Chemical synthesis of 2′(3′)-O-aminoacylribo-oligonucleotides: The preparation of cytidylyl-(3′→5′)-2′(3′)-O-glycyladenosine

BIOCHEMICAL

Vol. 22, No. 5, 1966

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

CE-EMICAL SYKTE-IESIS OF 2'(3'>-0-AMINOACYLRIEO-OLIGONUCLEOTIDES THE PREPARATION

OF CYTIDYLYL-(65%2'(3')-0-GLYCYLADENOSINE

S. Chladek, Institute

J.

of Organic

January

In amino son,

are

1957)

of the

vicinal

step bound

of

of

RNase

to

1958)

or by Tl

Smith,

free

(Hoagland

(Takanami,

Stephen-

or 3'hydroxyl

groups

2'

glycol

grouping

in

the

terminal

t-RNA's

(Hecht,

Stephenson,

t-RNA's

RNase

aminoacyl

Zamecnik,

can be degraded

have

been

Wilson,

Acs,

the

behaviour system

residue

investigated

Ishida, of

a some

Wilson,

Miura,

1965).

such fragments

polypeptide

of by paper

Smith,

and concluded to the

Lipmann,

The properties

(Herbert, 1964;

1959).

containing

CpCpA unit. type

1958;

by pancreatic

(Zachau,

and electrophoresis Herbert,

("acceptor")

to ribo-oligonucleotides

latter

has studied

the

, Zamecnik,

the

protein-synthesising

transfer

of proteins

to

of the

chromatography

Takanami

biosynthesis

linkage

2'(3')-O-aminoacyl

1964;

the

2'(3')-O-aminoacyladenosine

fragments

Czechoslovak

Prague

to t-RNA +)

The 2'(3')-O-aminoacyl

terminal

and Biochemistry,

of Science,

by an ester

CpCpA unit

and F. Sorm

31, 1966

an important acids

??emliEka

Chemistry

Academy

Received

:

that

in

a cell-

they

can

chain

1964).

+>Abbreviations

: t-RNA = transfer cytidylyl-(3ti5')-cytidylyl-(3+')-adenosin, nuclease, CpA = cytidylyl-(3'+5')-adenosin.

554

ribonucleic

acid, RNase

CpCpA = = ribo-

Vol. 22, No. 5, 1966

BIOCHEMICAL

Although of the

a number

simplest

of chemical

compounds

of this

aminoacylribonucleosides, 19651,

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

applicable

to

aminoacylribo-oligonucleotides

of

we have

for

&eneral

type,

has been

no procedure

Recently,

methods

described the

catalyzed esters

with

(??emlicka,

the

2',3'-cyclic give

grouping

1965).

orthoesters

(after

removal

the

preparation acid acid

ortho-

of ribonucleosides

readily

,of protecting

2'(3')-O-

so far.

amino

The resulting are

Feldmann,

of

on the

of N-protected

2',3'glycol

Chladek,

for

based

reesterification

2'(3')-O-

(Zachau,

reported

a new method

2'(3')-O-aminoacylribonucleosides

synthesis

i.e.

synthesis

has been

developed

the

ribonucleoside

hydrolysed groups)

in

acid

to

of

2'-

a mixture

and 3'-0-aminoacylribonucleosides. This

method

has been

applied

to

the

preparation

2'(3')-0-aminoacylribo-oligonucleotide, 2'(3')-0-glycyladenosine treated

with

III

(VI.

(ZemliEka,

methylformamide afforded

60 - 70 % yield

in

reaction longer isomerised --

(Scheme

time reaction

(5 % of the

in acid

linkage

in

III

(A +I ) orthoester showed the

expected

RNase

temperature;

2&5'isomer

after

of

the

when the however,

after

to be partially 2 h).

The ortho-

- ammonia - water (7 - water (5 : 2 : 3).

555

afforded ortho-

was detected

was found

di-

(80 equivalents

No isomerisation

at room

+> Solvent system A : 2-propanol B: 1-butanol - acetic acid

which

(II)

adenosine-2',3'-cyclic : 1.

III

salt)

anhydrous

by pancreatic

0.9

was 30 min. periods,

l>,

of III

ratio

3&5'internucleotidic

1965)

homogeneous

and the the

ammonium

trifluoroacetic

l?egradation

cytidine-3*-phosphate (VI)

the

ethylorthoester

chromatographically

UV spectrum.

ester

CpA (I,

Chladek,

containing

the

in

cytidylyl-(3L5')-

N-benzyloxycarbonylglycine

(80 equivalents)

of a

: 1 : 2),

Vol. 22, No. 5, 1966

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

C A Z = C6H,CH,0CODMF = lCH,l, N- CHO

I

CF,C%H in DMF. 30 min., 25 oc

OyOC,H5

0’

