Hydrolytic Equilibria and N7 Versus N1 Binding in Purine Nucleosides

11 Hydrolytic Equilibria and N 7 Versus N1

Downloaded by KTH ROYAL INST OF TECHNOLOGY on February 24, 2016 | http://pubs.acs.org Publication Date: January 26, 1983 | doi: 10.1021/bk-1983-0209.ch011

Binding in Purine Nucleosides of cis-Diamminedichloroplatinum(II) Palladium(II) as a Guide to Platinum(II) Reactions at Equilibrium R. BRUCE MARTIN University of Virginia, Department of Chemistry, Charlottesville, VA 22901 Antitumor Pt(II) amines are usually administered as cis dichloro complexes. This form persists in human blood plasma with its high 103 mM C1 con tent. The net zero charge on the complex fosters its passage through cell walls. Within many cells the C1 concentration is much lower, only ~4 mM. Substitution of C1 by H O then occurs. Depending upon pH deprotonation may also yield hydroxo con taining complexes. In neutral solutions of low C1 content the chloro-hydroxo complex predomi nates but aquo-hydroxo, chloro-chloro, aquo-chloro, and hydroxo-hydroxo complexes also appear. Of the three leaving groups H O is by far the best, followed by C1 and OH , which is inert. Most likely the aquo complexes react with cell consti tuents. At sufficient Pt(II) concentrations complexes with hydroxo bridges form at the ex pense of a water ligand, reducing the Pt(II) complex reactivity. Dihydroxo bridges were first observed on analogous Pd(II) complexes in solution and found to form more slowly with Pt(II). In purine nucleosides metal ion binding may occur at either N(7) or N(1). In guanosine and inosine the ratio of binding at N(1) to N(7) is pH dependent. At pH 7 the stability order for dienPd(II) binding at the common 5'-nucleotides is G7>I7>I1>G1>U3>T3>C3>A1>A7. This series represents a considerable promotion of guanosine and inosine N(7) stabilities over that found for H and CH Hg . Pt(II) appears to favor N(7) to N(1) even more than Pd(II). Even when binding at N(1) is favored, Pt(II) may become kinetically fixed at Ν(7) in the 6-oxopurines. Of the several common nucleoside binding sites both thermodynamic and kinetic factors favor Pt(II) binding at guanosine N(7). -

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0097-6156/83/0209-0231$06.00/0 © 1983 A m e r i c a n Chemical Society

In Platinum, Gold, and Other Metal Chemotherapeutic Agents; Lippard, Stephen J.; ACS Symposium Series; American Chemical Society: Washington, DC, 1983.


232

METAL CHEMOTHERAPEUTIC AGENTS

Cis (NH3)2PtCl2 and other P t ( I I ) complexes r e a c t only s l o w l y w i t h t h e n u c l e i c bases. The slowness may be e s s e n t i a l t o t h e i r e f f i c a c y as tumor i n h i b i t o r s , f o r i t provides i n t e g r i t y and n e u t r a l i t y during c i r c u l a t i o n and passage i n t o c e l l s . Equilibrium constants f o r a s s o c i a t i o n of c i s (NH3>2Pt(II) complexes w i t h n u c l e i c bases remain unknown. I t has been argued that p u b l i s h e d constants f a i l t o r e f l e c t systems a t e q u i l i b r i u m ( 1 ) . The aqueous chemistry of P t ( I I ) r e l e v a n t t o b i o l o g i c a l molecules has r e c e i v e d review ( 2 ) . As an i n d i c a t o r of e q u i l i b r i u m tendencies and constants f o r P t ( I I ) complexes, we have employed analogous P d ( I I ) complexes. Both metal ions form diamagnetic, planar complexes and p r e f e r n i t r o g e n t o oxygen dgnor atoms. They e x h i b i t s i m i l a r e f f e c t i v e i o n i c r a d i i of 0.60 A f o r P t ( I I ) and 0.64 A f o r P d ( I I ) ( 3 ) . However, complexes of d i e n P d ( I I ) r e a c t about 10^ times f a s t e r than those of d i e n P t ( I I ) ( 4 ) . At t h e same time P d ( I I ) exchanges s l o w l y among n u c l e i c base l i g a n d s i t e s so that i n -4î NMR s p e c t r a i n d i v i d u a l l i g a n d resonances appear f o r each k i n d of P d ( I I ) b i n d i n g s i t e . Because of i t s convenient r a t e of attainment of e q u i l i b r i u m c o n c e n t r a t i o n s , as w e l l as i t s diamagnetism and slow r a t e o f exchange on t h e NMR time s c a l e , P d ( I I ) o f f e r s one of the most a t t r a c t i v e metal ions f o r i n v e s t i g a t i o n o f i t s complexes. This a r t i c l e deals w i t h two separate aspects of P t ( I I ) b i n d i n g t o n u c l e i c bases. The f i r s t s e c t i o n d e s c r i b e s t h e complexes present i n aqueous s o l u t i o n s c o n t a i n i n g c i s (NH3>2Pt(II) over a range of pH i n media of h i g h and low c h l o r i d e i o n content. The second s e c t i o n considers aspects of t h e N ( l ) versus N(7) dichotomy f o r b i n d i n g a t purine n u c l e o s i d e s . To e l u c i d a t e t h e b i n d i n g of P t ( I I ) a t e q u i l i b r i u m , both s e c t i o n s r e l y on r e search performed on analogous P d ( I I ) complexes. S o l u t i o n P r o p e r t i e s of c i s (NH-Q2Pt(II) Complexes Antitumor P t ( I I ) complexes e x i s t i n human blood plasma i n an ambient c h l o r i d e i o n c o n c e n t r a t i o n of 103 mM. A much lower v a l u e of o n l y "4 mM CI"" occurs w i t h i n many c e l l s . E q u i l i b r i u m and r a t e constants have been reported f o r s u b s t i t u t i o n of CI"" by H2O i n c i s (NH3>2PtCl2. The s u c c e s s i v e e q u i l i b r i u m constants f o r formation of aquo and c i s diaquo complexes a r e 3.3 and 0.4mM, r e s p e c t i v e l y ( 5 ) . S i m i l a r values a r e found f o r enPtCl2 ( 6 ) . The above values a r e used h e r e i n as prototypes f o r c i s P t ( I I ) amines. The complex c i s (NH3>2Pt(H20>2^ undergoes two deprotonat i o n s t o g i v e f i r s t t h e aquo-hydroxo, and f i n a l l y t h e dihydroxo complexes. From t h e t i t r a t i o n data f u r n i s h e d by Jensen (7) a t 20°, I c a l c u l a t e from a n o n - l i n e a r l e a s t squares program of pH versus equiv base, ρΚχ = 5.5 and pK2 = 7.1. These values come out s l i g h t l y lower than those c a l c u l a t e d by Jensen. However, the newly c a l c u l a t e d values a r e i d e n t i c a l t o those reported by the Russian s c h o o l f o r s o l u t i o n s a t 25° w i t h 0.1 - 0.3 M NaN0 ( 8 ) . +

