One pot RP-diastereoselective synthesis of dinucleoside methylphosphonates using methyldichlorophosphine

Tetrahedron Printed in

ONE

Letters,Vol.30,No.41,pp Great Britain

POT

5587-5590.1989

Rr-DIASTBRBOSELBCTIVE

MBTHYLPHOSPHONATES

0040-4039/89 $3.00 Pergamon Press plc

SYNTHESIS

USING

OF

+ .oo

DINUCLEOSIDB

METHYLDICHLOROPHOSPHINB

Thomas L6schner and Joachim Engels’ J.W. Goethe-Universitlt, Institut fiir Organische Chemie, Niederurseler Hang, D-6000 Frankfurt am Main 50

Summary: Using diastereoselectivity

CHsPC12 and suitably protected is observed in the synthesis

Oligodeoxynucleoside use

methylphosphonates

as antisense

facilitates

their

hybridize

stronger

more

resistant

ODN-MePhos.

the

the

the

TpT case

outlined

Rp

containing

configurated The

synthesis

at

attractive

the

negative

and

on

electrostatic

based

DNA and

RNA. Their

. On the

other

diastereomers

at

substituted octamer

did

not

one for the

linkage

phosphorus phosphorus

binds

bind

at

favoured

analogues

stronger all

2.

Rp-isomer

for

on phosphorus

arguments

a distinct

each

n methyl

nucleotide charge

unnatural

hand

thymidine

Sp octamer

all nor

are

are missing

which

they

also

should

proved

disadvantage atom

to be

of these

resulting

atoms.

in

Recently

to

poly-A

Until

now

it was than

neither

is described,

2’ the any

except

for

3. this

goal

of a dinucleoside in Scheme

1.05equ.

1)

of two

ODN

an

Towards Rp-isomer

enzymes

anologue.

diastereoselective

cells

complementary

occurence all

phosphate

into

degrading

within

that

normal

with

is

diastereomers shown

penetration to

(ODN-MePhos.)

I. They

oligonucleotides

nucleosides at low temperature remarkable of dinucleoslde methylphosphonates.

1, (For

lequ.

we developed

a synthetic

methylphosphonate experimental

+4OC

under

conditions

procedure certain

see ref.

WI2

‘;

+4Tor-WC

+oon

2ichron~togr4~phy

Scheme

1

5587

which

conditions. abbreviations

favorably The

leads

general see

Table

to the

method 1).

is

5588

The

results

presented

synthesis

of

only

ratio

for

phosphorus.

The

TpT

Now

concerning

the

Table

1:

based

4.

At

we

the

have

time

and

RP-HPLC

up

the

absolute

other

configuration

bases

in

more

and

summarized induction

analysis

of the

in Table

1.

in the

synthesis

precipitated

Tr

MMTr

MMTr

MMTr

B’

T

T

T

T

CB2

CrIZ

CB =

AB 1

AB L

G’b’/NPE

B2

T

T

T

ABZ

T

AB Z

Gibn

T

A* =

T

RZ

32

Bz

Bz

BZ

TBDMS

Bz

BZ

BZ

and

plate

kinetic

The

aspects

with

small

from

moisture, observed

step

and

the

smaller

to

Rr-value

sillcagelon a silica

31P-NMR

with

purine

bases

showing

only

a small

are

fully

understood

more

not

of

free 1) are

exists,

one

another.

stable

and

even

isolatable investigated at

We found

phosphinic

-800, that

diesters

is

second

shift

induction yet.

This

(Fig.

leading

first

step

in all

cases.

Reactions

at

a trigonal-bipyramidal Furthermore because in as the 2&&

situ a l/l

by

mixture

are

formed

low

to

79%

even

at

trivalent

diastereomeric

sensitivity

ZIP-NMR

induction

at

transition

the

of their

reveals

highest

step

(A,G) in the

nucleophiles. not

were

laJlJ

within

used.

induction

is less

intermediates

the

The

reaction. first

the

with

nucleosides

in the

pseudorotations

attack

that

the

the

(T,C)

(Scheme

indicate

of

and

bases

&/a

equilibrium when

higher

with

after

isolated

the

temperature

reaction

due

the

and

Therefore

In rapid

were

one with

as eluent

is worst

of this

complicated

la/lb

diastereomers the

always

on the

Deoxyguanosine

resulting

second

and

aspects

are

individual is

diastereoselectivity

mechanistic

intermediates

depends

stereochemical

(-8O*C)

low temperatures. phosphorus

the

Rp-isomer

&-isomer.

excess

and

TBDMS = tert.-butyldlglethylsilyl. thymine, MMTr = monomethoxytrityl) Tr = trityl,

cytosine, T = Bz = benzoyl,

CHC1&.4eOH mixtures

to the

the

best.

The

using

relatively

temperatures

assignment

by

of dinucleoside

Tr

C =

detail

product

Tr

TBDMS TBDPS

on

stereochemistry.

