The specific synthesis of C3′–C5′-linked uridine oligonucleotides

BIOCHEMICAL

Vol. 8, No. 1, 1962

AND

THgSPElFICgThI%ESISOFC

3' -C +nim

D. H. Mer Institute

Received

May

BIOPHYSICAL

RESEARCH COMMUNICATIONS

UHIDIWE oLIlxmJcL&cTrmGs

aud H. 6. Khoraua

fer BUyme Research, uuiversit~ Hadim 6, WiscozMu

of Wisconsin,

1, 1962

to the specific

Indmebpingapproaches

~~~

oftheuaturally

intec~ethe~~~~has

cpy

been to prepare tibonucl~sJde-3'

phosphate derivatives

adjaaent2~-hydroqrl~~i%pretectedbg subsequentlyunder

1962;

couditious.

described

Ramler

in uhich the

agroupuhich

adequatelymild

used in the preyiously and Khorana,

quthesis

Thokeyiutermediates

approach (H. Smith,

and Khorana,

cauber~ed

1962)

wire

Ram&r, the

Goldberg

2~+tetrahydro-

pyraqylrib1muola~side-3~phosphates,thetetrahydroIyra@groupbelug rBIIy)vBc1 by an acidic

treatppesrt.

linkedribo-dinucleotidee

of C3'45'

necessaxy in the removal acidic

of the tetrahydropyraqrl

B&age.

vious observations isonerization accees

reaction,

The particularmerit

can be detected

In developing

this

approach,

phosphate and 2t+acetyhridine-3( Biouhm.

uuder alkaUne

(brow

et al.,

the preparation

c2*-c5f

to

of the 2'inthe from the pre-

conditioua

2r -C5,) of the inter-ribouucleotidic

the mlytic

Bkwh3n.

5'

approach to the above

group for the protection

which showedthatnhileunderacidic

isomerization

KhOrma,

group because prolonged

group for the purpose uaa inferred

(C3'-c5' --C

cautionwas

3'

Iu au alternative

next investigated.

use of an alkali-labile

syntheses of a number

of the C -C

isonerizatiou

the use of an alkali-labile

hydroz+qlf'unctionuas

specific

were thus accoqlished,

caused detectable

trbatmsPlt

inter-ribonucleotidic problem,

Although

scme linkage

conditioue

1956; Smith & &.,

no such 1962).

of 2~,5~-di-Q-acet@ridiue-~~

phosphate uas recorded

(mar

Research Coma. 1962) and in plodel m&euts 61

and

Vol. 8, No. 1, 1962

methyl

uritie-3’

obtainedwas

The present

approach to the first

t-

uxclusivelgthe

synthesis

of uridine

phosphate

1962) with 50% acetic

linkages. In the

(Raanler

Treatment

of Z*,!P-di+-tetra-

andKhorana,

d. Am. C&ma. Soa.,

acid at 0' for 3 hr. gave as the main products

phosphate.

phosphateand

The latter,

a

5'*tetrahydro-

which predominated

(75% of the

~sgrtrifiedbyaelectivereslcticmof2*~tetr~~l-

~&dine-3’

phosphate~thdinwthoqtritylchloride

bergandKhorana,1%2)

phosphate

axce88 of acetic

anhyxlride

andppidiae

@Jml.)iathe

products,

2'~acetyl-S'~te~~~3'

by addition

(0.28 Pole)

(20 ml.)

cyclic

uith

of uater

uaskept

resin)

(20 ml.)

atpE2.8

(pH

for 7.5 hr. at roomtmp. phosphate,

3’) phosphate and uridinb2*,3~

the other prodcyclic

phosphate.

uaspurifiedbypartitionchrcmatographyona

column using the solve&

a

Themixtureofresulting

phosphate,

The main product now was 2*+acetyluridSne-3'

Thedesiredproduct

uas treated

5*-&

phosphate and 5*4-

of Domx-50 acidic

being uridine-2T(or

colwmx.

in a mixkre

cold for 30&n.

tetrabydropyrauyluridine-2t,3* controlled

(Smith,F&&er,Gold-

and separationonaDEU%xllulose

Tetrshydropyraqyluridine-3'

system ethyl

alcohol-l34

In the secmd route to 2*+-acetyluridine-3'

the start&g

material

was St-w-~etho#zityluridine-3'

which nas prepared by direct

treatment

of au&line-3’

cellulose

aawmia acetate

(5-2, V/Y>.

(1)

can-

5*-Q-tutrahydropyranyluridine-39

mixtureof2~+tetrahydro~anylnr5d5ne-3~

ucts

oligonucleotides

phosphate IW~ prepared by two routes.

lgdropyranyl~e-3’

large

ribo-

records the successfU. application

the startingmaterialwas

pyranyluFid&e-31

The

degraded by pancreatic

phosphate and it ua8 prepared as follows.

ndxture),

RESEARCH COMMUNICATIONS

intexmediates.

C3,-C5, inter-nucleotidic

Zt4-Acetylurj.~e-3~ route,

cad&a

commpnication

of this

first

AND BIOPHYSICAL

phosphate ms prepared fronthese

~&hylesterthus lluchaSe.

BIOCHEMICAL

(pH 6.5)

phosphate, phosphate', phosphate with di-

Unpublished earperiBLent5 by Dr. Y. Lapidot of this laboratory showed that treatment of adenosine-3' phosphate with di-~m&hcurgtrityl chloride in pyridjne Under COJ.ltrO~ed ConditiOllS gave !?+-di-mthoq%rityladenosine-3Re-3' phosphate as the major prodwt. The latter 62

Vol. 8, No. 1, 1962

BIOCHEMICAL

p-methoqtrityl

chloride

IX34Lcellulose Acetylation

AND BIOPHYSICAL

in dry pyridiue

column (Smith,

followed

Ramler,

of the dimethoqyt,rityl

Q-tetrahydropyrsnyluridine-31

Goldberg

derivative

band synthesis, pyridine

2'+-acetyluridine-3'

