Electrochemistry of Reduced Pterin Cofactors - Advances in Chemistry

20 Electrochemistry of Reduced Pterin Cofactors GLENN DRYHURST, R. RAGHAVAN, DENIZ EGE-SERPKENCI, and LIONEL G. KARBER

Downloaded by UNIV OF ARIZONA on January 9, 2013 | http://pubs.acs.org Publication Date: June 1, 1982 | doi: 10.1021/ba-1982-0201.ch020

University of Oklahoma, Department of Chemistry, Norman, OK 73019

The redox chemistry of 5,6,7,8-tetrahydropterin (THP) and its 6-methyl and 6,7-dimethyl derivatives has been studied by electrochemical and related methods. The initial electrooxidation of the tetrahydro compounds is an almost reversible 2e-2H process giving an unstable quinonoid-dihydropterin, which rearranges in a first-order reaction to give the corresponding 7,8-dihydropterins. The latter species react with water to form an equilibrium mixture of covalently hydrated and non-hydrated 7,8-dihydropterins. The covalently hydrated 7,8-dihydropterins are structurally similar to the tetrahydropterins and are therefore electrooxidized at almost the same potentials. This reaction is also a quasi-reversible 2 e - 2 H process giving another unstable quinonoid. The latter compound undergoes a rather fast chemical rearrangement to another intermediate, thought to be an isomeric, but more stable quinonoid. The quinonoids formed from covalently hydrated 7,8dihydropterin break down to an equimolar mixture of pterin and 7,8-dihydroxanthopterin. The quinonoids formed by oxidation of the hydrated forms of 6-methyl and 6,7-dimethyl-7,8-dihydropterin break down to the corresponding methylated pterins. The non-hydrated forms of the 7,8-dihydropterins are electrooxidized to the corresponding pterins but at more positive potentials. +

+

e t r a h y d r o b i o p t e r i n (1) is t h e n a t u r a l c o f a e t o r for a n u m b e r o f h y d r o x y l a s e e n z y m e s that u t i l i z e m o l e c u l a r o x y g e n to i n t r o d u c e a 0065-2393/82/0201-0457$08.75/0 © 1982 A m e r i c a n C h e m i c a l Society In Electrochemical and Spectrochemical Studies of Biological Redox Components; Kadish, K.; Advances in Chemistry; American Chemical Society: Washington, DC, 1982.

BIOLOGICAL REDOX COMPONENTS

458

Ο

Η CHOHCHOHCH

3

Η


l h y d r o x y l g r o u p into the aromatic a m i n o acids p h e n y l a l a n i n e , tyrosine, a n d t r y p t o p h a n (1-3). F o l l o w i n g h i s i n i t i a l s t u d i e s o n t h e h y d r o x y l a tion o f p h e n y l a l a n i n e , K a u f m a n ( 4 - 6 ) proposed S c h e m e I to e x p l a i n the e n z y m i c h y d r o x y l a t i o n o f p h e n y l a l a n i n e b y o x y g e n i n the presence o f p h e n y l a l a n i n e h y d r o x y l a s e a n d a t e t r a h y d r o p t e r i n cofactor. T h u s , i n addition to oxidation o f p h e n y l a l a n i n e to tyrosine, a n oxidation o f the cofactor to a q u i n o n o i d - d i h y d r o form was proposed. A s e c o n d e n z y m e , d i h y d r o p t e r i d i n e reductase, catalyzes reduction o f the q u i n o n o i d to the tetrahydro l e v e l u s i n g the reduced form o f nico t i n a m i d e a d e n i n e d i n u c l e o t i d e ( N A D H ) as t h e r e d u c i n g a g e n t . I n t h i s s c h e m e , t h e r e is c l e a r l y n o o b v i o u s f u n c t i o n a l r o l e for t h e tet r a h y d r o p t e r i n c o f a c t o r o r a p l a u s i b l e r o u t e for a c t i v a t i o n o f o x y g e n . N u m e r o u s theories a n d suggestions s u b s e q u e n t l y have b e e n a d v a n c e d to e x p l a i n t h e r o l e o f t e t r a h y d r o b i o p t e r i n a n d o t h e r t e t r a h y d r o p t e r i n p s e u d o - c o f a c t o r s i n s u c h h y d r o x y l a t i o n r e a c t i o n s (4-14). M e c h a n i s t i c S c h e m e I I p r o p o s e d b y H a m i l t o n (10) t o e x p l a i n t h e h y d r o x y l a t i o n o f p h e n y l a l a n i n e b y oxygen i n the presence of p h e n y l a l a n i n e h y d r o x y lase a n d t e t r a h y d r o b i o p t e r i n appears to b e one o f the more w i d e l y a c c e p t e d . I n this m e c h a n i s m ( S c h e m e II), m o l e c u l a r o x y g e n is p r o p o s e d t o a t t a c k t h e t e t r a h y d r o p t e r i n c o f a c t o r (I, S c h e m e II) t o g i v e a h y d r o p e r o x i d e (II, S c h e m e I I ) . T h i s r e a c t i o n o r i g i n a l l y w a s s u g g e s t e d b y K a u f m a n (7-9). T h e p u t a t i v e h y d r o p e r o x i d e t h e n c l e a v e s across t h e C ( 4 a ) - N ( 5 ) b o n d g i v i n g a c a r b o n y l o x i d e (III, S c h e m e I I ) . T h i s " o x e n e " reagent was thought t o b e the active h y d r o x y l a t i n g agent b y transferring a n o x y g e n a t o m to t h e substrate S (i.e., a r o m a t i c a m i n o a c i d ) w i t h f o r m a t i o n o f t h e p y r i m i d i n e , IV ( S c h e m e I I ) . C y c l i z a t i o n o f t h e p y r i m i d i n e g i v e s q u i n o n o i d - d i h y d r o p t e r i n (V, S c h e m e I I ) , w h i c h t h e n is r e d u c e d t o t h e c o r r e s p o n d i n g t e t r a h y d r o p t e r i n b y N A D H i n t h e p r e s e n c e o f d i h y d r o p t e r i d i n e r e d u c t a s e . T h e fact t h a t 2 , 5 , 6 - t r i a m i n o 4 - p y r i m i d o n e a n d 5 - b e n z y l a m i n o - 2 , 6 - d i a m i n o - 4 - p y r i m i d o n e also c a n f u n c t i o n as c o f a c t o r s f o r p h e n y l a l a n i n e h y d r o x y l a s e a n d t h a t b o t h c o m p o u n d s a r e c l e a v e d t o a n o x i d i z e d p y r i m i d i n e a n d a n a m i n e (15) g i v e s s o m e s u p p o r t for t h e r i n g c l e a v a g e m e c h a n i s m (II —» III IV, S c h e m e II). Unfortunately, very little experimental

evidence

supports the

In Electrochemical and Spectrochemical Studies of Biological Redox Components; Kadish, K.; Advances in Chemistry; American Chemical Society: Washington, DC, 1982.

