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398 - Electrochemical behavior of mono- and oligonucleotides. VIII. Voltammetric studies on the interfacial behaviour of mono-, di- and triphosphate of cytidine
Bioelectroahemistry and Bioenergetic$ 7(~98o) 705--7°-2 J . Electroanal. Ckem. 116 (I98O) 7o5-7"2 Elsevier Sequoia S.A., Lausatme -- Printed in I t a l y
3 9 8 - Eleetrochemival Behavior of Mono- a n d OHgonucleotides. V I I L Voltammetric Studies on the Inter[aeial B e h a v i o u r of Mono-, Di- a n d Triphosphate of Cytidine * b y Y. M. TEMERK°, P- VALE NTA° a n d H . ~ ' . Nf3RNBERG° o Chemistry Department, F a c u l t y of Science, Assiut University, Assiut, E g y p t " I n s t i t u t e of Chemistry, Institute 4 Applied Physical Chemistry, Nuclear Research Center {KFA), D-StTO Jfilich, Federal Republic of Germatty ~Ianuscript received March 29th I980
Summary
A systematic study of the adsorption and interfacial behavior of the cytidine mononucleotides (5'-CMP, 3'-CMP, 3',5'-CMP, 5'-CDP and 5'-CTP) has been carried out. The adsorption was followed at the H M D E b y a . c . - v o l t a m m e t r y a n d single sweep v o l t a m m e t r y . Over a w i d e p o t e n t i a l r a n g e o n e h a s a dilute a d s o r p t i o n l a y e r w h i l e w i t h i n t h e potential range of m a x i m u m adsorption a t elevated bulk concentration f o r m a t i o n o f a compact film is o b s e r v e d . T h e a d s o r p t i o n c a n b e d e s c r i b e d q u a n t i t a t i v e l y for b o t h t y p e s of a d s o r p t i o n layers b y single a n d double s t e p FRU~tKIX i s o t h e r m s , - r e s p e c t i v e l y - T h e r e s u l t i n g a d s o r p t i o n p a r a m eters are evaluated and the conclusions on the respective interfacial behavior, orientations a n d interactions of these substances are discussed.
Introduction T h e investigation of the s t r u c t u r e a n d properties of nucleic acids is o f g r e a t b i o l o g i c a l i m p o r t a n c e f o r a b e t t e r u n d e r s t a n d i n g o f t h e m e c h a n i s m s b y w h i c h t h e g e n e t i c i n f o r m a t i o n is s t o r e d , d u p l i c a t e d a n d t r a n s m i t t e d i n l i v i n g cells. E x t e n s i v e r e c e n t s t u d i e s h a v e s h o w n t h a t n a t i v e DNA and other polynucleotides undergo potential dependent conformat i o n a l c h a n g e s w h e n a d s o r b e d a t a m e r c u r y elect_rode s u r f a c e [ I - 4 ] . A proper u n d e r s t a n d i n g of the interracial behaviour of polynucleotides a t b o t h , a n e l e c t r o d e s u r f a c e a n d a c h a r g e d biological i n t e r f a c e , relies o n a g e n e r a l k n o w l e d g e o f t h e i n t e r f a c i a l b e h a v i o u r of t h e c o n s t i t u e n t * Presented a t the 5th International Symposium on Bioelectrochemistry, 3--8 September I979, Weimar (D.D.R.).
0302-4598]80[0705-0722 ~ i98o Elsevier Sequoia S.A.
706
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e l e m e n t s of t h o s e m a c r o m o l e c u l e s , i.e. t h e i r c o n s t i t u e n t p u r i n e a n d p y r i m i d i n e bases, a n d t h e c o r r e s p o n d i n g nucleosides a n d nucleotides. I t w a s s h o w n t h a t bases, n u c l e o s i d e s a n d n u d e o t i d e s a r e a d s o r b e d a n d u n d e r g o a n association a t t h e c h a r g e d m e r c u r y - s o l u t i o n i n t e r f a c e [5-x9]. I n t h i s c o n t e x t it w a s s h o w n t h a t a d s o r b e d nucleosides a n d n u d e o t i d e s i n t e r a c t p r i n c i p a l l y z,/ar verti~-~al s t a c k i n g o f t h e bases. T h e y f o r m a t b u l k c o n c e n t r a t i o n s a b o v e a t r e s h o l d v a l u e a compact film. S u f ficient e v i d e n c e h a s n o w b e e n a c c u m u l a t e d t o s h o w t h a t t h e b a s e s t a c k i n g i n t e r a c t i o n s of a d j a c e n t h e t e r o c y d i c rings a l o n g t h e p o l y n u d e o t i d e s chain m a k e a significant c o n t r i b u t i o n t o t h e s t a b i l i t y a n d confo~mat i o n a l p r o p e r t i e s of nucleic acids, a s h a s b e e n also ~ s s u m e d b y o t h e r a u t h o r s [2o-23]. I t is t h e r e f o r e of i n t e r e s t t o s t u d y t h e a d s o r p t i o n a n d association in t h e a d s o r b e d s t a t e o f m o n o - , d i - a n d t r i p h o s p h a t e s o f c y t i d i n e a t t h e i n t e r f a c e m e r c u r Y l a q u e o u s e l e c t r o l y t e a n d t h e effect of t h e s t e r i c a l a r r a n g e m e n t a n d influence o f t h e p h o s p h a t e g r o u p .
Experimental Chemicals a n d solutions Cytidine-3'--monophosphate (3'-CMP), c y t i d i n e - 5 ' - m o n o p h o s p h a t e ( 5 ' - C M p ) , c y t i d i n e - 5 " , 5 ' - - m o n o p h o s p h a t e (cyclic CMP), cytidine-5"-d i p h o s p h a t e (5"-CDP), a n d c y t i d i n e - 5 " - t r i p h o s p h a t e (5"--CTP) w e r e o b t a i n e d f r o m SERVA (Heidelberg, F . R . G . ) . I n all m e a s u r e m e n t s t h e s u p p o r t i n g e l e c t r o l y t e w a s a MCILvAIN~ b u f f e r b r o u g h t t o c o n s t a n t ionic ~ h e n g t h of o-5 b y a d d i t i o n of KC1. All c h e m i c a l s w e r e r e a g e n t g r a d e , KCI w a s SUl~rapur, E . MERCK ( D a r m s t a d t ) . T h e c o n c e n t r a t i o n of t h e m o n o n u c l e o t i d e s in t h e s a m p l e s o l u t i o n s w a s d e t e r m i n e d w i t h t h e UNICAM S P 8oo s p e c t r o p h o t o m e t e r , p H w a s m e a s u r e d w i t h a n ORION p H m e t e r , M o d e l 7oz. A p p a r a t u s a n d methods All v o l t a m m e t r i c m e a s u r e m e n t s w e r e c a r r i e d o u t w i t h t h e m u l t i m o d e p o l a r o g r a p h i c a n a l y z e r P A R z7o in a t h e r m o s t a t e d METROHM ceil e q u i p p e d x~4th a 3 - e l e c t r o d e s y s t e m u n d e r p o t e n t i o s t a t i e control. P h a s e scns~.'tive a.c.-voltammograms c o r r e s p o n d i n g t o t h e o u t - o f - p h a s e c o m p o n e n t o f t h e t o t a l a.c. c u r r e n t (9 oo p h a s e angle) w e r e r e c o r d e d in a p s e u d o s t a t i o n a r y m ~ n n e r w i t h a s c a n r a t e o f t h e d . c . - r a m p o f 2 m V s -1. T h e a m p l i t u d e o f t h e a.c_ v o l t a g e w a s 5 mVpp a n d t h e f r e q u e n c y 33o H z , unless othexwise s t a t e d . T h e d e p e n d e n c e o f t h e o u t - o f - p h a s e a.c. c u r r e n t o n t h e a d s o r p t i o n t i m e ts w a s o b t a i n e d b y a d j u s t i n g t h e a d s o r p t i o n p o t e n t i a l , i.e. t h e m e a n e l e c t r o d e p o t e n t i a l , t o a p r e d e t e x m i n e d v a l u e , e x t r u d i n g t h e m e r c u r y d r o p a n d r e c o r d i n g a f t e r t h e r e s p e c t i v e :ts p e r i o d h a d e l a p s e d t h e o u t - o f - p h a s e a.c. c u r r e n t w i t h a s u i t a b l e t i m e base_
Electrochemical Behavior of Cytidine Phosphates
707
I n t h e single sweep mode, as well, t h e c u r r e n t - p o t e n t i a l c u r v e s and the corresponding charge-potential curves obtained were recorded with the built-in electronic integration circuit. For the evaluation of r e s p o n s e s a t s w e e p r a t e s a b o v e 2oo m V s -~ a d i g i t a l s t o r a g e oscilloscope, NICOLET, t y p e XO90, w a s a p p l i e d . T h e w o r "king e l e c t r o d e w a s a METROHM hanging mercury drop electrode (HMDE), type E4Io, with a surface a r e a o f L 7 5 X xo -z cmz. As r e f e r e n c e e l e c t r o d e s e r v e d a s a t u r a t e d c a l o m e l e l e c t r o d e f r o m I~GOLD, t y p e 3 o 3 - N S , a n d t h e a u x i l i a r y e l e c t r o d e w a s a c o i l e d p l a t i n u m wire_ T h e s o l u t i o n w a s d e a e r a t e d w i t h p u r e n i t r o g e n . T o avoid interference b y t h e c a t a l y t i c h y d r o g e n evolution, all measurem e n t s h a v e b e e n c a r r i e d o u t a t 5 °C w h e r e t h e c a t a l y t i c h y d r o g e n r e s p o n s e is s h i f t e d t o s u f f i c i e n t l y n e g a t i v e e l e c t r o d e p o t e n t i a l s . Results
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Phase sensitive a.c. v o l t a m m o g r a m s c o r r e s p o n d i n g t o t h e o u t - o £ phase c o m p o n e n t of t h e t o t a l a.c. response p r o v i d e an o v e r a l l p a t t e r n of the adsorption behaviour of the studied mono-, di- and triphosphates o f c y t i d i n e (Figs. ~-5)- T h e recorded c a p a c i t i v e a.c. c o m p o n e n t is p r o -
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Fig. z. a.c. voltammetric curve of cytidiue-3"-monophosphate ( 3 " - C ~ [ P ) a t t h e I-EtIDI~. o . 5 A I ~ C I L V A I N E b u f f e r , p H 3.°-4, 5 °C, a r e a o f H ~ I D E L 7 5 × I o -ffi c m ~. s c a n r a t e ~ m V s - : , f r e q u e n c y 3 3 o H z . a m p l i t u d e 5 m V p p . p h a s e a n g l e 9 o ° . C u r v e (1) o . o . (22 6, 9 x I o - S ; (3) = . i × x o -4 ; (4) S . S × z o - " : (5) L 5 × x o -~ ; (6) 5 × I o -~ ; (7) I . o × i o - ' - ~'r 3 - C . . ~ P .
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p o r t i o n a l t o t h e differential d o u b l e l a y e r c a p a c i t a n c e in p o t e n t i a l regions w h e r e no f a r a d a i c p r o c e s s o c c u r s [z4]- T h e p H o f t h e solution w a s a d j u s t e d t o s u c h a ~-alue ( p H 3.z4) t h a t t h e p r o t o n a t e d f o L ~ of c y t i d i n e p h o s p h a t e prevailed. A t l o w b u l k c o n c e n t r a t i o n s o n e o b s e r v e s a r o u n d t h e p o t e n t i a l of m a x i m u m a d s o r p t i o n (-o-65 V) a progressive d e c r e a s e of t h e c a p a c i t i v e a.c. s~gnal d u e t o increasing a d s o r p t i o n of m o n o - , d i a n d t r i p h o s p h a t e s of c y t i d i n e . T h i s d e c r e a s e c o r r e s p o n d s t o t h e p r o g r e s s i v e c o v e r a g e o f t h e e l e c t r o d e s u r f a c e b y t h e di&de a d s o r p t i o n layer.
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F i g . ~-. a.c.--voltammetric curve of cytidine-5*--monophosphate ( 5 " - - C M P ) a t t h e H~.~IDE_ Curve ( : ) o . o ; ( z ) 9 - 9 X Xo - ~ ; ( 3 ) 5 - 9 X x o - t ; ( 4 ) x - 7 x : o - a ; ( 5 ) 3 - I × x o -~ ; ( 6 ) z - 3 X : o - ' - ; ( 7 ) z - 5 × × XO-'- "1I 5 " - - C M P . o t h e r c o n d i t i o n s as in Fig. L
At more elevated bulk concentrations above the threshold value f o r t h e r e s p e c t i v e m o n o n u c l e o t i d e a p i t is o b s e r v e d . T h e p i t o c c u r s f o r 3 ' - C M P a t a significantly a n d for 5 ' - C M P a t a s o m e w h a t l o w e r t h r e s h o l d v a l u e t h a n f o r C T P . As h a s b e e n s h o w n b y VETTERL [5, 6, 9, X2] a n d K R z . ~ . ~ et ag. [xo, ~x, x4] t h e p i t reflects t h e f o r m a t i o n of a compact a d s o r p t i o n film d u e t o p r o n o u n c e d l a t e r a l i n t e r a c t i o n s of a d s o r b e d n u c l e o t i d e species in a c e r t a i n p o t e n t i a l r a n g e . A f t e r t h e p i t a r o u n d t h e p o t e n t i a l - o . 9 5 t o - L 2 5 V a n e x t e n d e d n o n - f a r a d a i c p e a k is o b s e r v e d c a u s e d b y t h e r e o r i e n t a t i o n o f t h e a d s o r b e d n u c l e o t i d e species. T h e
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F i g . 3a.c.-x-oltammetric curve of cytidine 3",5"-phospha.te (cyclic 3".5"-C:MP) at the HMDH. C u r x - e (z) o . o : (2) L S X x o - s : (3) 3 - 9 8 X I o - s : (4) 9 - 9 9 × x o - s ; (5) L 6 S × I o -a ; (6) 2 . 6 6 X z o -a : (7) 5 - 5 2 x x o - a : (S) 2 - x × x o - a : (9) 1-34Xio-±;'(x o) 2 . o × I o - ' - ; ( x I ) 3 _ 7 × I O - ' - ; (z2) 4.48 × zo -a 3I cyclic 3",5"-C~IP, other conditions as in Fig. L
analysis of t h e p e a k c u r r e n t in t e r m s of t h e phase a n g l e gives a v a l u e of 9 oo. Moreover, t h e p e a k c u r r e n t varies l i n e a r l y w i t h t h e f r e q u e n c y of t h e s u p e r i m p o s e d a.c. voltage. T h e s e c h a r a c t e r i s t i c s c o r r e s p o n d to a n o n - f a r a d a i c n a t u r e of t h e process c a u s i n g this peak. This a.c. p e a k o b s e r v e d also w i t h a d e n i n e m o n o n u e l e o t i d e s [1o, I x , / 4 ] , d e n a t u r e d [ 2 5 ] a n d n a t i v e DlqA [zT], a n d r e l a t e d b i o s y n t h e t i c p o l y n u c l e o t i d e s [26] is d u e to a s t r u c t u r a l r e a r r a n g e m e n t of t h e a d s o r b e d species. As will be discussed l a t e r b a s e d on t h e results g a i n e d w i t h s i n g l e sweep v o l t a m m e t r y t h e a d s o r p t i o n of all c y t i d i n e d e r i v a t e s in t h e d i l u t e s t a g e t a k e s place v i a t h e m o s t h y d r o p h o b i c m o i e t y of t h e molecule, i.e. t h e c y t o s i n e base. T h e ~ - e l e c t r o n i n t e r a c t i o n ~,ith t h e e l e c t r o d e xs411 f a v o u r for t h e r a n g e of positive c h a r g e o r zero c h a r g e a flat a d s o r p t i o n of t h e c y t o s i n e m o i e t y . I n t h e c o m p a c t s t a g e of t h e a d s o r p t i o n t h e c y t o s i n e a n d p h o s p h a t e moieties i n t e r a c t w i t h t h e e l e c t r o d e d e p e n d i n g on t h e i r c h a r g e a n d o n t h e sterical a r r a n g e m e n t of t h e molecule. F o r c y t i d i n e 3 ' - m o n o p h o s p h a t e (3'-CMP) t h e p h o s p h a t e g r o u p i n 3"-CMP r e m a i n s d i s t a n t f r o m t h e p r o t o n a t e d N(3) of t h e c y t o s i n e m o i e t y a n d its influence: o n t h e adsorption, o c c u r i n g h e r e p r e d o m i n a n t l y v i a t h e base, r e m a i n s u s u a l l y small, e x c e p t f o r r a t h e r positive potentials. T h u s , for 3--CMP (viz. Fig. x) t h e n e g a t i v e a s c e n d i n g p a r t of t h e dip e x t e n d s
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Fig. 4¢c.-voltammetr/¢ curve of cytidine--5"--diphosphat¢ (5"--CDP) at the HYDE. Curve (l) o . o : 2 - 9 × x o - s ; ( 3 ) 3 - 9 × I O - s ; ( 4 ) 5 - 9 × x o - s ; ( 5 ) x . 2 × x o - a ; ( 6 ) x - 7 × x o -~ ; ( 7 ) 4 - 5 × z o - 4 ; (8) 8 . 8 X 1 0 =4 : (9) I-9 X I O -= : (IO) 3 . 8 X I O -s ; (II) 8 . 2 X I O -s : (12} 2 . 8 X IO-'- ; (I3) 3 . 8 X I O -:E ; {Z4) 6 . Z X IO--" ; (XS) I-X X t O =I "~Ir 5 " - C D P . other conditions a s in F i g . I.
