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LIBRARY 5
Michigan State
Univwny

This is to certify that the

V thesis entitled
THE INTERACTION OF THE CIS— AND I§A§§7DICHLORODIAMMINE—

PLATINUM(II) COORDINATION COMPLEXES WITH DNA AND ITS

COMPONENTS IN VITRO 2
presented by

Sill: Abdel-Lntif Aly Mansy

has been accepted towards fulfillment
of the requirements for

Phi D. gjkgmwin__§122hlfiifis

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MCDAC. 1491974

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ABSTRACT
THE INTERACTION OF THE CIS- AND I§Afl§7DICHLORO-
DIAMMINEPLATINUM<II) COORDINATION COMPLEXES
wITH DNA AND ITs COMPONENTS IN VITRO
By

Samir Abdel-Latif Aly Mansy

Cis-Pt(II)(NH3)2Cl is a potent antitumor agent in many animals

2

while the trans-isomer exhibits no antitumor activity. Harder et al.22
demonstrated the selective and persistent inhibition by the

cis-Pt(II)(NH3)2Cl of new DNA synthesis, at SuM, in human amnion AV

2 3
cells. The tggngrisomer did not show any effect on DNA, RNA or protein
synthesis except at very high concentrations. It is believed, at present,
that the primary lesion leading to antitumor activity is an attack by the
gig:Pt(II)(NH3)2C12 on the cellular DNA.

As the c s-isomer has two active chlorine atoms on one side, while
the 55225 isomer has the two active chlorine atoms Opposite to each other
in the square planar complex, a comparative study of the interaction of
the two isomers with DNA and its components (nucleosides) could determine
the nature of binding of the gig—isomer with DNA, that is responsible
for the antitumor activity. The interaction between the platinum isomers
and the nucleosides, synthetic polynucleotides, and various DNA's with
different GC content, was studied in a medium of 0.1 M NaCln4 in the dark

at 37°C. The reaction occurs with the loss of chloride ions through

29

equation. The pH values of the solutions were in the range 5.2 to 7.2

except for the case of the pH dependent studies.

Samir Mansy

Difference spectra (U.V.) using four 1 cm cells on a model Cary 15
spectrophotometer was employed to determine the locations of the inter-
action. The sites of attachment of the metal ions on the nucleosides
can be identified by blocking the nitrogen atoms with protons, or alter-

natively with a methyl group. At a pH less than the pKa of N1 of adeno-

sine, the cis— and trans- platinum species gave identical difference
Spectra, indicating that the interaction was monofunctional at N7 of the

purine ring. At a pH 5-7, adenosine, with the cis-isomer, has a peak at

Azsonm and a shoulder at A nm, which grows with increasing concentra-

300

tion of the platinum species while the trans-isomer shows a peak at

Azaonm. lrMethyl-adenosine shows a broader band at Azgonm with the gig—

species than the trans-isomeric species at A nm which suggests the

280

possibility of chelation between N7 and 6—NH in the case of the cis-

2

isomer. The peak at A nm of the cis-isomer with adenosine could be

280
assigned to the monofunctional interaction at N1, and that at Azgonm

to the interaction at N7 chelating with the 6—NH2 group. Adenosine did
not show any interaction at a pH higher than 11.

Cytidine did not show any interaction with either isomer at a low
pH. At a pH higher than the pKa of N1 (N3 of the pyrimdidne ring is

named N1 in this thesis), cytidine has a peak at Azgonm with the cis-

isomer and a shoulder at 3300nm. This is probably due to chelation with

the neighboring amino group. The trans-isomer gives a sharp peak at

A290nm. Both isomers did not interact with l-methylcytidine, indicating

that the interaction is at N1 of the pyrimidine ring. At a pH higher

than 11, cytidine did not interact with either of the platinum isomers.

At a pH below the pKa of N guanosine has identical difference

1’
spectra with both platinum isomers. The fact that 1-methylguanosine

Samir Mansy

has a similar interaction with both isomers, while 7-methylguanosine does
not interact with either platinum isomer, leads to the conclusion that
both isomers of the platinum species interact similarly at N7 of the

purine ring, giving rise to a peak at A nm. At a pH higher than the

290
pKa of N1, the interaction at N1 produces a peak at 3265nm in addition
to that of N7 at Azgonm. At a pH higher than 11, there was no inter-
action between guanosine and either of the platinum isomers.

Inosine behaved similarly to guanosine at different pH values,

giving a peak at A nm due to the binding at N7 and a peak at A258 due

270
to the N1 interaction.

Uridine and thymidine show very little interaction with either gig:
or tggggrplatinum isomers over the entire pH range. This could be
eXplained by the low basicity of N1 of the pyrimidine ring.

The dimers A3'pS'A and A2'p5'C with both gig: and trans: platinum
isomers behave as the adenosine and cytidine monomers in the pH range
1-10. At a pH higher than 10, the gisfisomer showed a strong inter-
action with both A3'pS'A and A2'p5'C while the tggggrisomer did not
show any interaction, as in the case of the monomers. This interaction
at a high pH might be due to chelation between the two gigfactive groups
and the two 6-NH2 groups that lie over each other in the stacked form.
This was further proved by the change of the CD spectrum of the dimer
as a function of temperature due to the change of the stacking conforma-
tion. The gigrPt(II)-dimer peak did not show any change as a function
of temperature, while the tggggrisomer abolished the CD peak completely.

Job's method was used to determine the number of ligands in the

resulting complexes. Adenosine, l—methyladenosine, and cytidine show a

1:1 complex (PtL) with the cis-isomer. The trans-isomer also gave a

Samir Mansy

PtL complex with adenosine, l-methyadenosine, but more than one complex
with cytidine. Both isomers gave a 1:2 complex (PtLZ) with guanosine,
l-methylguanosine, inosine and l-methylinosine.

A series of difference spectra were taken of the interaction of
nucleosides with varying concentrations of gig: and giggg- platinum
isomers at a pH 5-7 where only one platinum-nucleoside species is
expected, in order to determine the formation constants (Kf). Adeno-
sine, l-methyladenosine and cytidine have Kf values in the range of 1 +5,
while guanosine, l—methylguanosine, inosine and l-methylinosine have Kf
values in the range of 10+9.

At a pH around 5, single stranded synthetic polynucleotides, except
polyuridylic acid (poly U), formed a white precipitate with both gig: and
igggg:platinum isomers, in the concentration range above 1:2 of the platinum
isomer to the phosphate while the doubly stranded polymers stayed in solu-
tion. The difference spectra of polyadenylic acid (poly A) with both
platinum isomers are different; the cis-isomer spectrum shows the lack of
the long wavelength shoulder characteristic of the chelation between N

1

or N7 with the 6-NH2 group. Poly U shows a strong interaction with the

cis-isomer, while only a weak interaction with the trans—isomer.
Poly A.Poly U showed similar difference spectra with both platinum isomers,
and as N1 of poly A is hydrogen bonded with N1 of poly U, we can conclude

that both platinum isomers interact monofunctionally at N of poly A.

7
Poly d(A-T).d(A-T) showed also similar differerence spectra with both
platinum isomers, thus excluding chelation between N1 and the 6-NH2 group
in the case of the gig:isomer. Polyguanylic acid (poly G) showed two
peaks at A265“. and Azgonm with both platinum isomers, indicating the

interaction is at N1 and N7 of the guanine base.

Samir Hansy

DNA, of different GC content, shows two peaks at 1270nm and Azgonm
at the beginning of the reaction, and these peaks then converge to one
peak at Azsnnm. The rate of the reactions increase as the GC content
increases, and there is also a break in the line describing the first
order kinetics indicating that there are two consecutive mechanisms.

In summary, as guanosine shows the highest formation constant,
and N7 of guanosine in DNA is available for the interaction with the
platinum species, we can conclude that the interaction in DNA is at N7
of the guanine base, followed by partial denaturation, where N1 of the
same base, and also cytidine and adenine will be then available. The
possibility of interstrand cross linking between the platinum isomers

and DNA, and also the possibility of intra—strand cross linking between

the cis-isomer and ApA, ApC or CpC could not be excluded.

 

22. Harder, H. C., and Rosenberg, 8., Int. J. Cancer Q, 207 (1970).

29. Banerjea, D., Basolo, F., and Pearson, R., J. Am. Chem. Soc. 79,
4055 (1957). '-

THE INTERACTION OF THE Q13: AND IBANS-DICHLORO-
DIAMMINEPLATINUM(II) COORDINATION COMPLEXES

WITH DNA AND ITS COMPONENTS IN VITRO

By

Samir Abdel-Latif Aly Mansy

A THESIS
Submitted to
Michigan State University
in partial fulfillment of the requirements

for the degree of

DOCTOR OF PHILOSOPHY

Department of Biophysics

1972

@QCopyright by
SAMIR ABDEL-LATIF ALY MANSY

1972

ii

Dedicated to my parents and to my ever beloved Ibtissam

iii

ACKNOWLEDGMENTS

The author wishes to express his deepest thanks to Professor
Barnett Rosenberg for his encouragement and interest during all
the different studies.

The author wishes also to express his sincere gratitude to
Dr. R. Williams, Department of Inorganic Chemistry, Oxford Univer-
sity, England and to Dr. Andrew Thomson, University of East Anglia,
England, for their stimulating discussions on the interaction of
metal ions with macromolecules, were responsible for my own interest
in this field.

Finally, I would like to express my indebtedness to Dr. James
Boeschele for many fruitful discussions and to Mr. Hassan El-Nabtity
for his technical assistance.

This research was supported by National Cancer Institute, grant

number CA-11349.

iv

TABLE OF CONTENTS

I 0 INTRODUCTION 0 O O O O O O I O O O O O O O O O O O O O O 0

Platinum Coordination Complexes in Cancer Chemo-
therapy. Review . . . . . . . . . . . . . . . .

The reactive centers in DNA . . . . . . . . . .
Interaction of Metal ions with Nucleic acids . . .

Approach to the problem . . . . . . . . . . . . . .

II. EXPERIMENTAL . . . . . . . . . . . . . . . . . . . . . . .

Monomers, Dimers, Polymers . . . . . . . . . . . .
pH Study . . . . . . . . . . . . . . . . . . . .
Determination of number of Ligands of nucleosides

per platinum complex . . . . . . . . . . . . . .

Determination of the sites of binding and its forma—

tion constants . . . . . . . . . . . . . . . . . .
Calculation of the formation constant . . . . . .
Kinetics of polynucleotides and DNA-Platinum
complex interaction . . . . . . . . . . . .

III 0 RESULTS 0 O O O O O O I O O O Q C O O O O O O O O O O O 0

(A)

(B)

(C)

The effect of the pH on the binding site of
nucleosides to the gig: and trans—Pt(II)(NH3)2C12 .
Monomers . . . . . . . . . . . . . . . . . . . .
Adenosine . . . . . . . . . . . . . . . . . . . .
l-Methyladenosine . . . . . . . . . . . . . . . .
Cytidine . . . . . . . . . . . . . . . . . . . .
Guanosine . . . . . . . . . . . . . . . . . . . . .
Inosine . . . . . . . . . . . . . . . . . . . . .
Uridine . . . . . . . . . . . . . . . . . . . . .
Thymidine . . . . . . . . . . . . . . . . . . . . .
Dimers . . . . . . . . . . . . . . .
Adenylyl (3' +5' ) Adenosine (A3' p5' A). . . . . . . .
Adenylyl (2' +5' ) Cytidine (A2' p5' C) . . . . . . . .
Polynucleotides . . . . . . . . . . . . . . . .
The stoichiometry of the cis- and trans-platinum
complexes in solution . . . . . . . . . . . . . . .
The formation constant of the gig: and trans-
platinum nucleoside complexes . . . . . . . . . . .
The interaction of Pt(II)(NH3)2C12 with poly-
nucleotides . . . . . . . . . . . . . .

Page

15
27

31
32

32
33

35

36
37

39

41

41
41
44
44
47
49
49
54
54
54
54
56
59

59

75

92

Poly Guanylic acid . . . . . . . . . . .
Poly Cytidylic acid . . . . . . . . . .
Poly Adenylic acid . . . . . . . . . . .
Pflly‘Uridylic acid . . . . . . . . . . .
Poly A. poly U . . . . .

Poly deoxyadenylic-poly deoxylthymidylic
(double stranded) . . . . . . . . . . .
DNA . . . . . . . . . . . . . . . . . .

Iv. DISCUSSION 0 O O O O O O O O O O O O O O C 0

Interaction at N and N of adenosine .
Interaction at tAe 6—NH of adenosine .
Bifunctional binding at 6-Nll2 and N7 of
adenosine . . . . . . . . . . . . . . .
Guanosine Reactions . . . . . . . . . .

l—Methyl Guanosine Reactions . . . . . .

Interaction at the N and the 6—NH2 cytidine
A study of the interaction of dinucleotides
with cis- and trans-Pt(II)(NH3)2C12 by circular

Dichroism spectoscOpy . . . . . . . . .
Kinetics of DNA with Pt(II)(NH ) C1 .

3 2 2
Conclusion . . . . . . . . . . . . .

BIBL IOGMPHY O O O O l O O I O O O O O O I O O O O 0

APPENDIX .

vi

Page

92
92
96
96
96

' 100

100

110

110
113

114
118
123
123

125
129
131
134

140

LIST OF TABLES

Table Page
191
-1 Average distribtuion of Pt among the various
classes of cellular compounds in three bacteria . . . . . 2
2 Regression of large solid Sarcoma 180 tumors in
Swiss white mice with injection of platinum
compound 8 0 O O O O O O O O O O O I O O O O O O 0 O O O O 4
3 The best results of the increase of life span

by cis-Pt(II)(NH3)2C12 . . . . . . . . . . . . . . . . . 5

4 The effect of the combination chemotherapy of
cis-Pt(II)(NH3)2C12 with other drugs on the sur-
vival of animals . . . . . . . . . . . . . . . . . . . . 5
5 The theoretical values of the coulombic integral

of the different sites of the purine and pyrimi-
dine bases . . . . . . . . . . . . . . . . . . . . . . . 18

6 The electronic characteristics of the amino groups
of the purine and pyrimidine bases . . . . . . . . . . . 25

7 The stoichiometry of the cis- and trans—platinum
nucleoside complexes in solution at a pH around 5 . . . . 76

8 The interaction of the cis- and trans-Pt(II)(NH3)2012

at the different sites of the nucleoside . . . . . . . . 88
9 The formation constants of Pt(II)+Nuc1eoside complexes. . 91
10 Rate of the reaction of Pt(II)-isomers with DNA at

different tempertures . . . . . . . . . . . . . . . . . . 105

11 Activation energy of interaction of the Pt(II)(NN

C12
"1 th DNA 0 O O O O O O I I O I O O O O O O O O

)
3020 O 0 109

vii

Figure

10

11

12

13

14

15

LIST OF FIGURES

Structure of platinum compounds . . . . . . . . .
The basicity of the pyridine derivatives . . .

The effect of the hydroxy-group on the basicity
Of the pyrimidine ring 0 O O O O O O O O O O I 0

Raman spectra of cis- and trans-Pt(II)(NH3)2Cl2 .
Absorption spectra of the Pt(II)(NH3)2C12 isomers

Suggested mode of binding of Pt(II)(NH3)2C12
isomers with adenosine at a pH 5. 6- 7. 3 . . . . .

Difference spectra of the Pt(II)(NH3 ) 2Cl2 isomers
with adenosine at various pH values3 . . . . . . .

Difference spectra of the Pt(II)(NH3)2C12 isomers
with 1-methyladenosine at pH = 3. 27 . . . . .

Difference spectra of the Pt(II)(NH 3)2C12 isomers
with cytidine at various pH values . . . . . .

Difference spectra of the Pt(II)(NH 3)2C12 isomers
with guanosine at various pH values3 . . . . . . .

Difference spectra of the Pt(II)(NH ) 22C1 isomers
with l-methylguanosine at various pEZ values . . .

Difference spectra of the Pt(II)(NH 3)2C12 isomers
with inosine at various pH values . . . . . . .

Difference spectra of the Pt(II)(NH ) C12 isomers
with l-methyl, 7—methyl-inosine at a pH - 3.27 .

Difference spectra of the Pt(II)(NH3)2C12 isomers
with A' 3p5' A at various pH values . . . . . . .

The structure of the A3'p5'A and A2'p5'C determined

by x-ray diffraction e e e e e e e e e e e e e 0

viii

Page

19

21
34

42

43

45

46

48

50

51

52

53

55

57

Figure

16

17

18

19

20

21

22

23

24

25

26

27

28

29

3O

31

Difference spectra of the Pt(II)(NH3)2C12 isomers
with A2' p5' C at various pH values . . . . . . . . . . .

Difference spectra of the Pt(II)(NH3 )C 13’ Pt(II),
isomers with adenosine, L, "Job' 3 metfiod . .

Difference spectra of the Pt(II)(NH3)2C12, Pt(II),
isomers with 1-methy1adenosine, L, "Job's method" . .

Difference spectra of the Pt(II)(NH ) 2C12, Pt(II),
isomers with cytidine, L, "Job's meghod"2 . . . . . .

Difference spectra of the Pt(II)(NH ) C12, Pt(II),
isomers with guanosine, L, "Job's met od . . . . . . .

Difference spectra of the Pt(II)(NH3)2 C12, Pt(II),
isomers with l-methylguanosine, L, Job' 3 method" . . .

Difference spectra of the Pt(II)(NH3)2C12, Pt(II),
isomers with inosine, L, "Job' 3 method" . . . . . . . .

Difference spectra of the Pt(II)(NH 3)2C12, Pt(II),
isomers with l-methylinosine, L, "Job 8 method . . . .

Graphical determination of composition of the
Pt(II)(NH3)2C12 - adenosine complex . . . . . . . . . .

Graphical determination of the Pt(II)(NH 3)2C12-
l-methyladenosine complex . . . . . . . . . . . . . .

Graphical determination of composition of the
Pt(II)(Nll3)2Cl2 - cytidine complex . . . .

Graphical determination of composition of the
Pt(II)(Nll3)2C12 - guanosine complex . . . .

Graphical determination of composition of the
Pt(II)(NH3)2C12 - methylguanosine complex . . . . . .

Graphical determination of composition of the
Pt(II)(NH3)2C12 - inosine complex . . . . . . . . . . .

Graphical determination of composition of the
Pt(II)(NH3)2012 - l—methylinosine complex . . . . . . .