DRYHURST ET AL.

Reduced Pterin C of actors


20.


459



460


20.

DRYHURST ET A L .

Reduced Pterin C of actors

461

e x i s t e n c e o f t h e i n t e r m e d i a t e s p e c i e s p r o p o s e d i n S c h e m e I I or i n d e e d in the other reaction m e c h a n i s m s proposed ( 4 - 1 4 ) . K a u f m a n (7) d e tected a transient intermediate ( X

m a x

= 250 and 290 nm) d u r i n g hy

d r o x y l a t i o n o f p h e n y l a l a n i n e at p H 8 - 8 . 2 i n t h e p r e s e n c e o f l o w c o n centrations o f tetrahydrobiopterin a n d h i g h h y d r o x y l a s e concentrations


(7-9). This intermediate

was proposed

to b e a 4 a , 5 - h y d r a t e (2).

H 2 H o w e v e r , other m e c h a n i s m s h a v e n o t f o u n d i t necessary to i n v o k e such a n intermediate

(e.g., S c h e m e I I ) . F u r t h e r m o r e , t h e s p e c t r a l l y

distinct intermediate noted b y Kaufman ( 7 - 9 ) apparently can b e ob s e r v e d o n l y w h e n t e t r a h y d r o b i o p t e r i n is t h e cofactor, not w h e n other pseudo-cofactors are e m p l o y e d . L o s s o f a m i n e f r o m p y r i m i d i n e c o f a c t o r s ( 1 5 ) is c o n s i s t e n t w i t h t h e r i n g o p e n i n g reaction o f the p t e r i n cofactors n o t e d i n S c h e m e I I . H o w ever, n o direct e v i d e n c e

supports

the existence

o f a ring-opened

i n t e r m e d i a t e o r , i n fact, a n y o x y g e n a t e d i n t e r m e d i a t e . fragmentation

Furthermore,

o f p y r i m i d i n e c o f a c t o r s is n o t p r o o f o f a s p e c i f i c a c t i

v a t e d o x y g e n r e a g e n t s u c h as v i n y l o g o u s o z o n e [ ( 1 5 ) S c h e m e I I ] . Mechanisms have been

p r o p o s e d f o r t h e a u t o o x i d a t i o n o f tet

r a h y d r o b i o p t e r i n a n d tetrahydrofolic a c i d that i n v o l v e t h e i n t e r m e d i a c y o f r a d i c a l s p e c i e s r a t h e r t h a n h y d r o p e r o x i d e s (11-13). R a d i c a l s a l s o w e r e r e p o r t e d as i n t e r m e d i a t e s i n c h e m i c a l o x i d a t i o n s o f t e t r a h y d r o p t e r i n s w i t h H 0 i n t r i f l u o r o a c e t i c a c i d (16) o r i o d i n e i n a l c o h o l (17). Q u i n o n o i d - d i h y d r o p t e r i n s h a v e b e e n p r o p o s e d as t h e i n i t i a l , u n stable d i h y d r o products o f c h e m i c a l oxidation o f tetrahydropterins (18-21). A r c h e r a n d S c r i m g e o u r ( 1 8 ) , f o r e x a m p l e , o x i d i z e d 6 , 7 - d i methyl-5,6,7,8-tetrahydropterin w i t h f e r r i c y a n i d e a n d p r o p o s e d that t h e c o r r e s p o n d i n g q u i n o n o i d - d i h y d r o p t e r i n is f o r m e d . Investigators r e p o r t e d (22) that 5,6,7,8-tetrahydropterin a n d its 6-methyl a n d 6,7-dimethyl derivatives give a single polarographic o x i d a t i o n w a v e at t h e d r o p p i n g m e r c u r y e l e c t r o d e ( D M E ) , w h i c h is c l a i m e d t o p r o c e e d b y a n EE m e c h a n i s m , t h e first s t e p b e i n g a o n e e l e c t r o n r e a c t i o n t o a c a t i o n r a d i c a l t h a t is o x i d i z e d f u r t h e r (~le, - 2 H ) to quinonoid-dihydropterin. A few cyclic voltammetric exper2

2

+



462

i m e n t s at a m e r c u r y e l e c t r o d e at p H 9 s h o w e d t h a t s o m e t e t r a h y dropterins give quasi-reversible couples a n d the oxidation peak was p r e s u m e d to b e a 2 e - 2 H r e a c t i o n t o g i v e q u i n o n o i d - d i h y d r o p t e r i n (19). T e t r a h y d r o f o l i c a c i d w a s r e p o r t e d to b e o x i d i z e d p o l a r o g r a p h i c a l l y at p H 6.8 i n a I e - 1 H r e a c t i o n to a n e u t r a l r a d i c a l t h a t c a n e i t h e r d i m e r i z e or u n d e r g o a d d i t i o n a l e l e c t r o o x i d a t i o n to q u i n o n o i d d i h y d r o f o l i c a c i d , w h i c h c a n rearrange to 7 , 8 - d i h y d r o p t e r i n a c c o m p a n i e d b y side c h a i n cleavage (23, 24). +


+

T h e a v a i l a b l e e v i d e n c e suggests that an unstable q u i n o n o i d d i h y d r o p t e r i n i n t e r m e d i a t e is f o r m e d d u r i n g e n z y m i c , c h e m i c a l , a n d e l e c t r o c h e m i c a l oxidation o f tetrahydropterins. T h i s two-electron defi c i e n t c o m p o u n d is u n s t a b l e a n d r a p i d l y r e a r r a n g e s to t h e c o r r e s p o n d i n g 7 , 8 - d i h y d r o p t e r i n (18). A careful r e v i e w o f the r e p o r t e d w o r k on the e n z y m i c o x i d a t i o n o f the a r o m a t i c a m i n o a c i d s r e v e a l s that the r o l e o f the t e t r a h y d r o p t e r i n c o f a c t o r is n o t r e a l l y u n d e r s t o o d . F u r t h e r m o r e , a n u m b e r o f c l i n i c a l a n d c h e m i c a l observations cannot b e r e c o n c i l e d w i t h any o f the pro p o s e d m e c h a n i s t i c schemes. F o r e x a m p l e , recent reports s h o w e d that e l e v a t e d l e v e l s o f a p t e r i n , b e l i e v e d to b e 7 , 8 - d i h y d r o x a n t h o p t e r i n (3), w e r e d e m o n s t r a t e d i n the u r i n e o f patients suffering from p h e n y l ketonuria ( P K U ) a n d w i t h d i h y d r o p t e r i d i n e reductase deficiency (25, 26). A l s o , a u t o x i d a t i o n ( 2 7 ) o f t e t r a h y d r o b i o p t e r i n at p H 7 . 5 , a n d c h e m i c a l o x i d a t i o n o f t e t r a h y d r o f o l i c a c i d (28) g i v e 7 , 8 - d i h y d r o x a n t h o p t e r i n . T h e o r i g i n o f the latter c o m p o u n d cannot b e r a t i o n a l i z e d on the basis o f t h e k n o w n c h e m i s t r y o f t e t r a h y d r o b i o p t e r i n or t e t r a h y d r o f o l i c a c i d . 1