t o p o t e n t / a l ~ b e y o n d --o.8 V. "ALso f o r 3 " - C M P t h e d i p e x t e n d s t o p o t e n tialg b e y o n d - o . 8 V_ a n d i t e x t e n d s o n t h e p o s i t i v e s i d e t o p o t e n t i a l s o f l e s s t h a n --o_3 V_ Th;~ is t o b e i n t e r p r e t e d a s a s i g n i f i c a n t c o n t r i bution to the adsorption by the negatively charged phosphate group. As model considerations reveal, the latter, under the electrostatic a t t r a c t i o n of t h e e l e c t r o d e , c a n b e t u r n e d i n t o a p o s i t i o n w h e r e t h e phosphate group comes into direct contact with the surface, thus cont r i b u t i n g t o t h e a n c h o r i n g o f t h e species. H o w e v e r , w i t h t h e p o s i t i v e l y c h a r g e d p r o t o n a t e d N(3), t h e b a s e is s u b j e c t e d t o r e p e l l i n g t e n d e n cies, a l t h o u g h o n e h a s t o b e a r i n m i n d t h a t p r o n o u n c e d t e n d e n c i e s f o r ~ - e l e c t r o n i n t e r a c t i o n xs~ll c o u n t e r a c t , t o s o m e e x t e n t , t o t h i s e f f e c t o n t h e p r o t o n a t e d b a s e . T h e p e a k a t - o . 5 V c o r r e s p o n d s t o a specific r e o r i e n tation to a position where the base comes into strong interaction with the surface, while the adsorption of the hydrophilic phosphate group attains a solution-sided position. The occurrence of this reorientation p e a k is t o b e e x p e c t e d as_ a t p H 3 . z 4 t h e n e t c h a r g e o f t h e 3 ' - C M P x.x-z~,~tterion is a p p r o x i m a t e l y b a l a n c e d . The considerable change in the dielectric properties of the interface ( a l t e r a t i o n o f d i r e c t i o n o f t h e d i p o l e , c h a n g e s i n h y d r a t i o n ) g i v e s rise t o a significant alteration in double layer capacitance which manifests
Electrochemical B e h a v i o r of Cytidine P h o s p h a t e s
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itself in the observed non-faradaic peak. From the fact that the reorienr a t i o n is o b s e r v a b l e f o r a f r e q u e n c y o f 330 H z , o n e c o n c l u d e s t h a t i t s r a t e is f a s t . ~ F u r t h e r t h e r a t h e r e x t e n d e d size o f t h e p e a k i n d i c a t e s t h a t the reorientation does not occur for each individual adsorbed monon u c l e o t i d e z w i t t e r i o n a t t h e s a m e p o t e n t i a l , b u t is d i s t r i b u t e d a r o u n d t h e
peak potential (-o.5 V). The negative phosphate group is in 5'-CMP (Fig. z) situated closer to t he p r o t o n a t e d N(3) of t h e cytosine unit a n d compensates partially its positive charge. At potentials more positive t h a n t h e potential of m a x i m u m adsorption (-o.65 V). one can distinguish th e following influences o n a d s o r p t i o n . T h e = - e l e c t r o n i n t e r a c t i o n w i t h t h e p o s i t i v e charge of the electrode would favour a rather flat adsorption of the cytosine moiety. However, the repulsion of the protonated N(3 ) acts antagonistically. This influence, in principle unfavourable for adsorption, may, however, be moderated to a certain extent for 5'-phosphate, where the negative phosphate group attains a position proximate to the prot o n a t e d N ( 3 ). T h u s , t h e r e is a r a t h e r g o o d p r o b a b i l i t y t h a t t h e a r r a n g e m e n t d e s c r i b e d will p r e v a i l f o r 5 ' - C M P a t p o t e n t i a l s m o d e r a t e l y m o r e positive than the potential of maximum adsorption U,. Due to this
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a.c.-voltammetric curve of cTtidine-5"-triphosphate (5"--CTP) at the I-L~[DE. Curve (i) o.o; (-~) x.99xxo -~; (3) 5-96xx° -s; (4) 9-9xxo -5; (5) t_2Sxxo - i ; (6) 2.~-4xIO-a; (7) 6-9xxo-a ; (8) 9 . 4 x I o - ~ ; (9) I-xSxIo-S: (io) 1.4~-xto-a: (ix) 1.67xio-:I; (I2) 2 . I X X IO -3 ; (13) 3-][ X IO "-3 ; ( I 4 ) 4 . 0 X IO -• ; ( I 5 ) 5 . 8 X IO -:$ ; (][6) S. 4 X I O -3 ; (17) I . I 6 X IO-'- ; ( I S ) 1 . 6 X X O - ' - ; (xg) 4 - 5 6 X X O - - ' ; (20) S . 7 6 X I o - ~ - : (21) 9 . 5 X I O ÷ ~ ; ( 2 2 ) i . o 3 x I o - 1 ; (~-3) L I 4 X I o - I z~I 5 " - C T P . o t h e r c o n d i t i o n s a s i n F i g . L
7z2
Temerk.
Valenta and Niirnberg
c h a r a c t e r i s t i c sterical a r r a n g e m e n t of 5"--CMP t h e r e o r i e n t a t i o n t o a n o t h e r p o s i t i o n a t m o r e p o s i t i v e p o t e n t i a l s , ~where t h e m o l e c u l e is a d s o r b e d via t h e p h o s p h a t e group, will n o t o c c u r in c o n t r a s t t o 3"-CMP. A r o u n d U , t h e n t h e = - e l e c t r o n i n t e r a c t i o n x,~ll b e m a i n l y r e s p o n s i b l e f o r t h e a d s o r p t i o n . A t m o r e n e g a t i v e p o t e n t i a l s t h e progressive r e p u l s i o n of t h e p h o s p h a t e g r o u p f r o m t h e i n t e r f a c e will c a u s e i t s p r o g r e s s i v e l y m o r e s o l u t i o n - s i d e d position. T h e n t h e c o m p e n s a t i n g influence o n t h e p o s i t i v e c h a r g e of t h e p r o t o n a t e d N(3) declines in p r o p o r t i o n t o t h e d i s t a n c e a t which the phosphate group becomes located. At the same time the positive c h a r g e a t t h e p r o t o n a t e d N(3 ) c a n d i s p l a y i t s influence g r a d u a l l y compensating more and more at not too elevated negative potentials t h e c o n c o m i t a n t repulsion b y t h e e l e c t r o d e of t h e = - e l e c t r o n s y s t e m o f c y t i d i n e . Y e t a t a n y r a t e , t h e p h o s p h a t e in 5 ' - p o s i t i o n s h o u l d a l w a y s p r e f e r a s o m e w h a t closer p o s i t i o n t o t h e c y t o s i n e m o i e t y t h a n in 3 " C M P . F o r cyclic 5',5"--CMP (Fig. 3), t h e a d s o r p t i o n r a n g e e x t e n d s t o more negative potentials than for a n y other cytidine phosphate. This i n d i c a t e s t h a t t h e r e is v i r t u a l l y n o c o u n t e r b a l a n c e b y t h e n e g a t i v e p h o s p h a t e t o t h e p o s i t i v e c h a r g e a t t h e p r o t o n a t e d N(3) of t h e a d s o r b e d c y t o s i n e moiety-_ I t m e a n s t h a t t h e n e g a t i v e l y c h a r g e d p h o s p h a t e g r o u p m u s t p r e f e r a m o s t d i s t a n t position f r o m t h e c y t o s i n e m o i e t y . Consid e r a t i o n s of m o l e c u l a r m o d e l s r e v e a l t h a t s u c h a n a [ l a n g e m e n t is e a s i l y possible if t h e h y d r o p h i l i c s u g a r a n d t h e 3 ' - 5 ' - e s t e r i f i e d p h o s p h a t e a t t a i n a r a t h e r s o l u t i o n - s i d e d position. F o r c y t i d i n e 5 " - d i p h o s p h a t e (5"-CDP) a n d c y t ] d i n e 5"--triphosphate (5"-CTD) t h e a d s o r p t i o n ~ia t h e p h o s p h a t e g r o u p o c c u r s a t a r a t h e r p o s i t i v e l y c h a r g e d e l e c t r o d e (w-z. Fig. 4 a n d 5)- D u e t o m o r e h y d r o p h i l i ¢ p h o s p h a t e g r o u p s in t h e m o l e c u l e s t h e i r a d s o r p t i v i t i e s a r e diminished. H o w e v e r , in t h e r a t h e r p o s i t i v e p o t e n t i a l r a n g e e l e c t r o s t a t i c a t t r a c t i o n of t h e n e g a t i v e p h o s p h a t e g r o u p will f a v o u r significantly- t h e a d s o r p t i o n of t h e m o n o n u c l e o t i d e s . ~Vhereas t h e a d s o r p t i o n v i a p h o s p h a t e g r o u p s r e a c h e s as n e g a t i v e e l e c t r o d e p o t e n t i a l s as - o . 7 V, so t h a t n o a d s o r p t i o n in t h e compact s t a g e ~r/a t h e c y t o s i n e m o i e t y t a k e s place f o r 5 " - C D P , t h e b e h a v i o u r of 5 " - C T P is different in t h e s a m e p o t e n t i a l r a n g e . T h e h i g h l y h y d r o p h i l i c p h o s p h a t e g r o u p s t e n d t o a t t a i n a solution s i d e d o r i e n t a t i o n as s o o n as t h e e l e c t r o d e ceases t o b e p o s i t i v e l y c h a r g e d . T h u s , t h e r e is a small region b e t w e e n - o . 6 a n d - o . 7 V w h e r e t h e a d s o r p t i o n v i a t h e c y t o s i n e b a s e t a k e s place as is i n d i c a t e d b y t h e cmTesponding s e c o n d p i t a t high b u l k c o n c e n t r a t i o n s . T h e p e a k a r o u n d - o - 5 V cmTes p o n d s t o a r e o r i e n t a t i o n , w h e r e t h e b a s e is a t t a c h e d t o t h e e l e c t r o d e a n d t h e hydxophilic p h o s p h a t e SToup a t t a i n s a s o l u t i o n - s i d e d position. T o p e d o r m q u a n t i t a t i v e l y a c o m p a r a t i v e s t u d y on t h e a d s o r p t i o n of m o n o - , d i - a n d t x i p h o s p h a t e s of c y t i d i n e a t t h e H M D E t h e e q u i l i b r i u m v a l u e s of t h e c a p a c i t i v e a.c. c m T e n t a t a g i v e n b u l k c o n c e n t r a t i o n w e r e m e a s u r e d a n d t h e degree of c o v e r a g e 0 w a s e v a l u a t e d [24] a c c o r d i n g to the relation
Co-- C 0
=
AI~ =
co ~
c.
(~t~.).
(~)
Electrochemical
Behavior
of Cytidine
Phosphates
7x3
w h e r e t h e C's axe differential c a p a c i t a n c e s , in t h e s u p p o r t i n g e l e c t r o l y t e C 0, a t t h e b u l k c o n c e n t r a t i o n c o r r e s p o n d i n g t o full c o v e r a g e Cm, a n d t h e o t h e r b u l k c o n c e n t r a t i o n s C ; A / ~ . is t h e d e c r e a s e in t h e c a p a c i t i v e a.c. c u r r e n t for a g i v e n b u l k c o n c e n t r a t i o n of t h e a d s o r b e d s u b s t a n c e w i t h r e s p e c t t o t h e / ~ . - v a l u e in t h e s u p p o r t i n g e l e c t r o l y t e a n d (AI~),u is t h e m a x i m a l d e c r e a s e c o r r e s p o n d i n g t o full c o v e r a g e 0mT h e r e s u l t i n g c o n c e n t r a t i o n d e p e n d e n c e of A / ~ . (Fig_ 6) h a s in t h e r a n g e o f t h e p o t e n t i a l of m a x i m u m a d s o r p t i o n t h e f o r m of a d o u b l e s t e p a d s o r p t i o n i s o t h e r m . F r o m t h e d e p e n d e n c e of Ala~. on t h e b u l k c o n c e n t r a t i o n , several i n t e r e s t i n g conclusions m a y b e drawn_ T h e first p l a t e a u c o r r e s p o n d s t o t h e dilute l a y e r a n d t h e s e c o n d p l a t e a u a t larger b u l k c o n c e n t r a t i o n s c o r r e s p o n d s t o t h e comgbact film_ T h e interfacial association of c y t i d i n e t a k e s place a t a l o w e r t h r e s h o l d c o n c e n t r a t i o n t h a n for 3~-CMP o r 5t-CMP_ T h e tendency for interracial association diminishes in t h e o r d e r c y t i d i n e > 3 t - C M P > 5~-CMP d u e t o t h e d e g r e e of c o m p e n s a t i o n of t h e effective c h a r g e on t h e p r o t o n a t e d N(3) b y t h e negatively charged phosphate group.
2.5 2.0
1.5 <] 1,0
O.5 w
I
c (M}
!
10.4
F i g . 6. I-lair l o ~ a r i t h m l c p l o t o f a d s o r p t i o n i s o t h e r n m f o r c y t i d i n e (1) : c)~tidine 3 " - m o n o p h o s p h a t e (z), a n d ¢ y t i d i n e 5 " - - m o n o p h o s p h a t e ( 3 ) : a d s o r p t i o n t i m e ~_4o s : a d s o r p t i o n p o t e n t i a l - o . 7 v" (z). 0.65. (2). (3); P[-~ 3--~4 -
O b v i o u s l y t h e d i m i n u t i o n of t h e effective c h a r g e on t h e p r o t o n a t e d N(3 ) d e c r e a s e s t h e s t a b i l i z a t i o n b y b a s e s t a c k i n g , w h i c h c a u s e s t h e int e r f a c i a l association of a d j a c e n t a d s o r b e d n u c l e o t i d e molecules a n d c o n s e q u e n t l y t h e f o r m a t i o n of t h e compact film.
