Bisulfite-catalyzed transamination of cytosine and cytidine

Vol.’ 40, No. 4, 1970

BIOCHEMICAL

BISULFITE-CATALYZED

TRANSAMINATION OF CYTOSINE AND CYTIDINE

Robert Department

of

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

Shapiro

Chemistry,

and Josef

New York

M. Weisgras

University,

New York,

N.Y.

10003

Received July 6, 1970 Summary :

Sodium bisulfite catalyzes the reaction of cytosine derivatives with amines to give N4-substituted cytosines. The reactions proceed in good yield, under mild conditions, with a number of aliphatic and aromatic amines. It is suggested that bisulfite may cause the cross-linking of nucleic acids and proteins by this mechanism.

It tives

under

a useful been

3 water

principal

mild

method

applied

acids. with

has recently

been

conditions for

the

to the

deamination

product

that for

NaHS03 reacts

the

in such

cytosine

of the

certain

amines

is

the

C4

N4 - alkyl

839

R

This

deriva-

constitutes and has

residues

can compete 2 (see

HN

cytosine

derivatives,

cytosine

intermediate, cases

with

1 + 2 + 3 + 4. 1,2 I -

of simple

deamination

to report

as a nucleophile

that

by the path,

specific

We now wish

reaction

reported

of nucleic successfully

Scheme 1).

The

- or arylcytosine,

Vol. 40, No. 4, 1970

6, presumably known

BIOCHEMICAL

formed

reactions

tives.

of

In these

serves

both

amino

group.

via -

in Table apart

I.

identities

spectra

synthesized

were

established

by satisfactory

properties

:

6h,

mp 290-293

10,700;

PH 7 A 280 mu, E max

400;

(decomp)

;A

PH 1

14,600;

increased

were: yield

rate

of the

(decomp);

R 2-propanol f ’

- water

tion

rate

effect

also

deamination

falls

of this

off

on the

anilinium

ion

(pKa 4.63).

cytosine

with

methylamine

at pH 7.3,

but

only

is

(pK,

uracil

very

was undoubtedly

upon the

equilibrium,

more

compensated

7.5

10.66).

was formed

above

This

reaction

resonance

for

1 + 2. 1 ..f -

the

(tic), spectra

for

the

than

when the

reactions

Thus,

the

840

yield

reaction

adverse

time

the

as the

effect

required

in the

1

The

decreased

The deaminabeneficial of aniline

to

reaction

of

of 6d was obtained -,was run at pH 5 .O. The

was avoided,

due to the

pH.

conversion

An excellent

of 6a.

to the

5, but

was seen

the

76%; pH 7, 80%.

acidic

effect

of

preparation

more basic

by the

6h -_ of 6h -I

The variation

at the

for

A more dramatic

use of pH values slow.

(80:20)

to pH 5, can be ascribed

at pH values

yield

PH 7 X 276 mu, E

pH 1 max A 288 rnp, E max

pH 3, 62%; pH 4, 64%, pH 5, 68%; pH 6,

competing

following

12,700;

6i, mp 205-206 m-.

at pH 7, as compared

samples

6h and i were II

283 mu,

6i, 0.80. The infrared and nuclear magnetic -and 6i were in accord with their assigned structures. -yield (determined by tic) with pH was investigated

and ultra-

authentic

and by the

0.75,

The results

infrared

max

pH 14” A 290 mn,c max

10,400;

The

with

analyses

are summarized

or uridine.