Difference spectra of adenosine, O. 55 x 10- ”M
in 0.1 M NaClOA , with various concentratiozm of
the Pt(II)(NH 322 C12 isomers; 5, 0.25 x 10

6, 0. 37x M; 27, O. 5 x 10-4 M; 8, O. 5 x
10—4 M; 20, 04 x IO-4N . . . . . . . . . . . . .

ix

Page

58

60

61

62

63

64

65

66

67

68

69

7O

71

72

73

76

Figure

32

33

34

35

36

37

38

39

40

41

42

43

Page

Titration curve of adenosine with Pt(II)(NH3)2C1

isomers plotted from Figure 31 . . . . . . . . 77
Difference spectra of l-methyladenosine, 0.41 x

10‘“ M in 0.1 M NaClOa, with various concentration

of the Pt(II)(NH3)2C12 isomers, 5, 0.25 x 10‘4 2;

6, 0.37 x 1 ”AM; 7, 0.5 x 10-4vN; 8, 0.75 x 10'

20, 4 x 10' . . . . . . . . . . . . . . . . . . . . . . 78

Titration curve of l-methyladenosine with
Pt(II)(NH3)2C12 isomers, plotted from Figure 33 . . . . . 79

Difference spectra of cytidine, 10-4 M in

0.1 M NaClO , with various concentration of

Pt(II)(NH3)2C12 isomers, 5, 0.25 x 10““ M; 6, 0.37

x 10-4 M; 7, 0.5 x 10-4 M; 8, 0.75 x 10" M; 20,

4 x 10‘4M . . . . . . . . . . . . . . . . . . . . . . . . 80

Titration curve of cytidine with Pt(II)(NH3)£ 12
isomers, plotted from Figure 35 . . . . . . . . . 81

Difference spectra of guanosine, 1.1 x 10'4 M in

0.1 M NaClO4, with various concnetration of

Pt(II (NH3)2C12 isomerz, 5, 0. 25 x 10 44M; 6.0.37

x 10‘ M, 7, 0.5x M, 8, 0.75 x 10’ M; 20, 4

x 10 “M . . . . . . . . . . . . . . . . . . . . . . . 82

Titration curve of guanosine with Pt(II)(NU3)2C12
isomers, plotted from Figure 37 . . . . . . . . . . 83

Absorption spectra of adenosine, 0.55 x 10 w:

cis-Pt(II)(NH) - adenosine, 0.49 x 10 _4

and trans-Pt(II§(NH3)2C12 — adenosine, 0.48 x M10 M . . 84
-144

Absorption spectra of 1-methyladenosine, 0. 41 x 10 M;
cis-Pt(II)(NH3 )C — 1 methyladenosine, 0.36 x 10 4M M;

and trans-Pt(II§(NH3)2CI2 — 1-methy1adenosine, 0. 37

x lO‘zM . . . . . . . . . . . . 85

Absorption spectra of cytidine, 10-4 M

cis-Pt(II)(NH3)2C12 - cytidine, 0.93 x 10‘“ M 86
Absorption spectra of guanosine, 1.1 x 10’“ M;
cis-Pt(II)(NH) - guanosine, 1.05 x 10'

and trans-Pt(Ilgcfi N3)2C12 - guanosine, 1.05 x

10- 0 O O O O O I O I O O O I O O O O O O O 87

Difference spectra of the Pt(II)(NH3 ) €12 isomers,
0.2 mM, with polyguanylic acid (poly C),20.1 mM p . . . . 93

Figure

44

45

46

47

48

49

50

51

52

S3

54

55

Difference spectra of the Pt(II)(NH3) C12 isomers,
0.2 mM, with polyguanylic acid (poly $0 0.1 mM P, in

0.1 M NaCIOA at 37°C 0 o o o o o o o o o o a

Difference spectra of the Pt(II)(NH ) 22C1 isomers,
0. 2 mM, with polycytidylic acid (poiy2 C), 0.1 mM P,

in 0.1 M NaC104 at 37°C, B, before precipitation of
the complex 0 I O O O O O C O O O O C C O O C O O 0

Difference spectra of the Pt(II)(NH3 ) C12 isomers,

O. 2 mM, with polyadenylic acid (poly 3X),2 0. 1 mM 9,

in 0.1 M NaClOa at 37° C, 8, before precipitation

of the complex . . . . . . . . . . . . . . . . . . .

Difference spectra of the Pt(II)(NH3 ) 2C12 isomers,
0. 2 mM, with polyuridylic acid (poly U0, 0.1 mM P,
in 0.1 M NaC104 at 37° C, 8, end of the reaction . .

Difference spectra of the Pt(II)(NH3)2C12 isomers,

0.2 mM, with doubly stranded poly A. poly U, 0.1 mM P,

in 0.1 M NaClOa at 37°C, 8, end of the reaction . . .
Difference spectra of the Pt(II)(NH )2 C12 isomers,
0.1 mM, with doubly stranded poly d(2
0.05 mM P, in 0.1 M NaClOa at 37°C, 8, end of the
reaction 0 O O O O O O O O O O O O O O O O O O O 0

First order kinetics of the reaction of the synthetic
polynucleotides with gig:Pt(II)(NH3)2C12 in

0.1 M NaClOa; Pt(II)/P - 2 . . . . . . . . . . . . .
First order kinetics of the reaction of the synthetic
polynucleotides with trans— Pt(II)(NU3)2C12 in

0.1 M NaClOA; Pt(II)/P - 2 . . . . . . . . .

Difference spectra of the Pt(II)(NH) C12 isomers,
0.15 mM, with DNA, 0.15 mM P, in 0.1 M NaClOa at 37°C

First order kinetics of the reaction of
cis-Pt(II)(NH3 )2012, 0.15 mM, with DNA, 0.15 mM P, in
0.1 M NaClOA at 37C . . . . . . . . . . . . . . . .

First order kinetics of the reaction of
trans-Pt(II)(NH3)C 0.15 mM, with DNA, 0.15 mM P,
in 0.1 M NaC1043a§ 37° °C . . . . . . . . . . . . . . .

Schematic representation of the existence of different

complexes in solution at differnt ph's . . . . . . .

xi

A- r). poly d(A-T),

Page

94

95

97

98

99

101

102

103

104

106

107

112

Fizu

A!»
’3

l\.J

Figure

S6

S7
58

59

Summary of the interactions of the Pt(II)(NH3)2C12
isomers with nucleosides at different pH's

CD spectra of A3'p5'A

CD spectra of A3'pS'A with Pt(II)(NH3)2C12 isomers

Structure of DNA

xii

Page

119
126
128

132

I. INTRODUCTION

Platinum Coordination Complexes in Cancer Chemotherapy: Review

In 1965 Rosenberg et a1.1 studied the effects of alternating elec-
tric fields on cell division in E. Coli B bacteria using platinum elec—
trodes. When the current was turned on at 1000 Cps, the cell division
was completely inhibited in a short while. Microscopic examination
revealed that the bacteria continued to grow, forming very long fila-
ments, even after terminating the current. They found that various
platinum species were synthesized by electrolysis from the platinum
electrodes and certain chemical species in the culture medium.

Rosenberg et al.2 found that the neutral compounds gigyPt(IV)(NH ) C1

3 2 4
and cis-Pt(II)(NH ) C1 was the most effective inhibitors of cell divi-

3 2 2
sion and inducers of filaments. The trans isomers and the other four
charged species [Pt(IV)NH3c15]', [Pt(IV)Cl6]--, [Pt(II)(NH3)C13]' and
[Pt(II)C14]= had little or no effect on cell division.
Renshaw and Thomson (1967)3 followed the distribution of labeled
platinum compounds (Ptlgj) among various bacterial cellular components.

The cis-Pt(IV)(NH C14 in filamentous E. Cali 8 was found associated

3)2
with metabolic intermediates, nucleic acids and cytOplasmic proteins
(Table 1). In E. Cali 8 whose growth was inhibited by [Pt(IV)c16]”
the platinum was bound only to cytOplasmic proteins, and, as inhibition
of cell division did not occur, they concluded that binding to cellular

proteins was responsible for growth inhibition but not for inhibition

of cell division. Furthermore, they found that the metal complex was

2

Table 1. Average distribution of Pt191 among the various classes of

cellular compounds in three bacteria

 

 

Percentage of radioactivity

 

 

Fraction
E. coli B. cereus S. aureus
Metabolic intermediates . . . . . 19 74 60
Lipids . . . . . . . . . . . . . 6 2 1
Nucleic acids . . . . . . . . . . 30 19 20

Cytoplasmic protein . . . . . . . 45 S 19

 

bound predominantly to metabolic intermediates in Bacillus Cereus and
Staphylococcus aureus, in which neither growth nor cell division were
inhibited. Rosenberg et al., 19694 showed that gingt(II)(NH3)2C12,
gigyPt(IV)(NH3)2C14 and Pt(II)(NH2)2(CH2)2C12 were potent inhibitors of
Sarcoma 180 tumors in mice. Successful regression of large solid Sarcoma
180 tumors by gig:Pt(II)(NH3)2C12 was about 1002, while that with

cis-Pt(IV(NH C14 was 832 (Rosenberg et al., 1970)5 (Table 2).

3’2

The gigrdichlorodiammineplatinum(II) compound exhibits antitumor
activity against a variety of tumor systems. Rosenberg et al.4’5
reported the growth inhibition of Sarcoma 180 and Leukemia L1210 tumor
in mice. The drug showed similar activity against the Reticulum Cell
Sarcoma,6 rat Dunning Ascites Leukemia and Walker 256 Carcinosarcoma.
Table 3 shows the best results of tumor tests reported by the National
Cancer Institute.8

DMBA (7,12 - dimethylbenzanthracene) deve10ps mammary tumors in

9’10’11 Mammary tumors

rats, which closely resembles human breast cancer.
are sensitive to hormonal chemotherapeutic agents, but are not respon-
sive to many cytotoxic agents. Welsch12 showed that the gigedichloro-
diammineplatinum(II) was highly effective in promoting the regression

of the DMBA-induced mammary tumors. The drug showed higher activity in
mammary tumors pretreated with cytoxan. Venditti13 disclosed that the
antitumor activity of gigfdichlorodiammineplatinum(II) in combination
with cyc10phosphamide (cytoxan), was superior to the single drug (svner~
gistic action). Speer et al.14 reported similar effects in combinations
of cis-dichlorodiammineplatinum(II) with cyclophosphamide (cytoxan) and

*with other substances. Table 4 summarizes the percent prolongation of

survival (maximal value of 200% means complete cures' of all animals)

Table 2. Regression of large solid Sarcoma 180 tumors in Swiss white
mice with injection of platinum compounds

 

 

 

 

 

 

E2.
2E. E2:
Compound Dose schedule mice deaths of_ Z_
(mg/kg) in_ cures cures
test
cis-Pt(II)(NH3)2012 2.0 daily 6 6 0 0a
cis-Pt(II)(NH3)2C12 4.0, day 8 6 s 1 17b
cis-Pt(II)(NH3)2C12 4.0, days 8 and 17 6 1 5 83
cis-Pt(II)(NH3)2C12 6.0, day 8 6 2 4 67
c s-Pt(II)(NH3)2Cl2 6.0, days 8 and 16 6 3 3 SO
cis-Pt(II)(NH3)2C12 8.0, day 8 6 0 6 100
cis-Pt(IV)(NH3)2Cla 4.0 daily 6 5 1 17b
cis-Pt(IV)(NH3)2C14 6.0, day 8 6 5 1 17b
cis-Pt(IV)(NH3)2C16 6.0, days 8 and 15 6 3 3 50
cis-Pt(IV)(NH3)2Cla 8.0, day 8 6 3 3 SO
gingt(IV)(NH3)2Cla 8.0, days 8, l6 and 23 6 l S 83
cis-Pt(IV)(NH3)2C14 10.0, day 8 6 s 1 17h
b
Controls 24 20 4 17

 

aDeaths due to platinum compound
bExpected spontaneous regression rate

All tumors weighed more than 0.5g size of day 8 tumors in 12 mice,
0.96 i 0.443. There were no "no takes" in this test.

Table 3. The best results of the increase of life span by
cis-Pt(II)(NH3)2C12

 

 

 

Tumor Host Best Results
Leukemia L-1210 BDF, Mice Z ILS - 3802; 4/10 cures
Lewis Lung Carcinoma BDF, Mice 1002 cures
B-l6 Melanocarcinoma BDF, Mice Z ILS - 2222; 1/6 cures

Walker 256 Carcinosarcoma Fisher 344 Rats 1002 cures

P 388 Lymphocytic Leukemia BDF. Mice z ILS ‘ 213

 

Table 4. The effect of the combination chemotherapy of
cis-Pt(II)(NH3)2C12 with other drugs on the survival of animals

 

 

Cis-Ptgii)(NH,12912

 

 

 

 

mglkg x da§§_f Other substance Prolongation
2.5 x 4 days Cytoxan, 100 x 1 day 2002

2 x 4 Thioguonine, 10 x l 2002
10 x l S-Fluorourocil, 12 x 4 1502

8 x l Vincristine, 0, 12 x 4 1002

8 x 1 Hydroxyurea, 80 x 4 902

10 x 1 None 852

 

6

of animals treated with gigrdichlorodiammineplatinum(II) in combination
with other antitumor agents.

Inhibition of cell division and the induction of filament formation
in some strains of E. Coli, could be achieved by low doses of UV (Witkens,
1967)15 and ionizing radiation (Adler and Hardigree, 1965).16 Hydroxy-
urea (an anti-tumor drug) also causes elongation in E. Coli (Rosenkranz
et al., 1966)17 and inhibits cell division in mammalian cells (Young et
al., l967)18. In the correlation of inhibition of cell division, and
elongation of E. Coli, with some of platinum complex compounds to the

17’19'20 Harder et al., 196921 reported

other agents mentioned above,
the inhibition of DNA synthesis in mammalian cells by platinum compounds.
Harder et al., 197022 tested the effect of I, gig:Pt(II)(NH3)2C12; II,
glgyPt(IV)(NH3)2C14; III, Pt(II)(NH2)2(CH2)2C12; IV, Pt(II)(NH3)4C12;
and V, tggggrPt(IV)(NH3)2Cl4, on overall DNA, RNA and protein syntheses
in mammalian cells in tissue culture (Figure 1).

Compounds I, II, and III are bacterial filament formers and inhibit
S-180 tumor growth, while compounds IV and V do not inhibit S-180 tumors
and are not bacterial filament formers. Harder et a1.22 followed the

3

DNA synthesis by incorporation of H — thymidine in human amnion AV3 cells

in tissue culture over a 24 hour period in the presence and absence of
the platinum coordination complexes. There was a concentration-dependent

effect of cis-Pt(II)(NH3)2Cl on DNA synthesis. At SpM and below of

2

cis-Pt(II)(NH3)2Cl DNA syntheis was selectively and persistently

2’
inhibited. At ZSuM and above, DNA synthesis was more rapidly inhibited
than RNA synthesis, (as measured by the uptake of 3H-uridine), and pro-
tein synthesis (as measured by the incorporation of 3H-leucine) in the

cell. After 24 hours of treatment all three were virtually completely

3)2C12;

II, cis—Pt(IV)(NH ) C1 ; and III, Pt(II)(NH ) (CH ) C1 has antitumor
““‘ 3 2 4 2 2 2 2 2

Figure 1. Structure of platinum compounds. I, cis-Pt(II)(NH

activity; IV, Pt(II)(NH3)4C12; and V, trans-Pt(IV)(NH3)2C14 do not show

antitumor activity.

cu H3

 

 

M
cu u3 cu
I u "I
cu
"a" H;
cu
Iv v

Figure l

8

inhibited. The complete inhibition of DNA synthesis after 6-10 hours

using ZSuM of cis-Pt(II)(NH3)2Cl , which is a good therapeutic dose in

2
mice for treatment of S-180, suggested that DNA was the primary target

in terms of overall cellular function.

22
Harder et al. found that the inhibition of DNA synthesis by anti-

tumor platinum compounds is almost irreversible, and occurs at a very

slow rate in contrast to the rate of inhibition of hydroxyurea,20’23

which does not inhibit RNA or protein.18’24’25

Howle et a1.26 studied the effect of gisrdichlorodiammineplatinum(II)
on the incorporation of ribo- and deoxyribonucleosides in DNA in vitro.
The incorporation of thymidine (Th, deoxyuridine (dud), deoxyadenosine
(dAdo) and deoxyguanosine (dGuo) was inhibited, and they have almost the

same trend of inhibition. The effect of the cis-Pt(II)(NH Cl2 on the

3)2
inhibition of deoxycytidine (dcyd) incorporation was delayed for two

hours at a 10-4 M cis-Pt(II)(NH3)2Cl2 and for three hours at 2.5 x 10‘5 M

cis-Pt(II)(NH3)2Cl and only 60% inhibition was achieved after five hours.

2

Cells from mice treated, in vivo, with cis-Pt(II)(NH3)2C12

incorporate thymidine, in vitro, at a rate much less than the control cells

before sacrifice,

for 96 hours, incorporate dcyd at a markedly enhanced rate during the
first 8 to 10 hours and then return to the control value after about 40
hours after treatment. Howle et al.26 suggested that the platinum species
interacts with the cell membrane which becomes more permeable to dcyd.
Although the DNA synthesis was diminished, the effect was not manifested
in the dcyd-rate of incorporation and dcyd may be the rate limiting step

of the diffusion through the membrane.

Harder et al.22 reported the lack of inhibition of 3H—thymidine uptake
into the acid-soluble pool after 6 hours in the human amnion AV3 cells
by cis-Pt(II)(NH3)2Cl2 and the incorporation of 3H-thymidine in DNA
was inhibited in two hours. As there is no inhibition of precursor
movement into the cell, DNA might be the primary lesion site. The
binding sites on DNA will be the controlling factor for the incorpora-
tion of the deoxynucleosides. If the gig-Pt(II)(NH3)ZClZ interacts
with deoxyadenosine at N1, deoxycytidine at N1 and deoxyguanosine at
N7, we expect that thymidine and deoxyguanosine incorporation will be
inhibited as the sites of hydrogen bonding will be blocked, and the deo-
xycytidine incorporation will be normal or enhanced according to the

reactivity of N of the platinum guanine complex

1
Howle et al.26 suggested a two-stage mechanism for explaining the
slow rate of DNA inhibition by these platinum compounds. The platinum
compound by itself is inactive and must be converted to an active
species which inhibits DNA, RNA and protein synthesis. Another species
inhibits only DNA synthesis. Harder et al.22 had a different interpre-

tation, their postulate was
Pro ———————e> Pt1-—————53> Pt2 (l)

where Pto is the initial platinum species; Pt1

species which is the active species for DNA inhibition but less so for

is a slowly increasingly

RNA and protein inhibition; Ptz, which is the equilibrium with Pt is

1’
responsible for RNA and protein synthesis inhibition. Their suggestion

for the slow formation rate of Pt1, and the low equilibrium constant

for Pt2 production, would eXplain the selective synthesis inhibition

10
of DNA, and the slower rate of RNA and protein synthesis inhibition.

Renshaw et al.3 found that an appreciable amount of the labelled
platinum compound was bound to the protein as well as to the nucleic
acids. Table 1 shows almost equal distribution of platinum complex
compounds between the nucleic acids and cytoplasmic proteins. The
selectivity of one or more species produced from the anti tumor platinum
complexes for DNA inhibition raises some important questions. What
ligand changes occur in this species that the other species lack, and
is the selectivity due to these changes? Does the geometry and the
stereochemistry of this species determine its interaction with DNA,

RNA. and proteins? Are these changed ligands more kinetically active?

