Penicillin interaction with guanosine

Vol. 46, No. 1, 1972

BIOCHEMICAL

PENICILLIN

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

INTERACTION WITH GUANOSINE

Lou S. Kan, Frank K. Schweighardt, Department

of Chemistry,

Pittsburgh,

Received

October

San Kao, and Norman C. Li

Duquesne University

Pennsylvania

15219

7, 1971

Because of the widespread use of penicillins as anti%?!!%%l agents the question of how penicillin affects the function and struct&e of nucleic acids becomes of biological importance. This communication reports a nuclear magnetic resonance study which shows that penicillin-G interacts with guanosine in dimethyl sulfoxide and can break the strong guanosine-cytidine pairing by forming a binary hydrogen-bonded complex of penicillinguanosine. The binding sites in penicillin are the carboxylatfb and the carbonyl groups, while the NH and NH2 groups of guanosine act as hydrogen donors. Because of the widespread agents,

the question

structure

of how penicillins

of nucleic

wish to report

a nuclear

guanosine-cytidine

penicillin-guanosine. NMR spectrometers, spectrometer

magnetic

multiplet

interacts

resonance

as antibacterial the function

and

significance.

We

(nmr) study which

with guanosine

and can break the

pairing '9' by forming a binary complex of Although we have used both 600 and lOO- MHz

we find

that only by using a higher-field

NMR

spectrum case, 250 MHe) can a well-resolved Figure 1 gives the nmr spectrum of 0.2 be obtained.

(in this

of penicillin M penicillin

affect

acids becomes of biological

shows that penicillin-G strong

use of penicillins

(250 MHz) in D20, and shows that the phenyl peak is a and the CH2 peak an AB quartet, J&l5 HE. Both the phenyl

and CH2 signals

appear as singlets

at 60- and lOO- MHz. decomposes in less In unbuffered aqueous solution , penicillin than 24 hours, whereas in dimethyl sulfoxide (DMSO), we have found it to be stable

for more than seventeen 22

Copyright

@ 1972 by Academic

Press, Inc.

weeks at 30°.

For this

Vol. 46, No. 1,1972

2.60

2.70

Figure

1.

reason,

BIOCHEMICAL

2.60

2.50

-0.90

0.60

0.70

-1.10

-,.oo

and guanosine

in penicillin

4 HZ, JBX = 9 HZ.

-

represent

-3.00

-3.10

-3.20

(250 MHs) in D20.

is not sufficiently

pH for nmr measurements,

between penicillin signals

-

--0.40 -0.50 l?P.M.

Nmr spectrum of 0.2 M penicillin-G

and because guanosine

at neutral

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

soluble

we have studied

in DME%d6.

in D20

the interaction

The CHA - CHB - NHX

an ABX system, with JAX y 0, JAB&

The CZCOO- signal

methyl groups are nonequivalent,

is a singlet,

giving

rise

and the m

to two separate

sing-

lets. When guanosine penicillin shift

proton

decreases

is added to 0.1 M penicillin peaks are shifted,

in the order:

C6H5, as shown in Figure

2A.

and the extent

of downf.ield

C&200-> CHB> NH, CH3, CH3> CHA> CH2, If

penicillin

guanosine field

in DMSO-d6, the

is added to 0.1 M

in DMSO-d6, the NH2 signal in guanosine is shifted downand slightly broadened, the G-NH signal is shifted downfield 23

Vol. 46, No. 1, 1972

BIOCHEMICAL

B

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

G-NH

AX

XYX X

/

/

G-8H 79oi)--x-o-

x-o-x-o-x

(I

HZ. 77a

1

o&t

380 lb0 -i -o-----oI 150 1

X

bao- a / 660 --o-

-0-e

I /x

ZH, b40;

140w

1

lGfx

To;

/

0-o

5M

b

7

*

9

0012345673

9

lo

(P EN’)x

lo’,

10

M

Figure 2A. Chemical shifts of penicillin-G protons at 100 MHz (TMS as internal reference) in DMSOsolutions containing 0.1 M penicillin and varying concentrations of guanosine. Figure 2B. Chemical shifts of guanosine protons vs. penicillin concentration in DMSO (x, with Of1 M guanosine; 03th 0.1 M guanosine and 0.1 M cytidine present). For numbering scheme, see scales used for G-NH and for G-NH2. Figure 3. Note the different at a pace double that signal

is only slightly

(see Figure

2B).

affected

the two NH2 protons, The other

broadened,

by the addition

The 10% increase

of G-NH compared with penicillin.

of G-NH2 and greatly

and the G-8H

of penicillin

in the rate of downfield

that of G-NH2 is understandable, only one is involved proton

because of

in hydrogen bonding with

is hydrogen bonded to DMSO. These

nmr data lead to the conclusion

that

a penicillin-guanosine

is formed in DMSO, in which two hydrogen

bonds are involved,

the binding

3.

cular

sites

shift

are as shown in Figure

models has shown that this

is reasonable. 24

Construction

complex and

of mole-

The G&H is at a

Vol. 46, No. 1, 1972

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

NH I c=o

GUANOSINE

PENICILLIN-G

Figure plex.