3


11.

MARTIN

Equilibria and Binding of cis-PtCh(NHs)B

233

The a c i d i t y constant f o r water deprotonation i n c i s (NH3>2Pt(Cl)(H2O) i s unknown and needs t o be estimated. For t h r e e examples (9) I note that t h e p K v a l u e f o r deprotonation from an aquo-chloro complex i s about t h e average o f t h e succes s i v e ρΚχ and pK2 values from the corresponding diaquo complex. Thus from t h e average o f t h e ρΚχ and pK2 v a l u e s i n the preceding paragraph I estimate f o r c i s (NH3>2Pt(Cl)(H2O) that p K = 6.3. T h i s v a l u e s i s i d e n t i c a l t o that reached i n r e f e r e n c e 9 by a s i m i l a r argument. Combination o f the above e q u i l i b r i u m constant values w i t h the ambient C I " c o n c e n t r a t i o n s y i e l d s the P t ( I I ) complex d i s t r i b u t i o n curves shown i n F i g u r e s 1-3. F i g u r e 1 shows mole f r a c t i o n P t ( I I ) versus pH f o r c i s (NH3>2PtX,Y i n t h e presence o f 103 mM C I " , as occurs i n blood plasma. From low pH t o pH 7.8 t h e c i s (NH3)2PtCl2 complex pre dominates. From 7.8^ 7.3 the dihydroxo complex becomes i n c r e a s i n g l y predominant. F i g u r e 3 shows mole f r a c t i o n P t ( I I ) versus mM c h l o r i d e c o n c e n t r a t i o n a t pH 7,0. The predominant complexes present a r e s t r o n g l y dependent upon c h l o r i d e c o n c e n t r a t i o n a t low concentra t i o n s . At zero c h l o r i d e c o n c e n t r a t i o n t h e aquo-hydroxo and hydroxo-hydroxo complexes account f o r >98% o f t h e P t ( I I ) ; t h e aquo-aquo complex occurs a t l e s s than 2%. A l l three complexes decrease i n amount as C I " i s added. Mole f r a c t i o n s o f t h e chloro-hydroxo and chloro-aquo complexes peak a t 9 mM C l ~ . Above 17mM C I " t h e c h l o r o - c h l o r o complex predominates. T h i s complex a l s o predominates i n c l i n i c a l f o r m u l a t i o n s c o n t a i n i n g 0.15 M NaCl. From the second order r a t e constants f o r s u b s t i t u t i o n by p y r i d i n e i n d i e n P t ( I I ) complexes (4-, 10), I estimate the r e l a t i v e l e a v i n g group a b i l i t i e s o f H2O t o C I " t o be 70 t o 1. Bound hydroxide i o n appears i n e r t and a p p a r e n t l y undergoes s u b s t i t u t i o n o n l y a f t e r conversion t o water i n a r a p i d p r o t o n i c e q u i l i b r i a . D i s t r i b u t i o n curves i n Figures 1-3 r e p r e s e n t i n g complexes w i t h t h e good l e a v i n g group H2O appear as s o l i d curves. Com plexes w i t h o n l y the poor l e a v i n g groups C I " and OH" a r e shown as dashed curves. At 103 mM C I " i n F i g u r e 1 o n l y t r a c e s o f complexes c o n t a i n the good water l e a v i n g group. Under c o n d i t i o n s such as occurs i n plasma, t h e P t ( I I ) complexes remain r e l a t i v e l y i n e r t . At t h e i n t r a c e l l u l a r 4 mM C I " shown i n a


a


234


1.0I

J


1

J—

cr, c r

Ζ

(NH )

g μ-

[CI"]