C o n v e n t i o n a l studies o f the e n z y m i c h y d r o x y l a t i o n reactions that u t i l i z e t e t r a h y d r o p t e r i n cofactors, a n d c h e m i c a l a n d s i m p l e p o l a r o g r a p h i c studies o f the oxidations o f tetrahydropterins h a v e not pro v i d e d the r e q u i r e d u n d e r s t a n d i n g o f the c h e m i s t r y o f these cofactor s p e c i e s . M e c h a n i s t i c S c h e m e s I a n d I I s h o w e d t h a t i n o r d e r to u n d e r s t a n d the roles o f r e d u c e d pterins i n n o r m a l a n d a b n o r m a l b i o l o g i c a l reactions, one m u s t u n d e r s t a n d the f u n d a m e n t a l redox a n d related chemistry o f these c o m p o u n d s . A c c o r d i n g l y , w e i n i t i a t e d an investiga tion into the redox a n d related chemistry o f b i o l o g i c a l l y significant r e d u c e d p t e r i n s to u n d e r s t a n d m o r e c l e a r l y t h e b i o c h e m i c a l f u n c t i o n o f t h e s e c o m p o u n d s as cofactors i n e n z y m i c h y d r o x y l a t i o n a n d o t h e r p r o c e s s e s . T h e first s t e p i n t h i s i n v e s t i g a t i o n w a s to s t u d y t h e r e d o x c h e m i s t r y o f 5,6,7,8-tetrahydropterin ( T H P , 4), 6-methyl-5,6,7,8-tetrah y d r o p t e r i n ( 6 - M T H P , 5) a n d 6,7-dimethyl-5,6,7,8-tetrahydropterin ( 6 , 7 - D M T H P , 6) u s i n g e l e c t r o c h e m i c a l a n d r e l a t e d m e t h o d o l o g i e s . T h e s e c o m p o u n d s are a l l s t r u c t u r a l l y s i m p l e r t h a n t h e n a t u r a l c o f a c t o r

1

When phenylalanine hydroxylase is missing, or is present in abnormally low levels, phenylalanine cannot be hydroxylated to tyrosine. This condition results in a form of mental retardation.


DRYHURST ET A L .


20.

Reduced Pterin Cofactors

463

ΗΝ

H N 2

t e t r a h y d r o b i o p t e r i n (1), y e t a l l t h r e e c a n f u n c t i o n as c o f a c t o r s

2

for t h e

phenylalanine hydroxylase-catalyzed hydroxylation o f phenylalanine ( 2 9 ) . H o w e v e r , T H P l e a d s to f o r m a t i o n o f o n l y a b o u t o n e - h a l f o f t h e amount

o f tyrosine formed w h e n

tetrahydrobiopterin

or t h e

other

c o m m o n p s e u d o - c o f a c t o r s are u s e d . T h i s b e h a v i o r o f T H P is s a i d t o u n c o u p l e t h e e n z y m i c p r o c e s s , t h a t i s , to c a u s e f o r m a t i o n o f H 0 2

2

as

w e l l as t h e n o r m a l p r o d u c t ( 3 0 ) .

Experimental Chemicals. Pterin (2-amino-4-ketopteridine) was obtained from A l d r i c h . 7 , 8 - D i h y d r o p t e r i n ( 7 , 8 - D H P ) was synthesized u s i n g a modification (31) of the procedure described elsewhere (32). T h e procedure o f Bobst and V i s c o n t i n i (33) was m o d i f i e d slightly (31) to prepare T H P . T h e c o m p o u n d s 6 - M T H P and 6-methyl-7,8-dihydropterin ( 6 - M D H P ) were obtained from C a l b i o c h e m . T h e c o m p o u n d 6 , 7 - D M T H P (monohydrochloride or d i h y drochloride) was obtained from A l d r i c h , a n d 6,7-dimethyl-7,8-dihydropterin ( 6 , 7 - D M D H P ) was synthesized according to V i s c o n t i n i (34). 6 , 7 - D i m e t h y l pterin ( 6 , 7 - D M P ) was obtained from S i g m a , and 7,8-dihydroxanthopterin was prepared b y a p r e v i o u s l y p u b l i s h e d method (35). Apparatus. C o n v e n t i o n a l electrochemical e q u i p m e n t was used. T h i n layer spectroelectrochemistry u t i l i z e d a quartz c e l l c o n t a i n i n g an optically transparent reticulated vitreous carbon ( R V C ) electrode (ca. 0.7-mm thick, 100 pores per inch, F l u r o c a r b o n C o . ) . A t m o s p h e r i c oxygen was e x c l u d e d from the thin-layer c e l l u s i n g a m o d i f i e d design o f Norris et a l . (36). T h i n - l a y e r spec troelectrochemical experiments u t i l i z e d H a r r i c k r a p i d scan spectrometers 2

They commonly are referred to as pseudo-cofactors.