The kinetics of the substitution of the ligand groups of
Pt(II)(NH3)2C12 with different ligands in aqueous solution will shed
some light on the reactivity of the platinum species towards the
reactive groups of the nucleic acids and the proteins.

Substitution reactions of square planar complexes are best explained

in terms of a biomolecular displacement mechanism.27’28

"20 11+ ..

m3x“+ + Y ——> mg + x (2)
A two-term rate law is followed:
Rate-kllMA3Xfi] + kleA3Xn+l [Y’] (3)

k1 - first-order rate constant

k2 - second-order rate constant

11
For the case of an excess of Y, a pseudo - first order condition is

obtained:
k - k + k (4)

A plot of k0 3 versus [Y] 13 linear with an intercept of k1 for the

b

reagent independent path, and a slape of k for the reagent path (see

2
below). Basolo et al.29 studied the rate of substitution of the
chlorines of gig: and 55225: dichlorodiammineplatinum(II) in aqueous
solution with a variety of nucleOphilic reagents. QigrPt(NH3)2C12
dissolved in water for some time, comes to an equilibrium within a
few days in which 452 of the original coordinated chlorine is ionic as
indicated in equations 5 and 6; while the Eggggrisomer

+ -
cis-Pt(NH3)2C12 + H 0 -————=>- cis-Pt(NH3)2(H20) C1 + C1 (5)

2

cis-Pt(NH3)2C12 + OH -————=*- cis-Pt(NH3)2(OH)2 + 2Cl (6)

on standing in solution for several weeks did not form any ionic chlo—
ride. When the tgggg_complex was treated with alkali all of the chlo-
rine was replaced. The rate of the reactions which are independent of
the concentration of the entering reagent is insensitive to the net
charge on the platinum complexes. gig_and‘£:ggg - Pt(NH3)2C12,
Pt(NH3)3Cl+, gigrPth(NH3)2Cl+, and PtCl:-, all react at the same rate
within a factor of about three. Thus, the 8N1 mechanism is not compa-
tible with the coulombic effects. A square planar complex will have
two further groups coordinated above and below the plane at a greater

distance than the four primary ligands.3o-.33 These groups may be solvent

12

or solute molecules .

MLA2X+Y————>MLA2Y+ X (7)

The substitution may occur via two different pathways.

Path I Patu II
(Solvent dependent) (Solvent independent)
5
M \
A/?\x “/E x
s 5
+x -x slow
+x1 -x slow 11
S
5 Y
~e—-— /
AL’/’ "\.5 ‘A;/' 3 “e S A» S
l s
5
\fi/ \ :/
a .M
5 (products) 5

13

This scheme explains the stereospecificity of the reactions, the effect
of the s-bonding group on those trans to it, the reactivity and second
order kinetics of n-bonding reagents, the small influence of electrosta-
tic factors on the rates, and the second order kinetics for some non-
bonding reagents such as NH3. Both bond making and bond breaking are

of comparable importance in the reaction of Pt(II)(NH3)2Cl2 with nucleo-
philic agents. Basolo et al.29 found that the rate of release of Cl-
from the interaction of transmisomer with aniline or pyridine (see

equations 8-11) was approximately twice that of the hydroxide ion or

glycine.
k1
-————‘. + _
Pt(NH3)2C12 + H20 ‘____ Pt(NH3)2(H20)C1 + Cl (8)
k2
k
Pt(NH) (H 0)Cl+ + Y 3 P (NH ) YC1+ + H 0 9
3 2 2 ’ t 3 2 2 ( )
kt.
Pt(NH ) YCl+ H 0 'T“—“‘ Pt(NH ) Y(H 0)++ + Cl- (10)
3 2 2 ‘= ' ' 3 2 2
k
5 k6
++ ++
P NH + - P H + H
t( 3)2Y(H20) Y ————=> t(N 3)2Y2 20 (11)

Thus the kinetics of Pt(II)(NH3)2C12 is in 8N2 (lim) caterorv, and the
rates show the behavior of displacement mechanism. The nature and con-
centration of the entering groups exert a major effect on the rates of
the reaction. According to the combined<3- and n-trans effect theory,
which suggests that good trans activators are strongly bonded to the
metal, the leaving groups, which are high in the trans effect series,

are slowly replaced. Therpoor trans effect groups, NH and 0H’, are

3
very difficult to replace in Pt(II) complex,34 since the amount of

14

covalent bonding into the as orbital must be small for electronegative
ligands because of the high energy of the up orbital of platinum. The
bond strength is determined mainly by as and op orbitals, and by ionic
interactions. The nucleOphilic agent is governed by its polarizability.
The order of nucleOphilic reactivity of agents to Pt(II) had been esta-

blished as follows:29’35

thiourea> I") 8011‘) N3) N02) Py> aniline> olefin) NH?
01‘) 1129011-

Thus the species that could be formed from Pt(II)(NH3)2Cl2 in aqueous
solution would be Pt(II)(NH3)2(H20)+C1 and/or Pt(II)(NH3)2(H20);+ and
both will interact with DNA, RNA and more strongly with proteins,
according to the nucleOphilic reactivity scheme above. Proteins will
be a stronger nucleOphilic agent than DNA and RNA due to the presence
of the sulphur bonds.

The hypothesis of Howle et al.26 and Harder et al.22 of two kinds
of Pt species, one of which interacts with DNA, RNA and protein generally,
and the other with DNA specifically, does not agree with the chemistry
of the square planar platinum(II) complexes.

Inhibition of DNA synthesis could be due to the intra- and or inter-
strand linkage with gingt(II)(NH3)2C12.

The cross linking between the two strands of DNA with gigrPt(II)(NH3)2C12
was investigated, and compared with the classical antitumor, bifunctional,

alkyalting agents.36 Roberts et al.37 develOped a new method for demon-

strating interstrand cross-links in DNA (hela cells) treated with mustard

15
gas. One strand of DNA bore a density label "heavy" and other strand

was "light". The cross linking between these two strands produced a
"hybrid" species. This hybrid species contains equal amounts of heavy
and light DNA which will show a peak in the center between the heavy
and the light single strands in 03012 gradient centrifugation.

To calculate the percentage of cross-linked DNA, the heavy stranded
DNA was radioactively labeled and the hybrid was assayed as a percentage
of the heavy DNA peak. The cross-linking of DNA was similarly observed
treated cells, and it is approximately of the

2

same order as of the mustard gas treated cells. It is worth noting that

in Ola-Pt(II)(NH3)ZCI

Roberts et al.36 did not observe cross—linking in the trans-Pt(II)(NH3)2C12
0

treated cells, which may indicate that the distance 3.3A between the two

active groups of the cis-isomer plays a role in cross-linking the two

strands.

The reacgive centers in DNA

Nucleic acids are polymeric molecules which are built up by the
extended repetition of a few fundamental units called nucleotides.
Each nucleotide is composed of nitrogenous purine or pyrimidine base,
a sugar residue and a phosphate group.

There are two kinds of nucleic acids, ribonucleic acid (RNA), whose
sugar is D—ribose, and deoxyribonucleic acid (DNA) whose sugar is
2-deoxy-D—ribose. Both nucleic acids contain two purines and two pyrim-
idines. The two purines, adenine and guanine and the pyrimidine cyto-
sine are common to both types of acids. RNA contains the pyrimidine
uracil and DNA contains the pyrimidine, thymine. The chemical preper-

ties of these heterogeneous bases depend on the distribution of the

16

electronic indices (electrical charges of the n-electrons, bond orders
and free valencies). The MO calculations indicate that all the carbon
atoms are positively charged and since the platinum complex compounds
used in this research are electrOphilic agents, we will not discuss
the properties of the carbon atoms. The nitrogen atoms of the purine
and pyrimidine bases can be classified into three groups.

1. Ring nitrogens which don't have a hydrogen atom attached
(pyridine-type)

2, Ring nitrogens which have a hydrogen atom attached (pyrrole-
type)

3. Nitrogen of extracyclic amino groups.

I. Nitrogens of the pyridine type:

The nitrogen atoms in heterocylic compounds contribute one n-elec—
tron to the pool of the mobile electrons, and, due to the conjugation
in the molecules, they carry an excess of electrons. They are negatively
charged and thus they are the centers of reactivity of the purines and
pyrimidines in the case of electrOphilic agents attach.

Protonation and alkylation of the purine and pyrimidine bases
occur at the nitrogens38-40 of the pyridine type. Consequently we may
expect the electrOphilic platinum species (hydrolysed products from
Pt(II)(NH3)2Cl2 to combine with the purine and pyrimidine bases at the

‘1 tried to find a

unportonated ring nitrogen. Pullman and Pullman
relation between the electronic charge of the ring nitrogen and its

basicity, but they met many difficulties. In 1958, by using the self-
consistent field molecular-orbital method, Nakajina and Pullman"2
showed that in polynitrogen heterocylics, the electronic charge of a

ring nitrogen cannot be taken as a measure of its basicity. Protonation

and other reactions involving the lone-pair electrons of the heterocylic

17

nitrogen depend not only on electronic charge of the nitrogens but also
on the values of the coulombic integrals between the "lone-pair" elec-

trons of this atom and the other n-electrons of the molecule.

pKa = B + I Q (ll/pp)
lip p
B - constant characteristic of a family of substances.
Q - the net charge of atom p

P
(ll/pp)- coulomb integral between an electron l of the lone pair of

nitrogen and the electrons of atom p
B, is obtained by a semi-empirical method, comparing the experimental
and theoretical values of the basicities of a series of related compounds.
Calculating the coulombic integral of every site, we can predict the most
basic site for interaction with the electrophilic agents as illustrated
in Table 5. The greater the absolute values of the summation of the
coulombic integral, the greater the pKa, and, as indicated in Table 5,
N of adenine is of the highest basicity and the electrOphilic attack

1

should occur at this site; while for guanosine, N is of the highest

7
basicity for this interaction. These theoretical results agree with
44-48
the exPerimental data for the cases of protonation and alkylation.
Pyrimidine, (VIl)49’50 shows weak basicity (pKa - 1.31) while pyridine,
(VI) shows a higher value (pKa - 5.2). The insertion of the electron-
attracting second nuclear nitrogen atom in the pyridine ring depletes
the n-electrons from the first nitrogen atom thus decreasing its basic-
51,52
ity. Pyrimidine could be correlated to B-nitrOpyridine, (V),

(pKa - 0.8), as a nitro-group is a strong electron attracting group.

18

Table 5. The theoretical values of the coulombic integral of the dif-

ferent sites of the purine and pyrimidine bases

 

 

 

 

 

 

2 Qp(11/pp) (ev)
1gp
pKa
Compound -51 £3 E? Experimental

Purine -1.52 -0.97 -0.32 2.4
Adenine -l.9l -l.72 —0.42 4.2
Guanine -l.ll -1&§l_ 3.3
Hypoxanthine -0.49 -1;12. 2.0
Xanthine -_(_)__._§_2_ 0 . 8
Cytosine —2 25

 

Figure 2. The basicity of the pyridine derivatives. VI, pyridine;
VII, pyrimidine; VIII, B—nitro-pyridine; IX, 4-methylpyrimidine;
X, 4,6—dimethy1pyrimidine, XI, 4-methoxy-pyrimidine; XII, S-hydro-

xypyrimidine.

19

v: \m vm
cu3 ca: 8‘3 o"
/ / u/
"0 1 . 1;) (I

IX x XI XII

Figure 2

20

Insertion of electron-donating groups, e.g. methyl, methoxy or
hydroxy-groups, will increase the charge layer on the nuclear nitrogen

53

atom and the basicity increases. 4-Methy1pyrimidine, Ix. has a

pKa - 2.0; 4,6-dimethylpyrimidine,54 X pKa - 2.8; 4-methoxypyrimidine,55

XI has a pKa - 2.5; and 5-hydroxypyrimidine,56 XII has a pKa = 1.87.

2. Ring nitrogens with a hydrogen atom attached:

A very interesting phenomenon appears in the case of hydroxy pyrim-
idines. An increase or decrease of the basicity of the nuclear nitro-
gen depends on the position of the hydroxyl-group in the ring. Figure 3
shows the change of the pKa of the nuclear nitrogen of the ring. In
aqueous solution 4-HydroxypyrimidineS7 XIII and 2-hydroxvpvrimidine58 XIV exist
as cyclic amide tautomers, and the strength of a ring nitrogen atom is
considerably weakened when it becomes part of such a cyclic amide. The
enhanced basic strength of the hydroxypyrimidines is due to the nitrogen
atom that is not involved as amide. When both nitrogen atoms are invol-
ved e.g. uracil XV and thymine XVI, the basic strength draps to zero.

Insertion of an amino-group at different positions shows a great
change of pKa's of the nuclear nitrogen. The pKa of S-aminOpyrimidine59
is 2.8, which is of the expected order compared to a pKa - 1.31 of the

49

unsubstituted pyrimidine. A large increase of the pKa's of 2- and 4-

49 (3.54 and 5.71) is due to the presence of the amino

aminOpyrimidine
groups which allow more resonance in the cations, as shown in the (next page
The 4-amin0pyrimidine shows a higher pKa than that of the 2-aminopyrimi—
dine due to the preferred p-quinonoid form that contributes to its

cationic hybrid than that of the o-quinonoid form of the 2-isomer. The
basic strength of the substituted pyrimidine depends upon the distribu—

tion of the double bonds in the ring.

Figure 3. The effect of the hydroxy-group on the basicity of the
pyrimidine ring. XIII, 4-hydroxypyrimidine; XIV, 2—hydroxypyrimidine;

XV, Uracil; XVI, thymine.

21

155.13 H”?

XIII XIV
O O
/"\ /u c":
"" 1 "J: .
a»): . :.

xv xvu

Figure 3

I'll:

1/\\ .{*

I -———I;
II

22

I

:z+
I:
N

J

/ :2
an:

Redistribution of the double bond by substitution in the ring

removes the base-strengthening resonance.

4-Amino—1,2-dihydro-l-

methyl-2-0x0pyrimidine (l-Methyl cytosine)6o has a pKa a 4.57 which

is below that of 4-amino—pyrimidine.

The basic strength is greatly

diminished in the case of 4-amino-, 1,6 dihydrO-l-methyl 6-ox0pyrimi~

dine51

lI/l‘
Ofl;\aa .
4...,

(pKa - 4.57)

(pKa - 0.98) the cause of which is not understood.

NH,

A
1,1»,

Cit:
(pKa - 0.98)

23

Uracil and thymine have very low pRa's while the basicity of N3 of

the cytosine is of the order of adenine, as the theoretical calculation
predicts.

In a polynucleotide (doubly stranded), like DNA, the involvement of
some of the ring nitrogens in hydrogen bonding decreases the value of
the theoretical predictions. N1 of adenine is hydrogen bonded with N1
of thymine, while the N1 and N of adenine are available for chemical

7

reactions. N1 of cytosine is hydrogen bonded with N

consequently N

1 of guanine and

of cytosine is blocked, and N of guanine is available

1 7
for interaction with electrOphilic agents. Alkylation of DNA and RNA

lead to the alkylation of the guanosine61 at N7.

3. Nitrogen of extracyclic amino groups:

I 3'"? t ' NH,
'1: \n \> "5'; MK) MN.
N T H2N A... OAN/
a I It
Adenosine Guanosine Cytosine

H fill/5

Isocytosine

24

The amino groups of adenosine and cytosine react readily with alkyl
groups forming N-alkyl derivatives.62 Guanine and isocytosine do not so
react. Freenkel-Conrat63 showed that formaldehyde (HCHO) is a specific
reagent for attacking the amino groups of the purine and pyrimidine
bases. In studying the interaction of formaldehyde with DNA and RNA,
Staehelin64 showed that RNA was much more reactive than DNA, and that
the reactions occurs with the amino groups that are not engaged in hydro-
gen bonds. The reaction of formaldehydewith the free amino groups forms
a Schiff's base63 and/or secondary condensation64 with other active

hydrogen atoms.

HCHO + HzN-Base _____a. CHi=N-Base (l3)

HCHO + 2H2N-Base ____€,. Base-g-CHz-fi-Base (14)

The reaction of formaldehyde with two free amine bases corresponds to
the formation of more complex bridges by bifunctional alkylating agents
of the nitrogen mustard type.65
The reactivity of adenine, cytosine, and the inertness of guanine
and isocytosine in the reaction with HCHO or any alkylating agents could
be interpreted in terms of the electronic characteristics of the amino
groups. Table 6 summarizes the ebctronic characters of the amino groups.
Among the purines and pyrimidines of nucleic acids we find that the N
atom of the NH2 group of adenine has the highest electroniccharge and
the greatest free valence. We expect that this base is the most reactive
one in the reaction with electrOphilic reagents. Guanine and cytosine

have the same order of electronic charge on the NH2 group, but the free

valence of cytosine is higher than that of guanine and less than that of

25

Table 6. The electronic characteristics of the amino groups of the

purine and pyrimidine bases

 

 

N. atom of the amino group

 

 

Compound ghgggg_ Free Valence

Adenine 1.810 0.960
Guanine 1.803 0.933
Cytosine 1.803 0.944
Isocytosine 1.800 0.925
2,6—Diaminopurine N2 - 1.829 0.978
N6 - 1.810 0.959

Adenine (in AT pair) 1.763 0.908
Cytosine (in CC pair with 2-H bonds) 1.751 0.828
Guanine (in CC pair with 2-H bonds) 1.809 0.943
Guanine (in SC pair with 3-H bonds) 1.773 0.906
Cytosine (in CC pair with 3-H bonds) 1.751 0.828

 

26
adenine, which explains why the reactivity of the amino group of cyto-

sine is less than that of adenine and of guanine, which is inert.

Adenine Cytosine Guanine
reactive less reactive inert
0.960 0.944 0.933

The order of decreasing reactivity of the amino groups of the bases is
shown above. The lowest value of a free base, which is guanine is
0.933. These bases, however, form hydrogen bonds with their complimen-
tary bases and the electronic charges and free valencies change. Thus
the electronic charge of the amino group of the adenine in an A-T pair
decreases to 1.763 and the free valence to 0.908. Cytosine, in a GC
pair with two hydrogen bonds, has a free valence 0.828. The amino group

of the guanine in the pair has a free valence of 0.943.

Guanine Adenine Cytosine
(G-C pair) (A—T pair) (G-C pair)
2-H bonds 2-H bonds

0.943 0.908 0.828
Adenine Guanine Cytosine
(A-T pair) (G-C pair) (G—C pair)
3—H bonds 3-H bonds

0.908 0.906 0.828

27

From the free valence calculation one would expect that in native DNA
the amino-groups of the purines and pyrimidines will be inert for any
reaction. In partially denatured DNA, the amino group of the guanine
will have the highest free valence and a reaction at this amino group
will occur. When the hydrogen bonds of an adenine-thymine pair are
broken, the free amino group of adenine will have a high free valence
for interaction. Also the free amino group of cytosine will be avail-

able for interaction with electrOphilic reagents.

