BIOCHEMICAL

Vol. 68, No. 2, 1976

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

AFFECT OF SPLEEN AND SNAKE VENOM PEOSPHODIESTERASES ON NUCLEOTIDES CONTAINING >WCLEOSIDES IN THE SYN CONFORllATION Kelvin K. Oqilvie Department of Chemistry McGill University Montreal, Quebec H3C 3Gl Frank H. Hruska Department of Chemistry University of Manitoba ?7innipeq, Manitoba Received

October

28,197s

SUMMARY: Dinucleotides containing 6-methyldeoxyuridine and inosine have been prepared and subjected to spleen and snake venom phosSpleen enzyme degrades all nucleotides tested phodiesterases. while snake venom causes very little degradation of nucleotides having 6-methyldeoxyuridine in the 3'-terminal position. The clear implication is that snake venom will not recognize nucleoside units in the syn conformation.

Recently catalyzing

several

authors

polynucleotide

phorylase

synthesis,

and RNA polymerase,

side

di-

This

has caused considerable

mation (4,5)

or triphosphates

of nucleosides

"wobble"

will

having

in the

not

the bases in the

calculations

indicate

the barrier

paper we show that

little uridine(A)

degradation but

nucleo-

have been In addition

state

to rotation

confornucleosides

has been shown to exist

in the crystalline

this

(6,7)

in solution.

conformations that

phos-

the

such as 8-halopurine

-svn and anti

enzymes

syn conformation.

in determining

syn conformation inosine

certain

use as substrates

and 6-methyluridine

nucleoside

that

such as polynucleotide

interest

and several,

and 6-methylcytidine

shown to exist the

have shown (l-3)

in both

(8) although is

snake venom phosphodiesterase

low

(9).

In

causes very

of deoxyadenylyl-(3'-5')-6-methyl-2'-deoxy-

readily

degrades

thymidylyl-(3'-5')-inosine(D)

and

BIOCHEMICAL

Vol. 68, No. 2, 1976

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

thymidylyl-(3'-5')-isopropylidineinosine(C). esterase

degrades

all

Spleen

of the above nucleotides

phosphodi-

and also

6-methyl-

2'-deoxyuridylyl-(3'-5')-thymidine(B). MATERIALS AND METHODS All priately

nucleotides

described

protected

were synthesized

nucleoside

units

using

method of Letsinger

and Ogilvie

(10).

used to protect

5'- hydroxyl

positions

synthesis protect

the

3'-hydroxyl

was used during removed prior

the

Spleen including Corp., of

synthesis

was obtained

the Department the

Biochemicals

were carried (12,13).

out

using

reactions

groups

were

and electrophoresis.

Corp.,

from several Worthington donated

of McGill

gave the

standard

procedures

were carried

out

sources Biochemicals

by Dr.

University.

same results.

Snake venon phosphodiesterase

Enzymatic

was used to

Protecting

sample kindly

of Biochemistry

above sources

during

enzymes and the pure nucleotides

phosphodiesterase

purified

was

Isopropylidineinosine

of C and D.

with

group

nucleosides

group(II)

chromatography

Nutritional

from all

of all

by paper

and a highly

phosphotriester

of deoxynucleosides.

to treatment

were characterized

the

appro-

The methoxytrityl

and the -t-butyldimethylsilyl the

from the

J.

Spencer

Enzyme

Degradations

at pH 6.5 and 37°C

was obtained using

standard

from Calbiochem. procedures

at

pH 9 and 37°C. RESULTS AND DISCUSSION We have recently possess side it

the

syn-conformation

and when incorporated

was clearly

this rapid

shown (14)

of interest

nucleotide. degradation

caused only

Spleen

by PMR that

in solution, into

both

nucleotides

to determine

after

such as A.

376

free

hours

nucleo-

As a result

of enzymes on

caused complete

However nine

in the

the effect

phosphodiesterase

of the molecule.

