86 $3.oo + .oo Pergamon Journals Ltd. 4705

Tetrahedron Letters,YoI.27,No. Printed in Great Britain

0040-4039/86 $3.oo + .oo P e r g a m o nJ o u r n a l s L t d .

39,pp 4705-47O8,1986

CHEMICAL 5' -PHOSPIIORYLATIONOF OLIGODEOXYRIBONUCLEOTIDES THAT CAN BE MONITOREDBY TRITYL CATION RELEASE

Thornas Horn and Mlckey

Chiron Research Laboratories,

S. Urdea'

Chiron Corporation,

Eneryvllle,

4560 Horton Sgreet,

CA 94608, USA

ABSTMCT: A new phosphoramidite-derived -O-4,41reagent, (2-cyanoethoxy)-2-(2r dinethoxytrityloxyethylsulfonyl)ethoxy-NrN-dilsopropylaminophosphine, for the 5,phosphorylation of oligodeoxyribonucleotides has been developed. Phosphorylation efflciency can be deEermined by the release of 4,4r-dinethoxytrltyl catLon in acld. 5r-Phosphorylation the

5r-hydroxyl

Ehis

ls

forrn is

conducted

Alternatively,

versus

purificaclon retenElon

of tlme

mobillty

between the

the coupllng synthesized

shlfE

This

for

phosphorylaElon

a phosphorylatlon cation is

reagents reactlon.

is

usually

as

Usually kinase.

fully

of

protected

Ehat employ

Also, for

afEer

acld

WheEher or not

an additional

polynucleoEides

(1)

purlficatlon

time of

the 5r-

and

1n Ehe rnobllity larger

or

than 20 bases.

be monitored

deprotection

that

the 5r-

after

work-up

che difference

deEermlned.

DMTCi DMT-OCH2CH2SO2CH2CH2 OH PYRIDINE

to dage is

determined

reagenE Ehat could

tradltionally

reported

the HPLC retention

requires

ollgoner.

Ewo forms ls minimal

DNA synthesis

applications.

ATP and To polynucleotide

or by a change in

species.

lhe unphosphorylated

HO CH2CH2SO2CH2CH2-OH

wlth

trrost chernical phosphorylaelon

the 5r-hydroxyl

durlng

ollgoners

which are syntheslzed

biologlcal

chemisEry (2).

the orange 4,4r-dfunethoxytrityl

nucleotldes

molecular

can be used on automated DNA synuhesizers

way to rnonltor

We chose Eo deslgn of

for

have been developed

fragrnent was actually

by an electrophoretlc phosphate

purlfled

phosphoramldite

no convenient

phosphorylated

oligodeoxyribonucleotldes

necessary

by treaLing

One drawback ltith ls

often

sorne of which

solid-supporced

there

synthetic

chernlcal nethods

(lr2),

fragments

of

by the release

the same way coupling

of

A1so, we were interesEed

ln

R = COCH2CH2CO NH CPG R'=H

ruco-r2cu2o-e-r( 14 ct

R

_1 OIPEA

DMT-O-CH2CH2SO2CH2CH20. TR P.N 'R NCCH2CH2O'

(3)

O B O B R =,P-o+---, F o ,,l-o cocx2cn2co'NH cPG o-cHs o,cH3

R= - 9 H c H 3 CH: B HO+OR '

,r,

Q

B

R= Po+ o" ====+=:TFTRAZoIF

A

q

o-P-o+oH o-

DMTO.CH2CH2SO2CH2CH2O.B P.OrrOR NccH2cH2o €)

D M T O C H 2 C H 2 S O 2-C H -'F-o1on 2CH2O\:?^B^^. NH4OH NCCH2CH2O

O

B

=oo|f,; oPotoq O-

Scherne I

4705

(4)

tt

,

LUTTD|NE

NH4oH 60"C.18h

o i o,lt o* o_

4706

fully

reagent

the

naklng

Since removal

of

protecting

of

more rigorous

requires

(4).

co hydrolysis

resistan!

being parlicularly

protecting

potenlial

Another

possible

is

phosphate diesters

in

former

the

in

protection

ether-conlaining

to our needs appeared to

be tailored

by Tesser et al

(5,6).