Interaction of Metal ions with Nucleic acids

0n polynucleotides, there are two main sites of interaction with

metal ions, the phosphate moieties of the ribose phosphate backbone

67’68 The reaction with

69-73
a

and the elctron donor groups on the bases.

phosphate leads to stabilization of the ordered structure nd

consequently, an increase of the melting temperature (Tm) with increase
of metal concentration. Reaction with the bases destabilizes the

ordered structures, which leads to denaturation and a decrease67 of Tm.

Using the phenomenon of variation of Tm with varying concentration of
metal ions, the interaction can be divided into two classes. Most mono-

valent ions and some divalent cations bind to the negatively charged

2+ 2+),

sugar phosphate backbone, (Na+, Mn , and Co while metal ions like

2+
Cd , Cu2+ and Pb2+ bind preferentially to the heterocylic bases. Bind-

ing of metal ions on polynucleotides is not an all or nothing interaction.7

Mg(II)> Co(II)> Ni(II)> Mn(II> Zn(II> Cd(1V)> Cu(II> Ag(I)>

HgIII)

The metal ion Mg(II) at the left of the series above increases the Tm of

28
DNA and cannot induce any renaturation of the double helix. Co, Ni,

and Mn cause partial denaturation, while Cu(II) at the right of the
series produces almost complete renaturation of the double helix. The
affinity of the metal ions for the base to that of the phosphate increa—
ses from left to right. Cu+2 which binds mainly to the nucleoside bases
can bind also to the phosphate”.80 depending upon the ionic strength
and the concentration of the free metal ions in solution.

Davidson et al.“"82

studied the binding of Hg(II) by DNA and its
components at an acid pH. Hg++ forms linear complexes of the type
L—-Hg--L, where L is the ligand (nucleoside base) in the presence of
excess of ligand. With excess of Hg++ there is a 1:1 complex. Simpson83
determined the site of mercury binding at various pH values using UV
difference spectra technqiue. The interaction of the CHBHgOH or CH3Hg+
with the nitrogen site depends on the pH, which determines which species

predominates. Also the pKa of the nitrogen site determines whether the

site is protonated or not e.g.
atiH<pK of nitrogen site fl)

CH HgOH + NH “—A CH HgN + H0 (15)

3 ‘= 3 2
the reaction is independent of pH.

at H K N

CH HgOH + N : CH

3 HgN + 0H" (16)

3

the reaction is favored as the pH decreases.

29
.2512K(N)E»2HT>2KIH21._

 

CH3Hg+ + NH ;—_.—‘* CHBHgN + 11* (17)
the reaction is favored as the pH increases.
at 3K LN) (pH >11KIH2)

011311; + N {—3 CH3HgN+ (13)

the reaction is pH independent.

As the different nitrogen sites of the nucleosides were blocked by a
proton (at different pH's) or by a methyl group (nucleoside derivative)
Simpson was able to determine the site of interaction and its formation

constants with CH HgOH and Hg(OH)2. He found that methyl mercuric ions

3
bind at N7 of guanosine and inosine. Mercuration occurs at N1 of aden-
osine and N1 of cytidine. X-ray diffraction study84 showed that copper

chloride reacts with cytosine at N forming 1:2 complex. Methyl mer-

l
curic ions reacted with the amino groups of adenine, cytidine and guano-
sine at pH 10-11. The binding of the Hg-ions at ”If which is protonated,
of uridine does not agree with the x-ray diffraction study of Uracil-

mercuric chloride complexes by Sundaralingam.85 X-ray studies show that

the binding is at 0 and the combination is with HgCl rather than with

A 2

ng+.

Nuclear magnetic resonance studies of metal ions bound with nucleo-
sides, nucleotides and polynucleotides show that the site of binding of

the metal to the purine or pyrimidine bases changes with the location

30
of the phosphate group on the sugar ring. Capper binds preferentially

to adenosine rather than to the ribose. The unchanged sharp resonance
peak of the 6-amino group of adenosine in the presence86 of Cu(II),

and the broadening of the H-8 and H-2 protons of the adenine ring may
indicate that Cu(II) does not bind to the 6-amino-group and that binding

is at the nitrogen atoms neighboring these positions (N7, N or N3).

1
Addition of Cu(II) to 5-AMP broadens preferentially the H-8 proton
resonance than H-2, indicating that binding at N is favored to N of

7 l
87 The effect was observed in the interaction of Cu(II) with

the ring.
3' -AMP. The addition of Cu(II) to 2' -AMP broadens the H-8 and H-2
peaks simultaneously. The lower H-l' (ribose proton) resonance broaden-
ing of 5' - and 3' -AMP and the broadening of H-l' resonance along with

the base protons of 2' -AMP,, indicates binding at N , and Cu(II) may

1
form a chelate structure between the phosphate group at the 2' - posi—
tion and N3.

In poly A, H-8 broadening was maximal and the interaction was at
the N7 position. In 5' -IMP, the H—8 and H-2 resonance were broadened
simultaneously,88 indicating the binding of Cu(II) near both protons,
at N7 and 06 or at N7 and N1. In poly I, Cu(II) produced preferential
broadening of H-8 rather than H-2 proton resonance. Thus binding is
also at N7 as in poly A. Binding of Cu(II) to poly C is at the N1 posi-
tion88 as 131the monomer.86 Cu(II) coordinates to the ribose hydroxyls
of uridine, UMP, and poly U, and as these are being occupied. binding
will be on the uracil ring in the vicinity of N1 and C5° Poly A, po1y u
did not show any interaction with Cu(II) at room temperature. As melt-

ing occurs in the presence of Cu(II), H-8 proton resonance of poly A was

mostly broadened; H-2 was less broadened, and H—5 of poly U was the

31

least effected. Cu(II) binds to N of poly A and N to a lesser extent,

7 l

and the least to N1 of poly U.

Approach to the problem.

It is believed that the active antitumor coordination complexes of
platinum exert their biological effect by producing a primary lesion in
cellular DNA. It is now necessary to investigate the nature of the
interaction between these complexes and DNA. Both interstrand cross-
1inking and intrastrand dimer formation have been suggested as the bio—
logically significant lesion. We have attempted here to investigate
these interactions, using both simple nucleosides and oligomers and
polymers of nuclear bases, to determine all possible classes of reac-
tions with the platinum coordination complexes.

In order to assess the biologically significant interactions out
of the total class of permissable reactions, we have used the simple
fact that gigrdichlorodiammineplatinum(II) exhibits marked antitumor
activity against a variety of tumor systems, while its isomer,
trans-Pt(II)(NH3)2Cl2 is inactive. Therefore, only those reactions
occurring with the gig complex--but not with the £ggp§_complex will be
significant. This assumes that the trgpg complex has the same fate in
an animal system as the gi§_complex up to the point of DNA attach.

While this is not yet completely proven, there are indications

(Hoeschele and Van Camp, personal communication)that this is so.

II. EXPERIMENTAL

All materials were obtained from Sigma Biochemicals and MILES

LABORATORIES were of highest purity and were used without further puri-

fication.

Monomers

Adenosine, sigma grade; l-methyladenosine; cytidine (pfs);
3-Iethy1cytidine methosulfate(l-ribofuranosvl-J-metnvlcvtosine)
3-methylcytidine will be named l-methylcytidine in this thesis to agree
with the numbering of the purine ring; thymidine (pfs), sigma grade;
uridine (pfs), sigma grade; guanosine (pfs), sigma grade; l-methylguano-
sine (pfs); 7-methylguanosine, grade II: 90-95%, crystalline; inosine
(pfs), sigma grade; l-methylinosine; 7-methylinosine (pfs), grade II:

85-95%, were obtained from Sigma Biochemicals.

Dimers
Adenylyl (3' + 5') adenosine (ApA), free acid (pfs), adenylyl (2'., 5')

cytidine (ApC), free acid (pfs), were also obtained from Sigma Biochemicals.

Polymers
The following synthetic polynucleotides (white fibrous), were

obtained from MILES LABORATORIES.

Polyadenylic acid (poly A), potassium; polycytidylic acid (poly C),
potassium; polyguanylic acid (poly G), sodium; and polyuridylic acid
(poly U). Ammonium , did not obtain olignucleotides, nucleotides, nucleo-

sides and inorganic salts.
32

33
Polyadenylate-polyuridylate (poly A. poly U) had a base ratio

A:U of 1:1.3, Tm 42.0°C, and hyperchromicity of 25.3). Polydeoxyadenylate-
thymidylate, Na salt [poly d(A—T). poly d(A-T)] had a Tm 61.0°C, and
hyperchromicity of 49.52.

Deoxyribonucleic acids (DNA) were obtained from Sigma Biochemicals.

Calfthymus DNA, type 1: sodium salt (pfs) G-C content is 422;
E. Coli DNA, type VIII (pfs) G-C content is 502;

Micrococcus Lysodeikticus DNA, type X (pfs), G-C content is 702.

The g_i_s_-- and _t_r__a_n.s isomers of Pt(II)(NH3)2Cl2 were prepared in this
laboratory by Dr. J. Hoeschele as described elsewhere (Lebedinskii and
Korabel'nik, 1947; Kauffman, 1963); the purity was verified by their
Laser-Raman spectra (Figure 4 )44. Each nucleoside in solution with
various concentrations of Pt(II)(NH3)2C12, were kept in the dark at
37’C to avoid any possible photo-reactions of platinum complexes. The
spectra of the solutions were measured at room temperature using a l-cm
cell in a Cary 15 Spectrophotometer until no further change of O.D.

occured (equilibrium was usually reached within two weeks).

pH study
One ml of lmJ‘of each nucleoside was diluted to 10 ml with 0.7,
0.07, and 0.001 M HClOa, and with 10-5, 10—4’ 10-2, 10"1 N NaOH to give

-2.

10 M nucleoside at the different pH's.

Cis- and trans-Pt(II)(NH3)2Cl2 were dissolved in 0.1 “M NaClOl‘
3

produce a solution of 10- M. Addition of equal volumes of nucleoside to
platinum complex allowed the reaction to proceed at various pH's. as

shown on the next page:

Figure 4. Raman spectra of cis- and trans-Pt(II)(NH3)2Cl The

2.
spectra were recorded using solid samples in capillary tubes, a
100 on He-Ne laser and a spectrOphotometer, spex 1400 Double Mono-

chromotor and ITT-FN-l30 "Star-tracker" photomultiplier, capable

of less than 1 cm"1 resolution.

34

a shaman

Tau ><

 

 

q
00-

 

 

N0.

#1 q
DON con

    
  

   

,2'
s—r'r

~ g. --'~- .'

 

on:

*N»

 

 

 

 

tun

Woumzzzetg

 

 

‘— ALISNSLNI

35

Egg 2!.
l 2 ml of nucleoside in 0.7 M HC104 + 2 m1 of Cis-, trans-Pt* 0.81
2 .. .. .. .. «0.07 n .. +" " " " " " 1.61
3 v " H " " 0.001 M " + " " " " " " 3.27
4 n n " " " 0.1 M Neelo4 + " " " ” " " 5.58
5 n n n v " 10‘5 N NaOH + " " " " " " 5-95
6 n n n H " 10" N " + " " " " " " 8.86
7 n n H H " 10-3 N " + " " " " " " 10.46
8 n n n H " 10'2 N " + " " " " " " 11.31
9 n H H H " 10'1 N " + " " " " " " 12.12

*Pt I Pt(II)(NH3)2C12

Determipagion of number of Ligands of nucleoside per platinum complex:
The composition of the formed nucleoside-platinum complex usually

one species, in solution was determined by the variation method, "Job's‘

method".90 For more than one species the compositions were determined

by the Voshburgh and C00per teChnique,104

mettle:

A mole fraction of 2 x 10-4M of each nucleoside, X, dissolved in
1, 2, 3, a, s, 6, 7, 8, and 9 m1 is diluted to 10 ml with 2 x 10‘“M
Pt(II)(NH3)2012. Thus (M) + (L) - C, where (M), (L), C are the molar
concentrations of Pt—complex, nucleoside complex and constant respec-
tively. The absorption spectrum of the formed complex is maximal when
the metal and the ligand are brought together in the same ratio in which
they exist in the complex.

In the case of more than one species, the absorption spectra of

1:1, 1:2, 1:3 mixtures of M:L were recorded. If there are three species

36
in solution, the wavelength at which 1:1 and 1:2 have the same extinc-
tion coefficient would be suitable for identifying the ML species, and
that at which 1:2 and 1:3 have the same extinction coefficient would be

suitalbe for the ML2 species.

_petermination of the sites of bindigggiand its formation constants:

The site of the interaction of gig: and iigpngt(II)(NH3)2C12 with
nucleosides was identified at different pH's. and with nucleosides
methyl-substituted at different sites. The formation constants at these
sites were measured using Simpson's technique.83 A series of spectra of
nucleosides with varying concentrations of gig: and iigpngt(II)(NH3)2C12,
at a certain pH where there is only one species to be expected were
measured. The ratio of the platinum metal to the nucleoside varied from
0.1 to 8 in the cases of adenosine and l-methyl adenosine, and from 0.05
to 4 in the cases of guanosine, l-methyl guanosine and cytidine.

The change in absorption at the wavelength of maximum change as
measured by difference spectra using four l-cm cell, was plotted against
log[Pt(II)(NH3)2C12] and a sigmoidal titration curve was fitted to the
points.

See next page for calculation of the formation constant.

Calculation of

(8)

[ML] '
[L] '

[M] -

[Lo]

["0]

At the midpoint

therefore

37

the formation constant:

M + L.-—-“__ ML (19)

f 2.1—. (20)

[M] [L]

concentration of the formed complex compound
free ligand in solution

free metal complex in solution

total concentration of ligand in solution

total concentration of metal complex in solution

of the curve we have the following:

[LM] - 1/2 [L0] - [L] (21)

Kf " __[_L_]_.... ' .1... (22)
[L] [M1 [M]

[M] - [fix] - 1/2 [L0] (23)

where [Mx] - [M0] corresponds to the midpoint of the
curve

K - 1 (24)

[MK] - 1/2 [L0]

38

(b) M + 2L (:3. ML2 (25)
[L12 [M]

At the midpoint:

ZIML2] - 1/2 L0 therefore [MLZ] - 1/4 L0 (27)
[L] - L0 - 2(1/4 L0) - 1/2 L0 (28)
[M] - Mx - 1/4 L0 (29)
Kf _ 1/4 [L0] . i74 [L0] (30)

 

 

T
[1/2 L01 [8, - 1/4 L0] [1/4 L31 [MK] - l/4[Lol

1 (31)

7<
I

therefore

 

[Lo] [Mx] - 1/4 [L0]

39

There are many sites on the mononucleoside where the gig: and ggggg
platinum species may interact. Both gig: and ggggg: isomers can inter-
act bifunctionally with the azanitrogen and the amino groups. Since the
lgig: and ggggg: isomers have dramatically different biological effects,
differences in the modes of binding of the isomers are of particular
interest. The gig: isomer has an additional mode of binding, not avail-
able to the ggggg isomer, by forming a bi-dentate chelation via N and

7

the 6—amino-group of adenosine; and the N and the 4-amino group of cy—

3
tidine. The evidence (see below) that the gig:platinum species can bind
to the 6-amino group of adenosine and the 6-amino-group of cytidine, led
to the investigation of the possibility that two adenosines or one adeno-
sine and one cytidine or two cytidines moieties could be linked together
by the gig:isomer. The inter—base separation in DNA is close to 3.4A,
and as the distance between the two cis-ligands of square planar platinum
compounds is about 3.3A, the linking of two bases by one platinum complex
is stereochemically possible.

The purine dimer A 3' p 5' A in solution has a separation between
the 6-amino groups of 3 to 4 A. The ApA, ApU and the platinum isomers
were dissolved in 0.1 M NaC104 at a molar ratio of 1:1 (dimer to Pt)
and incubated in the dark at 37°C until no further change was seen. The
circular dichrosim spectra were measured on a JASCO CD machine fitted

with a thermostatted cell holder. The temperature could be varied over

the range 5° to 45°C.

Kinetics of polynucleotides and DNA - Platinum Compiex Igieraction
One ml of DNA, (Calf Thymus, 382 CC; E. Coli, $02 GC; M. Lysodeik-

ticus, 721 CC; 0.3mM p) in 0.1 M NaC104 was mixed with one m1 0.3mM cf

freshly prepared cis-, and trans-Pt(II)(NH3)2Cl2 in a one cm cell fitted

40
into a thermostatted holder in the Cary 15 at 37°, 51.5° and 61.5°C.

The rate of the reaction was measured by the difference spectra technique.

Both the sample and reference compartment were held at the same tempera-

ture. The reaction was followed with time until precipitation occurred.
The kinetics of interaction of the single stranded poly A, poly U,

poly G, poly C and the double stranded poly A. poly U, and d(A-T),d(A-T)

with the platinum isomers were also followed at the same temperatures

and under the same conditions as for DNA-platinum interactions, using

the same technique.

III. RESULTS

The effect of theng on the binding,site of nucleosides to the cis- and

trans-Pt(II)(NH3)29i2.

The interaction between the isomers of Pt(II)(NH

 

3)2C12 and nucleo-
sides, substituted nucleosides, synthetic polynucleotides and DNA was
carried out in a medium of 0.1 M NaClOa until no further changes occur-
red in the absorption spectra. The medium provides the high salt con-
centration necessary to maintain the stability of the DNA. Perchlorate
ions do not bind the metal complexes so the medium will not compete with
the DNA for the platinum complexes. Aquation, with loss of chloride
ions occur rapidly under these conditions, so that the reactions will
occur via the aquated species.

Figure 5 shows the hydrolysis of gig: and giggg:Pt(II)(NH3)2C12
in 0.1 M NaClO4 overnight. The pH of the solution was not controlled
since a buffer may introduce potential ligands for the metal complexes.
In those experiments the pH of the solutions fell in the range 5 to 7,
except for the case of the pH dependent studies where the pH was adjusted
by the addition of cone. HClO4 or NaOH and measured at the time the
spectra was run.

As thegig:Pt(II)(NH3)2Cl2 can interact either monofunctionally or
bifunctionally, such as chelation with a neighboring group, while the
‘ggggg:isomer is a mono-functional agent (Figure 6), a comparison of the

difference spectra of the two isomers will reveal the nature of binding

with nucleosides, dinucleotides, and polynucleotides.

41

Figure 5. Absorption spectra of the Pt(II)(NH3)2Cl2 isomers.
The spectra of 1 mM cis- and trans- isomers in 0.1 M NaClO4 using
5—cm. cell, were recorded on cary 15, a, freshly prepared solution;

b, solution left overnight at 37°C.

42

m ouowam

 

 

 

$1.5QO zofiamOmmq <Ebmdm 20:.dmomm<
Es < are:
ooe on» can 03 One oov can 00» cow.
qaaAanfi—fidqq—fiaje— Tiaanallql--dnla+1qaa4naqu

 

 

 

mmq

a: ._o.l.xzv,.__zo

 

Figure 6. Suggested mode of binding of Pt(II)(NH3)2C12 isomers

with adenosine at a pH 5.6 - 7.3.