3

2

^

Pt X,Y

= 103

mM

Ï0.5h

V

y

UJ -I

A

χ cr.oH

ο

-

\ / \ / * / \ • S 7

CI",

OH"/

OH",

H 0

\ \ \ ^

2

~r

6

8

9

10

PH

Figure 1. Mole fraction of Pt(II) vs. pH for cis-(NH ) Pt(H) complexes in presence of 103 mM ambient Ct~. Solid curve represents complex with a water ligand. The mole fractions of the diaquohydroxo and aquohydroxo complexes never exceed 0.015. s

2

PH Figure 2. Mole fraction of Pt(U) vs. pH for cis-(Ν H ) Pt(II) complexes in presence of 3.5 mM ambient Cl~. Solid curves represent complexes with a water ligand. Polymerization of the aquohydroxo complex to give hydroxo-bridged dimers and turners is not considered. 3

2



11.

MARTIN

235

Equilibria and Binding of as~PtCh(NH )2 3

CI" CI"

(NH ) 3

2

Pt X,Y

pH = 7.0

y H f i O . O H - " ^ ^

c r , OH"

OH", OH" CI", H 0 2

20

40

60

80

100

120

140

160

mM CI" Figure 3.

Mole fraction of Pt(II) vs. [Cl~] for cis-(TVH ) Pt(11) complexes at pH 7.0. Solid curves represent complexes with a water ligand. 3 2



236


F i g u r e 2 t h e s o l i d c u r v e s i n d i c a t e a p p r e c i a b l e mole f r a c t i o n s o f c o m p l e x e s w i t h g o o d w a t e r l e a v i n g g r o u p s f r o m pH 4 « - 8 . In n e u t r a l s o l u t i o n s a b o u t 33% o f t h e P t ( I I ) c o m p l e x e s c o n t a i n good w a t e r l e a v i n g g r o u p s a t 4 mM C l ~ w h i l e o n l y a b o u t 3% do s o a t 103 mM Cl~. When e q u i l i b r i u m i s a t t a i n e d , a much g r e a t e r f r a c t i o n o f P t ( I I ) c o m p l e x e s c o n t a i n good w a t e r l e a v i n g g r o u p s w i t h i n c e l l s than i n t h e plasma. C o m b i n a t i o n o f t h e r a t e a n d d i s t r i b u t i o n r e s u l t s a t pH 7 i n d i c a t e s t h a t t h e P t ( I I ) complexes w i t h i n a c e l l a r e about 7 t i m e s more r e a c t i v e t h a n t h o s e i n t h e p l a s m a . Aquo c o m p l e x e s a r e almost t h e o n l y r e a c t i v e s p e c i e s w i t h i n a c e l l and t h e major r e a c t i v e species i n the plasma. Due t o t h e i r g e n e r a l p r e d o m i n a n c e , c i s d i c h l o r o c o m p l e x e s p r o v i d e a b o u t 1/3 o f t h e r e a c t i v i t y i n t h e plasma. Upon t i t r a t i o n o f 10 mM enPd(0H2)2 w i t h s t a n d a r d b a s e a n e n d p o i n t i s r e a c h e d a t pH 7 . 5 a f t e r a d d i t i o n o f o n e e q u i v b a s e . We d i s c o v e r e d , h o w e v e r , t h a t t h e r e v e r s i b l e t i t r a t i o n c u r v e f l a t t e n s o n t h e pH a x i s a n d c a n n o t b e f i t t e d w i t h a n e q u i l i b r i u m e x p r e s s i o n f o r a s i m p l e d e p r o t o n a t i o n ( 1 1 ) . We s u c c e e d e d i n f i t t i n g t h e t i t r a t i o n curve t o a combination deprotonation and dimerization process that y i e l d s a b i n u c l e a r , dihydroxo bridged dimer. F o r t h i s r e a c t i o n t h e o v e r a l l e q u i l i b r i u m c o n s t a n t was f o u n d t o b e Kd = 1 0 · M. H

enPd(OH ) 2

2 + 2

- 8

3

J

2 H 0 3

+

J>>d(en)

+ (en)Pd^

2+

^ 0 ^ H F o r t h e c o r r e s p o n d i n g P t ( I I ) c o m p l e x , enPt(OH2)2 > e n d p o i n t i s r e a c h e d a f t e r a d d i t i o n o f two e q u i v s b a s e . T h e d i m e r i z a t i o n proceeds s l o w l y , and i t i s p o s s i b l e t o r e s o l v e t h e t i t r a t i o n c u r v e o b t a i n e d a t 2 3 ° w i t h 0.2 M KNO3 i n t o two s u c c e s s i v e d e p r o t o n a t i o n s (11) w i t h p % = 5 . 8 f o r f o r m a t i o n o f t h e a q u o - h y d r o x o c o m p l e x a n d pK2 = 7 . 6 f o r f o r m a t i o n o f t h e dihydroxo complex. I f o n l y o n e e q u i v b a s e i s added t o enPt(OH2)2 and t h e s o l u t i o n c o n t a i n i n g predominantly t h e aquohydroxo complex i s a l l o w e d t o s t a n d , no t i t r a b l e groups r e m a i n ( 1 1 ) . Thus d i m e r i z a t i o n d o e s o c c u r , b u t more s l o w l y t h a n w i t h t h e corresponding Pd(II) complex. Presence o f b i n u c l e a r , dihydroxo b r i d g e d P d ( I I ) and P t ( I I ) complexes i n s o l u t i o n r e c e i v e s support from c r y s t a l s t r u c t u r e d e t e r m i n a t i o n s o f c i s diamine P t ( I I ) complexes (12,13). T r i n u c l e a r complexes w i t h t h r e e P t ( I I ) and t h r e e hydroxo b r i d g e s also occur i n c r y s t a l s (14,15). R e d u c i n g t h e c o n c e n t r a t i o n o f enPd(0H2)2 t o 1 mM makes i t p o s s i b l e t o r e s o l v e t h e p r e v i o u s r e a c t i o n i n t o component d e p r o t o n a t i o n and d i m e r i z a t i o n s t e p s . 2+

a

n

2 +


MARTIN

11.

