A new synthesis of oligodeoxynucleoside methylphosphonates on control pore glass polymer support using phosphite approach

Tetrahedron Letters,Vol.24,No.9,pp Printed in Great Britain

A

NEW

SYNTHESIS

CONTROL

OF

PORE

POLYMER

Sinha,

Institute University

0040-4039/83/090877-04$03.00/O 01983 Pergamon Press Ltd.

OLIGODEOXYNUCLEOSIDE

GLASS

N.D.

877-880,1983

V.

METHYLPHOSPHONATES

SUPPORT

USING

GroBbruchhaus

of

Organic

of

Hamburg, D-2000

Chemistry

PHOSPHITE

ON

APPROACH

and

H.

Kijster

and

Biochemistry

Martin-Luther-King-Platz

Hamburg

13,

6

FRG

ABSTRACT trimer and tetramer blocks of deoxynucleoside methylphosphonodiDimer, d(AT), d(ATG) and d(ATTT) were synthesized on CPG polymer esters d(AC), support using deoxynucleoside methylphosphonomonochloridite. Only dinucleotide methylphosphonates were phosphorylated at 5' -OH with T4-polynucleotide These phosphorylated dimers hydrolized quantitatively with NaOH kinase. (0.5 M) in 15 minutes at room temperature. Nucleic

acid

nucleotide properties cesses.

analogues

bonds and The

have to

al

. 4, and

and

phosphonodiesters Reftina5)

using

condensation sired

product

communication

of

deoxynucleoside

in

pure would

form

methylphosphonodiesters

with

trivalent

rapid

now

been

using very

have

in

of the

time.

general

report

(1 mmol) and

after

hydrochloride

30

purposes

this

been in

were

first

polymer

in

inter-

biological are

Phenyl

needed

and

to

T'SO

by

Agarwal where

isolate

and

longer

the

observation). of

et

methyl-

phase,

synthesis

support

Recently by

synthesized

solution

pro-

nucleoside

synthesized

group.

al,so

or

thiophosphates3).

been

unpublished

the

The

was

of

approach

basis

of of

In

dethis

oligodeoxynucleo-

synthesis

prepared

THF

of

this

minutes was

at

and

strategy

according

intermediates

is

the

-78' to and

(5 mmol)

C under warm the

a77

support

yields.

CPG

in

large

yield

outlined

in

scheme

1.

follows:

report

and

was

CPG

from

has

in

bound

our

dry

in

N-protected

laboratory

THF

(1 ml)

6) . was

methyldichlorophosphine

atmosphere

slowly

filtrate

large

polymer

products

dissolved

Argon up

as

methylphosphonodiesters

is as to

(CPG)

reasonably

pure

(1 mmol)

allowed

filtered

give

synthesis

2,6-lutidine

(1 ml)

in

glass

deoxynucleoside to

the

pore 6)

oligonucleotides

synthesis

procedure

a mixture in

acids

above

method

controlled

N-acyl-5'-0-trityldeoxynucleoside to

nucleic

Rothermund,

to

using

triester

deoxynucleoside

added

of

have

sugar

physico-chemical

the

purification

introduced

synthesis used

short

have

at

study

phosphorus. we

phosphite

The

by

triester

(D.

like

either to

nucleoside

studied

extensive

side

for

been

modified

we

used

diesters

have

and

for

phosphonates*),

CD

their

time

Recently

used

methylphosphonate

PMR

and

interactions

analogues

nucleoside

deoxynucleoside

modification

synthesized

understand

nucleotide

phosphites'),

possessing

been

to

room

over

15

- 20

temperature.

concentrated

under

minutes Lutidine

reduced

878

Scheme

pressure. (10 to

After

ml,

l:l),

give

0.2

the it

M

tion

(1 ml)

was

(IO-20

Fmol)

removed

with

the

sation than

by

95%

by

bound

activated

was

M

3-5

minutes.

in

CPG

by

remove

any

for

minutes.

10

or

by

was

complete.

and

then

cycle The

was

washing

THF

introduced

was

Finally alone by

washing

(30-50

repeating

minutes

nucleoside

to

1.1

The of

with

ml)

was

the

above

was was

ml).

and

in

through

was

of

the

the

oxidized

(5-10

THF

The

process

with

(2 ml)

(30-50

period

of

the ml)

high

to vacuum

(7:3)

the

detrity-

mixture

the

until

through

until

next

more

with

ml)

under

nBuOH/lutidine/THF

conden-

in

CH2C12/CH30H

beads

again

When

complete

dried

either

time

charged

passed

then

performed.

The

reagent

A mixture

was

g)

column

ZnBr2

was

yield

pyridine

(20-30 g,

(5 ml),

passed

methylphosphonomonochloridite. 45

1.0

pyridine

solution

(1OO:l)

lation

nucleoside

with

with

ml,

nucleo-

minutes

and

diester

by

washing

solu-

concentration.

oxidation

THF 10

(5 mmol)

containing 15

The

cation

(40:20:1),

pyridine/lutidine.