43

(i) MONO-FUNCTIONAL BINDING (TRANS)

H3N OH
\p{ H3N OH
/ \ \ /
NHszo NH3 NH2 /Pt\
RNI N —-> KNI '1‘ 2
R R
(ii) Bl-FUNCTIONAL BINDING (gig)
I“:
HO—Pt—NH3 "(”3
NH, HZIO HN—FIH—NH,
N’ ' N> __, N< | N> + 211,0
3N N N N

I I
R R

Figure 6

ad

th

511

Pee

the

he

44
(A) Monomers

Adenosine

Figure 7 shows the binding of the gig:Pt(II)(NH3)2C12, and
trans-Pt(II)(NH3)2C12, with adenosine (ratio of Pt Metal/base = 10) at
different pH's.

At a pH around 1, the gigrisomer with adenosine shows a peak of A280nm
which is similar to that of the giggg:isomer with adenosine at the same pH.
This suggests that both isomers interact with adenosine monofunctionally
at a low pH.

At a pH 5-7, in addition to the peak of Azgonm, the gig:isomer exhibits
a shoulder extending from Azaonm, to A350nm. The giggg:isomer shows a

peak only at A nm. The peak at 2280nm could be assigned to the same

280
mono-functional interaction of the gig; and the giggg:isomers with adeno-
sine as at the low pH. The appearance of the shoulder in the case of the
gig:isomer only suggests the possibility of the chelation with the neighbor
amino group.

At a pH around 11 both isomers did not show any interaction with
adenosine, and no precipitation was observed.

The binding of both isomers with adenosine could be at N or N of

l 7
the purine ring.

l-Methyladenosine

At a pH around 1, both the cis- and the trans-isomer show a weak
binding, while at a pH around 3, the cis-isomer shows a broad band, its
peak at Azgonm, which extends to A350nm (Figure 8), which could be due to

the binding at N and chelation with the neighbor 6-NH2 group of the pur-

7

ine ring. The trans-isomer shows a sharp peak at Azaonm and therefore,

Figure 7. Difference spectra of the Pt(II)(NH

adenosine at various pH values.

8, pH - 11.31; 9, pH - 12.12.

3)2C12 isomers with

1-6, pH 8 0.8-8.86; 7, pH - 10.46;

Dr‘

45

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emeomem 855.115 458%. mozmmmeaa
05:4 ctS‘
00m 000 Omm .00m 00m 000 .
.IITIT..-. ..-.8-1. . . . .4.....J.8-
)

.00-
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_
.8 A??? mzaozge ioo
Baku wIz .NAV

 

 

mm< mm<

Figure 8. Difference spectra of the Pt(II)(NH3)2Cl2 isomers with

l—methyladenosine at pH - 3.27.

46

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«Eomem wozwmmnna <Eomom mozummuna
Ass a 665 a
com omw om.» com com
1I 4 d J a I d 1 d u I J —I a a 4 J - q q a 11 - d 1J
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47

of the interaction at N7 only. The peak at 3280

compound could be correlated to the peak of the trans—isomer with adeno—

nm of the trans-complex

sine at pH around 1 (N1 was blocked by a proton), and to that of the gig:
isomer with adenosine at the same pH, at which the interaction is mono-
functional at N7.

The shoulder that appeared in case of the interaction of the gig:isomer
with adenosine at pH 5-7 could be assigned to the chelation effect with
the 6-NH group. The shoulder grew to a peak in the case of l-methyl-

2

adenosine to give the band that extends to A350nm.
At a pH above 5, it was not possible to study the interaction of
the cis- and the trans—isomers with l-methyladenosine due to its hydrolysis

to adenosine.

Cytidine

At a low pH, around 1, both cis- and trans-isomers did not show any
interaction, indicating that N1 and the 6-NH2 group are blocked by pro-
tons (Figure 9).

At a pH 5-7, the cis-isomer shows a band at 1 nm may be due to the

290
interaction at N and the shoulder is due to the chelation with the 6-NH

l 2
group. The giggg:isomer with cytidine shows a large band at Azgonm. The
absence of the shoulder would indicate that the interaction is monofunctional.

The fact that 1-methy1cytidine did not interact with either isomer
indicates that the interaction with cytidine is at N1 in case of the ggggg:
isomer, and at N1, chelating with the 6-NH2 group in the case of the gig:
isomer.

At higher pH's no interaction was observed between cytidine and either

isomers.

Figure 9. Difference spectra of the Pt(II)(NH3)2Cl2 isomers with
cytidine at various pH values. 1, pH - 0.81; 4-6, pH - 5.58—8.86;

7, pH = 10.46; 8, pH - 11.31; 9, pH = 12.12.

48

a omsmam

 

 

 

 

 

 

 

 

4.5.0QO mozmmmmmfi $10.0QO mozmmmmma
“EC: EE:
00m 000 00m own 000 00m
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49

Guanosine
The cis- and trans-isomers did not show any interaction with guano—
sine or l-methylguanosine at a low pH, around 1.

At a pH 5-7, N of guanosine is mainly protonated and the binding

1

will be at N7. The cis-Pt(II)(NH Cl2 will give the same spectra as

3)2
the trans-isomer when the interaction is with N7, since there is no
neighboring group to chelate with. The pH dependent spectra (FigurelO)

show the interaction at A nm in the acidic region and the other band

290

at A nm in the alkaline region. Figure Ilshows the similarity between

265
the gig: and g5ggg:isomers complexed with guanosine, and the gig: and
giggg:platinum complex with 1-methylguanosine, which have the same peak
at A290nm. There was no interaction between either of the platinum iso-
mers with 7-methy1gaunosine. Thus the interaction at pH 5.58 is at N7,
and both gig: and giggg:isomers act monofunctionally.

Guanosine and 1—methy1guanosine did not show any interaction with
either the gig: or giggg:isomers at a pH above 9.
The hydrolysis of 7-methylguanosine to guanosine in the alkaline

region, and to 7-methylguanine in the acidic region, hindered the study

of it with the platinum isomers at various pH's.

Inosine

The gig: and igggg:isomers gave similar peaks at 2262nm at a low
pH, around 1 (Figure12). At a pH 5—7, both isomers show a broad band
which has a peak at Xz7onm. l-methylinosine, with both isomers, showed
a band at 2260“” at a pH around 3 (Figure I». Thus we can assign the
band of l-methylinosine with the platinum isomers at 1260nm as due to

the interaction at N7. The difference spectra (Figure 13) of the cis-

and trans-isomers with 7-methylinosine show a band at A285nm which is

Figure 10. Difference spectra of the Pt(II)(NH3)2C12 isomers with

guanosine at various pH values. 1, pH 8 0.81; 2-4, pH - 1.61—5.58,

the binding is at N7(1290nm); 7, pH = 10.46, the binding is at N1

only; 8-9, pH = 11.31-12.12.

rt
)0

CH senses

<mkomam mozmmwmma $10.0QO mozmmmmma
ACE: AEE A
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H.245. ...
1 N0
.00 4 . .m0
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mmd mm<

Figure 11. Difference spectra of the Pt(II)(NH3)2Cl2 isomers with

l-methylguanosine at various pH values. pH - 3.3-8.9, the binding

is at N7 only.

51

HA ouowwm

 

 

  

 

 

 

<mkomam mozwmmmua <mkomam mozmmwuma
A55 < E5 4
COM COM OmN mdwmm . . . . own 4 i 400-
.NO-
. _.O-
0.0
5 e _.O . _.O
m2_mOZ<Doum_2-_ so» a o. wz_mOZ<Do
9,..‘
.3 4w. . No . No
oozwus
.mfiv -mdv

 

 

 

fl: mmd szfi 84

Figure 12. Difference Spectra of the Pt(II)(NH C1 isomers with

3)2 2
inosine at various pH values. 1—3, pH - 0.81-3.27; the binding is

at N7(1262nm); 4-7, pH = 5.58-10.46, the binding is at N7 and N1;

8-9, pH . 11.31-12.12.

NH UHDNHh

 

52

 

<mkomaw mozmmmmua <mkomam mozmmmmma
“Ea: flee:
00m 00m Omm Omm 000 00m
. . . . . n I . . . s . - _ . . . . . . . . . _ . .
Im.OI rem.0l

 

 

 

mm< 22 mmq
_
Azflxz: mz_moz_

Figure 13. Difference spectra of the Pt(II)(NH3)2Cl isomers with
l-methyl-, 7-methyl-inosine at a pH - 3.27. l-methylinosine shows

the binding at N7(1260nm), 7-methylinosine shows the binding at

N1(1280nm).

53

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on» “Seek“ <mkumamam wozwmmmma
L C
dqai-qe-1_q.om.m Iomm v4
— — a II — q . 4 «co-n OWN
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INC: 1
n._Aun anUI
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54

due to the interaction at N1.

We can conclude that the interaction of both platinum isomers with

inosine at a neutral pH would be at N1, but mainly at N7.

At a high pH, above 9, inosine, with both platinum isomers, shows a

band at A258nm which could be due to the interaction at N1.

Uridine

There were no interactions observed at any pH.

Thymidine

There were no interactions observed at any pH.
(B) Dimers

Adenylyl (g)..5') AdenosinejjA3'p5'A):
At a pH around 1, the cis- and trans-platinum isomers interacting

with A3'p5'A gave a similar peak at A nm, which could be correlated to

280

the one which appeared at A28Onm in the case of the monomers at the same

pH (Figure 14). As the peak of the monomer was assigned to the interaction
at N7 of the adenosine base, we can assign the peak of the dimer as due

to the interaction at N7 of each base.

At a pH 5-7 the cis-isomer gives a peak at A282nm and a shoulder

which extends from A3oonm to A350nm. The trans-isomer with A3'p5'A shows

only a peak at A nm which is the peak that was assigned for the inter-

282

action at N7. The difference spectra of the cis- and trans—platinum

isomers with A3'p5'A are similar to that of both isomers with adenosine.

‘Ve can conclude that the interaction of the gig-Pt(II)(NH C12 is at N

3'2
iind chelating with the 6-NH2 group in the case of the dimer, as in the

7

unanomer. The trans-isomer interacts at N7 in the case of the dimer, also

Figure 14. Difference spectra of the Pt(II)(NH3)2Cl2 isomers with

A3'p5A at various pH values. 1—7, pH = 0.81-10.46; 8, pH = 11.31.

55

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mecca
com

«a uuowwm

dmhomdm wozmmmmma

 

 

 

 

 

 

 

 

eccv‘
own 00m 00m
Jive-T I 1 . I 4 - . . . Jeo-
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1:0- .70-
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.no .nc
.eo .vo
L00 41:4 1&0
004 004

56

as in the monomer.

At a pH around 11, the gig:Pt(II)(NH3)2C12 showed an increase of the
shoulder from A300nm to A350nm, which was not observed in case of the
monomers. The.ggggg:isomer did not show any interaction with A3'p5A.

The lack of the interaction of the giggg:isomer with the dimers and mono-
mers at this high pH, and the appearance of a broad band in the case of
the gig:isomer with the dimer only, exhibits the possibility of a new

complex, formed between the cis-isomer and the two 6-NH groups that lie

2
above each other in the stacked form of the dimer (Figure 15).

Adenylyl(2'§5')Cytidine (A2'p5fC)

At a pH around 1, the cis-Pt(II)(NH3)2Cl interacting with A2'p5'C

2

shows a peak at 1 nm (Figure 16). The trans-isomer shows a peak at

278
Azaznm. As the cytidine monomers did not show any interaction with

either platinum isomers at the same pH, we can conclude that the inter-
action of the platinum isomers is monofunctional and it is at N7 of the
adenine base of the dimer.

At a pH 5-7, the cis-isomer shows a peak at 1283nm and a shoulder

which extends from x300nm and to ABSOnm. The peak at 1283nm could be

compared with, and assigned to the interaction at N7 of adenine base,

as the interaction at N1 of cytidine gives a peak at A Onm. The

29

shoulder A300 - A nm of the dimer could be assigned to that of the

350

chelation effect of N7 and the 6-NH2 group in the adenine base similar

to that of the adenine monomer.

At higher pH, above 9, the gig:isomer interacting with A2'p5'C
shows an increase in the band at Azgonm which could be assigned to the
chelation between the 6-NH2 group of the adenine base and the 6-NH2

group of the cytosine base. The trans-isomer did not show any interaction

Figure 15. The structure of the A3'p5'A and A2'p5'C determined

by X-ray diffraction.99

 

57

 

 

I? A2 p50

 

Figure 15

2
A2'p5'C at various pH values. 1-7, pH = 0.81-10.46; 8, pH - 11.31.

Figure 16. Difference spectra of the Pt(II)(NH3)2C1 isomers with

58

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A55 < AES <
000. 00m 00m Own 00m 0mm
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mm<

mm<

o .....<

a-

59
with A2'p5'C.

(C) Polynucleotides

The interaction of the gig: and trans-Pt(II)(NH3)2Cl2 could not be
studied with polynucleotides in the acidic or alkaline region due to the
denaturation which occurs in these pH ranges.

At a pH around 5, synthetic polyadenylic acid (poly A); polyguanylic
acid (poly G); polycytidylic acid (poly C); poly A. poly U; poly d(A-T);
poly d(A:T). poly d(A-T) and DNA formed a white precipitate with both

cis- and trans-Pt(II)(NH3)2Cl in the concentration range above 1:2 of

2
the platinum isomer to the phosphate. Polyuridylic acid did not form
any precipitate with either the gig;_or giggg:isomers. The details of
the interaction of the platinum isomers with polynucleotides are dis-
cussed in the kinetics section below.

The stoichiometry of the cis- and transeplatinum nugigoside complexeg_in
solution.

The difference spectra of the gig: and trans—Pt(II)(NH3)2Cl2 with
the nucleosides; adenosine, l—methyladenosine, cytidine, guanosine,
l-methylguanosine, inosine, l-methylinosine, at a pH around 5, are shown
in Figures 17-23. The plot of the change of the absorptivity of the

formed complex versus the ratio of the nucleoside concentration to the

sum of the concentration of the nucleoside and the Pt- complex,
[nucleoside]

[Pt(NH3)2C12] + [nucleoside]
complex. Figures 24-30 show the stoichiometry of the formed complexes.

 

, gives the number of ligands per Pt-

The presence of one or more species was determined by taking measurements
at different wavelengths. Table 7 summarizes the stoichiometry of the
complexes formed in solution. It shows thattil nucleosides, except

adenosine with cis-Pt(II)(NH3)2Cl2 and cytidine with the trans-isomer,

Figure 17. Difference spectra of the Pt(II)(NH3)2C12, Pt(II),

isomers with adenosine,L, “Job's method". The ratio of the molar

 

concentration of Pt(II) is, 0.1, 1; 0.2, 2; 0.3, 3; 0.4, 4;
Pt(II) + L

0.5, 5; 0.6, 6; 0.7, 7; 0.8, 8; 0.9, 9.

60

<m._.0mam mozmmwmma

Es ‘
00m

00m

 

A-II J 4'

ma ouowwm

Omm

<mH0mam mozmmmmmfi

AES <

00m
41

Omm
—

 

 

 

0.0
mmd

d 1

q

- d

 

 

mz_mozm0<

1

MO

00

L

 

L

0.0
mmd.

Figure 18. Difference spectra of the Pt(II)(NH3)2Cl Pt(II), isomers

2’
with l-methyladenosine, L, "Job's method". The ratio of the molar

concentration of ——-§££ll)———- is 0.1, l; 0.2, 2; 0.3, 3; 0.4, 4; 0.5,
Pt(II) + L

5; 0.6, 6; 0.7, 7; 0.8, 8; 0.9, 9.

61

0H ouswam

 

 

<me0mam mozmmmmmfi <E.0mam wozwmmmma
35 < 1&5 <
on... 8» ohm 8m 08 ohm

 

 

 

 

 

I

no :2 mzmozmoeéffizl
mm<

 

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L
a!
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Figure 19. Difference spectra of the Pt(II)(NH3)2C1 Pt(II), isomers

20

with cytidine, L, ”Job's methoc”. The ratio of the molar concentra-

 

tion of Pt(II) is 0.1, 1; 0.2, 2; 0.3, 3; 0.4, a; 0.5, 5; 0.6,
Pt(II) + L

6, 0.7, 7; 0.8, 8; 0.9, 9.

62

can

die-omam wozwmwmma

Es <
08

as .osnoe

nfim

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Es 4
SN

 

 

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wz_o_.r>0

 

L

no
mm<

Figure 20. Difference spectra of the Pt(II)(NH3)2C12, Pt(II),

isomers with guanosine, L, "Job's method”. The ratio of the molar

Pt(II)
Pt(II) + L

0.5, 5; 0.6, 6; 0.7, 7; 0.8, 8; 0.9, 9.

concentration of is 0.1, 1; 0.2, 2; 0.3, 3; 0.4, 4;

 

63

«#:0me mozwmwmma

2.5 a
8m

0m ouowwm

<mH0mam mozwmwmnzo

Es <
co...

 

 

 

 

 

 

 

 

 

Figure 21. Difference spectra of the Pt(II)(NH3)2C1 Pt(II), isomers

2’
with l—methylguanosine, L, "Job's method”. The ratio of the molar

Pt(II)
Pt(II) + L

0.5, S; 0.6, 6; 0.7, 7; 0.8, 8; 0.9, 9.

concentration of

 

is 0.1, 1; 0.2, 2; 0.3, 3; 0.4, 1.;

64

0mm

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ind fl
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mm 4

Figure 22. Difference spectra of the Pt(II)(NH3)2C12, Pt(II),
isomers with inosine, L, “Job's method". The ratio of the molar
concentration of Pt(II) is 0.1, l; 0.2, 2; 0.3, 3; 0.4, 4;

Pt(II) + L
0.5, 5; 0.6, 6; 0.7, 7; 0.8, 8; 0.9, 9.

 

65

NN ouswwm

dmkomam mozmmmmua 40.5QO wozmmmmma

AEE < AEE «
00m 00m 0mm 00m
1 q

+ - d — A d —

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1 — . a 1V0!

 

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q q T j q a 4 «

 

 

 

 

 

 

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L

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8 Azflnzz mzaoz. . so
mm< o mm<

Figure 23. Difference spectra of the Pt(II)(NH3)201 Pt(II)

29
isomers with 1-methylinosine, L, "Job's method". The ratio of

 

the molar concentration of Ptill) is 0.1, l; 0.2, 2; 0.3,
Pt(II) + L

3; 0.4, 4; 0.5, 5; 0.6, 6; 0.7, 7; 0.8, 8; 0.9, 9.

66

mm uuawwm

<m._-0wn_m wozwmwmnzo dmkomam mozmmwmua
AEE < AES <
00m 0mm

q 4 J 4 ‘- 4 d d d

 

 

 

 

 

 

 

 

 

 

 

 

 

mmd o 2.82.3252; mm<

Figure 24. Graphical determination of composition of the
Pt(II)(NH3)2012 - adenosine complex. The straight lines are the
relationships eXpected for PtL complex. The curved lines are the

experimental results.