464

( M o d e l s Β a n d C ) . A l l potentials are reported vs. the saturated c a l o m e l elec trode ( S C E ) at 2 5 ° C . Voltammograms were obtained at a p y r o l y t i c graphite electrode ( P G E ) h a v i n g a surface area o f about 0.02 c m . Unless otherwise stated, experiments were carried out i n phosphate buffers h a v i n g an ionic strength o f 0.5 M . 2


Results T h e w o r k r e p o r t e d i n t h i s c h a p t e r is c o n c e r n e d p r i m a r i l y w i t h u n s u b s t i t u t e d p t e r i n s p e c i e s , t h a t is, p t e r i n , 7 , 8 - D H P a n d T H P . H o w ever, w h e n appropriate, a n y significant differences b e t w e e n the b e h a v ior o f t h e v a r i o u s o x i d a t i o n states o f p t e r i n a n d t h e 6 - m e t h y l a n d 6,7d i m e t h y l derivatives w i l l be noted. T o u n d e r s t a n d t h e e l e c t r o c h e m i s t r y o f T H P , o n e m u s t first u n d e r stand the b e h a v i o r o f p t e r i n a n d 7 , 8 - D H P . P t e r i n is n o t o x i d i z e d e l e c t r o c h e m i c a l l y at t h e P G E . H o w e v e r , i t does g i v e t w o w e l l - d e f i n e d r e d u c t i o n peaks (peaks I I a n d I I I , F i g u r e 1A). A t least four o x i d a t i o n p e a k s ( I V , D , I I a n d I I I , F i g u r e 1A) are o b s e r v e d o n t h e r e v e r s e s w e e p a n d t w o n e w r e d u c t i o n p e a k s (Ic a n d I ) a r e o b s e r v e d o n t h e s e c o n d c a t h o d i c s w e e p . P e a k s I I , I I I , a n d Ic w i l l be discussed subsequently under D H P . Peak clipping experiments r e v e a l t h a t o x i d a t i o n p e a k s I V a n d D a r e f o r m e d as a r e s u l t o f t h e r e d u c t i o n peak I I process ( F i g u r e I B ) . P e a k I V increases i n height r e l a t i v e to p e a k I I w i t h i n c r e a s i n g s w e e p r a t e . T h e p e a k s e p a r a t i o n b e t w e e n r e d u c t i o n p e a k I I a n d o x i d a t i o n p e a k I V w a s 3 5 ± 10 m V at a s w e e p rate o f 5 V / s . T h i s b e h a v i o r i n d i c a t e s that p e a k s I I a n d I V constitute a quasi-reversible redox couple. S c a n n i n g through peaks II a n d I I I a p p e a r s t o c a u s e t h e c u r r e n t for p e a k D to i n c r e a s e a n d for q u a s i - r e v e r s i b l e r e d u c t i o n p e a k I to a p p e a r ( F i g u r e I C ) . A t l o w p t e r i n concentrations, oxidation peak D , formed on s c a n n i n g through peak II a l o n e , is s m a l l c o m p a r e d to p e a k I I ; b u t p e a k D g r o w s r e l a t i v e to p e a k II w i t h increasing pterin concentration. T h i s result i m p l i e s that a s e c o n d - o r d e r p r o c e s s is i n v o l v e d i n t h e p e a k I I r e a c t i o n , w h i c h g e n e r ates t h e s p e c i e s r e s p o n s i b l e for o x i d a t i o n p e a k D . C

a

a

C

a

c

a

a

a

C

a

C

C

a

C

a

C

C

c

C

C

C

C

A t t e m p t s to m e a s u r e c o u l o m e t r i c η - v a l u e s o n c o n t r o l l e d p o t e n t i a l r e d u c t i o n o f p t e r i n at p e a k I I p o t e n t i a l s w e r e u n s u c c e s s f u l . R a t h e r l a r g e b a c k g r o u n d c u r r e n t s w e r e n o t e d t h a t a p p a r e n t l y w e r e r e l a t e d to extensive c o a t i n g o f the e l e c t r o d e surface. T h e error thus i n t r o d u c e d i n c o u l o m e t r i c m e a s u r e m e n t s w a s c o m p o u n d e d f u r t h e r b y t h e fact t h a t o n l y e x t r e m e l y d i l u t e solutions o f p t e r i n c o u l d b e r e d u c e d , o w i n g to its v e r y p o o r s o l u b i l i t y . H o w e v e r , f o l l o w i n g c o n t r o l l e d p o t e n t i a l r e d u c t i o n o f p t e r i n at p e a k I I p o t e n t i a l s , t h e s p e c t r u m o f t h e e l e c t r o l y z e d s o l u t i o n (e.g., at p H 7, X = 325, 274, a n d 229 nm) was very s i m i l a r to t h a t o f 7 , 8 - D H P ( X = 330, 280, a n d 229 nm). Furthermore, c y c l i c v o l t a m m o g r a m s on the e l e c t r o l y z e d solution c o n f i r m e d the C

C

m a x

m a x


DRYHURST ET AL.


20.

Reduced

1.0

0.5

Pterin

0

Cofactors

-0.5

-1.0

465

-0.5

P o t e n t i a l (V 2

2

e

7,8-DHP

225 225 225 228

5.20 6.00 7.00 7.95

7.5 ± 0.5 11 ± 5

3.0 ± 0.5 1.8 ± 0.5 1.0 ± 0.2' 1.0 ± 0.2' B

6,7-DMDHP

3.0 4.6 5.6 6.8 7.3

254 263 262 262 266 a

51 ± 0.2 55 ± 3 33 ± 5 36 ± 27 106 ± 29

2.9 ± 1.0 7.0 ± 2.5 5.4 ± 2.5 3.7 ± 0.4 9± 3

Containing an optically transparent RVC electrode. Phosphate buffers having an ionic strength of 0.5 M. Wavelength emploved to monitor A vs. t curve. k Values obtained by fitting A vs. t data with a nonlinear least-squares program

b c d

(39). Dihydropterin. k and k were indistinguishable. 6,7-Dimethyl-7,8-dihydropterin.

e f

x

9

2



476

C o n t r o l l e d p o t e n t i a l e l e c t r o o x i d a t i o n o f 7 , 8 - D H P at p e a k b e t w e e n p H 7 a n d 9 i n v o l v e d the transfer o f 2.5 ± 0 . 1 electrons

III

a

per

m o l e c u l e . V o l t a m m e t r i c a n d spectrophotometric analysis o f the p r o d u c t i n d i c a t e d a m i x t u r e c o n s i s t i n g o f 7 8 ± 6 % p t e r i n a n d 2 1 ± 6 % o f 7. E l e c t r o l y s i s o f 6 - M D H P a n d 6 , 7 - D M D H P at p e a k I I I

a

at p H 2 - 5 g a v e

η - v a l u e s o f 1.9 ± 0 . 2 , w i t h t h e c o r r e s p o n d i n g p t e r i n as t h e

product.

F r o m these data, w e c o n c l u d e d that the r e d o x c h e m i s t r y o f the 7,8-dihydropterins m a y b e r a t i o n a l i z e d o n l y i f the c o m p o u n d exists i n aqueous solution in an e q u i l i b r i u m i n v o l v i n g a covalently hydrated ( 7 , 8 - D H P - H 0 , E q u a t i o n 3) a n d n o n h y d r a t e d ( 7 , 8 - D H P , E q u a t i o n 3)


2

(3)

H

H

7,8-DHP

7,8-DHP-H 0 2

f o r m s . T h u s p e a k Ia is, o v e r a l l , a 2 e - 2 H the

hydrated

species

+

quasi-reversible oxidation o f

to g i v e a n u n s t a b l e

q u i n o n o i d species.

t a u t o m e r i c s t r u c t u r e s a r e p o s s i b l e for s u c h a q u i n o n o i d ( 8 - 1 2 ) .