(5 ml)

then

by g,

A saturated

nitromethane/water

was

removed

washing

remaining

10 minutes.

phosphite

(0.6

followed

for

After

evacuated

the

followed

column

was

achieved

was

2,6-lutidine

column

mg).

trityl

remained,

colour

(1 ml)

toluene/THF-mixture

septum-capped (100-200

the

was

I2

a

with

containing

methylphosphonomonochloridite

system

THF/pyridine/water

Excess

foam,

(5 ml)

measuring

DMAP/THF/Ac20/lutidine

no

to beads the

nucleoside

determined

iodine

syringe to

to

THF

nucleoside

pressure,

condensation

0.1

in

Active

added

Argon

evaporation

dissolved

solution.

side was

second

was

(4/I/5)

nucleoside

unit

appropriate

for

one

complete

1 hour.

methylphosphonodiester

chain

was

removed

from

the

support

879

Table Oligomer

yield

(%)

Amax

(nm)d)

amin

(nm)d)

Rf

a)

d(AC)

93

265

245

0.12c)

d(AT)

94

264

234

0.26')

d(ATG)

85

271

243

0.21

0

d(ATTT)

76

268

243

0.32

0

Solvent systems: a) CH CN/H20 = 8:2 (v/v); b) CHCl /CH OH = 7:3 (v/v diastereomers were vis 3 ble but did not separate a!? distinct spots;d] in 10 mM Tris-HCl (pH 7.5), 1 mM EDTA.

with

NH3

moved

(25%,

and

2 ml)

the

beads

at

liquid

was

plates

(acetonitrile/water:

The

band

was

We

have

used

We

and

at

5' -OH

ditions.

Rf- values

Only

(r-P 32)ATP

d(AC)

utility

on

were

12

hour-incubation

in

traces

tic

purine

this

method

silica

(7:3).

The

methylphosphono-

and

pyrimidine

d(AC), The

deoxy-

d(AT),

overall

d(ATG)

yield,

UV-

table. trimer

and

tetramer

quantitatively

phosphorylated. with

where

as

obtained

under

usual Even

T4-polynucleotide

d(ATG)

could

not

be

conunder

kinase phosphory-

d(AT)

solution

in

and

d(AC)

were

found room

to

15 minutes

at

to

characterization

hydrolize

temperature

as

completely

with

demonstrated

in

2. is

sequence

This

work

Bundesminister

in

progress analysis

of

achieve

oligodeoxynucleoside

of

degradation

products

methylphosphonates.

was

supported

fUr

Forschung

by

the und

Deutsche

Forschungsgemeinschaft

and

Technologie.

References

3)

gel

material.

Acknowledgment

1) 2)

re-

combined

water.

polynucleotide-kinase

d(AT)

was The

CHC13/CH30H

bidistilled deoxynucleoside

dimers,

T4

purified

synthesized.

the

ml).

non-nucleotidic

bound

of

were in

of

liquid

(2-3

all.

NaOH

Work and

CPG

the

and

with

pyrimidine

and

water

with

as

phosphorylated

Phosphorylated M

and such

eluted

with

given

with

was

figure

are

phosphorylate

d(ATTT)

0.5

was

as

the

to

conditions

at

well

supernatant

removal

lyophilized

condensation

the

pressure

for

plate

demonstrate

drastic

lated

reduced

and as

hours,

bidistilled

methylphosphonodiesters

attempted

above

15

with

80:20) the

purine

To

d(ATTT)

absorption

on

for

nucleosides. and

under

concentrated

monochloridites

C for

rinsed

concentrated

nucleotide

product

35'

were

; c) Two Measured

A. Holy and F. Sorm, Collect Czech. Chem. Comm. 32, 1562 (1966). Rammler, D.H. L. Yenogoyan, A.V. Paul and P.C. Bax, Biochemistry 1828 (1967). M.E. Wolff and A. Berger, J. Amer. Chem. Sot. 3, 5337 (1957).

5,

880

Figure 1: Electrophoresis was carried out with polyacrylamide gel (20%). Lane 1, 2 and 3 contained oligo dT chain length standard, d(AC) and d(AT) respectively. Mobility for d(AT) phosphonate diester was in the range of d(T-T) phosphodiester, whereas d(AC) moving faster than d(AT) phosphonate diester according to Qss retention of C compared to T .

4) 5) 6) 7) 8)

The

phosphorylated d(AT) and after elution from polyacrylamide gel, were taken up in bidistilled water (100 ~1) and treated with 1 M NaOH soln. (100 il). A sample of 10 ~1 was taken out from this and the hydrolysis was stopped with dried Dowex 50 WX8 resin (pyridinium form) at different time interval. The samples were spotted on silica gel plate, developed in CH CN/H 0 : 8/2 mixture, and exposed ta X-r$y film. Lane 1, 2, 3 and 4 represent reaction times of 0, 2, 5 and 10 minutes, respectively.

P.S. Miller, J.E. Yano, C. Caroll, K. Jayaraman and P.O.P. T'so, chemistry 18, 5134 (1979). K.L. Agarwal and P. Reftina, Nucleic Acids Res. 6, 3009 (1979). H. Kbster, A. Stumpe and A. Wolter, Tetrahedron Let., submitted publication. bzA = benzoyldeoxyadenosine, tlC = 2-toluyldeoxycytosine, ibG = isobutyryldeoxyguanosine, T = deoxythymidine. R. Frank and H. Kbster, Nucleic Acids Res. 6, 2069 (1979).

(Received in Germany 16 November

1982)

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for