67

NI-IzN
ADENOSINE I
0.6- N | >
05*
0.4- '
A28 ,
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0.3“
2: ' A285
0.2"
1390‘
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0.6 t

0.5 ’

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0.4 I

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L/Pt=l

0.3 I °

0.2 '
A285

0.I-/ - .

 

 

0.0 l_ L l 1 l l L l 1
PI 0 2 4 6 8 IO
L l0 8 6 4

Figure 24

Figure 25. Graphical determination of composition of the
Pt(II)(NH3)2C12 — l-methyladenosine complex. The straight lines
are the relationships expected for PtL complex. The curved lines

are the experimental results.

68

NH
I—ME-ADENOSINE H,,C~NK I N>
‘N

 

 

 

 

AABS

 

 

 

 

Figure 25

Figure 26. Graphical determination of composition of the

Pt(II)(NH3)2C1 - cytidine complex. The straight lines are the

2

relationships expected for PtL complex in case of the gig: isomer

and for Pt2L3 in case of the trans: isomer. The curved lines are

the experimental results.

AA88

AA88

 

09

F CYTIDINE NJ}
04‘?!
P R

 

 

 

   
 

TRANS
L/Pt= l.5

 

 

   

 

0 2 4 6 8 I0
6

Figure 26

Figure 27. Graphical determination of composition of the
Pt(II)(NH3)2C12 - guanosine complex. The straight lines are
the relationships eXpected for Pth complex. The curved lines are

the experimental results.

70

0

Z

 

 

 

 

 

 

 

03_ GUANOSINE HNJ‘I >
' HZNkN If
(D 02}. 0 1292 R
2 1295 CIS
<1 0.! -
/ . 1&0 . L/Pt=2
0.0 l J— l 1 l l 1 l 1
Pt 0 2 4 6 8 :0
L IO 8 6 4 2 0
0.4
03
3’3
4 02
<1
0.:
0.0
Pt 0

 

Figure 27

Figure 28. Graphical determination of composition of the
Pt(II)(NH3)2C12-l-methylguanosine complex. The straight lines

are the relationships expected for PtL complex. The curved lines

2

are the experimental results.

AABS

AABS

 

 

71

 

 

 

 

 

 

0.20%-

0.5.

0.10- / TRANS
0.051» I. ””32
().C’ 1 1 4L. J_ l 41

p, o f 4 6 8 IO

L l0 8 5 ‘ 2 0

Figure 28

Figure 29. Graphical determination of composition of the
Pt(II)(NH3)2C12 - inosine complex. The straight lines are the
relationships expected for PtL2 complex. The curved lines are

the experimental results.

AABS

 

 

72

 

 

 

 

 

0
INOSI E ~ J N
0.3~ N ”'1 | \>
N '3‘
0265 R
0.2., A270
275
o... . 08.
23c,
\ L/Pt=2
0.0L. ‘1 l 1 l 1 1\
Pt 0 4 6 8 l0
L l0 8 6 4 2 0
0°5F
0.4+
8205
0.3-
. 275
\
/
0.2r-
TRAth
0" ' 1280 L/Pt= 2
(l‘>-' l J. 1 .4 l n\\
P? o 2 i 6 8 l0
L l0 8 6 4 2 0

Figure 29

Figure 30. Graphical determination of composition of the
Pt(II)(NH3)ZCIZ-l-methylinosine complex. The straight lines are

the relationships eXpected for PtL complex. The curved lines are

2

the experimental results.

0.3 '

AABS

 

73

O
I-ME-INOSINE HacmK'IN.)
kN N

:5

 

 

0.4 '

0.3 _

AABS

0.2 '

0.| ”

 

0.0 “'
Pt 0
L l0

 

2 4 6 8 l0
2

 

A262

1265

1270 .
\\\ TRANS

\ L/Pt=2

 

 

Figure 30

74

Table 7. The stoichiometry of the gig: and trans-platinum nucleoside
Couplexes in solution at a pH around 5

 

 

 

 

Nucleoside cis-Etjlljjflha trans- _ ‘.
__ 12212 __ W3.) 25.12
Adenosine PtL, PtZL PtL
l-Methyladenosine PtL PtL
Cytidine PtL PtL, PtL2
Guanosine Pth PtL2
l-Methylguanosine Pth Pth
Inosine PtL2 Pth
l-Methylinosine PtL2 PtLZ
7-Methylinosine Pt L Pt L

2 2

 

7S

lyre one species, only that contribute to the spectra. In the case of
adenosine, the formed complex PtL will be further attacked to form

Pt L as the concentration of the cis-Pt(II)(NH ) C1 increases. The

2 3 2 2
trans-isomer did not show this phenomenon with adenosine, but with

[L]
c tidine the ratio of . - 1.5 su eats the ossibilit of
Y [Pt] + [L] 88 P Y

the formation of a mixture of PtL and PtL2 which will hinder the measure-

 

ment of the formation constant.

The Formation constant of the cig: and trgn§:platinumégggleggide complexes
After determining the existence of one species and the nature of the
formed complex, the formation constant of the complex could be determined
by measuring the change of the O.D. versus the log[Pt(II)(NH3)2C12] and
taking the midpoint of the tiration curve as the point at which half the
complex has been formed (see appendix for calculation of the formation
constant). Figures 31-38 show the difference spectra of the nucleosides

with varying concentrations of the cis- and trans-Pt(II)(NH3)2C1 , at a

2
pH around 5, and also the titration curves.

The concentration of the free ligand and platinum complexes that
did not bind with the nucleosides could be calculated knowing the total
analytical concentrations and the formation constants (see Appendix).
Measuring a difference spectra of the Pt, nucleoside mixture versus the
free ligand and free metal gives the absorption spectra of the Pt-nucleo-
side only. The absorption spectra of adenosine-, l—methyladenosine-,
cytidine-, guanosine-Pt complexes are shown in Figures 30, an, 41 and 42

respectively. Table 8 summarizes the different sites of the interaction

with the platinum complexes, and Table 9 lists their formation constants.

Figure 31. Difference spectra of adenosine, 0.55 X lO—AM in 0.1 M

NaClOa, with various concentration of the Pt(II)(NH3)2Cl

2
4 4M; 7, 0.5 x 10“; 8, 0.5 x 10“”;

isomers;

5, 0.25 x 10‘

20, 4 x lo-AM.

M; 6, 0.37 X 10-

76

an muswwm

 

 

 

<mkowam wozwmwumfi_ <mkomam wozmmmmmRH
Es 4 A85 ‘

8m 1 8% . 8N .qoomlm . . . . own . . . 4 0mm 4 J ago-
. no. N, . no-
1 N.OI 1 NO!
1 +0- 4 #0.

co co

- _.o 1 _.o

.NO .NO

.mo ,m ,, inc

ON
.mo mz<mh .vd
/
, 1 DO .,.. 1 0.0
m
. p .
.8 a mzaozmoq , . mo
. £2
to . so

 

 

mm< mm<

Figure 32. Titration curve of adenosine with Pt(II)(NH3)2Cl2

isomers plotted from Figure 31.

77

mm muruwu

30.312053
ov Om ON 0. O._

 

gidd - - —«.-.-d u - —dqd-dd d - —dd¢dqdu d

 

.__
z z,
3.40
Ti - \ 2
\\ ~12
mZ<m._. mz_mozmo<

 

    

EVN

le

L

Nm

 

mm<<

Figure 33. Difference spectra of l—methyladenosine, 0.41 X 10-4%

in 0.1 M NaClOa, with various concentration of the Pt(II)(NH3)2C12

_ -4 -
isomers, 5, 0.25 X 10 1‘M; 6, 0.37 X 10 M; 7, 0.5 X 10 ‘‘H; 8, 0.75 X

4 4

10' M; 20, 4 x 10‘ M.

78

<mhowam mozwmwmmZU

2.5 K
com

com

mm «pawns

<mkowam mozmmwumzu

Es «
com com com

 

 

 

 

 

Lm.o m ,G_\\
_
Z Z/
mmq Azfiwzof mz_mozmo<-.;xfiz-_
IZ

 

l

mdv
mmeq

Figure 34. Titration curve of l—methyladenosine with

Pt(II)(NH3)2Cl° isomers, plotted from Figure 33.

79

em muswwm

Togfzxsag
0v Om ON 0. O._

 

  
 

1444‘: a u .d-udqd d u —-¢<4d - d 1 —uq¢u4d d d

   

 

4.
\ Z Z
. v
o\\\\\\ Adz n
Ill 12

wz_mozmo< -._>I._.ms_n_

-m.

LVN

l

 

smm
mm<<

 

Figure 35. Difference spectra of cytidine, 1074M in 0.1 M NaClOa,

isomers, 5, 0.25 X
4

with various concentration of Pt(II)(NH3)2C1
4

2
-4 - 4
M; 7, 0.5 X 10 M; 8, 0.75 X 10

10'4M; 6, 0.37 x 10‘ M; 20, a x 10’ M.

80

dmhommm wozwmwuma

¢ecV‘
com

com

mm ouswwm

<mkuwdm muzmmmmhzo
cecv<
0mm 00m 0mm

 

 

 

w a J - a d 4 4 a q — q 1

.vo-

 

 

mmq .Iz mzaEG 84

Figure 36. Titration curve of cytidine with Pt(II)(NH3)2Cl2

isomers, plotted from Figure 35.

81

Ge on

em eczema

Togfzxuzb

ON

0.

O._

 

qqdddfid d 1* M4‘4‘d1 d

d

11‘141 4

wz<mh

‘

d

\

#14441 d 4 d‘

o \
\
\

\

\
o

   
  

\

dddflddd d

 
 

‘

‘-___-—_-—-——-—F--_-

. ._.
0
Q
\z
«:2

mz_o:.>u

     

4ON

imN

.wn

10v

 

mmdd

 

-4
Figure 37. Difference spectra of guanosine, 1.1 X 10 M in 0.1 M NaClOA,

with various concentration of Pt(II)(NH3)2Cl2 isomers, 5, 0.25 X lO—4M;

4 4 4

6, 0.37 x 10’ M; 7, 0.5 x 10' M; 8, 0.75 x 10’ M; 20, a x 10‘4M.

ABS

82

 

GUANO$NE

 

 

 

350

300

1(nm)
WFFERENCE SPECTRA

250

300 350

250

1(nm)
MFFERENCE SPECTRA

Figure 37

 

Figure 38. Titration curve of guanosine with Pt(II)(NH3)2Cl isomers,

2
plotted from Figure 37.

83

mm musamm

Togfzxsé
on ON 0_ 04 e0

Huddefid d dddqdqq d d ddudddq d d --du<d“d o
‘\
\

O

 

s 4N.

.
1

ON

_
z 2 2f
.. A :4
‘0 /Z ZI 1Nm
0

9.1611111 m2.m02<30 1mm
wm<<

 

 

 

Figure 39. Absorption spectra of adenosine, 0.55 X 10-4M;

cis-Pt(II)(NH3)2C12 - adenosine, 0.49 X lO—4M; and trans—

Pt(II)(NH3)2C12 - adenosine, 0.48 X lO—AM.

84

on musmfim

 

 

 

 

 

2.5 <
008 com com 0mm oom
.IIIIIIIIIJHJHJHWIIlqwnu. 1 AV

. No
.. «o
. 0.0

J. 1
A2 _ J .. mo

xz \2

N12 ..

mz_mozmo< . o._

mm<

Figure 40. Absorption spectra of l—methyladenosine, 0.41 X lO-AM;

—4
gingt(II)(NHB)2ClZ-l—methy1adenosine, 0.36 X 10 M; and trangr

Pt(II)(N83)2C12-1-methy1adenosine, 0.37 X 10-4M.

85

cc muswwm

 

 

 

     

 

A65 4
00¢ Omm 00m OmN OON
1111IJmmHmu a
’I.’./ .s
l I z a ..
Jw_nv.Lw‘uA‘IAw<¢.u_./.z ./ .
I. .1 .\
I . 1
I I.
//.1 1 x . .
mr—Obfi / .
13.22.. a .
'I/ 1
a
’ ..

 

m
_
A2 .7] 13-22-. .
/2 2d,:l 7
12

mz_mozmo<....;I._.m_2 1. .

Nd

v.0

0.0

QC

 

O._

mmd

 

Figure 41. Absorption spectra of cytidine, lO—AM; gig—Pt(II)(NH3)2C12-

cytidine, 0.93 X lO—AM.

86

as «human

(mkomam mozmmmmma
ASS <
co».

 

d J 4 d fl d

 

NIZ

mz_o_._.»o

 

 

mm<

Figure 42. Absorption spectra of guanosine, 1.1 X lO-aM;

cis-Pt(II)(NH3)2Cl2 - guanosine, 1.05 X lO-AM; and trans-

Pt(II)(NH3)2C12 — guanosine, 1.05 X lO-4M.

87

Na muswfim

 

 

 

 

2.5 A
oov 0mm 00m 0mm ooN
m
_,__ 2/ 2f
6Q.
o
mz_moz<3o . .
- o._

mm<

88

 

 

 

 

Table 8. The interaction of the cis- and trans-Pt(II)(NH3)2Cl2 at the
different sites of the nucleosides
-NU :_ T128 0f
Purines pfl_ N cheia- complex Structural Formula
————— —1 ————
tion
Adenosine
l‘iflz
C18 5.6 + + PtL, PtzL
Il” I 5>
trans 5.6 + - PtL, Pt2L T
R
l-MefiAdenosine
NH
cis 3.27 - + PtL “\‘3 N
II | §h
trans 3.27 - - PtL EN N/
I
It
Guanosine
cis 5.6 - — Pth J?
"N
trans 5.6 - - Pth ;\ |
lizll II
I
It
l-Me—Guanosine
cis 5.6 - - 2th CM N§
trans 5 6 - - PtL klfl/
. 2 "2!!

Table 8 (cont'd.)

 

 

 

 

 

- H - Type of
Purines 2§_ 51 £7 che a complex Structural Formula
tion

 

 

5.6 - + PtL2

Inosine
cia 5.6 - + PtL
2 H 'N>
\ N
I .
Ii

trans

l-Me-Inosine

cis 5. 6 -— + PtL2 CQJIIN\
I 7

2 hi. If

R.

trans 5.6 - + PtL

7-Me-Inosine
CH3
3.27 + - PtL *
2 "3'15
km N

cis
trans 3.27 + - PtzL
I
I:

Table 8 (cont'd.)

90

 

 

 

-NH - Type of
Pyrimidines “RE £1 E] cheize complex Structural Formula
5.12.11
Cytidine
"Hz
cis 5.6 + + PtL ”
"' 1
trans 5.6 + — PtL2 g”?
I.
l—Me—Cytidine
cis 3.27 - - c" NH
‘12
trans 3.27 - — ) ‘7'
“I"
Uridine
(D
cis 5.6 T** )K
Her [I
trans 5.6 - 0/\~
R.
l-Me—Uridine CH3 0
\ )
cis 5.6 - - N |
trans 5.6 — - “‘7.
R.

 

* - Me (Methyl)
**- i (very weak)

91

Table 9. The formation constants of Pt(II)-Nucleoside Complexes

 

 

 

p§_ c 8 trans Type of
cogplex
Adenosine 5.6 0.24xlO+5 0.64x10+5 PtL
l-Me-Adenosine 3.27 0.22x10+5 0.1.1x1o+5 PtL
Cytidine 5.6 0.66x10+5 ------ PtL
Inosine 5.6 0.46x1o+9 0.72x10+9 PtL2
l-Me—Inosine 3.27 l.49x10+9 1.75x10+9 PtL2
Guanosine 5.6 1.06x10+9 l.06x10+9 m.2
l-Me-Guanosine 5.6 0.6xlO+9 0.6x10+9 PtL2

 

L - Ligand

92
The Interacgion of Pt(II)(NH 2912 with Polynucleotides

Studying the interaction of the platinum species with synthetic
polynucleotides may aid in understanding the interactions with nucleic

acids, which contain a multiplicity of bases and base sequences.

Poly Guanylic Acid
Poly G (single stranded) interacts with the cis- and trans-

platinum isomers in solution at 37°C giving two spectral peaks at 1 nm

290

and 1265nm. The cis- and trans-platinum isomers have been shown to

interact monofunctionally with guanosine. The peak at 1 nm could be

290
assigned to the one of guanosine monomers at N7, and the other peak at

1265nm assigned to attack at N Figure 43 shows the difference spectra

1.
of poly G with pip: and Egpppeplatinum isomers. Precipitation of

poly G occurs at the end of the reaction when the N1 (N1 of guanosine is
involved in the hydrogen bonding with the cytidine in the Watson and
Crick double helix of DNA) is fully attacked. As the spectra of the
supernate of the poly G-platinum complex (before complete precipitation)

shows an increase in the absorption band at A nm (platinum attack at

290
N7), the interaction at N1 must decrease the solubility of poly G in

solution (Figure 44).

Poly Cytidylic acid

The difference spectra of poly C with gig: and pgppprplatinum
isomers (Figure 45) show two peaks (Azsonm, 1295nm). The similarity
between the.gipr and pgppp: poly C spectra excludes the chelation
between the N1 and the 6-NH2 group in case of the cis-isomer, as com-
pared to the cytidine monomers. Precipitation also occurred at the end

of the reaction.

Figure 43. Difference spectra of the Pt(II)(NH3)2Cl isomers,

2
0.2 mM, with polyguanylic acid (poly G), 0.1 mM P.

93

m8 ounwam

 

 

 

 

<mhowam mozmmwmufig <mhomam mozmmwmmzu
Ea: Etc:
....omm. 1 . .omm . 100.8. 1 . 10mm, . . . .3845-
> .
/\.I\ /oo
._.o
.No

 

 

 

Figure 44. Difference spectra of the Pt(II)(NH3)2Cl2 isomers,
0.2 mM, with polyguanylic acid (poly G), 0.1 mM P, in 0.1 M NaClOa

at 37°C. A, before precipitation; B, precipitation of the complex.

94

ea muswam

<mhomam mozwmwmnzo

2.5 <
co».

 

J NO-

 

o zen.

 

1

NO

 

L

v.0
mm<

 

 

Figure 45. Difference spectra of the Pt(II)(NH3)2Cl2 isomers,
0.2 mM, with polycytidylic acid (poly C), 0.1 mM P, in 0.1 M NaClO

at 37°C, B, before precipitation of the complex.

4

95

0mm

<mkomam mozwmmeo

2.5 <
oom

0mm

me muswam

0mm
. a MO1W

dmkomam mozmmmmma

00m

A85 4

Com

 

 

 

1 8 d c 1

d

 

 

 

no
mm<

l

mz<mk

o 38

 

mmd.