Equilibria and Binding of

as-PtCh(NH k

237

3

H K 2 enPd(H 0)(OH)

+


2

D

2 H 0 +

t

(en)Pd

2

:Pd(en)

2+

H Consistent with the c r y s t a l structure reaction f o r formation o f t r i n u c l e a r ,

3

enPd(H 0)(0H) 2

+

-> «-

3 H 0 + 2

r e s u l t s we a l s o w r i t e a trihydroxo bridged trimer.

trimer

3+

A new n o n - l i n e a r l e a s t s q u a r e s a n a l y s i s o f pH v s . e q u i v b a s e f r o m t h e 1976 t i t r a t i o n d a t a a t 2 3 ° w i t h 0 . 2 M K N 0 (11) y i e l d s p K = 6 . 2 , l o g K = 3.7 ( M " ) , and l o g K = 6 . 5 ( M ~ ) . At t o t a l P d ( I I ) c o n c e n t r a t i o n s g r e a t e r t h a n 0 . 2 mM more P d ( I I ) o c c u r s a s d i h y d r o x o b r i d g e d dimer than a s t h e aquo-hydroxo complex. I t i s p o s s i b l e t o make a n a p p r o x i m a t e c o m p a r i s o n o f t h e t r i m e r i z a t i o n and d i m e r i z a t i o n c o n s t a n t s o f e n P d ( I I ) w i t h t h o s e for c i s (NH3)2Pt(II). From t h e c o n s t a n t s g i v e n above i n t h e e n P d ( I I ) s y s t e m , Κχ = 9 K ' . From t h e p a i r o f p o i n t s f o r d i m e r ( 0 . 6 0 ) a n d t r i m e r ( 0 . 4 0 ) P t ( I I ) m o l e f r a c t i o n s a t t h e end o f F i g u r e 7 i n r e f e r e n c e ( 1 6 ) I c a l c u l a t e t h a t Κχ = 4 Κ ρ / . (If the system i s not a t e q u i l i b r i u m , t h e numerical 4 f a c t o r should be g r e a t e r . ) T h e c o m p a r i s o n s u g g e s t s t h a t t h e two m e t a l i o n systems have comparable t e n d e n c i e s t o form hydroxo b r i d g e d dimers and t r i m e r s . The d i s t r i b u t i o n c u r v e s i n F i g u r e 2 a r e i n c o m p l e t e i n n e u t r a l s o l u t i o n s where t h e a q u o - h y d r o x o complex p o l y m e r i z e s t o y i e l d hydroxo b r i d g e d dimer and t r i m e r . The extent o f t h e d i m e r i z a t i o n depends upon t h e t o t a l P t ( I I ) c o n c e n t r a t i o n , and t h e d i m e r i z a t i o n e q u i l i b r i u m c o n s t a n t i s unknown. If the value f o r enPd(H20) ( 0 H ) o f Krj = 1 0 i s taken as r e p r e s e n t a t i v e , at a l l c o n c e n t r a t i o n s g r e a t e r t h a n 0 . 2 mM more P t ( I I ) o c c u r s a s dimer than a s t h e aquo-hydroxo complex. The d i h y d r o x o b r i d g e d dimer i s expected t o b e v i r t u a l l y i n e r t t o s u b s t i t u t i o n . B e cause i t i s l e s s s t r a i n e d , t h e hydroxo bridged t r i m e r promises t o b e e v e n more i n e r t t h a n t h e d i m e r . Thus t h e r e a c t i v i t y o f P t ( I I ) complexes i n n e u t r a l s o l u t i o n s i n t h e p r e s e n c e o f a low C l ~ b a c k g r o u n d d r o p s m a r k e d l y a t g r e a t e r t h a n 0 . 1 mM t o t a l Pt(II) concentrations. C o n c e n t r a t i o n s g r e a t e r t h a n 0 . 1 mM t o t a l P t ( I I ) a r e u n l i k e l y w i t h i n a c e l l , b u t t h e y a r e common i n many i n v i t r o e x p e r i m e n t s . 3