14% degradation

6-methyl-2'-deoxyuridine

and

snake venom enzyme at

37°C and 54% after

Vol.

68, No.

2, 1976

BIOCHEMICAL

AND

BIOPHYSICAL

RESEARCH

COMMUNICATIONS

HO

0 ‘C’

B

c

CH:

‘CH,

I-I

H

D

24

We therefore

hrs.

synthesized

thymidine

(B)

and found

molecule

as did

snake

Because

of

conformation

the

venom

test

conformation

solution. in

and have

-syn

barrier very venom

and anti less A

X-ray

fact

completely

that

snake

the

the

terminal

of

we decided

studies

have

shown

in

to

the

the

the

nucleoside

that

the

nucleotides

the

enzyme

inosine

since shown

C-N glycoside

bond

in

the

C and D show

barrier is

a period in

377

of the

exist (8,151

inosine

is

that

the

inosine that

is snake

nucleotides.

some degradation to

high

can

in

state

have

cause

to

a nucleotide

cr!ystalline

these

Asvn-

of

studies

venom did

and over

to

observation

degrades

that

effect

to use

attached

6-methyldeoxyuridine

an equilibrium

having

idea

the

position

on nucleotides

and

with in

3' -terminal

Theoretical

studies

degraded

when

about

and our

completely

inosine

importance

nucleoside.

consistent

the

of

or -anti-conformations

rapidly The

in

of

to rotation low

implication

phosphodiesterase,

implied

a "wobble"

enzyme

phosphodiesterase.

obvious

Several

either

6-methyl-2'-deoxyuridylyl-(3'-5')

spleen

venom

nucleoside

on snake the

that

0

but time

rotation that

of A is between

it

those

anti-conformation

is

syn

neverthe-

molecules will

of be

Vol. 68, No. 2, 1976

degraded.

BIOCHEMICAL

Thus this

observation

venom phosphodiesterase has shown here has freedom

that

phosphodiesterase

will inosine,

to exist

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

of an anti

have many important at

least

appear

for

snake

implications

and

in the dinucleotides

in the anti-conformation

does not

requirement

in

to have a similar

solution.

studied, Spleen

conformational

requirement.

ACKNOWLEDGEMENTS We thank Upjohn

the National

Company (KKO) for

Research financial

Council

of Canada and the

assistance.

REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9.

10. 11. 12. 13. 14. 15.

Kapuler, A.M., Monny, C., and Michelson, A.M. (1970) Biochem. Biophys. Acta, 217, 18-29. Kapuler, A.M., and Reich, E. (1971) Biochemistry, 10, 4050-4061. Ikehara, M., Tazawa, I., and Fukui, T. (1969) ibid, 8, 736-743. Tavale, S.S., and Sobell, H.M. (1970) J. Mol. m., 48, 109-123. Ikehara, M., Uesugi, S., and Yoshida, K. (1972) Biochemistry, 11, 830-836. Suck, D., and Saenger, W. (1972) Nature, 235, 333-334. Schweizer, M.P., Banta, E.B., Witkowski, J.T., and Robins, R.K. (1973) J. Amer. Chem. Sot., 95, 3770-3778. Subramanian, E., Madden, J.J., and Bugg, C.E. (1973) Biochem. Biophys. Res. Comm., 50, 691-696. Jordan, F. (1973) Biopolymers, 12, 243-255. Letsinger, R.L., and Ogilvie, K.K. (1967) J. Amer. Chem. Sot., 89, 4801-4803. Ogilvz, K.K. (1973) Can. J. Chem., 2, 3799-3807. Ralph, R.K., Connors, W.J., Schaller, H., and Khorana, H.G. (1963) J. Amer. Chem. Sot., 85, 1988-1997. Razzell, W.F., and Khorana, H.G.,71961) J. Biol. Chem., 236, 1144-1149. Manuscript in preparation. Munns, A.R.I., and Tollin, P. (1970) Acta. Cryst., E, 1101-1113.

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