Ο

Five The

Ο

N H - N

ΟΗ

OH Ν

Η Η

H,N

"Η

I

L / H

Η,Ν Η

10

11


20.

DRYHURST ET AL.


477

Ο


12 a c t u a l s t r u c t u r e for t h e p r i m a r y p e a k Ia q u i n o n o i d p r o d u c t , w h i c h is r e s p o n s i b l e for p e a k lé i n c y c l i c v o l t a m m e t r y , is n o t k n o w n . F o r p e a k I o f 7 , 8 - D H P , the k i n e t i c results support the v i e w that the p r i m a r y q u i n o n o i d p r o d u c t ( D H P - q u i n o n o i d - I , F i g u r e 2) u n d e r g o e s a firsto r d e r r e a r r a n g e m e n t to a s e c o n d , m o r e s t a b l e , i n t e r m e d i a t e , w h i c h w e s u s p e c t is o n e o f t h e o t h e r t h r e e q u i n o n o i d s p e c i e s ( D H P - q u i n o n o i d I I , F i g u r e 2). T h e s e i n t e r m e d i a t e s d e c o m p o s e to g i v e p t e r i n a n d 7,8d i h y d r o x a n t h o p t e r i n , as s h o w n i n F i g u r e 2 . A s n o t e d e a r l i e r , p e a k I I o f 7 , 8 - D H P is d u e t o 2 e - 2 H e l e c t r o o x i d a t i o n o f t h e l a t t e r s p e c i e s to x a n t h o p t e r i n , w h i c h i n t u r n is o x i d i z e d f u r t h e r i n t h e p e a k I V p r o c e s s . a

a

+

a

F o r t h e p e a k I p r o c e s s o f 6 - M D H P a n d 6 , 7 - D M D H P , i t is n o t p o s s i b l e to form 7 , 8 - d i h y d r o x a n t h o p t e r i n ; a n d h e n c e , f o l l o w i n g the c o n v e r s i o n o f q u i n o n o i d - I to q u i n o n o i d - I I , a s i m p l e d e h y d r a t i o n r e a c t i o n to t h e c o r r e s p o n d i n g p t e r i n m u s t o c c u r . a

+

O x i d a t i o n p e a k I I I is a 2 e - 2 H i r r e v e r s i b l e o x i d a t i o n o f t h e n o n h y d r a t e d 7 , 8 - d i h y d r o p t e r i n s p e c i e s to g i v e p t e r i n , as s h o w n i n F i g u r e 2. a

I n t h e c y c l i c v o l t a m m e t r y o f 7 , 8 - D H P , 7 is f o r m e d i n t h e p e a k I I r e a c t i o n ; a n d h e n c e , p e a k île a p p e a r s o n t h e r e v e r s e c y c l e ( F i g u r e 7 B ) . P e a k I I , w h i c h a p p e a r s o n l y at l o w p H , p r o b a b l y is d u e to e l e c t r o o x i dation o f a protonated form o f 7 , 8 - D H P - H 0 . a

a

2

L i n e a r s w e e p v o l t a m m e t r y o f T H P , 6 - M T H P , a n d 6 , 7 - D M T H P at t h e P G E i n d i c a t e d t h a t at l e a s t t h r e e p H - d e p e n d e n t o x i d a t i o n p e a k s are f o r m e d ( T a b l e I I I ) . F o r 6 - M T H P a n d 6 , 7 - D M T H P , p e a k I V ( T a b l e I I I ) r e s u l t s f r o m e l e c t r o c h e m i c a l o x i d a t i o n o f 6 - m e t h y l p t e r i n a n d 6,7d i m e t h y l p t e r i n (by c o m p a r i s o n w i t h the authentic material), respec t i v e l y , a n d w i l l n o t b e d i s c u s s e d further. P e a k I is a l w a y s t h e l a r g e s t p e a k at s l o w s w e e p rates f o l l o w e d , at m o r e p o s i t i v e p o t e n t i a l s , b y p o o r l y d e f i n e d p e a k s I I a n d I I I ( F i g u r e 1 0 A ) . A t s w e e p rates ^ 1 0 0 m V / s for T H P , for e x a m p l e , t h e d e c a y o f t h e p e a k I c u r r e n t f o l l o w s t h e t i m e c o u r s e e x p e c t e d for a s i m p l e , d i f f u s i o n - c o n t r o l l e d r e a c t i o n ( F i g u r e 1 0 B ) . H o w e v e r , at s l o w e r s w e e p rates, t h e rate o f c u r r e n t d e c a y is s i g n i f i c a n t l y l o w e r t h a n m i g h t b e e x p e c t e d for a n u n c o m p l i c a t e d diffusion-controlled reaction ( F i g u r e 10A). T h i s b e h a v i o r suggests that as a r e s u l t o f t h e p e a k I r e a c t i o n , a s p e c i e s is r a t h e r s l o w l y g e n e r a t e d a

a

a

a

a

a



478

Table I I I .

Peak Potential vs. p H Relationships for the Voltammetric Oxidation Peaks of 5,6,7,8-Tetrahydropterins

Peak

E p / V vs. SCE

pH Range" C

THP Ia Ia Ha IIIa

3-5.6 5.6-10.8 3-10.0 3-10.8

Ia Ha IIIa IV

2-11 2-5 3-11 3-11

0.404-0.073 0.180-0.033 0.510-0.040 0.980-0.058


6-MTHP"

a

0.320-0.056 0.420-0.055 0.730-0.051 1.62-0.064 e

6,7-DMTHP

0.309-0.053 0.442-0.055 0.847-0.078 0.630-0.021 1.51-0.052

2-11 2-4.1 2-4.1 4.1-10.6 2-11

Ia Ha IIIa IIIa IV a

a 6 c d e

Phosphate buffers having an ionic strength of 0.5 M. Measured at a sweep rate of 5 mV/s. 5,6,7,8-Tetrahydropterin. 6-Methyl-5,6,7,8-tetrahydropterin. 6,7-Dimethyl-5,6,7,8-tetrahydropterin.