The new products,

elemental

its

(tic),

of their

(80:20)]

displace

chromatography

uracil

by comparison - water

and to

and yields

layer

to the 536 deriva-

same nucleophile

cytosine,

6, were

7-11

analogous

the

used,

by thin

products,

by known procedures.

case,

conditions

observed

is

and semicarbazide

bond of

run,

product

process

the present

and Rf [Z-propanol

characterized

16,

reactions

This

hydroxylamine4

5,6-double

transamination

of 6a-g -,---

5.

unlike the

The only the

with

reactions,

to saturate

from

violet

intermediate

cytosine

The successful

AND BIOPHYSICAL RESEARCH COMMUNlCATldNS

rates

of increasing for

the

complete

became pH con-

8-C10H7CH3CH3-CH2C02H CH3-CH2CH2CH2CH2-CW~CH~CH~CH~-

H-

B-D-ribofuranosyl-

H-

H-

H-

B-D-ribofuranosyl-

CH3-

H-

H-

H-

H-

H-

B-D-rihofuranosyl-

H-

R"

B-D-ribofuranosyl-

R'

'gH5o-HOC6H4-

H-

R

36

21

45

5

30

30

10

10

30

mmo1es.a amine

PREPARATIONOF SUBSTITUTEDCYTGSINEDERIVATIVES

80

92

51

72

90

88

30

54

80

58

55

50

58

65

% yiedd % yiel$ (tic)(prep)-

6.7

7.4

7.2

7.4

7.1

7.3

5.0

4.1

7.0

pH

(ml)d

H20 (101

H20 (10)

H20 (101

H20 (7.5)

H20 (10)

H20 (12.5)

60% C2H50H (50)

57% C2H50H (35)

40% C2H50H (10)

solvent

48"

25'

4o"

25'

36'

35O

40"

5o"

37O

temp

168

108

144

96

288

66

24

6

69

time

aMmoles of cytosine (or cytidine) and NaHS03 were kept at 1 and 12.5, respectively. b The solution was brought to pH 12, with NaOH. The substances (1, 4, and 6) were separated by tic on cellulose in 2-propanol-water (80:20) for 9, b, and f; in 2-propanol-HCl-water (65:16.7:18.3) for 5 and d; in l-butanol-water-NH3 (86:14:1) followed by 2-propanol-water (80:20) for 9, g, h, and i. The amounts of 1, 4, and 6 were determined spectrophotometrically. Yields are based upon consumed 1. 2 The solution was brought to pH 8 and the product isolated by chromatography on Amberlite CG 120 resin. In the case of 6f, a subsequent crystallization from aqueous HC02H was needed. The yields are based upon the starting amounts of 1, and upon 6 isolated. d Where the workup was conducted both by tic and preparatively, the conditions given are those of the preparative reaction.

Product

TABLE I.

(hr)

Vol. 40, No. 4, 1970

sumption 25’)

of cytosine

in

its

at pH 6, and 40 hr,

after led

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

consumption

37”,

of the

to decreased

reaction

yields

(6+ 5 +2 ‘1)

was demonstrated.

a solution for

of

120 hr.

1.3

was allowed

of conditions, with

only

deamination, effects

dimethylamine

side

level,

from it

modification have

been

to

those

synthetic

acids.

Polynucleotides

only

the for

physical

of

Thus,

under

reactions

a variety

of cytidine apart

substances.

from

Steric

a-naphthylamine reactions

and

conducted

with

methylamine.

of this

reaction

possibilities

studies,

at the for

containing

of suitable

introduction

amines.

gave,

of the

and enzymatic

polymerization the

the

with

value

certain

of the

slowness

of nucleic for

to cytosine

or morpholine

failure

of important

chemical

NT - alkylcytosines l2 but

monomers.

“reporter

the

nucleo-

groups”

have

hitherto

The reaction 13,14

into

acids.

of hazard

paper

cytosinc to

to be a mutagen new possibilities

linking

as compared

with

Similarly,

the

the

sequence:

was obtained.

imidazole

up a number

of interest

deamination

damage.

for

obvious

In our previous

genetic

for

generally

by allowing

p-phenylazoaniline fluorescent

of unidentified

I)

the

be of use

nucleic

with

times

or cytidine,

an alkylcytosine

entirely

opens

been preparable may also

with

and also

(Table

Apart

to react

reaction

hr,

6d to react with II NH3 and 5 ml of H,O at pH 7.4, 25’,

(by tic) failed

been responsible

reactions,

of

was produced.

traces

may have

morpholine

cytosine

was 6.5

due to the

was achieved

ml cont.

and cytosine

only

cytosine

conversion

reaction

uracil

u-naphthylamine

2.5

of the

was undoubtedly

This

of

The transamination when cytosine

the

g NaHSOs,

A 74% yield

This

and bisulfite

Prolongation

materials,

of 6.

