Polymer support oligonucleotide synthesis VIII use of polyethylenglycol

Tetrahedron

Letters

No. 16,

pp 1535 - 1538,

POLYMER

SUPPORT USE

1972.

OLIGONUC OF

ftir Organische

Chemie

2 Hamburg

In our previous for

oligonucleotide

kinds

of polymers

might

occ:llr

the

meric

carrier.

of molecular recently mental

reaction

the s

,

for

given

be synthesized In order the chemical

this

the

deprotections

of

the determination cleavage

in buffer without

1

is bound material

systems

any damaging

penetrate

(PEG) Very

a few experi-

carrier

has

the

the condensation

in homogeneous by simply

in water

synthesis

or buffer

for

short

can be calculated

condensation

poly-

chain. reaction

and oligo-

by the Thus

dis-

relation

loss

of.

can be avoided

can be used

will

under remain

to the very

in order

very

mild

to drive conditions;

in the dialysis

surrounding sensitive

liquid.

the

tube, As

the while

dialysis

oligonucleotides

can

effects.

to anchor

the oligonucleotide

synthesis

( for

enzymatic

to the glycol

measured

synthesised

holds centre

group.

only

of this

chain

of the

by dialysis

temperature

bound

might

is necessary

component

will

this

can be done

in DNA of the

max

to the carrier

at low

yield

in a given

as it can be removed

which

but

Because

groups

The

same

the active

blocking

synthesis,

oligonucleotide

reaction

polyethylene

can be quickly

material

of the low-molecular-weight

product

we selected

occuring

yield

being

In these

problems

The

, where

of oligonucleotides

vessel.

nucleotidic

4)

substrate

peptide

insoluble.

2) , diffusion

species.

3,

blocking

at the

of the of

one

carriers

of a condensation

reaction

nucleotides

coefficients

completely

porosity

and dioxan.

bound

in a quartz

extinction

are

polymeric

as a macromolecular

synthesis

its

8 March 1972)

hydrophylic

carriers

with

phase

of the condensation with

of

the yield

problems

acting

the solid

For

all the four

the low-molecular-weight can be done

contact

pyridine

absorbance using

.

pubiication

which

-

Hamburg

Germany

of the phosphorylating

in water,

carrier

to completion,

condensation

for

5)

soluble

selective

consuming

An excess reactions

were

correct

as carrier

introduced

the polymeric

of the different

No time

000

and the yield

measuring

into

for

on polymeric

to circumvent

20

that it is

nucleotide

material

was

and all

reactions solving

In order

details

advantage

to come

weight

PEG

synthesis

1)

VIII

Universitit

6,

the use

reasoning

of the bulkiness

enzymatic has

accepted

supports

- on steric

Britain.

COL

1) or of permanent

swellable

in tieat

KCIster

we described

on polymer

are

in consequence

stepwise

of the enzyme

2)

synthesis which

Be side s this

be reduced for

1,

paper

LENGLY

Papendamm

1972;

printed

SYNTHESIS

und Biochemie,

13,

in UK 28 February

(Received

LEOTIDE

POLYETHY H.

Institut

Pergamon Press.

chain synthesis,

1535

to PEG see

we first 394)

)

tried

two methodes

for

No. 16

0 0

0 0

o=o I

0 I N II

+ w

-

II

,I

NO. 16

a)

1537

Monomethoxytrityl

reaction

group aa anchor

sequence which is rhown in the

tative yield ( 2 g k, mentary easily

analysis

4 mmol

tosyl

by infrared

coupled with the

of the

2 is dissolved

all PEG-bound

( 18 hrs.,

material

benzene or petrolether

in the cold,

against water or buffer.

phylized

in THF ( 2 hrs.,

groups

bound to the polymeric

form/methanol

= 9

using

ether ( 60 - 700).