Tr

diastereomeric

the

work are

with

resulting

the

for the

diastereomeric

Tr

The

being

after

assign

methods

unequal

Tr

layer

signals

to

new

an

Tr

thin

and

unable

we developed observed

R’

RP-chromatography.

state

where we

reaction

the

Temperature and base dependent methylphosphonates in TAF

identification

The

work Here

this and

obtained

spectroscopy

and

at

we were

excess

ratios

For

de.

earlier

investigated

(A = adenlne. G = guanine, TBDPS = tert.-butyldlphenylsilyl,

1, a)

on our

methylphosphonates.

diastereomeric

diastereomeric

31P/lH-NMR

are

dinucleoside

to

(Fig.1).

air The

of diastereomers observed from

arises &‘a

in after

5589

adding does

the not

second

change

nucleoside.

The

the absolute

la/lb

final

oxidation

stereochemistry

proceeds

2al2b

,-a

with

retention

of configuration

and

5.

3b

--J

+

3a RP

23

202

20:

236

Fig.

1 Diastereomeric intermediates la/& and a/& and the final dimers &fa in the synthesis of a TpT dimer measured at -8OoC by s1P-NHR at 121 HRz in THF with 8512 FIsPOI as external standard.

The

stereochemical

the

free

primary

oxidation

of

induction.

The

investigated The

the

stereochemical Even

induction

stereochemistry

diastereomers

of the 6.

of

the

(results

of

this

literature isomer

data was

large

pair.

additionally

TpT

l/l

demanding

CHJPCIZ. mixture

for

where

Adding

in

the

the

protection

at

of 6/l.

alcohol

changes

HO-T-062

Tr-T-OH

instead

secondary

of

TBDPS group

individual

investigation the

with

experiment

in

alcohol of the

This --la/lb protecting

3’-alcohol

with

-80°

and

shows

the

for

the

groups does

not

1).

Due

6’-nucleoside-part

for

following

in a

independant

dimers

We investigated

of a dimer

protons

the

(Table

first

results more

is

by the

reacted

&/a

stereochemistry

ROESY technique

supported

group

intermediates

so far. the

were

5‘-alcohol

of the

importance

diminish

aspects

will dimers

provided

the to

was

different

we

could

be

published

7 and

determined

environment

the

by P.S. Miller

by PD-NMR using

of

the

distances

assign

the

to

the

structure

as an authentic

The for

the

groups

one

sample

were

the

H3’ and

of the

results

NOE derived

within

neighbourlng

stereochemistry

elsewhere). X-ray

methyl

two H4’-

diastereomers compared

ApT dimer

s.

for comparison

This with

with ApT our

5590

corresponding

dimer.

prove the usefulness

Our results

are in Pull agreement

With this ROESY NMR method the stereochemistry were determined

with these

“standards”

and allow us to

of our method. from the dimers

so far. CpT and CpA were assigned

TpA. CpG is not known for sure

so far because

of

corresponding differences

of TpT, TpA, ApT and ApA to the induction

with literature

of TpT and

data

g, but is

under investigation.

AcknowledPement We thank Dr. P.S. Miller (Johns Hopkins University, Baltimore) for providing a sample of the X-ray ApT isomer and Dr. G. Zimmermann for recording the NMR spectra.

References Biochemistry 25, (1986) 62681) a) C.H. Agris, K.R. Blake, P.S. Miller, M.P. Reddy, P.O.P. Ts’o 6276. b) P.S. Sarin. S. Agrawal. MP. Civeira. J. Goodchild. T. Ikeuchi,P.C. Zamecnik. Proc. Natl. Acad. Sci. a, (1988) 7748-7451. c) For a comprehensive review see G. Zon in Pharmaceutical Research 3 (1988) 539-549. 2) W.J. Stec, presented at the Conference for Recognition Studies in Nucleic Acids, Sheffield, England, 1989. 3) Z.J. Lesnikowski, P.J. Wolkanin. W.J. Stec Tetrahedron Lett. 28s (1987) 5535-5538. 4) J. Engels, A. JHger Angew. Chem. Suppl. (1982) 2010-2015. Tetrahedron Lett. (1963), 2177-2180. 5) D.B. Denney. J.W. Hanifin 6) C.Grieslnger, R.R. Ernst Journal of Mag. Resonance 75, (1987) 261-271 and references cited therein. 7) Z.J. Lesnikowski. P.J. Wolkanin, W.J. Stec in “Biophosphates and Their Analogues-Synthesis, Structure, Metabolism and Activity”; K.S. Bruzik, W.J. Stec (Eds.); pp. 189-194, Elsevier, Amsterdam 1987. 8) K.K. Chacko. K. Lindner, W.Saenger, P.S. Miller Nucleic Acids Research ll, (1983) ??801-2814. 9) L. Callahan, F. Han, W. Watt, D. Duchamp, F.J. Kezdy, K. Agarwal Proc. Natl. Acad. Sci. & (1986) 1617-1621. (Received

in

Germany

5 July

1989)