80 %HC02H

‘CH,NH-Z

PH 7.5

20 min., O°C 1 CA

pancreatic

C A

IV 2’- isomer

2’-isomer VII

H2(PdO/BaS0,,1

80 o/oCH,C02H. 2h. O°C

C A

2’-isomer VIII

V A

OH OH 1 Ni2CH2C02 H

ester ely

is

III

in

stable

80 $ formic

isomerisation

to alkali; acid

it

during

was hydrolysed

20 min.

of 325'linkage)

to

at O°C (without

afford

cytidylyl-(3&Y')-

2'(3')-0-(N-benxyloxycarbonyl)glycyladenosine yield. the

The UV spectrum compound

phoretically

(pH 2.4)

(IV)

of IV closely

was chromatographically homogeneous

556

quantitativ-

resembled (system

and showed

in

that

90 '$ of

CPA;

B) and electroone half

of the

BIOCHEMICAL

Vol. 22, No. 5, 1966

mobility

of

CpA in

pancreatic

RNase

borate

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

buffer

at pH 4.7

(pH 6.8).

gave

Degradation

cytidine-3'-phosphate

2'(3')-0-(K-benzgloxycarbonyl)glycyladenosine ratio

1.1

removed

: 1.

(2 h at O'C+)) adenosine

to give

(V)in

to

B),

resembling

to

(VIII)

and at pH 7.5

in

ratio

at

to

polynucleotide the

orthoester

tool

for

the

to

method

introduction

particularly

possibilities, aminoacyl are

under

-+) No hydrogenation was observed

under

could

pancreatic

alkali

RNase

at

and adenosine

the

reaction

into active

model

Our results serve

residues

synthetic into

and lack of CpA with

for

this

of

indicate

as a useful

selectivity

promising

including residues

with

as a simple

of aminoacyl

in

observed it

W

chromatography

hydrolysis

with

of t-RNA.

reactions

method

pH 2.4,

some extent

The high

make

reaction,

cytidine-3'-phosphate

oligonucleotides.

II

PdO/BaSO4

and 2'(3')-O-glycyladenosine

chain

that

IV was

: 1.

CpA can be regarded the

over

of CpA, paper

cytidine-3'-phosphate

1.07

acid

the

The aminoacylnucleotide

and degradation

pH 4.7

of

ninhydrin

that

electrophoresis

CpA and glycine

the

yield.

by a positive

closely

(systlem

80 41; acetic

group

in

cytidylyl-(3;5')-2'(3')-O-glycyl-

quantitative

V was characterised spectrum

in

and

(VII)

The N-benzyloxycarbonyl

by hydrogenation

by

ribo-

of

side

the

orthoester

purpose.

These

attempts to introduce 2'(3')-O++) t-RNA by means of the orthoester investigation

the of cytosine residue these conditions.

++IRecently,

in

our Laboratory.

(Furukawa

et

the aminoacylation of t-RNA by N-triphenyl: methylglycln5 in the presence of dicyclohexylcarbodlimide in pyridine in 1.5 % yield has been reported (Sluckij, Gottich, 1965).

557

a1.,1965)

Vol. 22, No. 5, 1966

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

REFERENCES Drei .man, E.Ya., Dmitrieva, V.A., Kamzolova, S.G., Shabarova Z.A. , and Prokof'ev, A8.A., Zh. Obshch. Khim. 2, 3899 (1961). Furukawa, Y., Wizuno, Y., Sanno, Y., and Honjo, M., Chem. Pharm. Bull. El, 16 (1965). Hecht, L.I., Stephenson, M.L., and Zamecnik, P.C., Biochim. Biophys. Acta 2, 460 (1958). Hecht, L.I., Stephenson, M.L., and Zamecnik, P.C., Proc. Natl. Acad. Sci. U.S. a, 505 (1959). Herbert, E., Smith C.J., and Wilson, C.W., J. ILEol. Biol. 2, 376 (1964). Hoagland, M.B., Zamecnik, P.C., and Stephenson, M.L., Biochim, Eiophys. Acta 3, 215 (1957). Ishida, T. and Miura, K., J. Mol. Fiol. &l, 341 (1965). y;g;Tr, D.H. and Khorana, H.G., J. Am. Chem. Sot. Q, 1997 . Sluckij, D.I.snd Gottich, B.P., Biokhimiya & lo32 (1965). Smith, C.J., Herbert, E., and Wilson, C.W., Biochim. Biophys. Acta s, 341 (1964). Takanami, 1., Proc. Natl.' Acad. Sci. U.S. 2, 1271 (1964). Zachau, H.G., Acs, G., and Lipmann, F., Proc. Natl. Acad. Sci. U.S. a, 885 (1958). Zachau, H.G. and Feldmann, H., Progress in Nucleic Acid Research 4, 217 (1965). fsmlizka, J. and Chlldek, S., Tetrahedron Letters Q&, 3057.

558