96

Poly Adenylic acid

The difference spectrum of poly A with_c__i_s__-Pt(II)(NH3)2Cl2 shows two
peaks (1275nm, 1285nm), and precipitation occurs at the end of the reac-
tions. The pgppprisomer shows one peak at 1287nm, which keeps shifting
towards 1285nm, and precipitation also occurred at the end of the reaction.
Although the difference spectra of poly A with pig: and pyppprplatinum
isomers are different, the poly Aegiprisomer spectrum shows the lack of
the long wavelenth shoulder characteristic of the chelation between N1

or N7 with the 6-NH2 group in a monomer base (Figure 46)-

Poly Uridylic acid
Poly U shows one peak at Azssnm with the cis-platinum isomer, whereas

interaction with the trans-isomer produced the same peak at 1 nm, but

285
it was very slow to deve10p. Figure 47 shows the difference spectra of
the cis-isomer after eleven hours, and that of the trans—isomer after

nineteen hours from the start of the reaction at 37“C. The interaction

could be assigned to attack at the N of the uridine, and precipitation

1

did not occur.

PolygA. poly U

The Tm of the double stranded poly A. p01y U is 42“C, and as the
reaction was at 37“C, we eXpect the two stranded to be separated. The
nm and a small one at 1 nm.

290
In poly A. poly U the interaction of the platinum isomers is mainly with

difference spectra show a major peak at 1270
poly A, as the rate of the reactions with poly U is extremely slow com-
pared to the other poly-nucleotides. Although the interaction is mainly
with poly A in poly A. poly U, the difference spectra with the cis- and

trans-platinum isomers are different than those with the single stranded

 

J .. . d. I
‘ 1‘“ :14. 1.4."...—

Figure 46. Difference spectra of the Pt(II)(N83)2Cl2 isomers,

0.2 mM, with polyadenylic acid (poly A), 0.1 mM P, in 0.1 M NaClOa

at 37°C, B, before precipitation of the complex.

as enemas

(chomam wozmmwmmfi. <mhomam mozmmwmmfi.

97

 

 

 

 

 

 

 

ASS K AES <
OOn OmN Omn OOm OmN

1 . 1 . 1 1 . 160.. 1 . . . . . . 1 1 . 1.8-
1 NO- 1 NO-
1 _.O- 1 _.O-
0.0 0.0

4
1 _.O 1 _.O
.No wz¢mk .mo
1 no 1 no

a
L V0 L VO
>10

mm< < _a mmq

Figure 47. Difference spectra of the Pt(II)(NH3)2Cl2 isomers, 0.2 mM,
with polyuridylic acid (poly U), 0.1 mM P, in 0.1 M NaClOa at 37°C, B,

end of the reaction.

98

me shaman

<m5mam wozmmmmma $.5QO mozmmumma
AEeV< Acav<
0mm 08 com own 08 08
q 1 d u q A d d u q — q ”v.0|_1 - u q u A d1 d u q A d u 4v10l

 

 

mz<mh

mO

l

 

 

3 28

 

mm< mmd

Figure 48. Difference spectra of the Pt(II)(NH3)2Cl2 isomers,
0.2 mM, with doubly stranded poly A. poly U, 0.1 mM P, in 0.1 M

NaClOa at 37°C, B, end of the reaction.

99

<mkowew mozmmwmmzu
ES 4
8.1,

we shaman

<mhom1m wozmmmumRH

 

 

 

 

 

 

 

Es «
can com com
q a .4 a q q q u q d d J

oo 00
1 _.o 1 .0
1N0 1 1mo
.00 mz<mk 100
1 6.0 a 1 6.0
160 3 rod .4 20¢ Ho
mm< mm<

100

poly A. In the double stranded poly A. poly U, N1 of poly A is hydrogen

bonded with poly U and the interaction is at N The similarity of the

7.
difference spectra of cis- and trans-platinum—poly A. poly U (Figure (.3 ),
excludes the chelation between N7 and the 6-NH2 group of the adenine
bases. As most of the interaction is at the adenine bases, and poly U

is hydrogen bonded with poly A, the platinum complex with poly A. poly U

remains in solution.

Poly_deoxyadenylic-poly deoxythymidylic acid (double stranded)

Poly d(A—T).d(A—T) has an alternating adenine, thymine on each
strand. The interaction of d(A-T).d(A-T) with the pgppprplatinum isomer
is much faster than that with the gig:isomer. The gig:platinum isomer
interacts with poly d(A-T).d(A—T) only after 72 minutes from the start of
the reaction, whereas the pgppprisomer interacts immediately. The dif-
ference spectra of both gig: and ggpppfplatinum isomers with
poly d(A-t).poly d(A-T) are similar, they have the main peak at 1275nm,
and a small peak at 1285nm. Also there was no precipitation at the end

of the reaction (Figure 49).Fipums SO, 51 show the rates of the reactions

of the polynucleotides with cis- and trans-platinum isomers.

DNA
The difference spectra of DNA of different GC content reacting with

cis- and trans-platinum isomers show two peaks at 1 nm and Azgsnm in

270
the beginning of the reaction. These two peaks converge to one peak at
A280 (Figure 52). The trans-platinum isomer reacts much faster than the

cis-isomer (Table 10). The rate of the reaction of the cis- and trans-
isomers increases as the GC content increases. Figures 53, 54 show the

rates of the reaction of the platinum isomers with DNA of different GC

Figure 49. Difference spectra of the Pt(II)(NH3) Cl2 isomers,
2
0.1 mM, with doubly stranded poly d(A-T). poly d(A—T), 0.05 mM P,

in 0.1 M NaClOa at 37°C, B, end of the reaction.

101

0mm

ac unawam

<mhowam mozwmwmua

AES <

OOm OmN

 

<mkowam wozmmumnzo
2.5 <
00m

00

 

 

 

Lmo
mmd

m2<m._.

$.38 1.01 $-38 zen.

 

L

00
mm<

Figure 50. First order kinetics of the reaction of the synthetic

in 0.1 M NaClO ; Pt(II)/P - 2.

polynucleotides with pip-Pt(II)(NH3)2Cl2 4

102

1
rs» . 1:. “a .1.

cm shaman

 

 

    

 

 

- q d d a d d 4 d d W q q u d d u u u m.o
’0
, 4811.0. x 8.. 3..., 1 no
I 1.01. ..
v , . 1.1
70390. x _N no. I I x. 1 V0 U
¢ .5 , 1|]
0 _om /) / ox 9V «V
7830.986... / x. ,. 1 mo H.v mv
3143.2.136 / .hIhL
- / . 1 NO
/ I.
.8880. x mo. 1 .. x...
........ onOn. Arm 1 _.O
ooooooooooooooooo c I
................. I. I
L p b . F P p p . p _ ...P.....~...LtI4- - p n I p (r 0.0
ONN OON 09 02 03 ON. 00_ 0m 0m 0.» ON 0

“885$ 95 1..

Figure 51. First order kinetics of the reaction of the synthetic

Pt(II)/P . 2.

polynucleotides with trans—Pt(II)(NH3)2C12 in 0.1 M NaClOa;

103

Time (minutes)

Hm gunman

 

 

 

 

 

 

Figure 52. Difference spectra of the Pt(II)(NH3)2Cl2 isomers,

0.15 mM’with DNA, 0.15 mM P, in 0.1 M NaClOa at 37°C.

 

104

   
 

(f)
O

I.

300
1(nm)
DIFFERENCE SPECTRA

v ’
1 . .
25

 

m0 m a) 1x to 10 V m N — O —‘
<— o o o o o O O O O O (I)

     

 

 

DNA (M. LYSODEIKTICUS) at 515°C

. <<<<<<<<<<<<<<<<<<<<M

momwmwn’nN—Of
<—ooooo°ooooq

3 0
1(nm)
DIFFERENCE SPECTRA

 

Figure 52

105

 

 

 

 

 

 

Table 10. Rate of the reaction of Pt(II)-isomers with DNA at different
temperatures
Temperature k sec.1 Calf Thymus E. Coli M. Lysodeikticus
QipyPt(II)(NH3)2C12
37°C k1 3110‘5 3.71110”5 5110‘5
k2 5.11.1110‘S 5.951110“5 9.371110“5
. —4 -4 -4
51.5 0 k1 1.45x10 1.68x10 2.22x10
k2 2.34x10" 2.8x10-4 4.9x10‘4
61.5°c k1 2.85x10‘“ 5.5x10‘4 5.3x10‘4
k2 1.151110"3 1.21110"3 1.41110"3
Iran§;Pt(II)(NH3)2C12
o —S -5
37 c k 2.77x10 8.148x10 -5
1 5 6.21x10
k2 5.5x10-‘ 6.04x1o‘5
51.5°C k1 1.7x10-4 2.6x10—4 3.5x10_4
k2 6.6x10'“ 4.6x10‘“ 4.51110“4
. —4 -3 -3
61.5 0 k1 1.33x10 3.6x10 3.6x10
k 8.33x10“ 9.471110‘2 9.47111072

 

Figure 53. First order kinetics of the reaction of cis—Pt(II)(NH3)2C1

0.15 mM, with DNA, 0.15 mM P, in 0.1 M NaClo4 at 37°C.

29

“5‘. *"-.—~ ,

P;

106

mm shaman

 

 

 

II .
1 II 0 I.-

I I

II ./

.II .2-
1 I, I.
II I.
CI I.
1 II. I
I I
I, .1.
I I
1 I .II.
I I
1., ., .
‘I .I.
[III .I.-III
. N.- 3226633 2. .1/ I.-
. I .D.
on :8 ml! 11. 1...
. mm 6:52:60--.- .-. la.
.80 . .1...

ovm ON oom om 08 ON CNN 00 om. cm. 0! ON. 00. om om 0....

3235:: NEC-

 

QO
1 QC
1 NO
1 QO
P.
1 no 1M].
1.. 1..
_O .8
1 no
1 NO
1 _.O
,0.0
O

Figure 54. First order kinetics of the reaction of trans-

Pt(II)(NH3)2C12, 0.15 mM, with DNA, 0.15 mM P, in 0.1 M NaClO4 at

37°C.

107

Fig.1?“

 

on ousuwm

 

 

    

 

 

T 1.
1 111. J

. 3232082.. .21.!

__oo.mlol

. SEE-_- :oo 1.1
O¢N ONN OO 02 09 OE ON_ 00. Om OO 0.» ON O

32:55 DE:-

NO

108

content at 37°C. There are two consecutive mechanisms of interaction.
The rates of the reactions increase with increasing tempratures, and
precipitation occurs at the end of the reaction. Table 11 shows the
activation energies of the reactions with DNA of the pig: and pypppr

isomers.

 

109

 

 

 

 

 

 

 

 

 

Table 11. Activation energy of interaction of the Pt(II)(NU3)2Cl2 with
DNA
Activation epeggy Calf ThYflH§ E. Coli_ flb.ly§pggjktigp§
.0 8.1 @131
E1 9100 9100 9100

£2 10792 10800 10800

_-m-n————--——...-—— —--. - — — - —‘“——- _._.-—.— —— u-.. "—- " ——'~

IV. DISCUSSION

The interaction of the platinum isomers in solution with DNA and
its bases depends on the pK's of the active groups of both the base and
the platinum isomers. As earlier discussed in the chemistry of the
square-planar platinum complexes, the substitution reactions occur through

the aquated form, [Pt(II)(NH3)2(H20)2]2+, The cis-form has a pK - 5.6,

1

and a pK2 - 7.3 or 9.2. The trans-isomer has a pK1 - 6.3 and pK2 - 7.4

Thus, above pK it exists as [Pt(II)(NH3)2(OH)2]. At pH 5.6, which is

2’

between its pK and pKz, it exists as [Pt(II)(NH3)2(on)(H20)11+.

1

Interaction at N1 and N, of Adenosine
At a pH around 1 adenosine has pK of N1 of 3.5, and the reaction will

proceed as:

lng
$1“ I0 N "'
""2 NH, ‘d‘\+ "2 a
" II”| ‘f’

"*1?" > + “freak-NH; ‘—_": *3 N

a: w» .

110

I)

 

111

This reaction is suppressed by high concentrations of protons. At pH 4,

N1 of adenosine is not protonated and the reaction will be pH independent.

 

 

t W
8’ 6"
it../' ."H2
'1": } \’ / \> + u o
A e N l 2
"2'12N'> + .qo—H:||;_NH3—— v9“ =1 ‘ w h"

At pH 6, the Pt-species exists as [Pt(II)(NH3)2(H20XOH)]+1and.the reaction

will be suppressed as the OH- concentrations rise in case of reaction (a),

or be pH independent, as in reaction (b).

g ‘V
e” 5!.
a“! "“2
e v -
/ e’ \N’ + °"
1”
"2 If": I!
+ HO—PIt—Nl'la
g s. \ t
b *0 0
e?! * ""2
/ ’ a o
4? “ I ‘+- ‘2
e” |

112

 

U

.2.

C0

9 a:

g z 23 g? 3

Q‘E ”_5 NE «'5
N7 ‘2... it if 15

If: /

 

 

 

f?-

U

D

g I 3

l 1
’l «2.: _“HQEQFUNCT'WAL

3 1’ ¢\~,- I \HHH

t5 /’ ./' I \-\B'FUNCTIONAL

g I / .0, 2 '\\

0 13’)", :H \'
figerH’f"! . s. r .
0l 2 l2

Figure 55 Schematic representation of the existance of

different complexes in solution at different pH's.

Figure SSillustrates the interaction at different pH's.
If the interaction is monofunctional, the highest concentrations of

platinum-adenosine complexes (interaction at N1) will occur at pH . 3.5,

92,93

which is the pK of N of adenosine. The curve turns down at point

1
B if the OH‘ ion rather than the H20 molecule is replaced at pH 6, other-

wise it will turn down at the pH - pK2 of the platinum species. The pK2

of the platinum species is 7.2 or 9.2 (point C or D). N7 of adenosine

is never protonated, and the reaction will be suppressed at high pH due

only to the release of OH' ions from the platinum species, [Pt(II)(NH3)2(OH)2].

113

Interaction at the 6—NH2 of adenosine

*9
‘5
$2 4*
he
"N'l !l "\> + ago—mun, _.___=-. "Nm\ +
it' pflilt‘ \
I do I
a l H20

This reaction is suppressed at low pH, while that at pH 6 is neutral.

45‘?”
4§§g*
/‘\o
""2 T“: NH ~
._____25.
so + \——...... ~~ +~°
I “2°

114

At pH - 10, the reaction is pH independent.

 

 

{pfib
{pet
éMf.
‘C
129
/ _ _.__>. -
Q>+ "° ”“3 W kfi' >+"’°
N P "’
I " I
a I:
Bifunctional binding at 6-NH2_and N7 of adenosine:
At pII = 1
I“!
I: an" '
_— '—
’ ""2 "“3 "N ‘2‘ 3 H
l __:5 fiflfla’ ._+—
’"N~ II ‘j>'+' lfl!3"P"'N"H3 «<r-—""" '\ I :>
I Ifzb "'1 N T "2°
I.

115

At pH - 6

‘ fl":
""2 N" —"—NH3
N/| K) + uo-IL-fuu ——‘\————— / . 2“
lfimi ' Io '3 'fli GI qéfigl .+- if,
. "2°
I
At pH - 10
..
_ 9— ..
H 9| N": on

 

""2 I~‘II-I3
m "’ "°:'j,"""3 flit-HF: 1:39 "‘
I.

.1 ..,.

116

Reaction at pH - 1 is suppressed by high concentrations of H+ ions, at
pH - 6 it is pH independent, and at pH - 10 it is suppressed (above the

pK of the [Pt(II)(NH3)2(H20)2]++.

2
. In the case of dimers like A3'p5'A and A2'p5'C, more groups are

available for forming different complex compounds.

\\ +

illafii

 

117

 

 

_ Z I ..
u ”3 no A 1' V !" + ”‘29
‘ «.2 .3 .
+ I ~“Eu: 10 9“”: "-lwz nu;
...... 1..., g 2..-a: x
. é 2..“..1'“: ”0.02. z
"2 3 2

Reaction at pH - l is suppressed at low pH, while that at pH - 6, and

above the pK2 of the Pt(II)-complex is enhanced.

118

Figure 55 is a schematic representation of the existence of different

3)2012 has the

ability to chelate with a neighboring active group. Adenosine with

complexes in solution at different pH's. Cis-Pt(II)(NH

cis-Pt(II)(NH3)2C12, Figure 56 shows attack at N7 predominantly from

pH 1-3.2. At pH - pK (N1) - 3.5 the interaction is at N and N7

1
(pH - 3.5-5.6). In the region of the pKl-pK2 of the Pt(II)(NH3)2(H20)i++,

pH - 5.6-9.2, interaction at N1 and at N7 chelating with 6-NH2, occurs

as the predominant species with [Pt(II)(NH3)2(H20)(OH)]++ a pH higher
than the pK2 of Pt(II)(NH2)2(H20)2++ the interaction decreases to zero.

As the trans-isomer has a pK - 4.2, pK2 - 7.3, and no possibility of

1

chelation, the interaction is monofunctional. N7 is attacked in the
region pH - 0-4.2; N7 and N1 from pH - 4.2 to 7, and then a decrease in
the high pH region.

Cytidine follows the same path as the interactions of N and 6-NH

1 2

of adenosine, and also a chelate is formed with NHZ in the case of the
gggfisomer only. Cytidinegz’93 has a pK of N3 . 4.2. Thus the gingt(II)-
complex interacts at N3 and chelates with the amino group in the 6-position
in the pH region 4.2 to 9. The £5§2§_isomer interacts at N3 in the same
region, without chelation with the neighboring amino-group.

Guanosinega’gshas a pK of N7 8 2.4 and of N1 - 9.2. At low pH's, N7
of guanosine will be protonated and the interaction at N7 will not be
favored.

Guanosine Reactions (see next page)

 

Figure 56. Summary of the interactions of the Pt(II)(NH3)2Cl2 isomers

with nucleosides at different pH's.

 

 

119

 

em shaman

 

 

 

 

 

 

 

 

 

S
.—
m.
. w .
.. .. .. .. .N
. I I. .N .m _ 8m -_ ..
TIL... ....... .\ ... ZmOZ< 2 Is.
PM P. ........... I‘\.\. 1”
q‘ d 4 go 1 a < 4 40
4. .__
N .N
Ln .n
q 4 1 T 1 fl 1 4 a . q q a go

 

 

   

 

mzaozmoq .N
.F: Slides .. m
,I .. I 116.092 .e
mmammHH.

120
At pH I 1

 

 

NH ,
Hé-gfjflfla $4.5

DINO H9 01;; "Nfif’ ~I!)

A \ . 1"
W*}.' '> ‘91:] ranking?) ‘1'“
I I H20
At pH I 6
60‘s,“?!

121

At pH I 10

Reaction b at pH I 6, and both reactions at pH I 10 are not favored at
high pH's. From Figure56 we can conclude that the interaction with the
platinum isomers in the pH region 3-9 is at N7, and above pH I 9 the
interactions at both sites, N1 and N7, will be suppressed as the concen-
trations of the 0H” ions increases.

Inosine has the same route of interaction with the Pt isomers as
guanosine, the only differeence being in the pK's.96 The pl( of N1 is 8.8

and of N7 is 1.2. The interaction of the platinum isomers with guanosine

and inosine is monofunctional. The second active group of the platinum

isomers could interact with another molecule forming the Pth complex.

