

NMR data for 2c, 2d , 8, 9, 10; ¹H, ¹³C, and ³¹P NMR data for

4b, 4c, 4d , 4e, 5b, 5c, 5d , 5e, 6b, 6c, 6d , 6f, and 11; ³¹P NMR

data with respect to oxidative desulfurization; HPLC profiles

of oligomer 21, 22, and 24-28. This material is available free

of charge via the Internet at http://pubs.acs.org.

(30) Mohamadi, F.; Richards, N. G.; Guida, W. C.; Liskamp, R.;

Caufield, C.; Change, T.; Hendrickson, T.; Still, W. C. J . Comput. Chem.

1990, 11, 440-467.

J O991097E

Article Doi

Synthesis and Conformational Properties of Oligonucleotides Incorporating 2′-O-Phosphorylated Ribonucleotides as Structural Motifs of Pre-tRNA Splicing Intermediates

DOI: 10.1021/jo991097e

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Article abstract of DOI:10.1021/jo991097e

Full text of DOI:10.1021/jo991097e

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