In one reaction,

aniline

at pH 7.

starting

6 +5 +3 -+4. - I I I

with

The formation of protein

by sodium

X and for

the manner of 6f is _-

and nucleic

that

E. Coli.

in which a model acid

molecules.

a42

catalysis

of the

made the

latter

has subsequently 15,16

The results

bisulfite for

the

bisulfite

Bisulfite

organisms.’

phage for

was suggested

derivatives

living

for

it

a bisulfite Ultraviolet

been shown here

may inflict

a

raise

biological

- catalyzed light

crosshas been

Vol! 4Q, No. 4,197O

shown to link nucleic

BIOCHEMICAL

cysteine

and uracil

acid cross links

of microorganisms

residues

17

RESEARCH COMMUNICATIONS

, and the resultant

protein

-

are believed

by ultraviolet

6c suggests that bisulfite amines to nucleic

AND BIOPHYSICAL

to be a significant factor in the killing 18 Similarly, the formation of irradiation.

can catalyze

the binding

of carcinogenic

aromatic

acids. 7

Acknowledgements:

We wish to thank the National

Institutes

of Health

(grant

GM-11437) and the Damon Runyon Memorial Fund for Cancer Research

(grant

DRG-976) for their

support.

References 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18.

Shapiro, R., Servis, R. E., and Welcher, M., J. Amer. Chem. Sot. 92, 422 (1970). Hayatsu, H., Watayu, Y., and Kai, K., J. Amer. Chem. Sot. 92-, 724 (1970). Shapiro, R., Cohen, B. I., and Servis, R. E., Nature, in press (1970). Phillips, J. H., and Brown, D. M., Progr. Nucleic Acid Res. Mol. Biol. 7 349 (1967). Hayatsu, H., Takeishi, K.-I., and Ukita, T., Biochim. Biophys. Acta. 123, 445 (1966). Kikugawa, K., Hayatsu, H., and Ukita, T., Biochim. Biophys. Acta 134, 221 (1967). Shapiro, R., and Klein, R. S., Biochemistry 5, 3576 (1967). Szer, W., and Shugar, D., Acta Biochim. Pol. 13, 177 (1966). Fox, J. J., Van Praag, D., Wempen, I., Doerr, I. L., Cheong, L., Knoll, J. E., Eidinoff, M. L., Bendich, A., and Brown, G. B., J. Amer. Chem. Sot. t3& 178 (1959). Janion, C., and Shugar, D., Acta Biochim. Pol. Is, 261 (1968). Wempen, I., Duschinsky, R., Kaplan, L., and Fox, J. J., J. Amer. Chem. Sot. 83, 4755 (1961). Brimacombe, R. L. C., and Reese, C. B., J. Mol. Biol. 18, 529 (1966). Burr, M., and Koshland, Jr., D. E., Proc. Nat. Acad. Sx. U. S. 52, 1017 (1964). Hille, M. B., and Koshland, Jr., D. E., J. Amer. Chem. Sot. E, 5745 (1967) Hayatsu, H., and Miura, A., Biochem. Biophys. Res. Commun. 39, 156 (1970). Mukai, F., Hawryluk, I., and Shapiro, R., Biochem, Biophys. Res. Commun. 39, 983 (1970). sith, K. C., and Aplin, R. T., Biochemistry 2, 2125 (1966). Smith, K. C., Hodgkins, B., and O'Leary, M. E., Biochim. Biophys. Acta 114, 1 (1966).

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