Temerk,
714
Valenta
and
Nfiraberg
More q u a n t i t a t i v e conclusions i n c l u d i n g t h e v a l u e s of t h e a d s o r p t i o n p a r a m e t e r s c o m p u t e d f r o m t h e a.c.--voltammetric d a t a (Table I) will b e discussed t o g e t h e r w i t h t h o s e o b t a i n e d b y single sweep v o l t a m m e t r y .
Table z. AdsorptioIx parameters of dilute and compact layers of mono-, di-and triphosphates of cs~idine calculated from the results of out-of-phase a.c. voltammetry and single sweep voltammetry at the H~IDE_ 5 ° C , 0 - 5 ~-~CILVAINE buffer, pH 3.24, adsorption t i m e t s 2 4 0 s , U s - o . 6 5 V , _4 1 . 7 5 x IO - ± c m ~-
Substance
Dilute
IO 10 I"m ( m o l c m -2)
Sm ( r i m 2)
b (1 m o l - I )
-AG ° (KJ mol -l)
a
stage
Cytidine
x.34
1.24
I_24 x IO4
3I-x
o-54
3t_St--CM-P
x_Io
I_SI
8 . 5 0 X IO a
30-3
0-59
3t-C~IP 3"--C~IP
x.o 5 o.7x
1.64 2.33
7.o3 x xo = 6.9 x xo 3
29-9 29.8
o.6o o-63
5'-CDP
o.59
2.8o
5-7
x Io a
29-4
o-72
5t--CTP
0-53
3 .08
4-7
x IO a
28-9
o-84
Cytidine 3t.St--C~,lrP
4.90 4.00
0.34 0. 4 Z
3z.o °-2.0
z7. 3 16_ 9
z.xo 2.14
3r...C~,fp 5t-CMP
3.80 2.6o
0.44 o.63
19.o I7.8
I6. 7 I6.6
2.17 2.30
5t--CDP J--3TP
2.xo x -90
0.75
I4- 7 12_ x
I6-4 x6. x
2-56 2_ 8 6
Compact
stage
O-83
Single sweep volgammetric measurements T h e a d s o r p t i o n of m o n o - , d i - a n d t r i p h o s p h a t e s of c y t i d i n e a t t h e H M D E c a n b e also s t u d i e d in a v e r y efficient m a n n e r m-a t h e f a r a d a i c response d u e t o t h e r e d u c t i o n of t h e a d s o r b e d species, if a s u i t a b l e m e t h o d as single s w e e p v o l t a m m e t r y is applied. T h e use of t h e r e d u c t i o n p e a k t o s t u d y t h e a d s o r p t i o n is b a s e d on t h e fuHfillment of t h e following requirements. T h e ~ v e e p r a t e h a s t o b e r a t h e r high. T h e n t h e r e d u c t i o n response is or.ly d u e t o t h e r e d u c t i o n of t h e m a t e r i a l a d s o r b e d d u r i n g t h e a d s o r p t i o n t i m e t, elapsed a t t h e sweep s t a r t i n g p o t e n t i a l Us. C o n s e q u e n t l y
Electrochemical ]Behavior of Cytidine Phosphates -1.65
-1.45
•
uCv}
~ 4 i ~,~. -o
=
-
•
1.25
--1.05
, ~ / . . . ~ - - - - ~ - ' -"
'
-0.135
7I 5
-0.65
HiTl I
-20
/:1 !
t
/IL// i //,'.:-/ i ,:11
5
nE
..~[-4o .,..,
dil/ •e l : -
\3t I i "
/ I
-60
-80
Fig. 7-
Sweep voltammetric response of cytidine nucleotides (t) Cytidine, (z) Cydic 3".5--C-%[P. (3) 3"-CMP, (4) 5"-CMP. (5) 5"-CDP, (6) 5"-CTP ; pI~ 3.z4 ; adsorption time t s 2 4 0 - ~ ; ~-x -o.65 V ; sweep rate 2o X: s-t. bulk concentration z. 4 x to -a ;1I.
t h e d i f f u s i o n c o n t r o l l e d c o m p o n e n t o f t h e c u r r e n t b e c o m e s negligib l e [xO, 16, 25]- U n d e r t h e s e c o n d i t i o n s , t h e r e d u c t i o n r e s p o n s e r e f l e c t s not only t h e adsorption degree b u t with respect to slower interracial e v e n t s also t h e i n t e r r a c i a l s i t u a t i o n o f t h e a d s o r b a t e a t t h e s w e e p s t a r t i n g p o t e n t i a l Us, w h i c h c o r r e s p o n d s t h e e f f e c t i v e a d s o r p t i o n p o t e n t i a l l i t , zS]. O n l y v e r y r a p i d s t r u c t u r a l a l t e r a t i o n s o f t h e a d s o r b e d s u b s t a n c e will be a b l e to f o l l o w r a p i d s w e e p s [27]. T h e f u n d a m e n t a l c o n d i t i o n t h a t t h e sweep r a t e h a s to be h ig h e n o u g h to m a k e negligible t h e c o n t r i b u t i o n t o t h e faradaic response of material diffusing t o w a r d s electrode during t h e s w e e p , i m p o s e s i n t h e c a s e o f l o w m o l e c u l a r s u b s t a n c e s , s u c h as t h e mononucleotides, the application o f r a t h er high sweep rates. I t has been proved b y e x t e n d e d tests t h a t for t h e mononucleotides a sweep rate of a t V s - t o r m o r e is s u f f i c i e n t t o m e e t a l l t h e a f o r e - m e n t i o n e d r e q u i r e m e n t s . E x a m p l e s o f s i n g l e s w e e p v o l t a m m e t r i c r e s p o n s e s a t p H 3.24 f o r m o n o - , d i - a n d t r i p h o s p h a t e s o f c y t i d i n e a r e s h o w n i n F i g . 7- T h e decreasing peak height in the order cytidine > cyclic-3',5'-CMP > 3'C M P > 5 " - C D P ~ 5 " - C T P c o r r e s p o n d s t o t h e d e c r e a s i n g e f f e c t i v e posit i v e c h a r g e o n t h e p r o t o n a t e d lq(3 ) o f c y t o s i n e d u e t o t h e e f f e c t o f t h e c o m p e n s a t i n g n e g a t i v e p h o s p h a t e groups_ T h e n e g a t i v e s h i f t of t h e p o t e n t i a l of t h e reduction p e a k for t h e m o n o - , d i - a n d triphosphates of cytidine compared with cytidine itself indicates, that the attachment
7x6
Temexk. Valent~ and Nftrnberg
of t h e p h o s p h a t e g r o u p decreases t h e r e d u c i b i l i t y in t h e o r d e r c y t i d i n e > cyclic 3", 5"-CMP > 3"--CMP > 5"--CMP > 5" C D P > 5"-CTP. A n i m p o r t a n t f a c t o r i n d e c r e a s i n g r e d u c i b i l i t y is t h e p r e s e n c e of n e g a t i v e l y c h a r g e d p h o s p h a t e groups, w h i c h c a u s e g e n e r a l l y a t Us d u r i n g ts e l e c t r o s t a t i c repulsion of t h e c o m p o u n d s f r o m t h e similarly c h a r g e d H M D E surface a n d w e a k e n eIectron ~ i t h d r a w a l b y t h e ribose m o i e t y . This t r e n d is s u p p o r t e d b y t h e f a c t t h a t t h e d e c r e a s i n g r e d u c i b i l i t y of t h e c y t i d i n e p h o s p h a t e increases w i t h t h e n u m b e r of p h o s p h a t e groups. F o r m o n o - , d i - a n d t r i p h o s p h a t e s of c y t i d i n e different degrees of c o m p e n s a t i o n of t h e positive c h a r g e o n t h e p r o t o n a t e d lq(3 ) axe c o n n e c t e d w i t h sterical reasons d e t e r mining the distance between the protonated N ( 3 ) a n d the negative p h o s p h a t e s as p o i n t e d o u t before. As was sho~,aa earlier E25-29] for nucleic acids t h e a m o u n t of reducible s u b s t a n c e a d s o r b e d a t t h e e l e c t r o d e a n d f o r m i n g a m o n o m o l e c u l a r l a y e r is p r o p o r t i o n a l to t h e t o t a l c h a r g e Q c o n s u m e d d u r i n g t h e r e d u c t i o n . T h u s , t h e c h a r g e d e n s i t y Q[A c o r r e s p o n d s i m m e d i a t e l y t o t h e surface c o n c e n t r a t i o n U of t h e a d s o r b e d m a t e r i a l a t t h e sweep s t a r t i n g p o t e n tial U,. I t follows Q = n~r (2) A w h e r e n ~ 3 is t h e n u m b e r of electrons c o n s u m e d in t h e e l e c t r o d e react i o n [30, 3x], ~ is t h e F a r a d a y c o n s t a n t , A is t h e a r e a of t h e H M D E , a n d U t h e surface c o n c e n t r a t i o n of t h e r e d u c i b l e species. T h e potential d e p e n d e n c e of t h e s u r f a c e c o n c e n t r a t i o n I ~ for t h e a d s o r p t i o n of m o n o - , di-- a n d t r i p h o s p h a t e s of c y t i d i n e in t h e dilute s t a g e is s h o w n in Fig. 8. T h e r e s u l t s r e v e a l t h a t d e p e n d i n g o n t h e c h a r g e of t h e e l e c t r o d e a n d o n t h e c h a r g e caxried b y t h e xe s p y : t i r e a d s o r b e d species different orient a t i o n s of t h e moieties a r e c o n c e i v a b l e a c c o r d i n g t o t h e r e s p e c t i v e ads o r p t i o n p o t e n t i a l w h i c h equals t h e r e s p e c t i v e sweep s t a r t i n g p o t e n t i a l U~. T h e s e different o r i e n t a t i o n s are possible b y r o t a t i o n of t h e s u g a r a r o u n d t h e N(I) glycosidic b o n d w i t h t h e c y t o s i n e a n d (except for cyclic 3 ' , 5 ' CMP) b y free r o t a t i o n of t h e p h o s p h a t e s . T h e n e t result, s h o w n in Fig. 8, is a decrease in surface c o n c e n t r a t i o n o v e r t h e whole p o t e n t i a l r a n g e t h e larger b e c o m e s t h e n u m b e r of p h o s p h a t e g r o u p s i n t h e n u d e o t i d e _ T h i s indicates o n c e m o r e t h a t g e n e r a l l y a d s o r p t i o n occurs m a i n l y zr/a t h e c y t o s i n e as t h e r e l a t i v e l y m o s t h y d r o phobic m o i e t y , while t h e h y d r o p h i l i c p h o s p h a t e a n d s u g a r r e m a i n h y d r a t e d a n d a t t a i n t h u s a position a t t h e solution side of t h e a d s o r p t i o n layer. T h e s e effects d u e t o t h e different h y d r a t i o n t e n d e n c i e s of t h e different moieties x~ill d i s p l a y t h e i r influence m o s t c l e a r l y in t h e p o t e n t i a l r a n g e a r o u n d U , ~ , w h i c h corresponds t o t h e p l a t e a u of t h e c u r v e s in Fig. 8. F u r t h e r m o r e , a t m o r e n e g a t i v e potentials, e l e c t r o s t a t i c repulsion of t h e p h o s p h a t e groups x~aill h a v e g e n e r a l l y u n f a v o u r a b l e effect o n adsorption t o a n e x t e n t w h i c h d e p e n d s for t h e r e s p e c t i v e m o n o n u c l e o t i d e o n t h e a c t u a l d i s t a n c e of t h e p h o s p h a t e f r o m t h e e l e c t r o d e surface. Y e t , t h i s influence, e x e r t e d b y t h e p h o s p h a t e groups, is significantly m o d e r a t e d
Electrochemical
Behavior
of Cytidine
~
Phosphates
7z7
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F i g . 8. D e p e n d e n c e o f s u r f a c e c o n c e n t r a t i o n I" o n t h e a d s o r p t i o n p o t e n t i a l U s m e a - g u r e d b y s , v e e p v o l t a m m e t r y . (1) c_vtidine. (2) 3"--5"--cyclic C~I~Po (3) 3"-CM'Po (4) 5 " - C ~ I P . (5) 5 " - - C T P ; p H 3.~ 4 ; a d s o r p t i o n t i m e t s -24o s : s w e e p r a t e 20 V s - L b u l k c o n c e n t r a t i o n z_6 × xo -a .II.