As for

the 2groups from

and arylsulfonylethyl

alkyl-

of both

compleEe elimlnation

function,

cyanoechyl

elirnlnated

thaE could

function

group reported

be rhe alk(ar)ylsulfonylethyl

(2)'

from a phosphate diester

these funclions'

of

phosphorus based on elther

scheme for

can be --

groups that

protecting

a trityl

to derive

would however be difficult

It

NH4OH.

can be fully

it

since

attractive

are particularly

have proven useful

and p-nitrophenylethyl

such as 2-cyanoethyl

eliminated

the choice

deprotecEion,

phosphate diesters

Ehis reason that

for

forrn Cypically

fhe diester

agents has been an important

and sirnple alkyl

is

It

in

triesEer

than {nternucleotide

aromatic

Halogenated

consideration.

from phosphate

phosphorylating

for

These

groups where one

blocking

a dialkylaminophosphine.

functions

conditions

groups

blocking

suiEable

for

ether

a dimethoxytrityl

(3).

chernlstry

s u g g e s L e d t h e u s e o f a s e E o f N H 4 O T {s e n s i l i v e

requirenents carries

phosphoramidite

with

compatible

N H O O Hs o l u t i o n .

Thus, we designed and prepared (2-cyanoethoxy)-2'(2r-4,4'-dirnethoxyltiLyloxy and tested

ethylsulfonyl)eChoxy-N,N-diisopropylaminophosphine(3) (Scherne I).

reagent

To solid

standing.

in

stirring

chloride for

the dark

dissolved

residue

in

ethyl

phase was dried

the producE was purified

solvents

by silica

gel

was left in

was then concentrated

(500 n1) was extracted

acetate

(150 m1) was added

pyridine

50 rnrnole)and the mixture

solution

The reacEion

l8h.

on

which crystallized

58.6 nmole) in

(DMT-CI; I6.95 g,

aq. NaCl and the organic

salurated

(10.6 g,

sulfonyldiechanol

4,4'-dimethoxyrrityl

oil

to give a viscous

dry acetonitrile

with

coevaporation

by repeated

in HrO) was dried

(65% w/v

sulfonyldiethanol

available

Commercially

of

as a phosphorylatlon

it

with

The

vacuo.

5 % a q . N a H C O 3a n d 8 0 2 rernoval

After

over anhydrous Na2S0O'

10.0 g of

column chromatography to give

purel(TLC,si1lca]rnCH2CI,;Rf=o.015).Ch1oro-N,N-diisopropylarnino-2-cyanoethoxysolution of I (4.5 phosphine 2 (4.6 mrnole) was added rapidly under argon to a stirred (10 rn1) at (DIPEA; 4.6 mnole) in rnethylene chloride mnole) and N,N-diisopropylethylarnine acetate (50 w i L h e t h y l 0 C. The solution was allowed to warm to room temperature, diluted nl)

and washed with

acetonitrile

and then

(2 x 20 n1).

into

aliquoted

The organic

1.5 rn1 septum-sealed

was removed by evacuatlon

added to a detritylated iodine 90 sec.

and thorough

spectroscopy suggesting at

a coupling

NMR sEudles

indicated

collected

efficiency

2O C and the supernatant

obtained

of

96%.

synthesis

of

and

$7lth aqueous

5Z w/v DCA in

Cl{rCl,

for

absorption

the original

thymidine

support

was exposed to 2 m1 of NH4Otlfor 2h 3IP5h. TLC, HPLC..rd an additional

The polyner

was healed at 60 c for

quantitative

2 mL of

of

under

Purlfication. in dry acetonitrile

was compared by visible

from deprotection

each containing

was stored

sEandard oxidatlon with

Ehe supporE was treated

orange solution

to the naterial

After

C?G support.

thyrnidine

washing,

The brighC

0.75 ml of 0.5 M lH-teerazole

with

with

was dissolved

Wheaton vials

and the product

The solvent 100 {mo1e of 3. .argon at -2O C (7). This crude product was used $rithouc further Reagent 3 was activated

phase was dried

product

The oily

evaporation.

by rotary

N a T S O Oa n d c o n c e n t r a t e d

anhydrous in

aq. Nacl

8oz saturated

thymldine

5'-monophosphate

without

4707

TABLE I.

