Proton NMR study of iron(II)-bleomycin: Assignment of resonances by saturation transfer experiments

Vol.

96, No.

1, 1980

September

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Pages 341-349

16, 1980

PROTON NMR STUDY OF IRON(BLEOMYCIN: ASSIGNMENT OF RESONANCES BY SATURATION TRANSFER EXPERIMENTS Rajasekharan J. Michael

P. Pillai, Robert E. Lenkinski, Ted T. Sakai, Geckle, N. Rama Krishna and Jerry D. Glickson*

Physical Biochemistry and Pharmacology Program of the Comprehensive Cancer Center and Department of Biochemistry, University of Alabama in Birmingham, Birmingham, AL 35294

Received

June 7,198O

Summary The assignment bleomycin complex method. A number which are shifted complex in H20. with the formation Curie Law or the the valeric acid N=, the carbamoyl may be coordinated

of the paramagnetically shifted in D 0 has been accomplished using of a 2ditional resonances arising by the metal ion are observed in The temperature dependence of the of an isolated 1:l complex, but Curie-Weiss Law. The magnitude of hydroxyl (or carbonyl) group, the oxygen, the pyrimidine Nl and/or to the iron(I1).

The bleomycins with

DNA, their

metal

ions

drug

cause nucleic

with

suggest

putative

(1,2).

bleomycins of free

In the presence strand bases

acids serves

Oppenheimer

(7-8)

target,

of molecular

and with

(12)

The Fe(I1)

have

various

recently

complex complex

oxygen

for

metals

reported

and a diamagnetic exhibited

(17)

in making

1 Abbreviations:

B-amino alanine; THR: threonine;

the assignments

of the resonances

polyvalent

and iron(

the

complexes metal

release of the

ions

to nucleic

(9-14)

acids,

(15,16). 1 a H NMR study

while

of a

Bleo-A2-Fe(II)-CO

paramagnetically

both to low field and to high field of the spectrum In the present study, we report the use of saturation

complexes

various

polyvalent binds

form

the concommitant

of the binary

end of the molecule site

which

and with

of DNA with

Studies

as a chelation

--et al. Bleo-A2-Fe(II)l

in D20.

antibiotics

scission

(3-6).

the C-terminal

the N-terminus paramagnetic

1) are antitumor pharmacological

single acid

nucleic that

complex

(Fig.

resonances of the Fe(II)the transfer of saturation fromllabile NE protons the H spectrum of the chemical shifts is consistent does not obey either the the shifts suggests that a-amino group, the imidazole the secondary amino group

shifted

resonances

of the metal-free antibiotic. transfer experiments

of the paramagnetic

Fe(II)-

Bleo-A2: Bleomycin-A2; TSP: Trimethylsilyl propionate; PYR: Pyrimidinyl propionamide; HIS: B-hydroxyhistidine; G: Gulose; M: Mannose; VAL: valerate.

341

ALA:

0006-291X/80/170341-09$01.00/0 Copyright 0 1980 by Academic Press, Inc. All rights of reproduction in any form reserved.

Vol.

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+ BLEOYYCIW

A2 :

R= -N”(W~3S(C*$2

62

R=

H

:

-N”(W&+~-N’$

B-HYDROXYHISTIDIBE

Figure Bleo-A2

On the basis

complex.

hydrogens ligation

in close

proximity

structure

of these

of bleomycin

assignments

to the metal

congeners.

we are

and to suggest

able

to identify

a set

of probable

sites.

We have ermination

previously

demonstrates

employed

of the kinetics

of the bleomycin of exchange

antibiotics

of the peptide the

feasibility

tion

and dissociation

this

method

Materials

Primary

1

NY+

are

available

saturation

transfer

of dissociation (18,19) NE's

of the

as well

of peptide

of a similar

as in

Zn(I1) (20).

complexes

of the kinetics

The present

of the kinetics

complex.

in the literature

in the det-

and Ga(III)

the analysis

hormones analysis

of the Fe(II)-Bleo-A2

experiments

Detailed

study

of formatreatments

of

(17,20-22).

and Methods

Sample Preparation. Bleomycin-A2 chloride was purified from Blenoxane (Bristol Laboratories, Syracuse, NY), the commercial mixture of bleomycin congeners, by chromatography on carboxymethyl Sephadex C-25 (23). Due to the extreme sensitivity of Fe(II)-bleomycin to oxidation, all samples were prepared under strict oxygen-free conditions. Bleomycin-A2 (20.5 pmoles) lyophilized from D2Q was dissolved in 0.500 ml of ultrapure D2Q (99.96% D, One half equivalent of a solution Merck, Sharp and Dohme, Montreal, Canada).