b y t h e p o s i t i v e c h a r g e of t h e p r o t o n a t e d N(3) in t h e c y t o s i n e m o i e t y ; which is a l w a y s m u c h closer to t h e s u r f a c e o f a n e g a t i v e l y c h a r g e d electrode. N e v e r t h e l e s s , e v e n in t h e a b s e n c e of p h o s p h a t e , a progTessive d e s o r p t i o n o c c u r s also for c y t i d i n e w i t h increasing n e g a t i v a t i o n of t h e electrode. T h i s is t o b e a s c r i b e d t o t h e h y d r o p h i l i c c h a r a c t e r of t h e prot o n a t e d N(3) of c y t i d i n e . D e s p i t e its p o s i t i v e c h a r g e f a v o u r i n g t h e elect r o s t a t i c a t t r a c t i o n b y t h e n e g a t i v e e l e c t r o d e this i n t e r a c t i o n will b e p r o g r e s s i v e l y o v e r r u l e d b y t h e increase in t h e t e n d e n c i e s t o w a r d h y d r a t i o n of t h e e l e c t r o d e b~, t h e w a t e r d i p o l e s p r o g r e s s i v e l y a c c u m u l a t e d f r o m t h e solvent_ T h e w a t e r dipoles a r e g e n e r a l l y a t t r a c t e d m o r e a n d m o r e w i t h t h e increase in t h e c h a r g e o f t h e e l e c t r o d e on b o t h sides o f U ~ , . The concentration dependence of the surface concentration can be determined from the response obtained at various p H values with the single s w e e p v o l t a m m e t r / c m e t h o d . F r o m t h e p H - d e p e n d e n c e (viz. Fig. 9 a n d zo) t h e influence of t h e p r o t o n a t i o n on t h e N(3) of t h e c y t o s i n e m o i e t y a n d t h e sterical a r r a n g e m e n t of t h e m o l e c u l a r is c l e a r l y seen. W h e r e a s a t p H 3-24 t h e r e a r e differences in t h e s u r f a c e c o n c e n t r a t i o n v a l u e s f o r different CMP's, t h e r e is no difference a t all a t p H 4.8, w h e r e t h e c y t o s i n e m o i e t y carries no m o r e a charge. T h e d e g r e e o f c o v erage 0 is in this c a s e o b t a i n e d f o r a given b u l k c o n c e n t r a t i o n d i r e c t l y f r o m t h e s u r f a c e c o n c e n t r a t i o n ]P a n d t h e v a l u e of P= c o r r e s p o n d i n g t o
Temerk, V a l e n t a a n d Nfirnberg
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F i g . 9D e p e n d e n c e o f t h e s u r f a c e c o n c e n t r a t i o n I" o n t h e b u l k c o n c e n t r a t i o n f o r t h e d i l u t e l a y e r m e a s u r e d b y ~ , - e e p v o l t a m m e t z y a t pI-I 3.24 ; ~) C y t i d i n e o 2) 3 L S " - c y c l i c C ~ I P . 3) 3 - C~[P: 4) 5 "-C~IP- 5) 5 " - c D P - 6) 5"--CTP-
f u l l c o v e r a g e o r f r o m t h e c o r r e s p o n d i n g c h a r g e s Q a n d Qm, w h i c h axe i m m e d i a t e e x p e r i m e n t a l d a t a , a c c o r d i n g t o e q u a t i o n (3)Y
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a n d t h o s e o b t a i n e d b y single s w e e p v o l t a m m e t r y c a n b e i n s e r t e d i n t o t h e e q u a t i o n f o r t h e FRUMKI,X a d s o r p t i o n isother~tJ. bc = 0 (I - - 0) -x e x p (--2aO) (4) O n e o b t a i n s t h e a d s o r p t i o n c o e f f i c i e n t b a n d t h e i n t e r a c t i o n coefficient a in the usual manner [3z-34]. In addition the free enthalpy of adsorption - A G ° c a n b e c a l c u l a t e d f r o m t h e a d s o r p t i o n coefficient b. T h e c o n c e n t r a t i o n d e p e n d e n c e of t h e s u r f a c e c o n c e n t r a t i o n I~ for c y t i d i n e a n d its
Electrochemical Behavior of Cytidine Phosphates
7x9
mono--, d i " a n d , t r i p h o s p h a t e s is s h o w n in Fig. 9 a n d zo f o r t h e first s t e p o f t h e d o u b l e s t e p a d s o r p t i o n i s o t h e r m , i.e. f o r t h e dilute layer. T h e similar e v a l u a t i o n f o r t h e s e c o n d s t e p is n o t p o s s i b l e f o r m o n o nucleotides, b e c a u s e a t t h e h i g h b u l k c o n c e n t r a t i o n t o b e applied, t h e diffusion c o n t r i b u t i o n t o t h e s w e e p v o l t a m m e t r i c r e s p o n s e is no m o r e negligible. T h e r e f o r e a n o t h e r p r o c e d u r e [x4] h a s b e e n a d o p t e d . The l i m i t i n g v a l u e o f s u r f a c e c o v e r a g e 0m for t h e first s t e p of t h e a d s o r p t i o n i s o t h e r m w a s c o m p u t e d f r o m t h e c h a r g e Qm f o r t h e dilute layer. F r o m this v a l u e a n d t h e r a t i o o f t h e t w o s t e p s of t h e i s o t h e r m o b t a i n e d f r o m a . c . - v o l t a m m e t r i c m e a s u r e m e n t s (Fig. 6) t h e s u r f a c e c o v e r a g e c o r r e s p o n d i n g t o t h e s e c o n d step, i.e. COml~aCt film f o r m a t i o n , h a s b e e n e v a l u a t e d . T h e a d s o r p t i o n p a r a m e t e r s o b t a i n e d in t h i s m a n n e r f o r c y t i d i n e a n d its m o n o - , d i - a n d t r i p h o s p h a t e s a r e listed in T a b l e x. I n t h e dilute s t a g e o n e o b s e r v e s t h e following tendencies. The maximal surface concent r a t i o n F , d e c r e a s e s in t h e o r d e r c y t i d i n e > cyclic 3 ' , 5 ' - C M P > 3 ' CMP > 5 ' - C D P > 5'-CTP- Consequently the average surface area per m o l e c u l e S , increases in t h e s a m e order. A t t h e s a m e t i m e t h e a d s o r p t i o n coefficient b d e c r e a s e s b y a f a c t o r 2.6 3 f r o m c y t i d i n e t o 5 ' - C T P . T h i s l o w e r i n g o f a d s o r p t i v i t y c o r r e s p o n d i n g f o r 5 " - C T P t o a d e c r e a s e i n AG ° b y 2.2 k J t o o l - ' is c o n n e c t e d w i t h t h e increasing n u m b e r of h y d r o p h i l i c p h o s p h a t e g r o u p s a n d also w i t h increasing l a t e r a l repnlqion of t h e nega t i v e l y c h a r g e d p h o s p h a t e groups, T h e S e - v a l u e r e q u i r e d will t h u s b e c o m e l a r g e r in t h e series of m o n o - , d i - a n d t r i p h o s p h a t e s w i t h increasi n g n u m b e r o f p h o s p h a t e groups. F r o m t h e r a t h e r larger v a l u e s of S~ f o r t h e c y t i d i n e p h o s p h a t e s it c a n b e c o n c l u d e d t h a t in t h e dilute l a y e r
I1 /
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8
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720
Temerko Valenta and Nfirnberg
mono--, d i - a n d t r i p h o ~ p h a t e s of c y t i d i n e a r e a d s o r b e d v i a t h e h e t e r o cyclic rings flat o n t h e e l e c t r o d e surface. F o r t h e compact a d s o r p t i o n s t a g e t h e following t e n d e n c i e s e m e r g e (see also T a b l e I). T h e I*~-value f o r c y t i d i n e is b y a b o u t 46 ~o, 57 ~/o a n d 6x % l a r g e r t h a n f o r 5 " - C M P , 5 " - C D P a n d 5 ' - C T P , r e s p e c t i v e l y . T h e m a g n i t u d e of t h e a d s o r p t i o n coefficients b a r e significantly l o w e r t h a n in t h e d ilfde stage. T h i s significantly l o w e r a d s o r p t i v i t y of t h e c o m p ~ film c o r r e l a t e s x~4th its s e n s i t i v i t y t o laxger a l t e r a t i o n s o f t h e a d s o r p t i o n p o t e n t i a l a n d i t s t e n d e n c y for s u d d e n collapse a t c e r t a i n values: of t h e interracial electric field a t t h e p o s i t i v e a n d n e g a t i v e side of t h e region of m a x i m a l a d s o r p t i o n a x o u n d t h e o p e r a t i v e p o t e n t i a l of zero charge. N e v e r t h e l e s s , t h e m a g n i t u d e of - A G ° i n d i c a t e s t h a t still t h e s a m e u n i t of t h e molecules, i.e. t h e c y t o s i n e m o i e t y , r e m a i n s p r e d o m i n a n t l y r e s p o n s i b l e f o r t h e a d s o r p t i o n in t h e compact film s t a g e a t t h e potential of m a x i m u m adsorption. T h e i n t e r a c t i o n coefficient a h a s g e n e r a l l y i n c r e a s e d in t h e compact stage, d u e t o t h e e n h a n c e d possibilities for inte, m o l e c u l a r a t t r a c t i v e i n t e r a c t i o n s , r e s u l t i n g f r o m t h e p e r p e n d i c u l a r o r i e n t a t i o n a n d t h e g r e a t e r p o p u l a t i o n of t h e a d s o r b e d m o l e c u l e s in t h e compact stage. T h e r a t h e r l o w a v e r a g e s u r f a c e areas Sm f o r c y t i d i n e p h o s p h a t e s in t h e compact film s t a g e i n d i c a t e a d e n s e p a c k e d s t r u c t u r e of t h e b a s e r e s i d u e s of t h e n u c l e o t i d e o r i e n t a t e d p e r p e n d i c u l a r l y t o w a r d s t h e s u r f a c e of t h e electrode. I n g e n e r a l t h e conclusions a b o u t t h e a d s o r p t i o n o f c y t i d i n e a n d c y t o s i n e p h o s p h a t e s in t h e dilute a n d compact s t a t e a g r e e w i t h t h o s e o b t a i n e d prex~iously f o r a d e n o s i n e a n d a d e n i n e p h o s p h a t e s using t h e s a m e m e t h o d s [xo, ~x, x4]. R e c e n t l y o u r conclusions a b o u t t h e o r i e n t a t i o n a n d sites of a d s o r p t i o n of m o n o - a n d d i n u c l e o t i d e s b a s e d o n a d e n i n e h a v e b e e n s u p p o r t e d b y RA_X.IAN s p e c t r a of t h e s e s u b s t a n c e s a d s o r b e d a t a n e l e c t r o c h e m i c a l l y a c t i v a t e d silver e l e c t r o d e [35, 36JT h i s l e a d s t o t h e conclusion t h a t t h e s t a c k i n g of t h e b a s e moieties i n n a t u r a l a n d b i o s y n t h e t i c p o l y n u c l e o t i d e s is c o m p a r a b l e t o t h e compact film of t h e m o n o m e r i c u n i t s a d s o r b e d a t t h e e l e c t r o d e interface. Thtm, f o r t h i s a n d o t h e r b a s i c p h y s i c a l p a r a m e t e r s t h e e l e c t r o d e I a q u e o u s solut / o n i n t e r f a c e c a n b e r e g a r d e d as a r e l e v a n t m o d e l for t h e i n t e r a c t i o n of p o l y n u c l e o t i d e s w i t h c h a r g e d biological surfaces.
References [I] P_ V_~tx.~rs a n d H.'~V_ - ~ R - ~ S E R C . B i o p h y s _ S t r u c t . l~[ech. ! ( I 9 7 4 ) x7 [2] E . PALE~EK. Collect. Czech. C h e m . C o m m u n . 3 9 (x974) 3 4 4 9 [3] H.~V. N~RXBERC; a n d P . ~-.~LEXT.~, P r o c . z 9 t h Syrap_ CoL~ton Res_ Soc_ T h e Behat~iour o f I o n s i n z~lacromolecular a n d B i o l o g i c a l Sys'tems, B r i s t o l x977, D_H. EVEREI-r, B . VINCENT ( E d i t o r s ) , p . 2ox, S c i e n t e c h n i c a , B r i s t o l (I978) [4] J . M . SE~U_~R[S, P_ V.ALENTA, H . I V . ~ U R N B E R 0 a n d B . ~IALFOY, P r o c . 2 9 t h S y r u p . C o l s t o n Res_ Soc_ T h e B e h a v i o u r o f I o n s in. ~ [ a c r o m o l e c u l a r a n d B i o -
Electrochemical. Behavior
[5] [6] [7]
[8] [9] [xo] lit]
[I2] [I3] [i4] [15] [16] [17]
[18] [x9]
[2O]
[2I] [2--.] [23] [24] [25] [26] [27] [28] [29] [3o] [3x] [32]
of Cytidine Phosphates
721
iogical S y s t e m s , B r i s t o l i 9 7 7 , D . H . E V E R E ~ r , B . V I n C E n T ( E d i t o r s ) , p . 2 3 0 , S c i e n t e c h n i c a , B r i s t o l (1978) V. VETTERL, Collect. Czech. C h e m . C o n z m u n . 31 (1966) 2 1 o 3 V. VETTERL, J. Electroanal- Cltem. [ n t e r f a c i a l E l e c t r o c h e m . 19 ( I 9 6 8 ) r 6 9 T.~V. XVEBB, B . J A ~ t K a n d P . J . ELVING, J . 2{nz. C h e m . Soc. 95 ( I 9 7 3 ) 9 9 I J.~V. ~VEBB, B . JaNIK a n d P . J . ELVIXG, J . A , n . Chem_ Soc. 95 (1973) 8495 U . RETTER, H . JEHRI.XG a n d V. VETTERL, J . E l e c t r o a n a l - C h e m . [ n t e r f a c i a l E l e c t r o c h e m . 57 {I974) 391 D_ KRZ-~ARId, P . V&LE~T.~ -~nd H.XV. N~R.~BERG, J . E l e c t r o a n a l . C h e m . [ n t e r f a c i a l E l e c t r o c k e m . 65 ( I 9 7 5 ) 863 P_ V.~L~_~TA, H.~V. NUR~BERG a n d D . KRZ~ARIC, BioelectrocRem. Bioe~zerg. 3 (1976) 418 V- VETTERL, Bioelectrochen,. B i o e n e r g . 3 ( I 9 7 6 ) 338 M . A . JE.'qSE,x, T . E . CUMMINGS a n d P . J . ELVING, Bioelectrochem. B i o e n e r g . 4 (1977) 4 4 7 D . KRZ~.akRI~, P . V.~-LE~TA, H.v~V. ~L]R-~BERG a n d M. BRA,~IC_~,, f . E l e c t r o a n a l . C h e m . [ n t e r f a c i a l E l e c t r o c k e n L 93 (1978) 4 I V . BR.~m:c. S . D . CHRISTtA-X a n d I ) . DRX'ttURST. B i o e l e c t r o c h e m . B i o e n e r g . 5 ( I 9 7 8 ) 635 Y . M . TEMERK a n d P . VALEXTA, J. Electroanal- C h e m . [ n t e r f a c i a l Electroc h e m . 93 (197 S) 57 Y.L~[. TE.MERK, P . V.*,LE.~T_~, H . vt~r. ~UR.~BERG. J . Electroanal- C h e m . I~zt e r f a c i a l E l e c t r o c h e m . 10O (1979) 77 ~[_ K-~.'ITZ, T . M . CU~I3tt,XGS a n d P . J . ELVI.XG, B e t . B u n s e n g e s . P h y s . C h e m . 83 (1979) 614 Y . ~ I . THMHRK, P . V.~,LENTA a n d H.XV. ~URYBERG, i n E l e c t r o a n a l y s i s i n H y g i e n e , E n v i r o n m e n t a l , C l i n i c a l a n d P h a r m a c e u t i c a l Cizemistry, XV.F. ~3IYTH ( E d i t o r ) , E l s e ~ 4 e r , A m s t e r d a m (x9So) p p . x x 3 - - r 2 6 M. LE.~G a n d FELS~XFELD, J . . ' ~ l o l - B i o l . 15 (1966) 455 G . D . FASXtAN, C. LIXDBLOXV a n d L . GROSM&N, B i o c h e m i s t r y 3 (1964) 1 o t 5 J . P . ,XIcT.~,GUE, U . R o s s a n d J . H _ GIBBS, B i o p o l y m e r s (1964) t 6 3 P . O . P _ T s ' o a n d S . I . CHAX, J . A m . Cltem. Soc. 86 ( I 9 6 4 ) 4 t 7 6 H . JEHRIKG, E l e c t r o s o r p t i o n s a n a l y s e m i t der IVechselstrompolarographie, A k a d e m i e V e f l a g , B e r l i n 1974 P . VALENTA a n d P . GRAH.MAN.~, J . t~lectroanal- Cicero. I n t e r f a c i a l . t~lectroc h e m . 49 ( I 9 7 4 ) 41 P . VALE,~Ta, H.~V. ~URNBERG a n d P . KLAHRE, Bioelectroclzem. Bioetterg. 2 (1975) 204 B . ~I.~LFOY, J . ~ [ . ~EQUARIS, P . Xr_~,LE_~T.aka n d H.XV_ ~ 0 R N B E R G , B i o e l e c t r o c h e m . B i o e n e r g . • (1976) 44 ° J . M . SEQUARIS, g . MALFOY, P . VALENT_~- a n d H.XV. NURXB~'RG, Bioele6trothem_ Bioenerg_ ~ ( i 9 7 6 ) 4 6 I g . ~[ALFOY, J . M . SEQUARIS, P . VALEXTA a n d H_XV. ~ 0 R S B E R G , J . E l e c t r o anal. C h e m . I n t e r f a c i a l E l e c t r o c h e m . 75 (1977) 455 B . Jx,XIK a n d P . J . ELVI~G. J . . 4 m . C h e n . Soc. 92 ( I 9 7 o) 235 e . J A N I K a n d P . J . ELVING, C k e m . B e y . 68 ( I 9 6 8 ) 295 A . ~ . FRUMKIN, Z . P h y s . ~5 ( I 9 2 6 ) 792
7zz
-
_ T e m e r k . Xralent~ a n d N f i r n b e r g
[33] _A_,~. FguMgt,'¢o Ewgeb_ E x a k t e n N a t - ~ m i s s _ T (Z928) 235 [34] A.~-. FRUMKt.~, itX A d u a n c ~ s i n E l e c t r o c h e m i s t r y a n d E l e c t r o c k e m i c a l E n ~ n e e r z n g P . Dwx_~uAY a n d C . W . T o s z - ~ ( E d i t o r s ) . I n t e r s c i e n c e , N e w Y o r k ( x 9 6 I ) Vol_ 1, p . 65 [35] E_ KOGLI~, J . M . SEQUARIS a n d P . VALE,'~TA, f . ~l/[ol, S t r i c t . 60 (x98o) 4 2 I [36] K_M_ ERVIN, E_ KOGLIN, J.M_ SEQUARXS. P_ VALE~TA a n d H . W . NORNBERG, J. E l e c t r o a n a l . C h e m . I n t e r f ~ w l a l E l e c t r o c h e m . i n p r e s s
3 9 8 - Eleetrochemival Behavior of Mono- a n d OHgonucleotides. V I I L Voltammetric Studies on the Inter[aeial B e h a v i o u r of Mono-, Di- a n d Triphosphate of Cytidine * b y Y. M. TEMERK°, P- VALE NTA° a n d H . ~ ' . Nf3RNBERG° o Chemistry Department, F a c u l t y of Science, Assiut University, Assiut, E g y p t " I n s t i t u t e of Chemistry, Institute 4 Applied Physical Chemistry, Nuclear Research Center {KFA), D-StTO Jfilich, Federal Republic of Germatty ~Ianuscript received March 29th I980
Summary
A systematic study of the adsorption and interfacial behavior of the cytidine mononucleotides (5'-CMP, 3'-CMP, 3',5'-CMP, 5'-CDP and 5'-CTP) has been carried out. The adsorption was followed at the H M D E b y a . c . - v o l t a m m e t r y a n d single sweep v o l t a m m e t r y . Over a w i d e p o t e n t i a l r a n g e o n e h a s a dilute a d s o r p t i o n l a y e r w h i l e w i t h i n t h e potential range of m a x i m u m adsorption a t elevated bulk concentration f o r m a t i o n o f a compact film is o b s e r v e d . T h e a d s o r p t i o n c a n b e d e s c r i b e d q u a n t i t a t i v e l y for b o t h t y p e s of a d s o r p t i o n layers b y single a n d double s t e p FRU~tKIX i s o t h e r m s , - r e s p e c t i v e l y - T h e r e s u l t i n g a d s o r p t i o n p a r a m eters are evaluated and the conclusions on the respective interfacial behavior, orientations a n d interactions of these substances are discussed.
