A new class of condensing reagents for rapid internucleotide bond formation in the phosphotriester approach and preparation of n3-benzoylthymidine as a key intermediate in oligodeoxyribonucleotide synthesis

Tetrahedron Letters,Vo1.25,No.36,pp Printed in Great Britain

A NEW CLASS

OF CONDENSING

IN THE PHOSPHOTRIESTER AS A KEY

Jun-ichi

Matsuzaki,

0040-4039/84 $3.00 + .OO 01984 Pergamon Press Ltd.

REAGENTS

FOR RAPID

APPROACH

AND PREPARATION

INTERMEDIATE

Department

4019-4022,1984

Hitoshi

Mitsuo

Hotoda,

Midoriku,

Tokyo

Sekine

SYNTHESIS

and Tujiaki

Institute

Yokohama

BOND FORMATION

OF N3-BENZOYLTHYMIDINE

IN OLIGODEOXYRIBONUCLEOTIDE

of Life Chemistry, Nagatsuta,

INTERNUCLEOTIDE

Hata

of Technology

227, Japan

bond formation in the phosphotriester Summary: Rapid internucleotide approach has been achieved in high yield by use of bis(2,4,6_trihalophenyl) phosphorochloridates (TCP and TBP) as new condensing reagents and the benzoyl group as the N -imide protecting group of thymidine.

In the rapid

oligodeoxyribonucleotide

synthesis,

the essential

determing

step is the construction

linkage.

One of the exciting methods is the phosphoramidite approach 1) 2) This approach and the other research groups. by Caruthers

developed provide

an internucleotide

oxidation

bond

instability

disadvantages;

step after

rapidly

method

seems

phosphotriester

linkage.

phosphotriester

and quantitatively,

of the phosphoramidite

each coupling

phosphotriester

of an internucleotide

rate-

reagents

On the other

reaction.

to be yet intriguing

Only drawback

since

of the latter

but has

can

some

and a necessity hand,

the

it gives

method

of

directly

a

is a relatively

To accelerate slow rate of condensation. such as tetrazole 3a) and N-methylimidazole Condensations little

at elevated

attention

An plausible chlorides

explained with

mechanism

or azolides

pyrophosphate

as a valid

to exploration

of the condensation

was proposed

produced

the hydroxyl

temperatures

has been directed

the coupling reaction, catalysts 3b) have been employed. have also been reported. 4) However,

by Knorre

from bimolecular intermediate

function.

Such

of condensing reaction

using and coworkers: 5)

phosphodiester

TCP

by azoles

pyrophposphate

TBP

4019

A symmetric

components

that can be activated tetrasubstituted

reagents. arenesulfonyl

was to react species

can

4020

be prepared

more

monofunctional

the dialkyl

as a good

leaving

phosphorus focussed

group

condensing

reagents.

found When

hydroxyl

components

[DMTrT(SPhj21g),

min) .

However,

for the rapid

derived

the reaction

considerable obtained

the case of uridine into

residue

of the thymidine

presence

of diisopropylethylamine

be prepared

active

triester

from

the

asymmetric

bond

in the phosphoro-

internucleotide

(TBP)*)

bond

formation.

(NT) was employed (O-HNEtl)]

the previously

reported

in a short

of some byproducts

were

time

thymidine (within

observed

usuful

benzoyl

(2 equiv.)

synthetic

by a modification

0

we tried to introduce an as in the case of uridine. 12)

chloride

(2 equiv.)

in pyridine

in the

for 15 h gave 2 in

intermediate,

N3-benzoylthymidine 2, 13) of the procedure of Jones: Thymidine

91%

O=bt SPh)2

1 0

HO

on TLC

side reactions seemed to be of thymidine 10) as reported in

of thymidine

unit L with

Et3SiCI

5%CF3C02H

/ )f2NEt >

5

These

DMTrO

NH .A N ’

in the

and the

0

v

was

of the imide function and guanosine. 11) Therefore,

the imide

A more

our attention 6) as new

phosphorochloridate

was completed

amounts

to serve

on the

to react with

a more

[DMTrTp(SPh)

in 76% yield.