'Iron(IIji) complexes of bleomycin have been found to be in fast exchange on the H NMR chemical shift time scale. In contrast with the Fe(I1) system, the Fe(III) complex exhibits relatively small shifts, but substantial broadening of the bithiazole resonances.

342

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50

40

30

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20

lo

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0

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-IO

-20

8 wm

Figure

2

The 400 MH& 'H NMR spectrum of Fe(II)-Bleo-A2 in ;60=";.t3;; K; Bleo-A2 = 38.5 mM; Fe(II) = 19.3 mu; The assignments of the shifted peaks are also shown. Inset: Representative difference

spectrum from the transfer of saturation experiment at 303'K. The tail of the arrow represents the peak that is saturated and the head indicates the resonance to which magnetization is transferred. The HOD signal at 4.8 ppm is caused by signal "breakthrough." of FeSO 4. 7H 0 in D20 was added to this solution and the pH (the meter reading uncorrected z or the deuterium isotope effect) was adjusted to 6.30 with NaOD. The sample was transferred to a purged 5 mm NMR tube, which was immediately sealed. The absence of significant oxidation of Fe(II) to Te(III) was ascertained The Fe(II)from the characteristics of the NMR spectrum of the sample. bleomycin-A2 sample in H20 was prepared by an analogous procedure. NMR Spectra. Proton NMR spectra were obtained at 400 MHz on a Bruker All chemical shifts were calculated with respect NH-400 NMR spectrometer. For the transfer of saturation experiments, TSP as the internal standard. the shifted resonances were selectively saturated using gated homonuclear decoupling with a decoupling time of 2-3 set and ca. 15 db below .l watt in H20 was obtained using The spectrum of Fe(II)-bleomycin-A2attenuation. Redfield 2-1-4-1-2 sequence (24). Results NMR Spectrum in D20 is

shown

in D20. in Figure

The 'H NMR spectrum 2.

The spectrum

343

of the Fe(II)-Bleo-A2

is a superposition

complex

of the res-

to

a

Vol.

96, No.

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1, 1980

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b. 9

7

8

*** * IL JL

a. 0

9

L 2

7

6

6 Figure

3

of free

Bleo-A 2, which

(9-O ppm) and those

between

50 and -20

of the metal diamagnetic at 13.1

as shown the

spectrum resonance

r

0

3

to the normal

in Figure

also

and the

contains

inset

at 13.1

cluster

The assignment

saturation

in the

complex

complex,

transfer of Figure

ppm results

complex

were

the previously

made in an analogous of the free unassigned

drug

manner

(11,12,25) sugar

extend

a few resonances

using

resonances

344

which

occur

at

shifted A typical

Saturation

of the 3

transfer

of saturation

resonance resonances

of Valerie of the

the known

and our recent

of resonances of the

experiments. 2. in

(% 10%) to 1.13 ppm, which corresponds to the u-methyl acid of free bleomycin. The assignments of the other resonances

spectral

which

3. All the resonances outside the of a single proton, except the resonance

to 3 protons using

diamagnetic

iron

region

of 5 or 6 protons.

is shown

of the

limited

intensity

corresponds

consists

was accomplished

difference

are

of the paramagnetic The diamagnetic

have

ppm which

resonances

ppm.

complex region

Q -1 ppm which

proton

4

The diamagnetic region of the 400 MHz 1H NMK spectrum of Fe(II)-Bleo-A . (a) pH = 6.30, T = 303'K; (b) pH = 8.0, T 1 ',%%O* Asterisks (*) indicate resonances that belong to the complex.

onances range

8, ;pm

assignments

assignments between

of the of some of

3.5 and 4.1

ppm

Vol.

96, No.

(e.g.

:

1. 1980

BIOCHEMICAL

M-4 at 3.88

G-3 at 4.12 experiment

rature

were

diamagnetic

is

(15).

Curie

transfer

do not obey

Weiss

shown in Figure measured

metal

in

ion. are

Bleo-A2

Experiments

complicated

with

in

by

by extrathose

obtained

the chemical to l/T)

shifts or the

of Fe(II)-Bleo-A2

still

yet

bleomycin

which

in progress

including These

interact

to identify

relaxation

in H20 (25),

observed,

observed).

NH protons

by the shorter

in H 0 is 2 in the spectrum

observed

are

resonance from

are

agree

of free

by asterisks)

in H20 originate

as reported

is

+ constant)).

2 and 3) and those

field

to the

behavior

obtained

Law (A6 proportional

to the resonances

(indicated

lowest

only

l/(T

This

The changes

Curie

vs tempe-

to extrapolate

complex

do not

of the

shifts

shifts

The 'H NMR spectrum

in D20 (Figures

observed

temperature

In addition

of resonances

67 ppm (the

but

4.