Introduction T h e investigation of the s t r u c t u r e a n d properties of nucleic acids is o f g r e a t b i o l o g i c a l i m p o r t a n c e f o r a b e t t e r u n d e r s t a n d i n g o f t h e m e c h a n i s m s b y w h i c h t h e g e n e t i c i n f o r m a t i o n is s t o r e d , d u p l i c a t e d a n d t r a n s m i t t e d i n l i v i n g cells. E x t e n s i v e r e c e n t s t u d i e s h a v e s h o w n t h a t n a t i v e DNA and other polynucleotides undergo potential dependent conformat i o n a l c h a n g e s w h e n a d s o r b e d a t a m e r c u r y elect_rode s u r f a c e [ I - 4 ] . A proper u n d e r s t a n d i n g of the interracial behaviour of polynucleotides a t b o t h , a n e l e c t r o d e s u r f a c e a n d a c h a r g e d biological i n t e r f a c e , relies o n a g e n e r a l k n o w l e d g e o f t h e i n t e r f a c i a l b e h a v i o u r of t h e c o n s t i t u e n t * Presented a t the 5th International Symposium on Bioelectrochemistry, 3--8 September I979, Weimar (D.D.R.).
0302-4598]80[0705-0722 ~ i98o Elsevier Sequoia S.A.
706
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e l e m e n t s of t h o s e m a c r o m o l e c u l e s , i.e. t h e i r c o n s t i t u e n t p u r i n e a n d p y r i m i d i n e bases, a n d t h e c o r r e s p o n d i n g nucleosides a n d nucleotides. I t w a s s h o w n t h a t bases, n u c l e o s i d e s a n d n u d e o t i d e s a r e a d s o r b e d a n d u n d e r g o a n association a t t h e c h a r g e d m e r c u r y - s o l u t i o n i n t e r f a c e [5-x9]. I n t h i s c o n t e x t it w a s s h o w n t h a t a d s o r b e d nucleosides a n d n u d e o t i d e s i n t e r a c t p r i n c i p a l l y z,/ar verti~-~al s t a c k i n g o f t h e bases. T h e y f o r m a t b u l k c o n c e n t r a t i o n s a b o v e a t r e s h o l d v a l u e a compact film. S u f ficient e v i d e n c e h a s n o w b e e n a c c u m u l a t e d t o s h o w t h a t t h e b a s e s t a c k i n g i n t e r a c t i o n s of a d j a c e n t h e t e r o c y d i c rings a l o n g t h e p o l y n u d e o t i d e s chain m a k e a significant c o n t r i b u t i o n t o t h e s t a b i l i t y a n d confo~mat i o n a l p r o p e r t i e s of nucleic acids, a s h a s b e e n also ~ s s u m e d b y o t h e r a u t h o r s [2o-23]. I t is t h e r e f o r e of i n t e r e s t t o s t u d y t h e a d s o r p t i o n a n d association in t h e a d s o r b e d s t a t e o f m o n o - , d i - a n d t r i p h o s p h a t e s o f c y t i d i n e a t t h e i n t e r f a c e m e r c u r Y l a q u e o u s e l e c t r o l y t e a n d t h e effect of t h e s t e r i c a l a r r a n g e m e n t a n d influence o f t h e p h o s p h a t e g r o u p .
Experimental Chemicals a n d solutions Cytidine-3'--monophosphate (3'-CMP), c y t i d i n e - 5 ' - m o n o p h o s p h a t e ( 5 ' - C M p ) , c y t i d i n e - 5 " , 5 ' - - m o n o p h o s p h a t e (cyclic CMP), cytidine-5"-d i p h o s p h a t e (5"-CDP), a n d c y t i d i n e - 5 " - t r i p h o s p h a t e (5"--CTP) w e r e o b t a i n e d f r o m SERVA (Heidelberg, F . R . G . ) . I n all m e a s u r e m e n t s t h e s u p p o r t i n g e l e c t r o l y t e w a s a MCILvAIN~ b u f f e r b r o u g h t t o c o n s t a n t ionic ~ h e n g t h of o-5 b y a d d i t i o n of KC1. All c h e m i c a l s w e r e r e a g e n t g r a d e , KCI w a s SUl~rapur, E . MERCK ( D a r m s t a d t ) . T h e c o n c e n t r a t i o n of t h e m o n o n u c l e o t i d e s in t h e s a m p l e s o l u t i o n s w a s d e t e r m i n e d w i t h t h e UNICAM S P 8oo s p e c t r o p h o t o m e t e r , p H w a s m e a s u r e d w i t h a n ORION p H m e t e r , M o d e l 7oz. A p p a r a t u s a n d methods All v o l t a m m e t r i c m e a s u r e m e n t s w e r e c a r r i e d o u t w i t h t h e m u l t i m o d e p o l a r o g r a p h i c a n a l y z e r P A R z7o in a t h e r m o s t a t e d METROHM ceil e q u i p p e d x~4th a 3 - e l e c t r o d e s y s t e m u n d e r p o t e n t i o s t a t i e control. P h a s e scns~.'tive a.c.-voltammograms c o r r e s p o n d i n g t o t h e o u t - o f - p h a s e c o m p o n e n t o f t h e t o t a l a.c. c u r r e n t (9 oo p h a s e angle) w e r e r e c o r d e d in a p s e u d o s t a t i o n a r y m ~ n n e r w i t h a s c a n r a t e o f t h e d . c . - r a m p o f 2 m V s -1. T h e a m p l i t u d e o f t h e a.c_ v o l t a g e w a s 5 mVpp a n d t h e f r e q u e n c y 33o H z , unless othexwise s t a t e d . T h e d e p e n d e n c e o f t h e o u t - o f - p h a s e a.c. c u r r e n t o n t h e a d s o r p t i o n t i m e ts w a s o b t a i n e d b y a d j u s t i n g t h e a d s o r p t i o n p o t e n t i a l , i.e. t h e m e a n e l e c t r o d e p o t e n t i a l , t o a p r e d e t e x m i n e d v a l u e , e x t r u d i n g t h e m e r c u r y d r o p a n d r e c o r d i n g a f t e r t h e r e s p e c t i v e :ts p e r i o d h a d e l a p s e d t h e o u t - o f - p h a s e a.c. c u r r e n t w i t h a s u i t a b l e t i m e base_
Electrochemical Behavior of Cytidine Phosphates
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I n t h e single sweep mode, as well, t h e c u r r e n t - p o t e n t i a l c u r v e s and the corresponding charge-potential curves obtained were recorded with the built-in electronic integration circuit. For the evaluation of r e s p o n s e s a t s w e e p r a t e s a b o v e 2oo m V s -~ a d i g i t a l s t o r a g e oscilloscope, NICOLET, t y p e XO90, w a s a p p l i e d . T h e w o r "king e l e c t r o d e w a s a METROHM hanging mercury drop electrode (HMDE), type E4Io, with a surface a r e a o f L 7 5 X xo -z cmz. As r e f e r e n c e e l e c t r o d e s e r v e d a s a t u r a t e d c a l o m e l e l e c t r o d e f r o m I~GOLD, t y p e 3 o 3 - N S , a n d t h e a u x i l i a r y e l e c t r o d e w a s a c o i l e d p l a t i n u m wire_ T h e s o l u t i o n w a s d e a e r a t e d w i t h p u r e n i t r o g e n . T o avoid interference b y t h e c a t a l y t i c h y d r o g e n evolution, all measurem e n t s h a v e b e e n c a r r i e d o u t a t 5 °C w h e r e t h e c a t a l y t i c h y d r o g e n r e s p o n s e is s h i f t e d t o s u f f i c i e n t l y n e g a t i v e e l e c t r o d e p o t e n t i a l s . Results
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Phase sensitive a.c. v o l t a m m o g r a m s c o r r e s p o n d i n g t o t h e o u t - o £ phase c o m p o n e n t of t h e t o t a l a.c. response p r o v i d e an o v e r a l l p a t t e r n of the adsorption behaviour of the studied mono-, di- and triphosphates o f c y t i d i n e (Figs. ~-5)- T h e recorded c a p a c i t i v e a.c. c o m p o n e n t is p r o -
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Fig. z. a.c. voltammetric curve of cytidiue-3"-monophosphate ( 3 " - C ~ [ P ) a t t h e I-EtIDI~. o . 5 A I ~ C I L V A I N E b u f f e r , p H 3.°-4, 5 °C, a r e a o f H ~ I D E L 7 5 × I o -ffi c m ~. s c a n r a t e ~ m V s - : , f r e q u e n c y 3 3 o H z . a m p l i t u d e 5 m V p p . p h a s e a n g l e 9 o ° . C u r v e (1) o . o . (22 6, 9 x I o - S ; (3) = . i × x o -4 ; (4) S . S × z o - " : (5) L 5 × x o -~ ; (6) 5 × I o -~ ; (7) I . o × i o - ' - ~'r 3 - C . . ~ P .
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p o r t i o n a l t o t h e differential d o u b l e l a y e r c a p a c i t a n c e in p o t e n t i a l regions w h e r e no f a r a d a i c p r o c e s s o c c u r s [z4]- T h e p H o f t h e solution w a s a d j u s t e d t o s u c h a ~-alue ( p H 3.z4) t h a t t h e p r o t o n a t e d f o L ~ of c y t i d i n e p h o s p h a t e prevailed. A t l o w b u l k c o n c e n t r a t i o n s o n e o b s e r v e s a r o u n d t h e p o t e n t i a l of m a x i m u m a d s o r p t i o n (-o-65 V) a progressive d e c r e a s e of t h e c a p a c i t i v e a.c. s~gnal d u e t o increasing a d s o r p t i o n of m o n o - , d i a n d t r i p h o s p h a t e s of c y t i d i n e . T h i s d e c r e a s e c o r r e s p o n d s t o t h e p r o g r e s s i v e c o v e r a g e o f t h e e l e c t r o d e s u r f a c e b y t h e di&de a d s o r p t i o n layer.