1

x

2

D

T

3

2

D

3

+

3

,

2

7


238



2+

E f f o r t s t o use c i s (NH3)2Pt(H20>2 and other c i s diaquo complexes t o provide g r e a t e r r e a c t i v i t y than t h e c i s d i c h l o r o complexes may be s e l f - d e f e a t i n g under some circumstances. I n n e u t r a l s o l u t i o n s w i t h a low C l ~ background, s u b s t a n t i a l f r a c t i o n s o f i n e r t hydroxo bridged dimer and t r i m e r form depending upon t h e t o t a l P t ( I I ) c o n c e n t r a t i o n . Dimer and t r i m e r formation occurs v i a t h e aquo-hydroxo monomer which occurs i n maximum conc e n t r a t i o n i n n e u t r a l s o l u t i o n s . The r a t e o f monomer disappearance e x h i b i t s a maximum i n n e u t r a l s o l u t i o n s (16). At c o n c e n t r a t i o n s g r e a t e r than 0.1 mM t o t a l P t ( I I ) , t h e r a t e o f i n e r t dihydroxo bridged dimer formation may exceed that o f water s u b s t i t u t i o n by another l i g a n d . Thus c o n c l u s i o n s based on t h e r a t i o o f r e a c t i v e P t ( I I ) complex t o other l i g a n d s such as n u c l e i c bases may have t o be reconsidered. This c o n c l u s i o n appears e s p e c i a l l y r e l e v a n t t o r e a c t i o n o f supposed c i s diaquo P t ( I I ) complexes w i t h h e l i c a l n u c l e i c a c i d s , where t h e secondary s t r u c t u r e slows f u r t h e r t h e r a t e o f complex formation w i t h t h e ^ l i g a n d . An otherwise i n e x p l i c a b l e r e p o r t that c i s (NH3)2Pt(1^0)2 r e a c t s more s l o w l y a t pH 7 w i t h DNA than does c i s (NH3)2PtCl (17X i s probably due t o formation of i n e r t hydroxo complexes and hydroxo bridged dimers and t r i m e r s . A dimer w i t h a s i n g l e hydroxo b r i d g e forms w i t h dienPd(H20)? . The i r r e g u l a r nature of t h e t i t r a t i o n curve had been noted (11). A recent n o n - l i n e a r l e a s t squares a n a l y s i s r e s o l v e d t h e t i t r a t i o n curve obtained i n 0.5 M KNO3 and 21° i n t o a deprotonation w i t h pK = 7.74 + 0 - 0 1 and a d i m e r i z a t i o n r e a c t i o n (18). We can reformulate t h e d i m e r i z a t i o n r e a c t i o n as 2

+

a

K6 dienPd(H 0) 2

2 +

+ dienPd(0H)

+

t

(dien)Pd-OH-Pd(dien) + H 0 2

and from t h e constants a l r e a d y given i n r e f e r e n c e 18 c a l c u l a t e K$ = 132 M" . At pH 7.74 t h e c o n c e n t r a t i o n s of t h e two r e a c t ants a t t h e l e f t appear i n equal c o n c e n t r a t i o n s , and a t 60 mM P d ( I I ) i s d i v i d e d evenly between t h e two s i d e s o f t h e r e a c t i o n . Higher t o t a l P d ( I I ) c o n c e n t r a t i o n s favor t h e dimer. The analogous compound d i e n P t ( H 2 0 ) exhibits a regular t i t r a t i o n curve w i t h p K = 6.13 a t 25° i n 0.1 M NaC104 (19). The slow d i m e r i z a t i o n r e a c t i o n permits easy e v a l u a t i o n of t h e a c i d i t y constant. The d i m e r i z a t i o n r e a c t i o n i n d i e n P t ( H 2 O ) awaits study. Hydroxo groups on P t ( I I ) complexes might a l s o r e a c t as n u c l e o p h i l e s . Though i t i s 1 0 ^ · times l e s s b a s i c than unbound hydroxide, t h e hydroxide bound t o dienPtOH * i s only 450 times l e s s r e a c t i v e i n promoting h y d r o l y s i s o f p - n i t r o p h e n y l a c e t a t e (13). Metal i o n bound hydroxide o f g r e a t l y decreased b a s i c i t y but only modestly reduced n u c l e o p h i l i c i t y occurs commonly w i t h metal ions and provides a r e l a t i v e l y h i g h c o n c e n t r a t i o n o f potent hydroxide n u c l e o p h i l e s i n n e u t r a l and even a c i d i c s o l u t i o n s (20). 1

2 +

a

2+

2

4


11.