3.

distance

Schematic representation

away from the hydrogen-bonding

frequency

is only slightly

Figure containing

2B further

affected

0.1 M guanosine,

standable

because of hydrogen-bonding On the addition

0.1 M guanosine

alone.

of penicillin. in a solution

in DMSOis downfield in DMSO. This is under-

between guanosine

of penicillin

and

to a solution

containing

the G-NH2 signal shifts the rate of downfield shift is not as great on adding penicillin

Furthermore

the trend

in a guanosine-cytidine taining

its

and 0.1 M cytidine,

however,

rate observed

0.1 M guanosine

com-

and therefore

by the addition

0.1 M cytidine

in a solution

cytidinelt2.

sites,

shows that the G-NH2 signal

from that

field:

of penicillin-guanosine

guanosine

alone,

mixture

to a solution

containing

is that the G-NH2 signal approaches

25

guanosim

frequency

that of a solution

at high concentration

downas the

of penicillin.

con-

Vol. 46, No.

1,1972

BIOCHEMICAL

These results

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

lead us to conclude

guanosine-cytidine

pairing,

that penicillin

a binary

disrupts

the

complex penicillin-guanosine

is formed. From the chemical taining

shift

0.1 M guanosine

G, as shown in Figure

and varying

for

1:l

guanosine-cytidine

cytidine guanosine

higher

therefore

ing 0.1 M penicillin+

K = 4 l./mole

uridine,

penicillin

and nucleoside

tween penicillin+

is strong

nmr spectra

and thymidine).

the guanosine-

enough to disrupt

the

cytidine

data

in penicillin-G,

= cytidine,

obtained

for

both

show that the interaction or adenosine unlike

be-

is significantly

The reason can very well

and adenosine,

the carboxylate

contain-

(nucleoside

guanosine,

both NH and NH2 groups in the same molecule. site

for

and is

for DMSOsolutions The

protons

and cytidine

weaker than with guanosine.

thymidine,

constant

in DMSO. The penicillin-

and 0.1 M nucleoside

adenosine,

that

The formation

des-

pairing,

We have also obtained

fact

constant

complex is about 30 l./mole,

complex192 , also determined

guanosine-cytidine

of penicillin-

the formation

complex2.

than the value

interaction

con-

complex, in the manner previously

of the penicillin-guanosine considerably

concentrations

2B, we have determined

of 1:l penicillin-guanosine cribed

data for G-NH2 in solutions

lie

in the

do not have

With uridine

and

group appears to be the only binding

with the NH group in uridine

and thymidtne

the hydrogen donor, Using an infrared formation

constant

guanosine

and cytidine

method, Pitha,

of over 105 for

have reported

et al.3,

hydrogen-bonding

derivatives in chloroform. the formation constant acceptor,

between Since DMSOis of the guanosine-

a strong

proton

cytidine

complex is only about 4 in this medium 192 .

vertical

stadcfng occurs for

mononucleosides4, 26

a

In water

and there

is little

as

Vol. 46, No. 1,1972

evidence

for

BIOCHEMICAL

formation

of specific

tween mononucleosides the structure certainly double

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

in water.

According

of the guanosine-cytidine

DNA molecule

The experiments carried

hydrophobic

reported

in this

in DMSOis almost

scheme known to occur in

DNA, since the interior

is largely

of the double

and is therefore

communication

out in DMSOmedium, and have relevance

biological

systems.

Certainly

"nonaqeous."

therefore

were

to behavior

complex in chloroform

one has to conclude that

in DMSOis far

stranded

in

if one compares the formation

stantha of guanosine-cytidine DMSO (k=4),

complexes be-

to Newmark and Cantor',

pair

the same as the base-pairing stranded

bonding

hydrogen-bonded

more similar

con-

(Kv1105)

and in

the degree of hydrogento interactions

in water than

in chloroform. The nmr results with a Varian

in this

communication

A-60, a Varian HA-100 or a spectrometer

a superconducting

solenoid,

The data illustrated spectrometer. viously. 5

described

at a frequency

in Figure

Sample preparation

were obtained incorporating

of 250 MHz for

2 were obtained

protons.

with a Varian

was the same as described

Ha-100 pre-

Acknowledgment: This work was supported by Grant GM 10539-09 from the National Institute of General Medical Sciences, U.S. Public Health Service, and used in part the NMR Biomedical Facilities at Carnegie-Mellon University supported by Public Health Service grant RR 00292. References: 1. 2.

3. 4. 5.

Newmark, R. A. and Cantor, C. R., 3. Amer. Chem. Sot., 90, 5010 (1968). Schwei hardt, F. K., Moll, C., and Li, N. C., J. Magn. Resonance, 2, 35 71970). Yitha J., Jones, R. N., and Pithova, J., Can. J. Chem., & 1044 k$%ker M P Chan, S. I., and Ts'o P.O.P., sot., 8 , '52r;l ii968). Wang, Is . M. and Li, N. C., J. Amer. Chem. Sot.,

27

J. Amer. Chem. 88, 4592 (1966).