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F i g . ~-. a.c.--voltammetric curve of cytidine-5*--monophosphate ( 5 " - - C M P ) a t t h e H~.~IDE_ Curve ( : ) o . o ; ( z ) 9 - 9 X Xo - ~ ; ( 3 ) 5 - 9 X x o - t ; ( 4 ) x - 7 x : o - a ; ( 5 ) 3 - I × x o -~ ; ( 6 ) z - 3 X : o - ' - ; ( 7 ) z - 5 × × XO-'- "1I 5 " - - C M P . o t h e r c o n d i t i o n s as in Fig. L
At more elevated bulk concentrations above the threshold value f o r t h e r e s p e c t i v e m o n o n u c l e o t i d e a p i t is o b s e r v e d . T h e p i t o c c u r s f o r 3 ' - C M P a t a significantly a n d for 5 ' - C M P a t a s o m e w h a t l o w e r t h r e s h o l d v a l u e t h a n f o r C T P . As h a s b e e n s h o w n b y VETTERL [5, 6, 9, X2] a n d K R z . ~ . ~ et ag. [xo, ~x, x4] t h e p i t reflects t h e f o r m a t i o n of a compact a d s o r p t i o n film d u e t o p r o n o u n c e d l a t e r a l i n t e r a c t i o n s of a d s o r b e d n u c l e o t i d e species in a c e r t a i n p o t e n t i a l r a n g e . A f t e r t h e p i t a r o u n d t h e p o t e n t i a l - o . 9 5 t o - L 2 5 V a n e x t e n d e d n o n - f a r a d a i c p e a k is o b s e r v e d c a u s e d b y t h e r e o r i e n t a t i o n o f t h e a d s o r b e d n u c l e o t i d e species. T h e
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F i g . 3a.c.-x-oltammetric curve of cytidine 3",5"-phospha.te (cyclic 3".5"-C:MP) at the HMDH. C u r x - e (z) o . o : (2) L S X x o - s : (3) 3 - 9 8 X I o - s : (4) 9 - 9 9 × x o - s ; (5) L 6 S × I o -a ; (6) 2 . 6 6 X z o -a : (7) 5 - 5 2 x x o - a : (S) 2 - x × x o - a : (9) 1-34Xio-±;'(x o) 2 . o × I o - ' - ; ( x I ) 3 _ 7 × I O - ' - ; (z2) 4.48 × zo -a 3I cyclic 3",5"-C~IP, other conditions as in Fig. L
analysis of t h e p e a k c u r r e n t in t e r m s of t h e phase a n g l e gives a v a l u e of 9 oo. Moreover, t h e p e a k c u r r e n t varies l i n e a r l y w i t h t h e f r e q u e n c y of t h e s u p e r i m p o s e d a.c. voltage. T h e s e c h a r a c t e r i s t i c s c o r r e s p o n d to a n o n - f a r a d a i c n a t u r e of t h e process c a u s i n g this peak. This a.c. p e a k o b s e r v e d also w i t h a d e n i n e m o n o n u e l e o t i d e s [1o, I x , / 4 ] , d e n a t u r e d [ 2 5 ] a n d n a t i v e DlqA [zT], a n d r e l a t e d b i o s y n t h e t i c p o l y n u c l e o t i d e s [26] is d u e to a s t r u c t u r a l r e a r r a n g e m e n t of t h e a d s o r b e d species. As will be discussed l a t e r b a s e d on t h e results g a i n e d w i t h s i n g l e sweep v o l t a m m e t r y t h e a d s o r p t i o n of all c y t i d i n e d e r i v a t e s in t h e d i l u t e s t a g e t a k e s place v i a t h e m o s t h y d r o p h o b i c m o i e t y of t h e molecule, i.e. t h e c y t o s i n e base. T h e ~ - e l e c t r o n i n t e r a c t i o n ~,ith t h e e l e c t r o d e xs411 f a v o u r for t h e r a n g e of positive c h a r g e o r zero c h a r g e a flat a d s o r p t i o n of t h e c y t o s i n e m o i e t y . I n t h e c o m p a c t s t a g e of t h e a d s o r p t i o n t h e c y t o s i n e a n d p h o s p h a t e moieties i n t e r a c t w i t h t h e e l e c t r o d e d e p e n d i n g on t h e i r c h a r g e a n d o n t h e sterical a r r a n g e m e n t of t h e molecule. F o r c y t i d i n e 3 ' - m o n o p h o s p h a t e (3'-CMP) t h e p h o s p h a t e g r o u p i n 3"-CMP r e m a i n s d i s t a n t f r o m t h e p r o t o n a t e d N(3) of t h e c y t o s i n e m o i e t y a n d its influence: o n t h e adsorption, o c c u r i n g h e r e p r e d o m i n a n t l y v i a t h e base, r e m a i n s u s u a l l y small, e x c e p t f o r r a t h e r positive potentials. T h u s , for 3--CMP (viz. Fig. x) t h e n e g a t i v e a s c e n d i n g p a r t of t h e dip e x t e n d s
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t o p o t e n t / a l ~ b e y o n d --o.8 V. "ALso f o r 3 " - C M P t h e d i p e x t e n d s t o p o t e n tialg b e y o n d - o . 8 V_ a n d i t e x t e n d s o n t h e p o s i t i v e s i d e t o p o t e n t i a l s o f l e s s t h a n --o_3 V_ Th;~ is t o b e i n t e r p r e t e d a s a s i g n i f i c a n t c o n t r i bution to the adsorption by the negatively charged phosphate group. As model considerations reveal, the latter, under the electrostatic a t t r a c t i o n of t h e e l e c t r o d e , c a n b e t u r n e d i n t o a p o s i t i o n w h e r e t h e phosphate group comes into direct contact with the surface, thus cont r i b u t i n g t o t h e a n c h o r i n g o f t h e species. H o w e v e r , w i t h t h e p o s i t i v e l y c h a r g e d p r o t o n a t e d N(3), t h e b a s e is s u b j e c t e d t o r e p e l l i n g t e n d e n cies, a l t h o u g h o n e h a s t o b e a r i n m i n d t h a t p r o n o u n c e d t e n d e n c i e s f o r ~ - e l e c t r o n i n t e r a c t i o n xs~ll c o u n t e r a c t , t o s o m e e x t e n t , t o t h i s e f f e c t o n t h e p r o t o n a t e d b a s e . T h e p e a k a t - o . 5 V c o r r e s p o n d s t o a specific r e o r i e n tation to a position where the base comes into strong interaction with the surface, while the adsorption of the hydrophilic phosphate group attains a solution-sided position. The occurrence of this reorientation p e a k is t o b e e x p e c t e d as_ a t p H 3 . z 4 t h e n e t c h a r g e o f t h e 3 ' - C M P x.x-z~,~tterion is a p p r o x i m a t e l y b a l a n c e d . The considerable change in the dielectric properties of the interface ( a l t e r a t i o n o f d i r e c t i o n o f t h e d i p o l e , c h a n g e s i n h y d r a t i o n ) g i v e s rise t o a significant alteration in double layer capacitance which manifests
Electrochemical B e h a v i o r of Cytidine P h o s p h a t e s
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itself in the observed non-faradaic peak. From the fact that the reorienr a t i o n is o b s e r v a b l e f o r a f r e q u e n c y o f 330 H z , o n e c o n c l u d e s t h a t i t s r a t e is f a s t . ~ F u r t h e r t h e r a t h e r e x t e n d e d size o f t h e p e a k i n d i c a t e s t h a t the reorientation does not occur for each individual adsorbed monon u c l e o t i d e z w i t t e r i o n a t t h e s a m e p o t e n t i a l , b u t is d i s t r i b u t e d a r o u n d t h e
peak potential (-o.5 V). The negative phosphate group is in 5'-CMP (Fig. z) situated closer to t he p r o t o n a t e d N(3) of t h e cytosine unit a n d compensates partially its positive charge. At potentials more positive t h a n t h e potential of m a x i m u m adsorption (-o.65 V). one can distinguish th e following influences o n a d s o r p t i o n . T h e = - e l e c t r o n i n t e r a c t i o n w i t h t h e p o s i t i v e charge of the electrode would favour a rather flat adsorption of the cytosine moiety. However, the repulsion of the protonated N(3 ) acts antagonistically. This influence, in principle unfavourable for adsorption, may, however, be moderated to a certain extent for 5'-phosphate, where the negative phosphate group attains a position proximate to the prot o n a t e d N ( 3 ). T h u s , t h e r e is a r a t h e r g o o d p r o b a b i l i t y t h a t t h e a r r a n g e m e n t d e s c r i b e d will p r e v a i l f o r 5 ' - C M P a t p o t e n t i a l s m o d e r a t e l y m o r e positive than the potential of maximum adsorption U,. Due to this
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a.c.-voltammetric curve of cTtidine-5"-triphosphate (5"--CTP) at the I-L~[DE. Curve (i) o.o; (-~) x.99xxo -~; (3) 5-96xx° -s; (4) 9-9xxo -5; (5) t_2Sxxo - i ; (6) 2.~-4xIO-a; (7) 6-9xxo-a ; (8) 9 . 4 x I o - ~ ; (9) I-xSxIo-S: (io) 1.4~-xto-a: (ix) 1.67xio-:I; (I2) 2 . I X X IO -3 ; (13) 3-][ X IO "-3 ; ( I 4 ) 4 . 0 X IO -• ; ( I 5 ) 5 . 8 X IO -:$ ; (][6) S. 4 X I O -3 ; (17) I . I 6 X IO-'- ; ( I S ) 1 . 6 X X O - ' - ; (xg) 4 - 5 6 X X O - - ' ; (20) S . 7 6 X I o - ~ - : (21) 9 . 5 X I O ÷ ~ ; ( 2 2 ) i . o 3 x I o - 1 ; (~-3) L I 4 X I o - I z~I 5 " - C T P . o t h e r c o n d i t i o n s a s i n F i g . L
7z2
Temerk.
Valenta and Niirnberg
c h a r a c t e r i s t i c sterical a r r a n g e m e n t of 5"--CMP t h e r e o r i e n t a t i o n t o a n o t h e r p o s i t i o n a t m o r e p o s i t i v e p o t e n t i a l s , ~where t h e m o l e c u l e is a d s o r b e d via t h e p h o s p h a t e group, will n o t o c c u r in c o n t r a s t t o 3"-CMP. A r o u n d U , t h e n t h e = - e l e c t r o n i n t e r a c t i o n x,~ll b e m a i n l y r e s p o n s i b l e f o r t h e a d s o r p t i o n . A t m o r e n e g a t i v e p o t e n t i a l s t h e progressive r e p u l s i o n of t h e p h o s p h a t e g r o u p f r o m t h e i n t e r f a c e will c a u s e i t s p r o g r e s s i v e l y m o r e s o l u t i o n - s i d e d position. T h e n t h e c o m p e n s a t i n g influence o n t h e p o s i t i v e c h a r g e of t h e p r o t o n a t e d N(3) declines in p r o p o r t i o n t o t h e d i s t a n c e a t which the phosphate group becomes located. At the same time the positive c h a r g e a t t h e p r o t o n a t e d N(3 ) c a n d i s p l a y i t s influence g r a d u a l l y compensating more and more at not too elevated negative potentials t h e c o n c o m i t a n t repulsion b y t h e e l e c t r o d e of t h e = - e l e c t r o n s y s t e m o f c y t i d i n e . Y e t a t a n y r a t e , t h e p h o s p h a t e in 5 ' - p o s i t i o n s h o u l d a l w a y s p r e f e r a s o m e w h a t closer p o s i t i o n t o t h e c y t o s i n e m o i e t y t h a n in 3 " C M P . F o r cyclic 5',5"--CMP (Fig. 3), t h e a d s o r p t i o n r a n g e e x t e n d s t o more negative potentials than for a n y other cytidine phosphate. This i n d i c a t e s t h a t t h e r e is v i r t u a l l y n o c o u n t e r b a l a n c e b y t h e n e g a t i v e p h o s p h a t e t o t h e p o s i t i v e c h a r g e a t t h e p r o t o n a t e d N(3) of t h e a d s o r b e d c y t o s i n e moiety-_ I t m e a n s t h a t t h e n e g a t i v e l y c h a r g e d p h o s p h a t e g r o u p m u s t p r e f e r a m o s t d i s t a n t position f r o m t h e c y t o s i n e m o i e t y . Consid e r a t i o n s of m o l e c u l a r m o d e l s r e v e a l t h a t s u c h a n a [ l a n g e m e n t is e a s i l y possible if t h e h y d r o p h i l i c s u g a r a n d t h e 3 ' - 5 ' - e s t e r i f i e d p h o s p h a t e a t t a i n a r a t h e r s o l u t i o n - s i d e d position. F o r c y t i d i n e 5 " - d i p h o s p h a t e (5"-CDP) a n d c y t ] d i n e 5"--triphosphate (5"-CTD) t h e a d s o r p t i o n ~ia t h e p h o s p h a t e g r o u p o c c u r s a t a r a t h e r p o s i t i v e l y c h a r g e d e l e c t r o d e (w-z. Fig. 4 a n d 5)- D u e t o m o r e h y d r o p h i l i ¢ p h o s p h a t e g r o u p s in t h e m o l e c u l e s t h e i r a d s o r p t i v i t i e s a r e diminished. H o w e v e r , in t h e r a t h e r p o s i t i v e p o t e n t i a l r a n g e e l e c t r o s t a t i c a t t r a c t i o n of t h e n e g a t i v e p h o s p h a t e g r o u p will f a v o u r significantly- t h e a d s o r p t i o n of t h e m o n o n u c l e o t i d e s . ~Vhereas t h e a d s o r p t i o n v i a p h o s p h a t e g r o u p s r e a c h e s as n e g a t i v e e l e c t r o d e p o t e n t i a l s as - o . 7 V, so t h a t n o a d s o r p t i o n in t h e compact s t a g e ~r/a t h e c y t o s i n e m o i e t y t a k e s place f o r 5 " - C D P , t h e b e h a v i o u r of 5 " - C T P is different in t h e s a m e p o t e n t i a l r a n g e . T h e h i g h l y h y d r o p h i l i c p h o s p h a t e g r o u p s t e n d t o a t t a i n a solution s i d e d o r i e n t a t i o n as s o o n as t h e e l e c t r o d e ceases t o b e p o s i t i v e l y c h a r g e d . T h u s , t h e r e is a small region b e t w e e n - o . 6 a n d - o . 7 V w h e r e t h e a d s o r p t i o n v i a t h e c y t o s i n e b a s e t a k e s place as is i n d i c a t e d b y t h e cmTesponding s e c o n d p i t a t high b u l k c o n c e n t r a t i o n s . T h e p e a k a r o u n d - o - 5 V cmTes p o n d s t o a r e o r i e n t a t i o n , w h e r e t h e b a s e is a t t a c h e d t o t h e e l e c t r o d e a n d t h e hydxophilic p h o s p h a t e SToup a t t a i n s a s o l u t i o n - s i d e d position. T o p e d o r m q u a n t i t a t i v e l y a c o m p a r a t i v e s t u d y on t h e a d s o r p t i o n of m o n o - , d i - a n d t x i p h o s p h a t e s of c y t i d i n e a t t h e H M D E t h e e q u i l i b r i u m v a l u e s of t h e c a p a c i t i v e a.c. c m T e n t a t a g i v e n b u l k c o n c e n t r a t i o n w e r e m e a s u r e d a n d t h e degree of c o v e r a g e 0 w a s e v a l u a t e d [24] a c c o r d i n g to the relation
Co-- C 0
=
AI~ =
co ~
c.
(~t~.).
(~)
Electrochemical
Behavior
of Cytidine
Phosphates
7x3
w h e r e t h e C's axe differential c a p a c i t a n c e s , in t h e s u p p o r t i n g e l e c t r o l y t e C 0, a t t h e b u l k c o n c e n t r a t i o n c o r r e s p o n d i n g t o full c o v e r a g e Cm, a n d t h e o t h e r b u l k c o n c e n t r a t i o n s C ; A / ~ . is t h e d e c r e a s e in t h e c a p a c i t i v e a.c. c u r r e n t for a g i v e n b u l k c o n c e n t r a t i o n of t h e a d s o r b e d s u b s t a n c e w i t h r e s p e c t t o t h e / ~ . - v a l u e in t h e s u p p o r t i n g e l e c t r o l y t e a n d (AI~),u is t h e m a x i m a l d e c r e a s e c o r r e s p o n d i n g t o full c o v e r a g e 0mT h e r e s u l t i n g c o n c e n t r a t i o n d e p e n d e n c e of A / ~ . (Fig_ 6) h a s in t h e r a n g e o f t h e p o t e n t i a l of m a x i m u m a d s o r p t i o n t h e f o r m of a d o u b l e s t e p a d s o r p t i o n i s o t h e r m . F r o m t h e d e p e n d e n c e of Ala~. on t h e b u l k c o n c e n t r a t i o n , several i n t e r e s t i n g conclusions m a y b e drawn_ T h e first p l a t e a u c o r r e s p o n d s t o t h e dilute l a y e r a n d t h e s e c o n d p l a t e a u a t larger b u l k c o n c e n t r a t i o n s c o r r e s p o n d s t o t h e comgbact film_ T h e interfacial association of c y t i d i n e t a k e s place a t a l o w e r t h r e s h o l d c o n c e n t r a t i o n t h a n for 3~-CMP o r 5t-CMP_ T h e tendency for interracial association diminishes in t h e o r d e r c y t i d i n e > 3 t - C M P > 5~-CMP d u e t o t h e d e g r e e of c o m p e n s a t i o n of t h e effective c h a r g e on t h e p r o t o n a t e d N(3) b y t h e negatively charged phosphate group.
2.5 2.0
1.5 <] 1,0
O.5 w
I
c (M}
!
10.4
F i g . 6. I-lair l o ~ a r i t h m l c p l o t o f a d s o r p t i o n i s o t h e r n m f o r c y t i d i n e (1) : c)~tidine 3 " - m o n o p h o s p h a t e (z), a n d ¢ y t i d i n e 5 " - - m o n o p h o s p h a t e ( 3 ) : a d s o r p t i o n t i m e ~_4o s : a d s o r p t i o n p o t e n t i a l - o . 7 v" (z). 0.65. (2). (3); P[-~ 3--~4 -
O b v i o u s l y t h e d i m i n u t i o n of t h e effective c h a r g e on t h e p r o t o n a t e d N(3 ) d e c r e a s e s t h e s t a b i l i z a t i o n b y b a s e s t a c k i n g , w h i c h c a u s e s t h e int e r f a c i a l association of a d j a c e n t a d s o r b e d n u c l e o t i d e molecules a n d c o n s e q u e n t l y t h e f o r m a t i o n of t h e compact film.
Temerk,
714
Valenta
and
Nfiraberg
More q u a n t i t a t i v e conclusions i n c l u d i n g t h e v a l u e s of t h e a d s o r p t i o n p a r a m e t e r s c o m p u t e d f r o m t h e a.c.--voltammetric d a t a (Table I) will b e discussed t o g e t h e r w i t h t h o s e o b t a i n e d b y single sweep v o l t a m m e t r y .