MARTIN

Equilibria and Binding of cis-PtCh(NH )i

239

3

N ( l ) versus N(7) Dichotomy i n P u r i n e Nucleosides (21) When s u b s t i t u t e d a t N(9) as i n n u c l e o s i d e s and d e r i v a t i v e s , the purine bases o f f e r two p o t e n t i a l metal i o n b i n d i n g s i t e s . I n n e u t r a l s o l u t i o n s o f adenosine both N ( l ) and N(7) deprotonate w h i l e w i t h the 6-oxopurines i n o s i n e and guanosine only N(7) deprotonates. N(1)H deprotonations occur w i t h p K values o f 3.6, 8.7, and 9.2 i n adenosine, i n o s i n e , and guanosine, r e s p e c t i v e l y (21). The r e l a t i v e extent o f metal i o n b i n d i n g t o N(7) and N ( l ) i n n e u t r a l s o l u t i o n s o f i n o s i n e and guanosine depends on pH. Despite r e p o r t s on s t u d i e s o f many metal i o n s , the N(7)/N(l) metal i o n b i n d i n g r a t i o s a t e q u i l i b r i u m i n purine nucleosides are known q u a n t i t a t i v e l y f o r only two cases, owing t o an e a r l y study on CH3Hg (22) and a more recent one on d i e n P d ( I I ) (18). The i n t r i n s i c N(l)/N(7) metal i o n b i n d i n g r a t i o s o f C^Hg"*" approach more c l o s e l y those o f the proton than do the b i n d i n g r a t i o s o f d i e n P d ( I I ) , which e x h i b i t a s t r o n g b i a s toward the l e s s b a s i c N(7) (18). This research seeks t o c l a r i f y the i n t r i n s i c N(l)/N(7) b i n d i n g r a t i o s f o r c i s diamine P t ( I I ) . Owing t o the d i f f i c u l t y of a t t a i n i n g and j u d g i n g attainment of e q u i l i b r i u m w i t h P t ( I I ) (1), we performed experiments w i t h P d ( I I ) . The enPd(II) binds a t both N ( l ) and N(7) t o produce extensive polymer formation (23). We f i n d no evidence f o r any N(7)-0(6) c h e l a t i o n o f enPd(II) i n 6-oxopurines. With o n l y a s i n g l e s i t e f o r s u b s t i t u t i o n , d i e n P d H 2 0 avoids the c o m p l e x i t i e s o f f e r e d by e n P d ( H 2 0 ) 2 and provides an i n t r i n s i c measure o f P d ( I I ) b i n d i n g a t i n d i v i d u a l n u c l e i c base s i t e s . Studies w i t h t r i d e n t a t e d i p e p t i d e s i n s t e a d of d i e n c h e l a t e d about P d ( I I ) y i e l d s i m i l a r r e s u l t s (24,25). A comprehensive i n v e s t i g a t i o n o f dienPdH20 + b i n d i n g t o nucleosides and 5-nucleotides has been reported (18); t h i s des c r i p t i o n w i l l h i g h l i g h t o n l y a few p o i n t s . From the separate peaks i n *H NMR s p e c t r a i t i s p o s s i b l e t o f o l l o w the chemical s h i f t s of each k i n d o f d i e n P d ( I I ) complex as a f u n c t i o n o f pH. F i g u r e 4 shows a p l o t o f H(8) chemical s h i f t versus pH f o r s e v e r a l species i n a s o l u t i o n o f 5 -GMP and d i e n P d ( I I ) . Upon p r o t o n a t i o n o f the phosphate group, (designated by Hp) the H(8) chemical s h i f t moves u p f i e l d , r e v e r s i n g the u s u a l d i r e c t i o n . For i n o s i n e and adenosine and t h e i r 5'-nucleotides, the H(2) chemical s h i f t has a l s o been i d e n t i f i e d over a wide pH range (18). Chemical s h i f t data such as t h a t shown i n F i g u r e 4 a l l o w a c a l c u l a t i o n o f a c i d i t y constants f o r deprotonations from f r e e l i g a n d and complexes. ^ Comparison o f areas under i n d i v i d u a l peaks i n the H NMR spectrum of d i e n P d ( I I ) - n u c l e o s i d e and n u c l e o t i d e complexes permits e v a l u a t i o n o f e q u i l i b r i u m s t a b i l i t y constants f o r complex formation. F i g u r e 5 shows the species d i s t r i b u t i o n i n a n e a r l y equimolar s o l u t i o n o f d i e n P d ( I I ) and 5 -GMP and F i g u r e 6 does the same f o r 5 -AMP. The d i f f e r e n c e between the d i s t r i b u t i o n s i n F i g u r e 5 and 6 r e s u l t s l a r g e l y from the d i f f e r e n t absolute


a

+

2+

2+

2

f

T

f




240


11.

MARTIN

Equilibria and Binding of cis-PtCl (NH )2 2

1

I

1

1

241

3

1

ι


BN^Hp

L

BHjHp

0.4

MB

/Â

7

M M BH 7

*Β"Μ7_

zr

P

A / ' Δ ^?

\

π

•

9

MyBM^p

M BMf

,^'ΒΜ,Ηρ

7

1

1

2

π Β

§

3

4

I

I

I

5

6

7

ι

8

9

PH 2

Figure 5. Species distribution of 5'-GMP binding by dienPd * presented as ligand mole fraction (LMF) vs. pH at 0.10 M dienPd * and 0.11 M GMP. Individual points are calculated from H-NMR intensities. Curves are derived from equilibrium constants given in Ref. 18. (Reproduced from Ref. 18. Copyright 1981, American Chemical Society.) 2

1

1.0 ι

1

1

1

1

1

1

Γ

2

Figure 6. Species distribution of 5'-AMP binding by dienPd * presented as ligand mole fraction (LMF) vs. pH with both total Pd(II) and total AMP at 0.10 M . Individual points are calculated from H-NMR intensities. Curves are derived from equilibrium constants given in Ref. 18. (Reproduced from Ref. 18. Copyright 1981, American Chemical Society.) 1