Reaction

91% yield.

selectively

bis(2,4,6-trichlorophenyl)

from reactivity

acyl group

could

expected

the desired

the phosphodiester

unit L

was

may be expected

each of TCP and 3-nitrotriazole

between

[Tp(SPh)2]

effective

with

electron-withdrawing

Therefore,

to produce

smoothly

component

phosphorochloridates

phosphorochloridates

were

Consequently,

to be very

and the dimer

reagents

give

occurs

and bis(2,4,6-tribromophenyl)

1.3 equiv.

condensation

diary1

in pyridine

that could

(TCP)')

with

[(R0)2P(0)O-]

component.

hindered These

of an azole.

chloridate

caused

is substituted

so that condensation

component

pyrophosphate

of the phosphodiester such as dialkyl

residue

of the phosphodiester

phosphodiester

were

agents

phosphate

on sterically

presence

by reactions

If the R group

[(RO)2P(0)Cll. groups,

directly

phosphorylating

> OH 2

87%

402 1

1

DMTro$

+

Hog2

cond;;::

reagents

I3

a : Thb’

DMTro--Q2

b : Cyan ?

O=b-SPh

iPh

(Bz)

5

0

O=bfSPh& (Bz)

5 Table

I. Results

of Coupling

hydroxyl component

phosphdiester (equiv.)

treated

with

chloride

1 h, the mixture

evaporated

pyridine

was

in CH2C12-CH30H silica

(7:3, v/v,

gel column

trimethylsilyl stage.

The N3-benzoyl

and neutral

50 ml)

conditions

resulted group

was

triethylamine-water-pyridine smoothly

removed

tritylation

phosphinic gave

removal

acid

the diester

components

were

A mixture

in pyridine

was monitored

on TLC.

was quenched

protected experiments enough

dimer

of 42

A. spot of s by addition

was obtained

are summarized

to shorten

all the protective

groups

from

2 by treatment

yield.

with

Treatment 2

of 2_ in

in 86% yield.

5/a (0.2 mmol),

dissappeared

was

within

The results

I.

Only

a small

reaction

with

good

the thymidylate

added

of NT (0.26

dry pyridine and the reaction

5 min,

and the

manner,

the fully

of the other

excess

yields.

dimer

and NT

with

In the usual

5 M

at 40 'C for 15 min

component

coevaporations

of water.

in Table

be

The usual

by TCP or TBP in the presence

in 95% yield.

the coupling

and 2% TFA/CHC13) with

salt in 88% yield.

(0.26 mmol)

by

at the acylation

(2:1, v/v)

(0.24 mmol),

TBP

followed

The use of

(80% AcOH

from

for

(TFA)

gave 2_15) in 85% overall

group

by repeated

(2 ml).

workup

at r. t. for 1 h.

the hydroxyl

condensed

stirred

to remove

at r. t., but yet could

NH40H

for 30

5% CF3C02H

by treatment

soln.)-triethylamine

anhydrous

and dissolved

reaction

cont.

as triethylammonium

rendered

with

mixture

in acidic

released

by phosphorylation

$ and 2 were

in good yields. mmol)

stable

being

toluene

treated

in a complicated

at 0 "C for 5 min gave

2% TFA/CHCl, These

s

with

The usual for 20 min. gave J_14) in 87% yield.

with

88 88 95 87 99

in pyridine

After

added.

further

of one phenylthio

(pyridine

was

(2:1:2, v/v/v)

by treatment

of 3_ followed

The selective

was

and gradually

yield (%)

10 15 15 15 10

(13 mmol)

and coevaporated

chromatography

chloride

chloride

(7.5 mmol)

The residue

completely.

TCP or TBP time (min)

3'-0,N3-Dibenzoylthymidine

trietylsilyl

min and then benzoyl

Using

NT(1.3) NT(2.0) NT(1.3) NT(l.O) NT(l.O)

TCP(1.3) TBP(1.3) TBP(1.3) TBP(1.3) TBP(1.3) *5'a:

was

Reactions

condensing reagents (equiv.)

5'a* 5a 5'a* 5a 5a

4a(1.2) 4b(1.2) 4a(1.2) 4a(1.2) 4b(1.2)

(5 mmol)

0

O:P4 SPh$

4 ti,NE13

amount

of TBP was

Deprotection

was performed

as

of

4022

follows:

1) 0.2 M NaOH-pyridine

neutralization 3) I2

(30 equiv.)

followed acetic

with

Dowex

5OW-X8

treatment

by removal

(l:l, v/v)

in pyridine-water

of the excess

acid at r. t. for 15 min.

(HCO; form)

gave

the pure

14 mg of the protected

at 0 "C for 20 min

(PyH+ form):

iodine

purification

dimer,

NH40H

(2:1, v/v)

by extraction

Finally,

thymidylate

2) cont.

TpTp,

followed at r.t.

by

for 1 h;

at r. t. for 1 h

with

benzene;

4) 80%

by DEAE-Sephadex

(103 A260

0. D. units

A-25 from

dimer) .