with two equivaleuts

lished

results

bodiimide

material

degraded by pancreatic

synthesized

was thus exclusively

2~+acetyluridine-tylrulidine-3' phosphate

1961).

monium hydroxide a combination preparative

After

Products cgclo-,

(0.066 mole)

for 6 hr.,

in the previous

uupub-

of mall

(6%) isolated

was

bond

For polymerization,

was reacted

in anhsdroug

ful.lY ide.dfied

treatmut

with 9h sm-

theproductswere

separatedby

ou a DFdE-cellulose

aud, wherever necesssry, the pattern studies

1961) axcept that

of the nucleotide

di-,

(I)(cf.

The inter-nucleotide

of the C3' -C 5' tyPe*

In general,

(Khoraua,

centration

phosphate

and the formation

ribonuclease.

paper chromatography

merizations

under these con-

uridylyl-uridiue

of ion exchsuge chromatography

obtained

Although

cyclic

a work-up including

at rocmtezp.

paper-electrophoreris. to that

iu the presence

with au excess of DCC (175 III&> for 5 days (Khorana

(O.lml.)

and Vizsolyi,

@CC).

laboratory)

was noted,

completely

ppkiine

on inter-nucleotide

phosphate was reacted in anhydrous

of M. Smith in this

amouuts of polymeric

the pH to

product was again purified

sxperizmt

was uridbae-3*,5t

the major product

by lowering

of 2',3'4.-Q-benzoyluridine

of an excess of dicyclohwlcar ditions

above for 5'-

phosphate aud uridine-2~,3*

The desired

In the first

chromatography.

on a

1962).

Khorana,

as described

phosphate followed

phosphate as the products.

by partition

by separatiou aud

about 2.5 for 7 min. gave 2t-Q-acetyluridiue-3~ cyclic

RESEARCH COMMUNICATIONS

column,

preparative

of results

was &uilar

on the deoxyribonucleotide due, presumably,

the polymerimtion

to the low con-

did not go as far.

were 1tridine-3~,5* cyclic phosphate (I),

aud tri-nucleotides

represahd

poly-

by the general

The the

structure

was characterized by ultraviolet absorption spectrum, by its quantitative conversion to the corresponding If,gl-cyclic phosphate on treatment withdicyclohexylcarbodiimide in aqueous pgridine andby reconversion to pure adenosins-3' phosphate after very mild acidic treatment. 63

(III)

Vol. 8, No. 1, 1962

BIOCHEMICAL

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

0

OH

640

H2

-/

uracil

0

$52 o=Fpo-

2

0

6-I

~~

H

3

OH

Uracil 0 H

6-P&

G-=0 60 -I

I

L P

dca

Urac 1

r)

-/

/

=2

Uraeil

0

v-0

III

II

andthelinearti-Padtri-nucleotides hozdogs, ization

(II).

rrhich raaain to be ide&ified, of the oligonucleotides

ribonuclease, followed

by degradation

be mphasized

nuclease

included

that

complete degradation

ribouuclease

phosphmomeaterase and detteradnation

involviag

of the internucleotide

The characterby pancreatic

uridzine and aridine-31 tzcQdmmts

in all

degradation alkaB?w

with pancreatic

of the two products,

ofthehigher

were also present.

dephosphorylationbybacterial

of the ratio should

(II)

Smalleramxmto

phosphate. pancreatic

It ribo-

bonds occurred.

Pr~s~ohQndoalasthodaforpolgmariaat~ofribos#lcleotidea have afforded

mmpl~zixtures

of oligomcleotides

C2,-C5, and C3,-C5, inter-nucleotide 1959; schralmn et al., the first

specific

bonds (Smith et al.,

The developaentnowreported

1962).

synthesis

l&&ages.

Theproceduresused

general.

Esrtemtion of this

of mixed oligonucleotides

ccmtaMngrandm

of rib+oligonucleotides

1958; Michelscw, constitutes

containing

C3,-C5,

are sbgiLe andofferprolniseofbeing work to the synthesis

is in progress. 64

of larger

polymers and

Vol. 8, No. 1, 1962

BIOCHEMICAL

AND BIOPHYSICAL

This work has been supportedbygrants Institute vice

of the National

and the National

Institutes

RESEARCH COMMUNICATIONS

fromtheNationalCsncer

of Health,

Science Foundation,

U. S. Public

Health

Ser-

WashIngton.

References Brown, D. M., &path, m, u-24 (19%).

D. I.,

Neilson,

A. H. and Todd, A. R., Nature,

H. G. Some Recent Developuemts in the Chdstry of Phosphate Esters of Biological Interest. JohnW5ley snd Sons, Inc., 1. Y., 1961.

Khorana,

Khorana, Echelson, Ranmler,

H. G. aad Vissolyi,

J. P., J. Am. Chezn. Sot.,

A. M., J. Chern. Sot., D. H. and Kborana,

Raumler* D.B.andKhorana, No. 2, l47 (1962).

(London),

M., Nofiatt, (19%).

Smith,

I., a.,

84, (1962),

in l%ess.

E!iechem.Bio~s.ResearchCarm.,~

schranln, G., Grotsch, H. and Pdlmam, Smith,

675 (1961).

1371 (1959).

H. G., J. Am. Charm. Sot., LG.,

&

W., Awmr. Chemiq., &

6. G. and Kborana, H. G., J. Am. Chm. Sot.,

Ram&w, D. H. Goldberg, &, 430 (1962).

I.

65

53 (1962). &

6204

II. a& Xhorana, H. G., J. Am. Ghan.