242


b a s i c i t i e s o f N ( l ) ; the two 5'-nucleotides possess a s i m i l a r i n t r i n s i c P d ( I I ) N(7)/N(l) b i n d i n g r a t i o o f -1.4. This r a t i o d i f f e r s r a d i c a l l y from t h a t f o r the proton, which amounts t o 1 0 · f o r 5 -GMP and 10 .7 f o r 5'-AMP (18). Thus the extent o f d i e n P d ( I I ) b i n d i n g t o N(7) o f GMP and guanosine g r e a t l y exceeds what might be expected from N(7) b a s i c i t y . I n n e u t r a l s o l u t i o n s our r e s u l t s show t h a t the d i e n P d ( I I ) complex d i s t r i b u t i o n favors MyBH^ f o r 5 -GMP and BMi f o r i n o s i n e and adenosine w h i l e f o r 5'-IMP and 5'-AMP both N(7) and N ( l ) complexed s p e c i e s occur i n comparable amounts (18). Presence o f the 5'-phosphate group f a v o r s formation o f the N(7) complexes. The s t a b i l i t y order o f decreasing d i e n P d ( I I ) b i n d i n g strengths a t the common 5'-nucleotides a t pH 7 i s G7>I7>I1>G1>U3>T3>C3>A1>A7 (18). Thus o f the common n u c l e o s i d e s , N(7) o f a guanine base provides the strongest d i e n P d ( I I ) b i n d i n g s i t e i n n e u t r a l solutions. How do the i n t r i n s i c N(l)/N(7) b i n d i n g r a t i o s f o r d i e n P t ( I I ) compare w i t h those now known f o r d i e n P d ( I I ) ? Because i t i s d i f f i c u l t t o achieve e q u i l i b r i u m w i t h P t ( I I ) complexes o f t h e N ( l ) protonated 6-oxopurines, we approached e q u i l i b r i u m w i t h an equimolar s o l u t i o n o f d i e n P t H 2 0 and 5'-AMP a t pH 5.1 (26). The s o l u t i o n sat f o r two months, during which time i t was heated a t 50° f o r 13 days. L e v e l i n g o f f i n t h e slow growth o f the ! H NMR peaks due t o the ΒΜχ complex suggests t h a t e q u i l i b r i u m may have been obtained. The l a s t r a t i o of M7BHP t o BMiHp complexes i s 1.9 (26). This v a l u e i s 4.4 times g r e a t e r than t h a t found f o r d i e n P d ( I I ) t o AMP (18). I f we g e n e r a l i z e the r e s u l t f o r AMP t o the other n u c l e i c bases, we conclude t h a t P t ( I I ) favors N(7) over N ( l ) b i n d i n g t o an even g r e a t e r extent than P d ( I I ) . Thus the N(7) b i n d i n g s i t e o f guanosine a l r e a d y atop the s t a b i l i t y order f o r P d ( I I ) becomes r e l a t i v e l y s t r o n g e r f o r P t ( I I ) compared to s i t e s on the other bases. We conclude t h a t the predominant P t ( I I ) b i n d i n g s i t e i n n u c l e o s i d e s and n u c l e o t i d e s i s N(7) o f guanosine. During a study o f the H(8) promoted exchange by P t ( I I ) bound t o N(7) i n p u r i n e s , we noted a t pH 5-6 a m i g r a t i o n o f d i e n P t ( I I ) from i n o s i n e N(7) t o N ( l ) (27). F i r s t d i e n P t H 0 r e a c t s a t N(7). 4

6

f

2


f

2+

+

2

Μ + ΒΗχ + MyBH-L The metal i o n a t N(7) promotes the a c i d i t y o f the proton a t N ( l ) by up t o 2 l o g u n i t s (18). M BH! + 7

H

+

+

M B~ 7

The g r e a t e r f r a c t i o n o f N ( l ) deprotonated s p e c i e s w i t h M7BH1 than w i t h ΒΗ^ provides a f a c i l e pathway f o r metal i o n c o o r d i n a t i o n a t N ( l ) a t lower pH than occurs w i t h unbound l i g a n d


MARTIN

11.

243

Equilibria and Binding of c\s-PtCU(NHn)z

M

+

M B~

Μ ΒΜ

7

?

1

2+

Our experiments w i t h equimolar i n o s i n e and d i e n P t H 2 0 produced a complete r e a c t i o n t o g i v e M7BH1, f o l l o w e d by appearance o f t h e b i n u c l e a r complex M7BM1 and reappearance o f f r e e l i g a n d ΒΗχ. F i n a l l y the metal i o n bound a t N(7) may be r e l e a s e d . M BM Downloaded by KTH ROYAL INST OF TECHNOLOGY on February 24, 2016 | http://pubs.acs.org Publication Date: January 26, 1983 | doi: 10.1021/bk-1983-0209.ch011