Reference and Notes Lett., 22, 1859 (1981): 1) S. L. Beaucage and M. H. Caruthers, Tetrahedron L. J. McBride and M. H. Caruthers, ibid., 2, 245 (1983r 2) S. P. Adams, K. S. Kavka, E. J. Wykes, S. B. Holder, and G. R. Galluppi, J. Am. Chem. Sot., 105, 661 (1983): T. Dorper and E. L. Winnacker, Nucleic Acids Res., 11, 2575 (1983): J. L. Fourrey, and J. Varenne, Tetrahedron Lett., 24, 1963 (1983). 3) a) J. Stawinski, T. Hozumi, and S. A. Narang, Can. J. Chem., 54, 670 (1976). A. Seth and E. A. Jay, Nucleic Acids Res., 8, 5445 (1-O). and 0. G. Chakhmakhcheva, Tetrahedron b) V. A. Efimov, S. V. Reverdatto, and S. Tachibana, Chem. Pharm. 961 (1982): T. Wakabayashi Lett., 2, Bull., 30, 3951 (1982). T. A. Millican, C. C. Bose, R. C. Titmas, G. A. Mock, and M. 4) T. P. Pxel, A. W. Eaton, Nucleic Acids Res., l&, 5605 (1982). V. P. Romanenko, and V. F. Zarytova, 5) E. M. Ivanova, L. M. Khalimskaya, Tetrahedron Lett., 23, 5447 (1983): V. F. Zarytova and D. G. Knorre, Nucleic Acids Res.,-72, 2091 (1984). diphenyl phosphorochloridate has 6) Since a long time ag;jS unsubstituted been used as a condensing reagent by Todd and Michelson. For a and Nucleotides", comprehensive review see "The Chemistry of Nucleosides A. Il. Michelson, Academic Press, London, pp. 400-443 (1963). 7) R. Anschiitz, Ann., 454, 106 (1927). N. P.xlesink, L. G. Dubenko, and M. I. Povolotskii, Zh. 8) E. S. KOZ~OV, Obshch. Khim., 49, 769 (1979). 9) M. Sekine, J. Matsuzaki, and T. Hata, Tetrahedron Lett., 22, 3209 (1981). 10) C. B. Reese and A. Ubasawa, Nucleic Acids Res. Symposium Series, 1, 5 (1980). recently, p-nitrophenylethyl has been reported as the protecting 11) Quite group of the thymine residue by F. Himmelsbach, B. S. Schulz, T. R. Charubala, and W. Pfleiderer, Tetrahedron, 40, 59 (1984). Trichtinger, 12) T. Kamimura, T. Masegi, K. Urakami, S. Honda, M. Sekine, and T. Hata, and J. Chattopadhyaya, Acta Chem. Lett., 59 (1983): C. J. Christopher Chem. Stand., B 37, 147 (1983). and R. A. Jones, J. Am. Chem. Sot., 104, 1316 13) G. S. Ti, B. Lxffney, (3982). 1 H NMR (cDc~,-CD~OD)~ 7.88-7.31(6H, m, ArH and 2: 14) N -Benzoylthymidine 4'-H), 6-H), 6.22(1H, t, J=GHz, l'-H), 4.39(1H, m, 3'-H), 3.91(1H,19, 3.78 (2H, m, 5'-H), 2.32(2H, m, 2'-H), 1.93(3H, S, 5-CH ). C NMR (CDCl -CD OD)d 169.15(C=O), 163.24(4-C), 149.54(2-C), rj6.81(6-C) 135.2$(4-&Bz)), 131.44 (l-C(Bz)), 130.46(2,6-C(Bz)), 129.28(3,5-&Bz)), 110.91(5-c), 87.34(4'-C), 85.82(1'-C), 70.81(3'-C), 61.77(5'-C), IR (KBr)ul749 (PhC=O), 1700 and 1653 (imide 40.42(2'-C), 12.46(5-CH ). Anal. calcd for 2 17~1806~2: c, 58.96; H, 5.24: N, 8.09. found C, C=O). 53.47; H, 5.40; N, 7.93. thymidine unit 2: 'H NMR(CDC13)6 7.22-7.96(25H, m, ArH), 15) N -Benzoylated 6.84(4H, d, J=8Hz, 4,5-H(DMTr)), 6.36(1H, t, J=6Hz, l'-H), 5.36(1H, m, 3'-H), 4.1O(lH, m, 4'-H), 3.78(3H, s, CH O(DMTr)), 3.41(2H, m, 5'-H), calcd for C50H450gN S P: C, 2.42(2H, m, 2'-H), 1.39(3H, s, 5-CH ). d 65.78; H, 4.97; N, 3.07; S, 7.02. faund C:aig.03; H, 5.20; N, 3.08;2S, 7.13. (Received

in Japan

26 May

1984)