122
AtpHI6

”$6
91’ ‘\\o
I~= as; fir
"Git-NH; H2
2 fig?“ ‘51: 6 A

 

 

._+__

The formation of Pth complex is also favored in the case of the trans-

isomer of the platinum species.

a:
”2»:
was. __ rm,
“04-"? l-Vt" Thu: 2-8
u u ,

 

 

123
Reactions in both isomers are suppressed at high concentrations of OH-

ions in the region below the pK of the platinum species.

 

2
l-Methzl Guanosine Reactions:
qui dy’
é‘e’é
O O
“'1‘ng * 2““
no '31“ NH "’Nk l 'I' ""2
g " 3 I! ll
2 “'3‘ w
_______________;:s
lflzhl “:;flH=H6 '-1-

Interaction at the N1 and the 6-NH2 of cytidine

Cytidine forms PtL complexes with the trans-isomer only; the amino group

2
in the six position chelates with the platinum in the case of the cis-

isomer. See next page.

124

I!
so 74'
pug,“ \n an,
[5” I] / \. HO
\ 2” gfl A I ‘1" 2
h’x”
2° \ '

125

A study of the interaction of dinucleotides with cis- and

trans-PthIlSNHalzglz by Circular Dichroigm spectrosCOEy

The two isomers of Pt(II)(NH3)zClz can bind to the 6-amino group of
adenosine and cytidine forming PtL and/or Pth complexes. This led to
the investigation of the possibility of the formation of the intra- and
inter-strand complexes in DNA in case of the gig:isomer, and inter-strand
complexes only in case of the Eggggrisomer. The inter-base separation in
DNA97 is close to 3.4; and as the distance between the two gigfligands of

98 is about 3.3A, the linking of two bases

square planar platinum compounds
to one platinum ion is stereochemically possible.

The purine dimer (A3'p5'A) provides an ideal model compound. The
6-Amino groups of the bases of ApA in solution lies vertically above one
another90 with a separation of 3 to 4A. Thus the amino groups are in an
ideal conformation to bind to a metal ion. Since it is the object of this
study to detect all possible types of binding with all sites, a circular
dichroism technique would reveal the inter-base links. Dinucleotides in
solution at room temperature are in an equilibrium between the stacked
form (Figure 57) and an unstacked or random conformation. The stacked
conformer has a characteristic circular dichroism spectrum which is more
intense than that of the unstacked form. The temperature dependence of
the equilibrium between stacked and unstacked conformers has been examined
by studying the temperature dependence of the CD spectrum characteristic
of the stacked conformer.

If the binding of gigyPt(II)(NH3)2C12 to A3'p5'A results in a cross-
link, then the dimer will be locked into the stacked conformation. Such
a dimer will not pass into the unstacked form as the temperature is raised,

since the platinum-base complexes are stable in solution at these higher

Figure 57.

M NaClOa.

CD spectra of A3'p5'A. Conc. dimer, 1 X 10-

l.

M, in 0.1

 

 

 

126

 

 

 

 

300

 

127

temperatures. The absorption spectrum of A3'p5'A, and the temperature depen-
dence of its CD spectrum agreed well with previously published results.
The absorption spectrum of the incubated A3'p5'A with trans-Pt(II)(NH3)2C12,

molar ratio of 1:1 (Dimer to Ft), is shifted to A nm (Figure 58) and

270
the characteristic double peaked CD spectrum is abolished. Thus the
binding of the transfisomer has brought about unstacking of the dimer.
This effect is similar to that observed with A3'p5'A at low pH. At a

pH below 3.5 (pK of N1 of adenosine),N 1is protonated and each base

will carry a positive charge at pH 1 and the repulsion between the bases
will lead to unstacking. Metallation at N1 of adenosine will place a
positive charge on the base. If both bases of A3'p5'A are metallated,
repulsion and complete unstacking will take place.

In contrast to this result, the CD spectrum of the complex between
the gig:isomer and A3'p5'A shows a remnant of the double peaked CD curve
characteristic of a stacked conformation, and is temperature independent,
confirming that this component of the solution does not arise from a small
residual quantity of stacked, but unmetallated, conformer. Since most of
the binding of the gig:isomer is of a mono-functional type, the strength
of the CD signal is low due to the small concentration of the dimer-
complex in solution. This species is most likely to be a form of A3'p5'A
with both bases linked to one platinum ion via their 6-amino groups.

In DNA itself the 6-amino-groups of two adenine bases are vertically

above one another with a separation of 3.4A. The binding of
cis-Pt(II)(NH3)2 Cl2 to two adenine moieties via their 6-amino-groups

could introduce an inter— and intra-strand cross-link into DNA. Since the

platinum isomers could interact at many sites with almost all the bases

of DNA, the kinetics of the interaction of the two isomers with DNA and

Figure 58. CD spectra of A3'p5'A with Pt(II)(NH3)2Cl2 isomers.
Conc. dimer, 1 X lO-aM; dimer: Pt(II)(NH3)2C12,———-, l X 10-4M;

_-_ , 1:1; _.- , 1; 3.0; -~-- ; 1:5; ---- , 1:10; in 0.1 M NaClOa.

126'

is. K

.\
-\Oossooo
lo“... ' O {A
I _

 

 

mz<mk

 

 

I no.»

I. 0.0.

O
7::

 

mm NHDNHW

.Is. «

"" ‘ I"

\
. I d d I 1 d
fl .1!

 

.8... e.n <

 

 

0.0.

 

129

synthetic polynucleotides provide some information on the most reactive

sites on the DNA.

Kinetics of DNA with Pt(II)(NH3zzglz

The formation constant of the two isomers of Pt(II)(NH Cl2 with

3)2
guanosine is of a higher order than those with other nucleosides (Table 9).
Guanosine might be the first target of DNA in the interaction with the
Pt(II)-complexes. The kinetics of three kinds of DNA having different
concentrations of GC pairs were followed to reveal the role of those

bases. Figure 53 and 54 show the rate of the reaction of DNA (Calf

Thymus I 382 CC, E. Coli I 502 CC, M. Lysodeikticus I 722 CC), with

Pt(II)(NH C12 at 37°C. The rate of the reactions increases as the GC

3)2
content increases. In Figures 53 and 54 there is a break in the line
indicating there are two consecutive mechanisms. The experimental rate
constant k1 is most probably due to the interaction of the platinum
isomers with the N7 of guanosine which is not involved in the hydrogen
bonding of the Watson Crick model. In the case of the gig:platinum
species, the unreacted hydroxyl group could chelate with the 6—oxygen,
breaking the hydrogen bonding between lI-NH2 group and 6-0 of the
cytidine-guanosine pair, and cytidine will be exposed for interaction.
Interaction of the platinum species with the guanosine-cytidine base
pair will lead to partial denaturation of DNA and exposure of other pos-
sible sites of the other bases for further interaction. The movement of
the strands around the platinum-guanine base complex due to the partial
denaturation, brings another base from the other strand close to the
active group of the platinum-guanine, and cross—linkage could be formed

between the two strands via the guanine bases. Poly d(A-T), double

stranded, in which the bases are alternating in sequence, did not react

130

with gigrPt(II)(NH3)2C12 except after 72 minutes at 37°C, which is in

the range of the start of the second reaction in DNA by extrapolating the
lines to zero time in Figure 50. This delay of the interaction of the
.gi§;platinum species suggests that denaturation of the A-T bases is a
first step before the interaction, since the N1 of adenosine is hydrogen
bonded with N3 of thymidine. Furthermore N7 is either not involved in

the reaction, or occurs at a very low rate. The cross linkage between the
two adenines from both strands of DNA could also be formed after the par-
tial denaturation occurs.

Poly U and poly dT, single stranded, has a very low rate of inter-
action with the gingt species, and an extremely low rate with the 552237
isomer. The N1 of the uridine base or thymidine is blocked by a hydrogen
atom, in addition to the steric hinderance of the -NH3 groups of the trans
isomer. It is difficulty to correlate the rates of the interactions of
the single and double stranded polynucleotides with the Pt(II)(NH3)2Cl2
and that of DNA, as the Tm's of the synthetic polynucleotides differs
drastically from one to other. Poly A. Poly U has a Tm I 42.0’C,
poly dG. poly dC I 81.7°C.; and poly dC. poly dC is insoluble in 0.1 M
NaClOA, which hindered the study of its kinetics at the same conditions.
Howle et al.61 studied the interaction of the gigrdichloro (dipyridine)-
platinum(II), labelled at the hydrogen [pyridine-3H] which is, with
nucleic acids in vivo as well as in vitro. The binding to DNA was mostly
inhibited by the addition of poly-Guanylic and Uridylic acid. Their con-
clusion was that the platinum species binds predominantly on the guanine

1.103

and/or uracil (thymie) rings. Horacek et a studied the effect of the

cis-Pt(II)(NH on the renaturation of the DNA (calf Thymus). An

3’2012
Optimal ratio of Pt/P I 0.05 for renaturation of DNA in SmM NaN03,

131
suggests the ability of the cis-Pt species to form ligand bonds between

bases of both chains in the double-helix structure. Horacek et al.103

reported eXperimental rate constants of the cis-Pt(II)(NH ) Cl with DNA

3 2 2
(calf-Thymus) at various temperatures. Their values at 37°C, 52°C, 65°C
are 4.0 x 10-5, 2.3 x 10" and 6.8 x 10-4 which agree with the experi-

mental values of K2 of this work,except for the last one.

Conclusion

The Pt(II)(NH3)2Cl does not react with DNA and its components in

2
the unaquated form. The two species of the aqua-form, [Pt(II)(NH3)2(OH)(H20)]+
and [Pt(II)(NH3)2(H20)]++ are the active forms for interaction with the
amino-groups and the aza-nitrogens of the bases. The gig: and Eggggrisomers

of the platinum species can interact with two bases forming PtLZ comugxes,

and both can cross link DNA. The gigrspecies forms intra- and inter-

strand cross links on DNA, while the Eggggrisomer forms inter-strand

cross links only. The Eggggrspecies acts monofunctionally with one base,

while the‘gig-isomer can act mono- and bifunctionally, chelating with the
active neighboring group. In DNA, guanosine is the most reactive site

for the interaction with the platinum species which leads to denaturation

and exposure of the cytidine, adenine and thymine for further attack.

A cross link between the double strands of DNA could be via two bases of

of the other, or N of both

1 7

bases. There is also a possibility of forming the cross link between the

guanosine involving N] of one base and N

two bases of adenine and cytidine (Figure 59).

The possibility of forming the intra—strand complexes on adenines,'
cytidines or an adenine-cytidine complex should not be excluded. The forma-
tion of the cross linking between the double strands is more favored in

case of the trans-isomer than the cis-isomer of the square platinum(II)

Figure 59. Structure of DNA.

132

O I
H H H H o
H H
(I) H G c H H I
o-IID=o o o
| o- H.N,H" 3’”, CH2
CH2 N N \ (I)
0 41"" H’N\H O=P—O
H H HH N 0" H Cl)
G H H
O H C ’0
I H H,’ N» H H
O—|'3=O \N, H/NKN O
O. I \ "7 FN CH2
CH2 NA ‘,oH’N\H O.
o I
O 0:}?—
H H H H A T H O
O H CH3 H H
I H H, 0 \ H H
O‘P=O \N/ N N O
0' N \N" HI T CH2
< ' a 0 0'
O O=[|3—O
H H HH H o
H H
(I) H T A H H
O-P=O o
I |_ (N N
o o ”N . > _CH.
CH. JL ,H— \ N 0|
O N N ’N\ O-FID-O
H 'fi/LO/H H I
H CH3 1

Figure 59

133

complexes. The gig:isomer is more likely to form an intra-strand complex
than the Eggggrisomer.

All the possible complexes could be formed with both the gig: and
.gggggrisomers of the square planar dichlorodiammineplatinum(II). However
the traggrisomer does not inhibit DNA synthesis and has no antitumor
action. Further work needs to be carried out to investigate whether the

trans-Pt(II)(NH Cl2 binds to DNA in the cell or not. Another area to

3’2
be investigated is to determine which complex compound is responsible for
the antitumor activity in the case of the cis-platinum isomers. Additional
work needs to be carried out on the kinetics of the reaction of DNA with

the platinum complexes, and the isolation of these complexes by chroma-

tographic techniques.

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APP END IX

Calcu1gtion of the formation constants of the Pt(II)-nucleoside complexes.

Adenosine

absortivity A

A ltt (1C t C10 . -
as y C1 oncen ra n Extinction coefficient x cell path 8 x b

 

E of adenosine I 14.9 x 10+3

b 1 cm

A of adenosine I 0.82

C I total analytical concentration of the ligand in solution

0-82 = 0.55 x 10‘“ a

14.9 x 10+3

therefore C

Figure 24 shows that the formed complex in solution of the cis- and

trans-Pt(II)(NH3)2012 is PtL
l

. (24)
[Mxl- l/2[LO]

 

therefore Kf

Kf I the formation constant of the Pt(II)-nucleoside complex

Mx I concentration of the Pt(II) at the midpoint of the curve (Figure

(1) cis-Pt(11)-adenosine comglex, (PtAZCis

Figure 32 shows Mx 6.5 x 10"5 M

 

1
= 0.24 3 10+5

 

therefore Kf I 5

6.5 x 10‘ - 1/2 x 0.55 x 10‘“

K (PtA)Cis

f a "
[CA ' (PtA)cis] [CPt ’ (PtA)cis]
140

 

- 0.24 x 10+5

141

CA , total concentration of adenosine in solution I 0.55 x 10-4 M

CPt’ total concentration of cis-Pt(II)(NH3)2012 in solution I 4 x 10-4 M

Therefore (PtA) ' 0.24 X 10+5 [2.2 x 10-8 ’ 4'55 x 10-Z.(P':A)cis

 

 

cis
2
+ (PtA) ]
cis
(PtA) - 0 49 x 10" M
cis °
and
L I the free ligand = 0.55 x 10-4 - 0.49 x 10-4 I 0.06 x 10-4 M
Cis-Pt(II) = the free cis-Pt(II) = 4 x 10'4 - 0.49 x 10‘“ . 3.51 x 10'° M
(ii) Trans-Pt(I1)—adenosine complex, (PtA)T
Figure 32 shows Mx = 4.2 x 10"5 M
Kf = 1 - 0.69 x 10“5
-5 —5
4.2 x 10 - 2.75 x 10
(PtA)T
K = = 0.69 x 10+5

 

CPt’ total concentration of trans-Pt(II)(NH3)2C12 in solution I 4 x 10‘4 M

Therefore (ptA)T = 0.69 x 10+5[2.2 x 10‘8

- 4.55 x 10'4 (pm)T
+ (PtA)2]
T

(Pt/I)T = 0.48 x 10‘“ M

4

and the free ligand I 0.55 x 10‘“ - 0.48 x 10- I 0.07 x 10_4 M

the free trans-Pt(II) I 4 x 10'“ - 0.48 x 10-4 I 3.52 x 10-4 M

142

As illustrated in the previous case, the concentration of the free
ligand (L) of the following nucleosides and the free platinum species
(Pt(II)) in solution, could be calculated. The total concentration of

the platinum species in 4 x 10-4 M in all cases of the nucleosides.

IIMethyladenosine

 

The formed complex is of the PtL type (Figure 25) in both cases of
the cis- and trans-Pt(II)(NH3)2C12. Knowing the analytical concentration
of the nucleoside (0.41 x 10—410 the following physical constants were

calculated.

(i) cis-Pt(11)-methy1adenosine complex

5 M (obtained from Figure 34)

5

M = 6.5 x 10—
X

Kf . 0.22 x 10+

L . 0.05 x 10“ M

g1§ePt(II) = 3.65 x 10‘“ M

(ii) grans-Pt(11):methy1adgposine

Mx = 4.5 x 10.5 M (obtained from Figure 34)
x, - 0.41 x 10+5
L - 0.04 x 10‘“ M

trans-Pt(II) I 3.63 x 10'4 M

gggnosine

The formed complex is of the PtL2 type in both isomers of

the Pt(II)(NH3)2CIZ. Using equation 31 and knowing the

143

analytical concentration of the nucleoside (1.1 x 10"4 M ) and the

following physical constants were calculated.

K . 1 1 (31)

f [LO] [Mx] - 174 [L0]

 

(i) cis—Pt(I1)_guanosine comp1ex

- 3.6 x 10'5 M (obtained from Figure 33)

Z
I

+9

74‘
II

1.06 x 10

0.05 x 10“ M

1'"
I

Cis-Pt(II) - 2.05 x 10‘“ M

(ii) trans-Ptjll):guanosine complex

5 M (obtained from Figure 38)

9

Mx a 3.6 x 10‘
Kf - 1.06 x 10*

L - 0.05 x 10‘“ M

[Egflg—Pt(11) = 2.05 x 10-4 M

1—Methy1gpanosine

The formed complex is also of the Pth type in both isomers of

the Pt(II)(NH Knowing the analytical concentration of the

l.

3)2012°
nucleoside (1.27 x 10- M), the formation constant of the following

complexes were calculated.
(1) cis-Pt(11)-methylguanosine complex

M - 4.5 x 10‘5 M
x

14f - 0.6 x 10+9

144
( ii) trans-Pt(II)-methylguanosine complex

-5
MK I 4.5 x 10 M

Kf . 0.6 x 10+9

Inosine

The formed complex is of the Pth type in both isomers of the

Pt(II)(NH C1 Knowing the analytical concentration of the nucleo-

3)2 2'

side( 1.097 x 10-4

M) the formation constant of the following complexes

was calculated.

(1) cis-253112;;nos1ne comp1ex

M a 4.7 x 10"5 M
X

Kf - 0.46 x 10+9
(ii) trans-Pt(11)-inosine complex

M I 4 x 10"5 M
x

xf - 0.72 x 10+9 M
1—Methy1inosine

The formed complex is also of the PtL2 type in both isomers of the

Pt(II)(NH3)2C1 Knowing the analytical concentration of the nucleoside

2.
(0.96 x 10" M). The formation constant was calculated.

(1) cis-Pt(II):methylinos1ge complex

M - 3.1 x 10‘5 M
X

Kf - 1.49 x 10“9

145

(ii) trans-Pt(11)-methylinosine com21ex

M - 3.0 x 10‘5 M
x

xf - 1.75 x 10+9

gxtidine

The formed complex is of the PtL type in case of the cis—Pt(II)(NH3)2C12.

There was more than one complex formed in case of the trans-Pt(II)-isomer

and thus its formation constant could not be calculated. Knowing the

analytical concentration of the nucleoside (10-4 M), the physical constants

of the following complex were calculated.

cis-P2(II)-qytidine complex
M I 6.5 x 10-5
x

Kf - 0.66 x 10+5

L - 0.07 x 10'“ a

cis—Pt(II) I 3.07 x 10-4 M

 

 

 

 

"‘IIIIIIIIIIIIIII“