7

+ Μ

1

+ ΒΜχ

Even w i t h d i e n P d ( I I ) and i n o s i n e the o v e r a l l t r a n s f e r o f m e t a l i o n from N(7) t o N(.l) i s slow (18). With N ( l ) protonated n u c l e i c bases, metal i o n c o o r d i n a t i o n at N ( l ) occurs v i a the b i n u c l e a r M7BM1 complex because o f t h e g r e a t e r a c i d i t y o f M7BH1 compared t o ΒΗχ, For a l l f i v e l i g a n d s s t u d i e d w i t h d i e n P d ( I I ) , the greater f r a c t i o n o f N(.l) depro tonated species when the l i g a n d i s metalated a t N(7) more than o f f s e t s the decrease i n s t a b i l i t y f o r metal i o n b i n d i n g a t N ( l ) i n M7B compared t o B, ( 7 l / a 7 B l / a B * r e f e r e n c e 18). The above mechanism provides the main pathway f o r metal i o n c o o r d i n a t i o n a t N ( l ) i n a c i d i c and n e u t r a l s o l u t i o n s o f i n o s i n e , guanosine, and d e r i v a t i v e s c o n t a i n i n g comparable amounts o f metal i o n . P t ( I I ) does d i s p l a c e the b a s i c N(3) proton from u r i d i n e (pK = 9.2); the s t a b i l i t y constant f o r d i e n P t ( I I ) b i n d i n g t o u r i d i n e has been estimated as a t l e a s t 10 times greater than t h a t f o r d i e n P d ( I I ) (28). For d i e n P d ( I I ) and u r i d i n e i n H 0 the s t a b i l i t y constant i s l o g Κ = 8,1 (29), Since d i e n P t ( I I ) y i e l d s a g r e a t e r adenosine N(7)/N(l) s t a b i l i t y constant r a t i o than d i e n P d ( I I ) , the s t a b i l i t y constant f o r P t ( I I ) a t N(7) o f purines should be about 50-100 times greater than t h a t f o r P d ( I I ) , D i s placement o f the N ( l ) proton o f guanosine and i n o s i n e and d e r i v a t i v e s by P t ( I I ) i s d i v e r t e d by p r i o r c o o r d i n a t i o n and k i n e t i c f i x a t i o n at N(7), Of the s e v e r a l common n u c l e o s i d e b i n d i n g s i t e s , both e q u i l i b r i u m and k i n e t i c f a c t o r s favor P t ( I I ) b i n d i n g a t guanosine N(7), Reactions o f P d ( I I ) compounds w i t h n u c l e i c bases serve as a thermodynamic r e f e r e n c e f o r r e a c t i o n s o f analogous P t ( I I ) compounds a t e q u i l i b r i u m . Large d i f f e r e n c e s between r e a c t i o n products o f analogous P d ( I I ) and P t ( I l ) com pounds may be a t t r i b u t e d t o i n t r o d u c t i o n o f k i n e t i c e f f e c t s i n the l a t t e r . K

K

V

S

e

K

K

n

a

2

Acknowledgments This research was supported by a research grant from the N a t i o n a l Science Foundation.


244


Literature Cited


1. 2.

Vestues, P.; Martin, R.B. J. Am. Chem. Soc. 1981, 103, 806. Howe-Grant, M.E.; Lippard, S.J. Metal Ions Biol. Syst. 1980, 11, 63. 3. Shannon, R.D. Acta Crystallogr. 1976, A32, 751. 4. Basoĺo, F.; Gray, H.B.; Pearson, R.G. J. Am. Chem. Soc. 1960, 82, 4200. 5. Reishus, J.W.; Martin, Jr., D.S. J. Am. Chem. Soc. 1961, 83, 2457. 6. Coley, R.F.; Martin, Jr., D.S. Inorg. Chim. Acta 1973, 7, 573· 7. Jensen, K.A. Z. Anorg. Allg. Chem. 1939, 242, 87. 8. Grinberg, Α.Α.; Stetsenko, A . I . ; Mitkinova, N.D.; Tikhonova, L.S. Russ. J. Inorg. Chem. 1971, 16, 137. 9. LeRoy, A.F. Cancer Treat. Rep. 1979, 63, 231. 10. Gray, H.B.; Olcott, R.J. Inorg. Chem. 1962, 1, 481. 11. Lim, M.C.; Martin, R.B. J. Inorg. Nucl. Chem. 1976, 38, 1911. 12. Faggiani, R.; Lippert, B.; Lock, C.J.L.; Rosenberg, B. J. Am. Chem. Soc. 1977, 99, 777. 13. Lippert, B.; Lock, C.J.L., Rosenberg, B.; Zvagulis, M. Inorg. Chem. 1978, 17, 2971. 14. Faggiani, R.; Lippert, B.; Lock, C.J.L.; Rosenberg, B. Inorg. Chem. 1977, 16, 1192. 15. Faggiani, R.; Lippert, B.; Lock, C.J.L.; Rosenberg, B. Inorg. Chem. 1978, 17, 1941. 16. Rosenberg, B. Biochimie 1978, 60, 859. 17. Mansy, .S.; Chu, G.Y.H.; Duncan, R.E.; Tobias, R.S. J. Am. Chem. Soc. 1978, 100, 607. 18. Scheller, K.; Scheller-Krattiger, V.; Martin, R.B. J. Am. Chem. Soc. 1981, 103, 6833. 19. Alcock, R.M.; Hartley, F.R.; Rogers, D.E. J. Chem. Soc. Dalton 1973, 1070. 20. Martin, R.B. J. Inorg. Nucl. Chem. 1976, 38, 511. 21. Martin, R.B.; Mariam, Y.H. Metal Ions Biol. Syst. 1979, 8, 57. 22. Simpson, R.B. J. Am. Chem. Soc. 1964, 86, 2059. 23. Sovago, I.; Martin, R.B. Inorg. Chem. 1980, 19, 2868. 24. Vestues, P.I.; Martin, R.B. Inorg. Chim. Acta 1981, 55, 99. 25. Scheller-Krattiger, V.; Scheller, K.H.; Martin, R.B. Inorg. Chim. Acta 1982, 59, 281. 26. Kim, S-H.; Martin, R.B. 1981, unpublished results. 27. Noszal, B.; Scheller-Krattiger, V.; Martin, R.B. J. Am. Chem. Soc. 1982, 104, 1078. 28. Lim, M.C.; Martin, R.B. J. Inorg. Nucl. Chem. 1976, 38, 1915. 29. Lim, M.C. J. Inorg. Nucl. Chem. 1981, 43, 221. RECEIVED October

4, 1982


Hydrolytic Equilibria and N7 Versus N1 Binding in Purine Nucleosides

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