28.7

( l0).

the above b)

After

mentioned

Uridine

carrier,

benzene/acetic

f with petrol pmol/g

tic

spot remains

acid chloride

molecules

on silica

70’

could be bound to $

all UV-absorbing

material

remains

extensive

reacted an alkali carrier

(2) 0.2

in a yield of

system.

dialysis bound/g

only to a very slight extent.

being described

can be coupled

solved in trisbuffer

TIPS,

dissolved

( 14)

anhydrous

in water

to protect

using

between Land 2’, 3’-

pyridine,

( TIPS ) as

20 hrs.,

and the extinction

all OH-groups

The 3’-0-acetyl

condensation

(0.1 After

g

s,

0.2

dialysis

determined

of ethyl vinylether

groups were removed can be followed

qualitatively

0. 5 mm01 TIPS,

and lyophyllimation

M pH 7. 5 which leads to 127. 5 umol nucleotidic

to give of the

by dissolving

step the first deoxynucleotide mm01 2,

22O).

which might not have

group 6) , and which will alter the properties

( 1 hr. , 0’ ) ; this reaction

to the carrier. 0.1

mmol

12is reacted with an excess 3c

In a subsequent

acetyl-thymidine-5’-phosphate 22O)

was

In order

acid labile blocking

spectroscopy.

triisopropylbensene

0.5

reaction,

12 in 0.1 N sodium hydroxide infrared

2,

a sample carrier.

in the condenration stable but

using

mmol

Conversion

at the origin using

reaction

78.2 umol gis

with p-

) and by precipitating

sulfonylchloride

After

lyo-

gel ( chloro-

O-acetyl-uridine-5’-phosphate agent ( 1 g !,

and to

reaction

Sephadex LH 20 as carrier 1) . It is introduced in a condensation

condensing

chloroform,

at the origin.

( 6 hrs.,

The use of uridine as anchor has already

:

a sample

by 3’-O-

4 hrs., is dis-

material/g

from this remult a greater than 98% conversion of 2 to g can be r265nm); max calculated ( E 265 of uridine -. 8200, E 265 of thymidine : 9400 ). After protection of the

(2

free OH-group removed

can

The usual

tube is then

in a Grignard

as can be shown by

3’-0-Acetyl-thymidine

dialysis

chromatographic

as anchor

organic

and

of

generated

1660 cm-‘).

The content of the dialysis

2 is now converted

can

(THF)

salts with ether,

low-molecular-weight

: 1, v/v ) ; the only trityl positive

to i is possible

( acetylation

which is freshly

reflux ; C = 0

and inorganic

by ele-

to give 7 The conversion can be shown by the absence = and by the color test with 10% perchloric acid. All trityl

band at 1660 cm

are

in quanti-

into 2 with an excess

in tetrahydrofuran ( 4).

in a

reflux) -1

of the C=O

of 2

to remove

and is ready for the next reaction.

bromoanisol

acetylation

salt of p-hydroxy-benzophenone methanol

1 could be tosylated =

= 2 could be converted

dialyze

the precipitate

chart.

group was introduced

, 22O) ; this could be established

product towards

reflux).

in absolute

work up is to precipitate

2 hrs

spectroscopy).

( 24 hrs.,

sodium

with sodium methoxide

accompanying

chloride,

and the resistance

be detected

sodium iodide in acetone be

The monomcthoxytrityl

:

of the uridine moiety

and the next condensation

with ethyl vinylether, step can take place.

the 3’-0-acetylgroup

can be

:

No. 16

13 =

0

15 =

14 =

oP

PEG-backbone

OAc

U = Uracil T = Thymine AC = Acetyl

The chemical under extensive Acknowledgement

Re ferencee

OH

2’-

and enzymatic investigation

: The

synthesis

of oligonucleotides 3, 4)

in this laboratory

author wishes

Forechungegemeinechaft

technical

assistance

of Mrs.

using this carrier

is now

.

to thank Professor

Deutsche

or 3’-linkage

for

C.

K. Heyns financial

Woldtmann

for hi6 intereqt

and the

support.

The excellent

is gratefully

acknowledged.

:

1)

Part VII in this eerier

H. Ki%ster and K.

2)

Part VI in thin eerie6

H. Kbeter,

3)

H.

4)

H. Ktrnter and K. Albersmeyer,

5)

M.

Mutter,

6)

A.

Holy and J. Smrt,

KUster and D. Skroch,

H. Hagenmaier Coil.

Heyns.

Tetrahedron

TetrahedronLetters -= Letter6

l9&,

1972,

preceding

to be publiehed to be publiehed

and E.

Bayer,

Angew.

Chem.

Czech,

Chem.

Comm.

2,

2&

883 (1971)

3800 (1966)

preceding

paper

paper