1:l

the diamagnetic

simple

in H2g.

ppm and

of saturation

dependence

temperature.

experiments.

Law (A6 linear

NMR Spectrum

number

the

transfer

and appear

of an isolated

However, to infinite

saturation

of the

of the chemical

298OK and 333OK,

the formation

of the plot

(A&) also

Plots

(9 to 0 ppm) at infinite

with

COMMUNlfATlONS

ppm, M-2 at 4.06

The temperature

I.

region et al.

the

of Shifts. in Table

between

Sausville from

shown

RESEARCH

I.

linear

consistent polation

The results

in Table

Dependence

shifts

BIOPHYSICAL

ppm, G-2 at 4.04

pH = 6.3).

summarized

Temperature chemical

ppm, G-4 at 3.91

ppm at 303'K, are

AND

times

resonances

with these

of these

a

one at the paramagnetic

resonances,

protons.

Discussion The quantitative would

require

dipolar are

interpretation

estimates

mechanisms

to each of

not available,

present

data.

shifts

originate

shifts,

useful It

since

reviews

qualitative

likely

do not

numerous

of the

the paramagnetic

that

Although

information

in close

shifts

proximity

imply

very

remoteness

from

the magnitude

of paramagnetic

shifts

have

the

paramagnetic ion.

the metal

of the shifts. appeared

and

estimates from

large

to the metal

identified

contact

these

can be obtained

exhibiting

can diminish

herein

of the Fermi

shifts.

resonances

necessarily

effects

analysis

contributions

the observed

from hydrogens

however,

site

is

of

of the relative

in the

Small binding Detailed literature

(26-28). It

can be seen from

sulfonium

group

in agreement C-terminus NTI.

It

observed

is

with

previous

C4EJ is noteworthy

either

I that

and the bithiazole

of bleomycin

the imidazole

Table

protons

studies in metal

consistent that

by saturation

the shifts

which

are all

a shifted transfer

relatively

indicate

minimal

(9-11).

The large

binding with

exhibited

the metal

coordination

imidazole

C2g resonance

experiments

345

by the dimethylsmall.

This

involvement shift

of the exhibited

of the has not

or by comparing

is by

imidazole been the spectrum

Vol.

96, No.

1, 1980

BIOCHEMICAL

II

AND

9

IO

BIOPHYSICAL

RESEARCH

8

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7

6

I

30

r-7 70

Figure

with

4

that

The close 3

Bleo-A2

25

65

60

20

55

50 6, pm

Ii

45

40

IO

35

30

The 400 MH; 'H NMR spectrum of Fe(II)-Bleo-A in FjiOEa;.;;; K, Bleo-A2 = 46.3 mM, Fe(II) = 237.2 XVM, Asterisks (*) indicate shifted NH - resonances. of a sample proximity

in which of the

in D20 was heated

the imidazole

imidazole for

C2-H is

replaced

C21J to NT could

readily

12 hours

at 85'C.

346

by deuterium. shift

its

3

resonance

Vol.

96, No.

1, 1980

BIOCHEMICAL

Summary of Saturation Saturated

AND

Transfer

Peak

BlOPHYSlCAL

Table I Experiments

RESEARCH

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at 303'K and pH = 6.3

Saturation Transfer observed at ~-

Assignment'

d6/dT

l/2

-0.26 -0.23 -0.41 -0.22 -0.16

45.2 ppm+ 42.7 39.6 32.9+ 24.7 20.3

2.83

3.98

VAl.

13.1

1.21

VAL Cag3 BITHIAZOLE C H 5BITHIAZOLE C5'lJ VAL C'H3 THR C"! DIMETHYLSULFONIUM Cfi3 M-l

7.38

2.55 3.80

1.13 4.22

2.8

2.94

a.12 7.83

5.01 of peaks)

Peaks ranging 3.8

-5.1 -6.6 -11.6

from

-0.12

-0.12

Sugar ClJ's, ALA or PYR CH M-2 (or ALA Call) ALA C"fl (or G-2) G-l (HIS Call or CBH) M-4 M-3

4.80

t Temperature

C'H-

ppm

3.89

-15.1+

+p, = 8.0,

- 4.1

4.13 4.10 5.37

-3.9

C4bJ

VAL CaH l/2 PYR CE2 VAL CBH -

2.58

7.9 + 7.7 i 7.1 6.5 9 0.6 + -1 ppm (cluster

PYR Cg2 HIS

0.05 0.07 0.07 0.10

0.10

= 323'K Temperature

= 313'K

5The uncertainty in the assignment of some of these resonances arises out of the complexity of the spectrum of the free drug between 3.8 and 4.1 ppm where several Cg resonances overlap. out of the observed observed

for

carbamoyl this

several

group

equivalent

spectral

shifts

range

resonances

or broaden

of the mannose

may be coordinated of the

it

beyond residue

to the metal

two distinct

pyrimidine

ion.