Table z. AdsorptioIx parameters of dilute and compact layers of mono-, di-and triphosphates of cs~idine calculated from the results of out-of-phase a.c. voltammetry and single sweep voltammetry at the H~IDE_ 5 ° C , 0 - 5 ~-~CILVAINE buffer, pH 3.24, adsorption t i m e t s 2 4 0 s , U s - o . 6 5 V , _4 1 . 7 5 x IO - ± c m ~-
Substance
Dilute
IO 10 I"m ( m o l c m -2)
Sm ( r i m 2)
b (1 m o l - I )
-AG ° (KJ mol -l)
a
stage
Cytidine
x.34
1.24
I_24 x IO4
3I-x
o-54
3t_St--CM-P
x_Io
I_SI
8 . 5 0 X IO a
30-3
0-59
3t-C~IP 3"--C~IP
x.o 5 o.7x
1.64 2.33
7.o3 x xo = 6.9 x xo 3
29-9 29.8
o.6o o-63
5'-CDP
o.59
2.8o
5-7
x Io a
29-4
o-72
5t--CTP
0-53
3 .08
4-7
x IO a
28-9
o-84
Cytidine 3t.St--C~,lrP
4.90 4.00
0.34 0. 4 Z
3z.o °-2.0
z7. 3 16_ 9
z.xo 2.14
3r...C~,fp 5t-CMP
3.80 2.6o
0.44 o.63
19.o I7.8
I6. 7 I6.6
2.17 2.30
5t--CDP J--3TP
2.xo x -90
0.75
I4- 7 12_ x
I6-4 x6. x
2-56 2_ 8 6
Compact
stage
O-83
Single sweep volgammetric measurements T h e a d s o r p t i o n of m o n o - , d i - a n d t r i p h o s p h a t e s of c y t i d i n e a t t h e H M D E c a n b e also s t u d i e d in a v e r y efficient m a n n e r m-a t h e f a r a d a i c response d u e t o t h e r e d u c t i o n of t h e a d s o r b e d species, if a s u i t a b l e m e t h o d as single s w e e p v o l t a m m e t r y is applied. T h e use of t h e r e d u c t i o n p e a k t o s t u d y t h e a d s o r p t i o n is b a s e d on t h e fuHfillment of t h e following requirements. T h e ~ v e e p r a t e h a s t o b e r a t h e r high. T h e n t h e r e d u c t i o n response is or.ly d u e t o t h e r e d u c t i o n of t h e m a t e r i a l a d s o r b e d d u r i n g t h e a d s o r p t i o n t i m e t, elapsed a t t h e sweep s t a r t i n g p o t e n t i a l Us. C o n s e q u e n t l y
Electrochemical ]Behavior of Cytidine Phosphates -1.65
-1.45
•
uCv}
~ 4 i ~,~. -o
=
-
•
1.25
--1.05
, ~ / . . . ~ - - - - ~ - ' -"
'
-0.135
7I 5
-0.65
HiTl I
-20
/:1 !
t
/IL// i //,'.:-/ i ,:11
5
nE
..~[-4o .,..,
dil/ •e l : -
\3t I i "
/ I
-60
-80
Fig. 7-
Sweep voltammetric response of cytidine nucleotides (t) Cytidine, (z) Cydic 3".5--C-%[P. (3) 3"-CMP, (4) 5"-CMP. (5) 5"-CDP, (6) 5"-CTP ; pI~ 3.z4 ; adsorption time t s 2 4 0 - ~ ; ~-x -o.65 V ; sweep rate 2o X: s-t. bulk concentration z. 4 x to -a ;1I.
t h e d i f f u s i o n c o n t r o l l e d c o m p o n e n t o f t h e c u r r e n t b e c o m e s negligib l e [xO, 16, 25]- U n d e r t h e s e c o n d i t i o n s , t h e r e d u c t i o n r e s p o n s e r e f l e c t s not only t h e adsorption degree b u t with respect to slower interracial e v e n t s also t h e i n t e r r a c i a l s i t u a t i o n o f t h e a d s o r b a t e a t t h e s w e e p s t a r t i n g p o t e n t i a l Us, w h i c h c o r r e s p o n d s t h e e f f e c t i v e a d s o r p t i o n p o t e n t i a l l i t , zS]. O n l y v e r y r a p i d s t r u c t u r a l a l t e r a t i o n s o f t h e a d s o r b e d s u b s t a n c e will be a b l e to f o l l o w r a p i d s w e e p s [27]. T h e f u n d a m e n t a l c o n d i t i o n t h a t t h e sweep r a t e h a s to be h ig h e n o u g h to m a k e negligible t h e c o n t r i b u t i o n t o t h e faradaic response of material diffusing t o w a r d s electrode during t h e s w e e p , i m p o s e s i n t h e c a s e o f l o w m o l e c u l a r s u b s t a n c e s , s u c h as t h e mononucleotides, the application o f r a t h er high sweep rates. I t has been proved b y e x t e n d e d tests t h a t for t h e mononucleotides a sweep rate of a t V s - t o r m o r e is s u f f i c i e n t t o m e e t a l l t h e a f o r e - m e n t i o n e d r e q u i r e m e n t s . E x a m p l e s o f s i n g l e s w e e p v o l t a m m e t r i c r e s p o n s e s a t p H 3.24 f o r m o n o - , d i - a n d t r i p h o s p h a t e s o f c y t i d i n e a r e s h o w n i n F i g . 7- T h e decreasing peak height in the order cytidine > cyclic-3',5'-CMP > 3'C M P > 5 " - C D P ~ 5 " - C T P c o r r e s p o n d s t o t h e d e c r e a s i n g e f f e c t i v e posit i v e c h a r g e o n t h e p r o t o n a t e d lq(3 ) o f c y t o s i n e d u e t o t h e e f f e c t o f t h e c o m p e n s a t i n g n e g a t i v e p h o s p h a t e groups_ T h e n e g a t i v e s h i f t of t h e p o t e n t i a l of t h e reduction p e a k for t h e m o n o - , d i - a n d triphosphates of cytidine compared with cytidine itself indicates, that the attachment
7x6
Temexk. Valent~ and Nftrnberg
of t h e p h o s p h a t e g r o u p decreases t h e r e d u c i b i l i t y in t h e o r d e r c y t i d i n e > cyclic 3", 5"-CMP > 3"--CMP > 5"--CMP > 5" C D P > 5"-CTP. A n i m p o r t a n t f a c t o r i n d e c r e a s i n g r e d u c i b i l i t y is t h e p r e s e n c e of n e g a t i v e l y c h a r g e d p h o s p h a t e groups, w h i c h c a u s e g e n e r a l l y a t Us d u r i n g ts e l e c t r o s t a t i c repulsion of t h e c o m p o u n d s f r o m t h e similarly c h a r g e d H M D E surface a n d w e a k e n eIectron ~ i t h d r a w a l b y t h e ribose m o i e t y . This t r e n d is s u p p o r t e d b y t h e f a c t t h a t t h e d e c r e a s i n g r e d u c i b i l i t y of t h e c y t i d i n e p h o s p h a t e increases w i t h t h e n u m b e r of p h o s p h a t e groups. F o r m o n o - , d i - a n d t r i p h o s p h a t e s of c y t i d i n e different degrees of c o m p e n s a t i o n of t h e positive c h a r g e o n t h e p r o t o n a t e d lq(3 ) axe c o n n e c t e d w i t h sterical reasons d e t e r mining the distance between the protonated N ( 3 ) a n d the negative p h o s p h a t e s as p o i n t e d o u t before. As was sho~,aa earlier E25-29] for nucleic acids t h e a m o u n t of reducible s u b s t a n c e a d s o r b e d a t t h e e l e c t r o d e a n d f o r m i n g a m o n o m o l e c u l a r l a y e r is p r o p o r t i o n a l to t h e t o t a l c h a r g e Q c o n s u m e d d u r i n g t h e r e d u c t i o n . T h u s , t h e c h a r g e d e n s i t y Q[A c o r r e s p o n d s i m m e d i a t e l y t o t h e surface c o n c e n t r a t i o n U of t h e a d s o r b e d m a t e r i a l a t t h e sweep s t a r t i n g p o t e n tial U,. I t follows Q = n~r (2) A w h e r e n ~ 3 is t h e n u m b e r of electrons c o n s u m e d in t h e e l e c t r o d e react i o n [30, 3x], ~ is t h e F a r a d a y c o n s t a n t , A is t h e a r e a of t h e H M D E , a n d U t h e surface c o n c e n t r a t i o n of t h e r e d u c i b l e species. T h e potential d e p e n d e n c e of t h e s u r f a c e c o n c e n t r a t i o n I ~ for t h e a d s o r p t i o n of m o n o - , di-- a n d t r i p h o s p h a t e s of c y t i d i n e in t h e dilute s t a g e is s h o w n in Fig. 8. T h e r e s u l t s r e v e a l t h a t d e p e n d i n g o n t h e c h a r g e of t h e e l e c t r o d e a n d o n t h e c h a r g e caxried b y t h e xe s p y : t i r e a d s o r b e d species different orient a t i o n s of t h e moieties a r e c o n c e i v a b l e a c c o r d i n g t o t h e r e s p e c t i v e ads o r p t i o n p o t e n t i a l w h i c h equals t h e r e s p e c t i v e sweep s t a r t i n g p o t e n t i a l U~. T h e s e different o r i e n t a t i o n s are possible b y r o t a t i o n of t h e s u g a r a r o u n d t h e N(I) glycosidic b o n d w i t h t h e c y t o s i n e a n d (except for cyclic 3 ' , 5 ' CMP) b y free r o t a t i o n of t h e p h o s p h a t e s . T h e n e t result, s h o w n in Fig. 8, is a decrease in surface c o n c e n t r a t i o n o v e r t h e whole p o t e n t i a l r a n g e t h e larger b e c o m e s t h e n u m b e r of p h o s p h a t e g r o u p s i n t h e n u d e o t i d e _ T h i s indicates o n c e m o r e t h a t g e n e r a l l y a d s o r p t i o n occurs m a i n l y zr/a t h e c y t o s i n e as t h e r e l a t i v e l y m o s t h y d r o phobic m o i e t y , while t h e h y d r o p h i l i c p h o s p h a t e a n d s u g a r r e m a i n h y d r a t e d a n d a t t a i n t h u s a position a t t h e solution side of t h e a d s o r p t i o n layer. T h e s e effects d u e t o t h e different h y d r a t i o n t e n d e n c i e s of t h e different moieties x~ill d i s p l a y t h e i r influence m o s t c l e a r l y in t h e p o t e n t i a l r a n g e a r o u n d U , ~ , w h i c h corresponds t o t h e p l a t e a u of t h e c u r v e s in Fig. 8. F u r t h e r m o r e , a t m o r e n e g a t i v e potentials, e l e c t r o s t a t i c repulsion of t h e p h o s p h a t e groups x~aill h a v e g e n e r a l l y u n f a v o u r a b l e effect o n adsorption t o a n e x t e n t w h i c h d e p e n d s for t h e r e s p e c t i v e m o n o n u c l e o t i d e o n t h e a c t u a l d i s t a n c e of t h e p h o s p h a t e f r o m t h e e l e c t r o d e surface. Y e t , t h i s influence, e x e r t e d b y t h e p h o s p h a t e groups, is significantly m o d e r a t e d
Electrochemical
Behavior
of Cytidine
~
Phosphates
7z7
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F i g . 8. D e p e n d e n c e o f s u r f a c e c o n c e n t r a t i o n I" o n t h e a d s o r p t i o n p o t e n t i a l U s m e a - g u r e d b y s , v e e p v o l t a m m e t r y . (1) c_vtidine. (2) 3"--5"--cyclic C~I~Po (3) 3"-CM'Po (4) 5 " - C ~ I P . (5) 5 " - - C T P ; p H 3.~ 4 ; a d s o r p t i o n t i m e t s -24o s : s w e e p r a t e 20 V s - L b u l k c o n c e n t r a t i o n z_6 × xo -a .II.