t h a t u n d e r g o e s f u r t h e r e l e c t r o o x i d a t i o n at p e a k I p o t e n t i a l s . H e n c e , t h e d e c a y o f p e a k I , p a r t i c u l a r l y at s l o w s w e e p rates, is r e t a r d e d b y e l e c t r o o x i d a t i o n o f t h e n e w s p e c i e s . W i t h T H P , s w e e p rate s t u d i e s ( 0 . 2 - 5 0 V / s ) s h o w t h a t t h e p e a k c u r r e n t f u n c t i o n (i /ACv ) f o r p e a k I is 1 6 2 0 ± 1 3 0 μ Α c m m M v~ s , w h i c h is c l o s e t o t h e v a l u e e x p e c t e d for a r e v e r s i b l e , t w o - e l e c t r o n e l e c t r o d e r e a c t i o n ( 1 7 0 0 μ Α c m m M V " s ) . T h e l a t t e r v a l u e w a s c a l c u l a t e d u s i n g a v a l u e for t h e diffusion coefficient o f T H P o f 5 x 1 0 " cm /s. a

a

112

p

-

2

1/2

a

1/2

- 2

l / 2

1 / 2

6

2

C y c l i c v o l t a m m o g r a m s o f T H P ( F i g u r e 11) r e v e a l t h a t p e a k I is w e l l d e f i n e d at a s w e e p rate o f 2 0 0 m V / s , y e t t h e o t h e r o x i d a t i o n p e a k s are r a t h e r s m a l l . S i m i l a r b e h a v i o r w a s n o t e d for 6 - M T H P a n d 6 , 7 D M T H P ( F i g u r e 12). T h e b e h a v i o r o f T H P is, h o w e v e r , t h e m o s t c o m p l e x a n d t h e r e f o r e h a s b e e n s t u d i e d i n g r e a t e s t d e t a i l at t h i s t i m e . H a v i n g s c a n n e d t h e v a r i o u s o x i d a t i o n p e a k s o f T H P , at l e a s t f o u r reduction peaks are observed o n the reverse c y c l e ( I , I I , H e , IIIc, a n d s o m e t i m e s 1^, F i g u r e 11). N o n e o f t h e s e p e a k s is o b s e r v e d u n l e s s t h e o x i d a t i o n p e a k s a r e first s c a n n e d . R e d u c t i o n p e a k I f o r m s a n a l m o s t reversible couple w i t h oxidation peak I . A t relatively l o w p H values (e.g., < p H 4 . 3 f o r T H P , < p H c a . 5 for 6 - M T H P , a n d < p H 4 f o r 6 , 7 a

c

C

c

a


20.

DRYHURST ET A L .

Reduced

Pterin

479

Cofactors

D M T H P ) , a n a d d i t i o n a l c o u p l e ( p e a k s I a n d U, F i g u r e s 1 1 A a n d 1 2 A ) a

a p p e a r s o n t h e first c y c l e . W i t h T H P , o x i d a t i o n p e a k D a p p e a r s o n t h e second cycle. Peak I

c

decreases r e l a t i v e to p e a k I

a

w i t h d e c r e a s i n g s w e e p rate

for a l l t e t r a h y d r o p t e r i n s , i n d i c a t i n g t h a t t h e p r i m a r y p e a k I

a

p r o d u c t is

unstable a n d undergoes a c h e m i c a l follow-up reaction. C o n t r o l l e d p o t e n t i a l c o u l o m e t r i c o x i d a t i o n o f T H P at p e a k

III

a

p o t e n t i a l s at p H 7 i n d i c a t e d t h a t f o u r (3.8 ± 0.2) e l e c t r o n s p e r m o l e c u l e are transferred. V o l t a m m e t r y a n d s p e c t r a l analysis o f the p r o d u c t revealed

t h a t T H P is c o n v e r t e d

almost

quantitatively

(>90%)

to


pterin. C o m p o u n d s 6 - M T H P and 6 , 7 - D M T H P exhibited almost identi c a l b e h a v i o r . C o u l o m e t r y o n t h e r i s i n g p o r t i o n o f p e a k I for a l l t e t r a h y a

d r o p t e r i n s s t u d i e d i n d i c a t e d t h a t 2 ± 0.3 e l e c t r o n s p e r m o l e c u l e w e r e transferred. V o l t a m m e t r y a n d U V s p e c t r o s c o p y o f the p r o d u c t r e v e a l e d

Figure 10. Voltammograms at the PGE of 0.53 m M THP in phosphate buffer, pH 5.85, at sweep rates of A, 5 mVIs and B, 200 mVls. Points show i - t decay calculated from the Cottrell equation.




480

1.0

0.5

0

-0.5

-1.0

-1.5

Potential (V vs. S C E ) Figure 11. Cyclic voltammograms at the PGE of 0.54 m M THP in phosphate buffers at pH 3.4 (A), 7.05 (B), and 9.98 (C). Sweep rate: 200 mV/s. that the c o r r e s p o n d i n g 7,8-dihydropterin was formed. H o w e v e r , elec t r o o x i d a t i o n at, o r s l i g h t l y p o s i t i v e , o f p e a k I p o t e n t i a l s at a r o u n d p H 3 r e s u l t e d i n f o r m a t i o n o f a m i x t u r e o f p t e r i n a n d 3. T h e o t h e r t w o tet r a h y d r o p t e r i n s gave the c o r r e s p o n d i n g pterins. a

B e c a u s e c y c l i c v o l t a m m e t r y o f a l l t e t r a h y d r o p t e r i n s ( F i g u r e s 11 a n d 12) i n d i c a t e d t h a t t h e p e a k I r e a c t i o n g i v e s a n u n s t a b l e i n t e r m e diate, t h i n - l a y e r s p e c t r o e l e c t r o c h e m i c a l e x p e r i m e n t s w e r e c a r r i e d out. a

F i g u r e 1 3 A s h o w s t h e s p e c t r u m o f T H P at p H 7 ( X = 300 a n d 2 2 0 n m ) i n a t h i n - l a y e r c e l l . C u r v e 1 i n F i g u r e 1 3 B is t h e s p e c t r u m o b t a i n e d a f e w s e c o n d s after i n i t i a t i n g t h e e l e c t r o l y s i s , a n d C u r v e 2 is the s p e c t r u m a b o u t 55 s later. T h u s , o n e l e c t r o l y s i s , the T H P b a n d at 2 2 0 n m d e c r e a s e s a n d s h i f t s to l o n g e r w a v e l e n g t h s , a n d t h e b a n d at 3 0 0 n m shifts t o s h o r t e r w a v e l e n g t h s . A f t e r s c a n n i n g C u r v e 2 ( F i g ure 13B), the electrolysis was t e r m i n a t e d a n d the spectral changes m a x



Figure 12. Cyclic voltammograms at the PGE of 1.1 m M 6,7-DMTHP in phosphate buffers at pH 3.1 (A), 5.0(B), and 11.0 (C). Sweep rate: 50 mV/s.