The shifts

detection. indicate The very

Cg2 resonances

that large

the and non-

suggest

that

group

may be adjacent to the metal ion binding site due to ligation of The perturbation of the CH the secondary amine. Nl and/or These the a-amino group as a ligand. of the alanine moiety implicates

the pyrimidine proton results

are

complexes

compatible in the

The shifts the valeric

acid

literature exhibited residue

with

the

results

of other

studies

of metal-bleomycin

(9-13). by the Cull, suggest

that

C$,

C'Ij,

this

residue

347

CaI13 and CylS3 resonances may also

be ligated

of to the

Vol.

96, No.

metal

the valeric

acid

the

imidazole

data

tends

analogue

ligation

site

to favor group

The above Fe(I1)

the

cc-amino

such a complex. premature

group,

structure

with

Zn(I1)

shifts (which

of this

the hydroxyl

of might

residue

of the metal

However,

to formulate

the following

imidazole

over

ion.

group

to construct

Our

or the

are now in progress of iron(bleomycin

model

in

this

for

valeric space

data,

the structure

laboratory

to further its

acid

magnetic

for

with it

of this

the

filling

structures

associated

experimental

and to determine

for

With

of plausible

as other

sites

oxygen,

group.

the uncertainties

as well

any detailed

amino

a number

of

coordination

Nn, carbamoyl

the secondary

in view

of the present

Experiments

with

to placement

explanation

COMMUNICATIONS

the anomalous

coordination

suggest

Nl and/or

we have been able

interpretation

RESEARCH

as the ligand.

considerations

complex:

of Fe(II))

or to direct

latter

serving

OH or CO, pyrimidine models,

BIOPHYSICAL

--et al. (11) have attributed resonances of the Bleo-A2 complex

as a diamagnetic

carbonyl

AND

Oppenheimer

ion.

serve

BIOCHEMICAL

1, 1980

the is

complex.

define

the

and kinetic

properties. Acknowledgement We thank the generous Service (TTS)

Grants

Drs. gift

W.T.

Bradner

of Blenoxane.

CA-13148

and by a Faculty

and S.T. This

(John

R. Durant

Research

Award

Crooke

research

of Bristol was supported

and JDG), FRA-162

Laboratories

CA-24411

(JDG) from

by Public (JDG),

for Health

GM-27900

the American

Cancer

Society. REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14.

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Sausville, E.A., Peisach, J. and Horwitz, S.B. (1976) Biochem. Biophys. Res. commun. 73, 814-821. Sausville, E.A., Stein, R.W., Peisach, .I. and Horwitz, S.B. (1978) Biochemistry 17, 2746-2753. Gupta, R.K. and Redfield, A.G. (1970) Biochem. Biophys. Res. Commun. 41, 273-281. Lenkinski, R.E. and Dallas, J.L. (1979) J. Amer. Chem. Sot. 101, 5902-5906. Lenkinski, R.E., Peerce, B-E., Dallas, J.L. and Glickson, J.D. (1980) J. Amer. Chem. Sot. 102, 131-135. Krishna, N.R., Huang, D.H., Glickson, J.D., Rowan, R. III and Walter, R. (1979) Biophys. J. 26, 345-366. in NMR Spectroscopy, Vol. 1, Hoffman, R.A. and Forsen, S. (1966) in Progress ed. Emsley, Feeney and Sutcliffe (pergamon Press, New York) pp. 15-204. Krishna, N.R., Huang, D.H. and Goldstein, G. (1980) Applied Spectroscopy, 34, 460-464. Fujii, A., Takita, T., Maeda, K. and Umezawa, H. (1973) J. Antibiotics 26, 396-397. Redfield, A.G. (1978) Methods Enzymol. 49, 253-270. Chen, D.M., Hawkins, B.L. and Glickson, J.D. (1977) Biochemistry 16, 2731-2738. Wiithrich, K. (1976) NMR in Biological Research (American Elsevier, New York) Chapter 6, pp. 221-286. J.P. (1973) in NMR of Paramagnetic Molecules, ed. La Mar, Horrocks Jesson, and Holm (Academic Press, New York) pp. l-52. Resonance Eaton, D.R. and Phillips, W.D. (1965) in Advances in Magnetic ed. J.S. Waugh Academic Press, New York) pp. 103-148.

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