Conpound

sol

T

Ho+oH 9 r Ho,?o+oH H

o

o

r

"u.o[,oio" rio.j-oa no

ra

sol

(R- :min)

rra

o'90

o'77

1'66

0.59

0.14

0.67

0.69

0.29

1.84

0.70

0.30

l.l0

o r our-o.esr-o,F,o.l-on ieo-'-

a ) N H a o H , 2 o . 0c . 5 9 , 0 . 6 9 ' b)NH4oH,6ot

0.59

g i -O+OH oMT-O.ESE,O.f cE.o

a)NHaoH,2o.c

N.D.

b )N H 4 o H , 6 o ' c

0.70-

3lp-Nlg"

HPLcb

T L C' (Rr. )

0.33',0.29,0.14

( oom)

4.01 +0.7(pyridinium salr)d -6.0,-5.9(Nnf-sa1t)d

I.94,O.67

0.14

0.67

N.D.

4.10,4.50

0.22"

0.70

N.D. 4.025 N.D.

a

_43.25

cE - NCCH2CH2. ESE--CH2CH2SO2CH2CF12. a) Sol I and Sol 7.5, respectively;

(v/v) are 8:2 and 9:l b) Cl8 reverse phase;

II

isopropanol: 0.1 M Triethylanrnonium 2.5 to l)'i( CH"CN in 0.05 M NH/HrpO/,

c) relarive 8.8; (10) d) DTNB in I M NMR spectrum

ro I M H"Po, in D"o; measured in Dro,"reTative'to H.Po4; e) Tris,-pH 8.0; f) DI"ffp5sitive was obEained if detritylation

concomitant

producEion of phosphonate or partially

When deprotection only,

a mixture

thyrnidine

If

5t-phosphorylated

sulfur

? l

evidenced by'^P-NMR

for

(f0).

naleirnido-

(the

of

to

Lhe 5'-phosphorothioate

coupling

efflciency

purlfied kinase

The use of permils

phosphorus

a low coupling. unmasking of phosphorylation investigatlng

Although

a new hydroxyl is other

of T4 DNA ligase

easily sinilar

the material

Thus lt

by the lack

is

of startlng

noiety

not

that

ideal

Ehe side-product

since

obtained

protecting

with

by

was deprotected

and

T4 polynucleotide

the chemically

if

and

ligatlon. reporEed here concomitant

rnoniloring results

in

reveals the

an additional

upon irork-up.

groups for

(12).

to check the

after

reaction

detritylation

and

Both sequences are near

materlal

the

as

on an automated

can be removed without

to repeat

removed frorn the oligonucleolide blfunctional

l.

I)

manner.

chemistry

group such as sulfonyldiethanol

possible

compound I is group,

lhis

k'ith

using

Figure

(8).

exchange can be

3, detritylated

reactions

are presenced in

protecting

of a noniEoring

deprotection.

of

(tabl.e

Solid-supported

phosphoranidite

reagent

The second half

fragments

as evidenced

a bifunctional

che introduction

(11).

The producE was then purified

deprotected.

The PAGE analyses

phosphorylated

fully

and disulfide

forrn which was then 5r-phosphorylated

phosphorylated

enzyr:natically

rrith

60 C NHaOH was obtained

a reasonably good

ls

sequence GGATCCGGATC wC as synthesized

electrophoresis.

as the 5r-hydroxyl and ATP.

reagents

at Ehis functlon

was phosphorylated

20 C

the 5r-phosphltylated

probes have been produced in

oligonucleotide

and fu1ly

gel

with

derlvative

in phosphorothioates

speciflcally

BarnHI linker

Lhe support

step (9),

converted

Geno-O-Matlc) using a solid-supported

One-half

polyacrylarnide

\.ras deprotected

the oxidation

and bromoacetyl-concaining

N H 4 O Ha t

and 2-cyanoethyl-5r-phosphate

in

Slnce the sulfur

labeled

The palindrornic instrument

was conducted with

iodine

used to modify oligonucleo!ides non-radioactively

thymidine

(Table I).

nucleotide

a mixEure of both 5r-phosphonate and nucleoslde

was quantitaLively

nucleophile,

protected

thymidine

5r-phosphitylated

oxidation,

By substltuting thymidine

in 502 D,o add 507. I v1 Tris-frc1-0105M ED'|A, pH becomes'bright ye11ow when sprayed with l0 mM a s e v i d e n c e d b y 1 0 7 "H ' S O , s p r a y ; g ) a n i d e n t i c a l was perforrned prior to NIIooH treatnent.

of boEh 2-(2r-hydroxyethylsulfonyl)ethyl-

was noted.

prior

without

of

pH

acetate pH 4.0;

We are

chernical phosphorylatlon