482

Β

îi Γ\λ

i i.

0.4 AU

jfflr

1


Λ

\ \ 400

300

200 400 W a v e l e n g t h (nm)

1

ι

I

300

200

Figure 13. Spectra of 0.6 m M THP in phosphate buffer, pH 7, electrolyzing on the rising portion of peak I in a thin-layer cell containing a RVC electrode. A: spectrum of THP. B: Curve 1 is the spectrum a few seconds after initiation of the electrolysis; Curve 2 is the spectrum after 55 s electrolysis; Curve 3 is the spectrum after complete decay of the intermediate and corresponds to 7,8-DHP. In B, each scan was 18.8 s, with no significant interval between scans. a

s h o w n b e t w e e n C u r v e s 2 a n d 3 w e r e o b s e r v e d . T h u s , t h e p e a k s at 2 3 0 a n d 281 n m g r o w w h i l e the absorbance a r o u n d 330 n m decreases. T h e arrows i n F i g u r e 1 3 B p o i n t i n the d i r e c t i o n o f the latter absorbance c h a n g e s . C u r v e 3 is t h e s p e c t r u m o f t h e final p r o d u c t ( X = 332, 281, a n d 2 3 2 n m ) , w h i c h is c l o s e to t h a t e x p e c t e d for 7 , 8 - D H P . C l e a r l y , a n u n s t a b l e i n t e r m e d i a t e is f o r m e d t h a t s p o n t a n e o u s l y d e c o m p o s e s i n t o 7 , 8 - D H P . T h e kinetics o f this transformation were s t u d i e d b y monitor i n g either the d e c a y o f the U V absorbance o f the i n t e r m e d i a t e or the increase i n product absorbance w i t h time. A t a l l p H values studied, t h i s p r o c e s s w a s first o r d e r . S i m i l a r b e h a v i o r w a s o b s e r v e d o n e l e c t r o c h e m i c a l oxidation o f 6 - M T H P a n d 6 , 7 - D M T H P . Values o f the ob s e r v e d r a t e c o n s t a n t s a r e p r e s e n t e d i n T a b l e I V . T h e e x p e r i m e n t a l rate c o n s t a n t w a s i n d e p e n d e n t o f c o n c e n t r a t i o n for i n i t i a l T H P c o n c e n t r a t i o n s r a n g i n g f r o m 0.3 to 3 m M . T a b l e I V s h o w s t h a t a b e l l - s h a p e d r e l a t i o n s h i p b e t w e e n & o a n d p H o c c u r s for a l l t h r e e t e t r a h y d r o p t e r i n s , w i t h fcobs b e i n g m a x i m a l at p H 5 . 6 - 6 . 0 . T h e l a t t e r fact s u g g e s t s t h a t H P 0 ~ catalyzes the c h e m i c a l f o l l o w - u p reaction. I n d e e d , e x p e r i m a x

b s

2

4


DRYHURST ET A L .

20.


483

Table IV. Observed First-Order Rate Constants for the Chemical Reaction of the Intermediate Species Generated on Electrochemical Oxidation of Tetrahydropterins at Peak I a

a

2

pH

lb

k xl0 lsob8

c

THP


3.1 4.1 5.1 5.6 5.9 6.4 7.0 8.4

2.5 2.7 4.3 5.5 5.5 2.9 1.8 0.7

6-MTHP" 1.1 1.2 1.7 1.5 3.5 2.9 1.2 0.3 0.3 0.2

1.98 3.10 3.96 4.58 5.60 6.02 6.95 8.05 9.30 9.85 e

6,7-DMTHP 2.0 3.1 4.0 4.6 5.6 6.0 6.8 7.0 8.0 9.3

1.4 2.3 2.9 3.0 4.3 4.8 2.0 1.8 0.7 0.5

Note: Electrolysis carried out in a thin-layer cell containing an optically transparent RVC electrode. Phosphate buffers having an ionic strength of 0.5 M. Mean of at least three replicate determinations. 5,6,7,8-Tetrahydropterin. 6-Methyl-5,6,7,8~tetrahydropterin. 6,7-Dimethyl-5,6,7,8-tôtrahydropterin. a

6

c

d

e



484

m e r i t s r e v e a l t h a t for H P 0 ~ c o n c e n t r a t i o n s r a n g i n g f r o m 0 . 0 4 to 0.4 M 2

4

i n p H 5.45 s o l u t i o n s m a i n t a i n e d at a c o n s t a n t i o n i c s t r e n g t h o f 2 . 0 M w i t h N a C 1 0 , t h e v a l u e o f Kbs m e a s u r e d f o l l o w i n g o x i d a t i o n o f T H P 4

2

i n c r e a s e s s y s t e m a t i c a l l y f r o m 0 . 6 9 x 1 0 " to 3.5 x 1 0 "

2

B a s e d on these results, w e c o n c l u d e d that p e a k I hydropterins

+

is a n a l m o s t r e v e r s i b l e 2e-2H

1

s" . a

o f the tetra

o x i d a t i o n to g i v e

an

i n t e r m e d i a t e q u i n o n o i d - d i h y d r o p t e r i n ( F i g u r e 2). T h e r e a r e five p o s


s i b l e s t r u c t u r e s for s u c h a q u i n o n o i d ( 1 3 - 1 7 ) , b u t i n s u f f i c i e n t i n f o r m a -

H—Ν

H N

H N

2

2

14

13

Ο

Η 15

H N 2

Η

Η

ΗΝ 17

16

t i o n is a v a i l a b l e to d e c i d e w h i c h o f t h e s e s p e c i e s is p r e f e r r e d . H o w e v e r , t h e q u i n o n o i d i n t e r m e d i a t e r e a r r a n g e s i n a first-order r e a c t i o n t o g i v e the c o r r e s p o n d i n g 7 , 8 - d i h y d r o p t e r i n ( F i g u r e 2). T h i s c o m p o u n d t h e n p a r t i a l l y h y d r a t e s t o g i v e 7 , 8 - D H P - H 0 , w h i c h is o x i d i z e d i n t h e p e a k I p r o c e s s . T h e l a t t e r r e a c t i o n o c c u r s at p o t e n t i a l s v e r y c l o s e to p e a k I a n d a c c o u n t s for t h e u n u s u a l c u r r e n t d e c a y o f v o l t a m m e t r i c p e a k I at s l o w s w e e p rates (see F i g u r e 1 0 A ) . T h e c h e m i s t r y a s s o c i a t e d w i t h the r e m a i n d e r o f the peaks o b s e r v e d o n l i n e a r a n d c y c l i c s w e e p 2

a

a

a


DRYHURST ET A L .