b y t h e p o s i t i v e c h a r g e of t h e p r o t o n a t e d N(3) in t h e c y t o s i n e m o i e t y ; which is a l w a y s m u c h closer to t h e s u r f a c e o f a n e g a t i v e l y c h a r g e d electrode. N e v e r t h e l e s s , e v e n in t h e a b s e n c e of p h o s p h a t e , a progTessive d e s o r p t i o n o c c u r s also for c y t i d i n e w i t h increasing n e g a t i v a t i o n of t h e electrode. T h i s is t o b e a s c r i b e d t o t h e h y d r o p h i l i c c h a r a c t e r of t h e prot o n a t e d N(3) of c y t i d i n e . D e s p i t e its p o s i t i v e c h a r g e f a v o u r i n g t h e elect r o s t a t i c a t t r a c t i o n b y t h e n e g a t i v e e l e c t r o d e this i n t e r a c t i o n will b e p r o g r e s s i v e l y o v e r r u l e d b y t h e increase in t h e t e n d e n c i e s t o w a r d h y d r a t i o n of t h e e l e c t r o d e b~, t h e w a t e r d i p o l e s p r o g r e s s i v e l y a c c u m u l a t e d f r o m t h e solvent_ T h e w a t e r dipoles a r e g e n e r a l l y a t t r a c t e d m o r e a n d m o r e w i t h t h e increase in t h e c h a r g e o f t h e e l e c t r o d e on b o t h sides o f U ~ , . The concentration dependence of the surface concentration can be determined from the response obtained at various p H values with the single s w e e p v o l t a m m e t r / c m e t h o d . F r o m t h e p H - d e p e n d e n c e (viz. Fig. 9 a n d zo) t h e influence of t h e p r o t o n a t i o n on t h e N(3) of t h e c y t o s i n e m o i e t y a n d t h e sterical a r r a n g e m e n t of t h e m o l e c u l a r is c l e a r l y seen. W h e r e a s a t p H 3-24 t h e r e a r e differences in t h e s u r f a c e c o n c e n t r a t i o n v a l u e s f o r different CMP's, t h e r e is no difference a t all a t p H 4.8, w h e r e t h e c y t o s i n e m o i e t y carries no m o r e a charge. T h e d e g r e e o f c o v erage 0 is in this c a s e o b t a i n e d f o r a given b u l k c o n c e n t r a t i o n d i r e c t l y f r o m t h e s u r f a c e c o n c e n t r a t i o n ]P a n d t h e v a l u e of P= c o r r e s p o n d i n g t o
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F i g . 9D e p e n d e n c e o f t h e s u r f a c e c o n c e n t r a t i o n I" o n t h e b u l k c o n c e n t r a t i o n f o r t h e d i l u t e l a y e r m e a s u r e d b y ~ , - e e p v o l t a m m e t z y a t pI-I 3.24 ; ~) C y t i d i n e o 2) 3 L S " - c y c l i c C ~ I P . 3) 3 - C~[P: 4) 5 "-C~IP- 5) 5 " - c D P - 6) 5"--CTP-
f u l l c o v e r a g e o r f r o m t h e c o r r e s p o n d i n g c h a r g e s Q a n d Qm, w h i c h axe i m m e d i a t e e x p e r i m e n t a l d a t a , a c c o r d i n g t o e q u a t i o n (3)Y
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a n d t h o s e o b t a i n e d b y single s w e e p v o l t a m m e t r y c a n b e i n s e r t e d i n t o t h e e q u a t i o n f o r t h e FRUMKI,X a d s o r p t i o n isother~tJ. bc = 0 (I - - 0) -x e x p (--2aO) (4) O n e o b t a i n s t h e a d s o r p t i o n c o e f f i c i e n t b a n d t h e i n t e r a c t i o n coefficient a in the usual manner [3z-34]. In addition the free enthalpy of adsorption - A G ° c a n b e c a l c u l a t e d f r o m t h e a d s o r p t i o n coefficient b. T h e c o n c e n t r a t i o n d e p e n d e n c e of t h e s u r f a c e c o n c e n t r a t i o n I~ for c y t i d i n e a n d its
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mono--, d i " a n d , t r i p h o s p h a t e s is s h o w n in Fig. 9 a n d zo f o r t h e first s t e p o f t h e d o u b l e s t e p a d s o r p t i o n i s o t h e r m , i.e. f o r t h e dilute layer. T h e similar e v a l u a t i o n f o r t h e s e c o n d s t e p is n o t p o s s i b l e f o r m o n o nucleotides, b e c a u s e a t t h e h i g h b u l k c o n c e n t r a t i o n t o b e applied, t h e diffusion c o n t r i b u t i o n t o t h e s w e e p v o l t a m m e t r i c r e s p o n s e is no m o r e negligible. T h e r e f o r e a n o t h e r p r o c e d u r e [x4] h a s b e e n a d o p t e d . The l i m i t i n g v a l u e o f s u r f a c e c o v e r a g e 0m for t h e first s t e p of t h e a d s o r p t i o n i s o t h e r m w a s c o m p u t e d f r o m t h e c h a r g e Qm f o r t h e dilute layer. F r o m this v a l u e a n d t h e r a t i o o f t h e t w o s t e p s of t h e i s o t h e r m o b t a i n e d f r o m a . c . - v o l t a m m e t r i c m e a s u r e m e n t s (Fig. 6) t h e s u r f a c e c o v e r a g e c o r r e s p o n d i n g t o t h e s e c o n d step, i.e. COml~aCt film f o r m a t i o n , h a s b e e n e v a l u a t e d . T h e a d s o r p t i o n p a r a m e t e r s o b t a i n e d in t h i s m a n n e r f o r c y t i d i n e a n d its m o n o - , d i - a n d t r i p h o s p h a t e s a r e listed in T a b l e x. I n t h e dilute s t a g e o n e o b s e r v e s t h e following tendencies. The maximal surface concent r a t i o n F , d e c r e a s e s in t h e o r d e r c y t i d i n e > cyclic 3 ' , 5 ' - C M P > 3 ' CMP > 5 ' - C D P > 5'-CTP- Consequently the average surface area per m o l e c u l e S , increases in t h e s a m e order. A t t h e s a m e t i m e t h e a d s o r p t i o n coefficient b d e c r e a s e s b y a f a c t o r 2.6 3 f r o m c y t i d i n e t o 5 ' - C T P . T h i s l o w e r i n g o f a d s o r p t i v i t y c o r r e s p o n d i n g f o r 5 " - C T P t o a d e c r e a s e i n AG ° b y 2.2 k J t o o l - ' is c o n n e c t e d w i t h t h e increasing n u m b e r of h y d r o p h i l i c p h o s p h a t e g r o u p s a n d also w i t h increasing l a t e r a l repnlqion of t h e nega t i v e l y c h a r g e d p h o s p h a t e groups, T h e S e - v a l u e r e q u i r e d will t h u s b e c o m e l a r g e r in t h e series of m o n o - , d i - a n d t r i p h o s p h a t e s w i t h increasi n g n u m b e r o f p h o s p h a t e groups. F r o m t h e r a t h e r larger v a l u e s of S~ f o r t h e c y t i d i n e p h o s p h a t e s it c a n b e c o n c l u d e d t h a t in t h e dilute l a y e r
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Temerko Valenta and Nfirnberg
mono--, d i - a n d t r i p h o ~ p h a t e s of c y t i d i n e a r e a d s o r b e d v i a t h e h e t e r o cyclic rings flat o n t h e e l e c t r o d e surface. F o r t h e compact a d s o r p t i o n s t a g e t h e following t e n d e n c i e s e m e r g e (see also T a b l e I). T h e I*~-value f o r c y t i d i n e is b y a b o u t 46 ~o, 57 ~/o a n d 6x % l a r g e r t h a n f o r 5 " - C M P , 5 " - C D P a n d 5 ' - C T P , r e s p e c t i v e l y . T h e m a g n i t u d e of t h e a d s o r p t i o n coefficients b a r e significantly l o w e r t h a n in t h e d ilfde stage. T h i s significantly l o w e r a d s o r p t i v i t y of t h e c o m p ~ film c o r r e l a t e s x~4th its s e n s i t i v i t y t o laxger a l t e r a t i o n s o f t h e a d s o r p t i o n p o t e n t i a l a n d i t s t e n d e n c y for s u d d e n collapse a t c e r t a i n values: of t h e interracial electric field a t t h e p o s i t i v e a n d n e g a t i v e side of t h e region of m a x i m a l a d s o r p t i o n a x o u n d t h e o p e r a t i v e p o t e n t i a l of zero charge. N e v e r t h e l e s s , t h e m a g n i t u d e of - A G ° i n d i c a t e s t h a t still t h e s a m e u n i t of t h e molecules, i.e. t h e c y t o s i n e m o i e t y , r e m a i n s p r e d o m i n a n t l y r e s p o n s i b l e f o r t h e a d s o r p t i o n in t h e compact film s t a g e a t t h e potential of m a x i m u m adsorption. T h e i n t e r a c t i o n coefficient a h a s g e n e r a l l y i n c r e a s e d in t h e compact stage, d u e t o t h e e n h a n c e d possibilities for inte, m o l e c u l a r a t t r a c t i v e i n t e r a c t i o n s , r e s u l t i n g f r o m t h e p e r p e n d i c u l a r o r i e n t a t i o n a n d t h e g r e a t e r p o p u l a t i o n of t h e a d s o r b e d m o l e c u l e s in t h e compact stage. T h e r a t h e r l o w a v e r a g e s u r f a c e areas Sm f o r c y t i d i n e p h o s p h a t e s in t h e compact film s t a g e i n d i c a t e a d e n s e p a c k e d s t r u c t u r e of t h e b a s e r e s i d u e s of t h e n u c l e o t i d e o r i e n t a t e d p e r p e n d i c u l a r l y t o w a r d s t h e s u r f a c e of t h e electrode. I n g e n e r a l t h e conclusions a b o u t t h e a d s o r p t i o n o f c y t i d i n e a n d c y t o s i n e p h o s p h a t e s in t h e dilute a n d compact s t a t e a g r e e w i t h t h o s e o b t a i n e d prex~iously f o r a d e n o s i n e a n d a d e n i n e p h o s p h a t e s using t h e s a m e m e t h o d s [xo, ~x, x4]. R e c e n t l y o u r conclusions a b o u t t h e o r i e n t a t i o n a n d sites of a d s o r p t i o n of m o n o - a n d d i n u c l e o t i d e s b a s e d o n a d e n i n e h a v e b e e n s u p p o r t e d b y RA_X.IAN s p e c t r a of t h e s e s u b s t a n c e s a d s o r b e d a t a n e l e c t r o c h e m i c a l l y a c t i v a t e d silver e l e c t r o d e [35, 36JT h i s l e a d s t o t h e conclusion t h a t t h e s t a c k i n g of t h e b a s e moieties i n n a t u r a l a n d b i o s y n t h e t i c p o l y n u c l e o t i d e s is c o m p a r a b l e t o t h e compact film of t h e m o n o m e r i c u n i t s a d s o r b e d a t t h e e l e c t r o d e interface. Thtm, f o r t h i s a n d o t h e r b a s i c p h y s i c a l p a r a m e t e r s t h e e l e c t r o d e I a q u e o u s solut / o n i n t e r f a c e c a n b e r e g a r d e d as a r e l e v a n t m o d e l for t h e i n t e r a c t i o n of p o l y n u c l e o t i d e s w i t h c h a r g e d biological surfaces.
References [I] P_ V_~tx.~rs a n d H.'~V_ - ~ R - ~ S E R C . B i o p h y s _ S t r u c t . l~[ech. ! ( I 9 7 4 ) x7 [2] E . PALE~EK. Collect. Czech. C h e m . C o m m u n . 3 9 (x974) 3 4 4 9 [3] H.~V. N~RXBERC; a n d P . ~-.~LEXT.~, P r o c . z 9 t h Syrap_ CoL~ton Res_ Soc_ T h e Behat~iour o f I o n s i n z~lacromolecular a n d B i o l o g i c a l Sys'tems, B r i s t o l x977, D_H. EVEREI-r, B . VINCENT ( E d i t o r s ) , p . 2ox, S c i e n t e c h n i c a , B r i s t o l (I978) [4] J . M . SE~U_~R[S, P_ V.ALENTA, H . I V . ~ U R N B E R 0 a n d B . ~IALFOY, P r o c . 2 9 t h S y r u p . C o l s t o n Res_ Soc_ T h e B e h a v i o u r o f I o n s in. ~ [ a c r o m o l e c u l a r a n d B i o -
Electrochemical. Behavior
[5] [6] [7]
[8] [9] [xo] lit]
[I2] [I3] [i4] [15] [16] [17]
[18] [x9]
[2O]
[2I] [2--.] [23] [24] [25] [26] [27] [28] [29] [3o] [3x] [32]
of Cytidine Phosphates
721
iogical S y s t e m s , B r i s t o l i 9 7 7 , D . H . E V E R E ~ r , B . V I n C E n T ( E d i t o r s ) , p . 2 3 0 , S c i e n t e c h n i c a , B r i s t o l (1978) V. VETTERL, Collect. Czech. C h e m . C o n z m u n . 31 (1966) 2 1 o 3 V. VETTERL, J. Electroanal- Cltem. [ n t e r f a c i a l E l e c t r o c h e m . 19 ( I 9 6 8 ) r 6 9 T.~V. XVEBB, B . J A ~ t K a n d P . J . ELVING, J . 2{nz. C h e m . Soc. 95 ( I 9 7 3 ) 9 9 I J.~V. ~VEBB, B . JaNIK a n d P . J . ELVIXG, J . A , n . Chem_ Soc. 95 (1973) 8495 U . RETTER, H . JEHRI.XG a n d V. VETTERL, J . E l e c t r o a n a l - C h e m . [ n t e r f a c i a l E l e c t r o c h e m . 57 {I974) 391 D_ KRZ-~ARId, P . V&LE~T.~ -~nd H.XV. N~R.~BERG, J . E l e c t r o a n a l . C h e m . [ n t e r f a c i a l E l e c t r o c k e m . 65 ( I 9 7 5 ) 863 P_ V.~L~_~TA, H.~V. NUR~BERG a n d D . KRZ~ARIC, BioelectrocRem. Bioe~zerg. 3 (1976) 418 V- VETTERL, Bioelectrochen,. B i o e n e r g . 3 ( I 9 7 6 ) 338 M . A . JE.'qSE,x, T . E . CUMMINGS a n d P . J . ELVING, Bioelectrochem. B i o e n e r g . 4 (1977) 4 4 7 D . KRZ~.akRI~, P . V.~-LE~TA, H.v~V. ~L]R-~BERG a n d M. BRA,~IC_~,, f . E l e c t r o a n a l . C h e m . [ n t e r f a c i a l E l e c t r o c k e n L 93 (1978) 4 I V . BR.~m:c. S . D . CHRISTtA-X a n d I ) . DRX'ttURST. B i o e l e c t r o c h e m . B i o e n e r g . 5 ( I 9 7 8 ) 635 Y . M . TEMERK a n d P . VALEXTA, J. Electroanal- C h e m . [ n t e r f a c i a l Electroc h e m . 93 (197 S) 57 Y.L~[. TE.MERK, P . V.*,LE.~T_~, H . vt~r. ~UR.~BERG. J . Electroanal- C h e m . I~zt e r f a c i a l E l e c t r o c h e m . 10O (1979) 77 ~[_ K-~.'ITZ, T . M . CU~I3tt,XGS a n d P . J . ELVI.XG, B e t . B u n s e n g e s . P h y s . C h e m . 83 (1979) 614 Y . ~ I . THMHRK, P . V.~,LENTA a n d H.XV. ~URYBERG, i n E l e c t r o a n a l y s i s i n H y g i e n e , E n v i r o n m e n t a l , C l i n i c a l a n d P h a r m a c e u t i c a l Cizemistry, XV.F. ~3IYTH ( E d i t o r ) , E l s e ~ 4 e r , A m s t e r d a m (x9So) p p . x x 3 - - r 2 6 M. LE.~G a n d FELS~XFELD, J . . ' ~ l o l - B i o l . 15 (1966) 455 G . D . FASXtAN, C. LIXDBLOXV a n d L . GROSM&N, B i o c h e m i s t r y 3 (1964) 1 o t 5 J . P . ,XIcT.~,GUE, U . R o s s a n d J . H _ GIBBS, B i o p o l y m e r s (1964) t 6 3 P . O . P _ T s ' o a n d S . I . CHAX, J . A m . Cltem. Soc. 86 ( I 9 6 4 ) 4 t 7 6 H . JEHRIKG, E l e c t r o s o r p t i o n s a n a l y s e m i t der IVechselstrompolarographie, A k a d e m i e V e f l a g , B e r l i n 1974 P . VALENTA a n d P . GRAH.MAN.~, J . t~lectroanal- Cicero. I n t e r f a c i a l . t~lectroc h e m . 49 ( I 9 7 4 ) 41 P . VALE,~Ta, H.~V. ~URNBERG a n d P . KLAHRE, Bioelectroclzem. Bioetterg. 2 (1975) 204 B . ~I.~LFOY, J . ~ [ . ~EQUARIS, P . Xr_~,LE_~T.aka n d H.XV_ ~ 0 R N B E R G , B i o e l e c t r o c h e m . B i o e n e r g . • (1976) 44 ° J . M . SEQUARIS, g . MALFOY, P . VALENT_~- a n d H.XV. NURXB~'RG, Bioele6trothem_ Bioenerg_ ~ ( i 9 7 6 ) 4 6 I g . ~[ALFOY, J . M . SEQUARIS, P . VALEXTA a n d H_XV. ~ 0 R S B E R G , J . E l e c t r o anal. C h e m . I n t e r f a c i a l E l e c t r o c h e m . 75 (1977) 455 B . Jx,XIK a n d P . J . ELVI~G. J . . 4 m . C h e n . Soc. 92 ( I 9 7 o) 235 e . J A N I K a n d P . J . ELVING, C k e m . B e y . 68 ( I 9 6 8 ) 295 A . ~ . FRUMKIN, Z . P h y s . ~5 ( I 9 2 6 ) 792
7zz
-
_ T e m e r k . Xralent~ a n d N f i r n b e r g
[33] _A_,~. FguMgt,'¢o Ewgeb_ E x a k t e n N a t - ~ m i s s _ T (Z928) 235 [34] A.~-. FRUMKt.~, itX A d u a n c ~ s i n E l e c t r o c h e m i s t r y a n d E l e c t r o c k e m i c a l E n ~ n e e r z n g P . Dwx_~uAY a n d C . W . T o s z - ~ ( E d i t o r s ) . I n t e r s c i e n c e , N e w Y o r k ( x 9 6 I ) Vol_ 1, p . 65 [35] E_ KOGLI~, J . M . SEQUARIS a n d P . VALE,'~TA, f . ~l/[ol, S t r i c t . 60 (x98o) 4 2 I [36] K_M_ ERVIN, E_ KOGLIN, J.M_ SEQUARXS. P_ VALE~TA a n d H . W . NORNBERG, J. E l e c t r o a n a l . C h e m . I n t e r f ~ w l a l E l e c t r o c h e m . i n p r e s s