20.


485

voltammetry of T H P (I , I I , I I , I I I , I V , I , I > He, He, H i e , I V , and a

a

a

a

a

c

c

a

D ) a n d d i s c u s s e d p r e v i o u s l y are s u m m a r i z e d i n F i g u r e 2. P e a k s I I , a

I V , a n d l i e a r e o b s e r v e d o n l y w i t h T H P b e c a u s e o n l y for t h i s c o m a

p o u n d c a n 3 b e f o r m e d . C o m p o u n d 3, w h i c h is r e s p o n s i b l e for p e a k I I , g i v e s 7, w h i c h i n t u r n is o x i d i z e d i n t h e p e a k I V a

a

process

and

r e d u c e d at p e a k l i é ·


Conclusions T h e e l e c t r o c h e m i c a l a n d related studies just reported indicate t h a t t h e r e d o x c h e m i s t r y o f T H P , i n p a r t i c u l a r , is q u i t e c o m p l e x . T h e i n i t i a l , quasi-reversible peak I process o f the tetrahydropterins stud i e d gives an unstable q u i n o n o i d o f uncertain structure. T h i s t w o e l e c t r o n - d e f i c i e n t s t r u c t u r e t h e n r e a r r a n g e s to t h e c o r r e s p o n d i n g 7,8d i h y d r o p t e r i n . T h e s e i n v e s t i g a t i o n s o f T H P , 6 - M T H P , a n d 6,7D M T H P g i v e n o e v i d e n c e for a n i n i t i a l o n e - e l e c t r o n r e a c t i o n g i v i n g a r a d i c a l i n t e r m e d i a t e , as p r o p o s e d b y o t h e r s ( 2 2 ) o n t h e b a s i s o f p o l a r o g r a p h i c s t u d i e s at a D M E . a

I n c o n c l u s i o n , t h e 7 , 8 - d i h y d r o p t e r i n finally f o r m e d as t h e e n d p r o d u c t o f t h e p e a k I r e a c t i o n r e a c t s w i t h w a t e r to f o r m a n e q u i l i b r i u m mixture o f covalently hydrated and nonhydrated species. T h i s h y d r a t i o n r e a c t i o n c l e a r l y is f a v o r e d i n a c i d s o l u t i o n b u t for 7 , 8 - D H P , t h e v o l t a m m e t r i c p e a k c o r r e s p o n d i n g to o x i d a t i o n o f 7 , 8 - D H P - H 0 ( p e a k I ) m a y b e o b s e r v e d at p H 7, i n d i c a t i n g t h a t t h e l a t t e r s p e c i e s e x i s t s to a n a p p r e c i a b l e e x t e n t at a r o u n d p h y s i o l o g i c a l p H . T h e h y d r a t e d f o r m s o f t h e 7 , 8 - d i h y d r o p t e r i n s a r e s t r u c t u r a l l y v e r y s i m i l a r to t h e t e t r a h y d r o c o m p o u n d s (e.g., c o m p a r e 7 , 8 - D H P - H 0 a n d T H P i n a

2

a

2

F i g u r e 2) a n d t h e r e f o r e , n o t s u r p r i s i n g l y , a r e e l e c t r o o x i d i z e d at n e a r l y t h e s a m e p o t e n t i a l as t h e t e t r a h y d r o c o m p o u n d s . T h i s o x i d a t i o n is a l s o a quasi-reversible 2 e - 2 H reaction g i v i n g another unstable q u i n o n o i d . T h e l a t t e r p u t a t i v e i n t e r m e d i a t e u n d e r g o e s a fast c h e m i c a l r e a c t i o n to g i v e a s e c o n d i n t e r m e d i a t e , t h o u g h t to b e a n i s o m e r i c , b u t m o r e s t a b l e , q u i n o n o i d . T h e q u i n o n o i d s f o r m e d from the c o v a l e n t l y h y d r a t e d forms o f 6 - M D H P a n d 6 , 7 - D M D H P r e a r r a n g e to g i v e t h e c o r r e s p o n d i n g m e t h y l a t e d pterins. H o w e v e r , the q u i n o n o i d intermediate generated o n p e a k I o x i d a t i o n o f 7 , 8 - D H P - H 0 b r e a k s d o w n to g i v e a m i x t u r e o f p t e r i n a n d 7 , 8 - d i h y d r o x a n t h o p t e r i n . A s n o t e d earlier, the latter c o m p o u n d h a s b e e n f o u n d i n p a t i e n t s s u f f e r i n g from P K U or a d i h y d r o pteridine reductase deficiency (25,26). I n addition, tetrahydrobiopterin (1) a n d t e t r a h y d r o f o l i c a c i d a r e t h o u g h t t o u n d e r g o c l e a v a g e at t h e C ( 6 ) - C ( l ' ) b o n d d u r i n g o x i d a t i o n ( I I , 4 0 , 41). S u c h a r e a c t i o n s h o u l d result i n the formation o f 7 , 8 - D H P a n d 7 , 8 - D H P - H 0 . T h e elec t r o c h e m i c a l results r e p o r t e d i n this chapter i n d i c a t e that the a l m o s t r e v e r s i b l e o x i d a t i o n p o t e n t i a l o f t h e l a t t e r s p e c i e s is v e r y s i m i l a r t o t h a t +

a

2

2


486


of T H P . Thus, 7 , 8 - D H P - H 0 quite possibly could function as a cofac tor in the aromatic amino acid hydroxylation with the formation of DHP-quinonoid-I/DHP-quinonoid-II intermediates (see Figure 2). The latter intermediates may not be reducible by dihydropteridine reductase and, in the case of a deficiency of the latter enzyme, could not be reduced. The result would be the formation of 7,8dihydroxanthopterin. This proposal is only one scheme to account for the appearance of the latter compound in the urine of patients with P K U or dihydropteridine reductase deficiency and must be regarded as speculative at this time. This fact is particularly true because the detailed redox chemistry of tetrahydrobiopterin, tetrahydrofolic acid, and their 7,8-dihydro derivatives is not known. However, as noted previously (41 ), 7,8-dihydrobiopterin might play an important role in many of the reactions of tetrahydrobiopterin.


2

Acknowledgments This work was supported by Grant No. GM-25842-03 from the National Institutes of Health. Additional support was provided by the Research Council of the University of Oklahoma. Literature

Cited

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Electrochemistry of Reduced Pterin Cofactors - Advances in Chemistry

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