Grupos de usuarios

In an attempt to f i n d a s u b s t r a t e f o r the monooxygenase from

Mathyloooccua capnulatuo

w it h the s t r u c t u r e shown in F i g . 3.6 (t h e reason f o r which w i l l be e x p la in e d in Chapter 4) i t was decided to t r y « - m e t h y l s t y r e n e . T h is s u b s tr a te was chosen on account o f i t s c o n ve n ie n t m o le c u la r s i z e and hence h a n d lin g c h a r a c t e r i s t i c s o f i t s

l i k e l y p r o d u c t ( s ) o f o x i d a t i o n .

R

F i g . 3.6

O x id a t io n o f « - m e t h y l s t y r e n e wit li the monooxygenase from

M. oapaulatua

gave a product which could not be i d e n t i f i e d by A. K. Wong'^, al th oug h a number o f l i k e l y products o f o x i d a t i o n (2- m e t h y l -2-p h e n y lo x ir a n , 2- p h e n y lp r o p -2- e n - l - o l and 2- p h e n y l p r o p -2- e n - l - n l ) had been s y n th e s is e d

to p r o v id e a u t h e n t i c standards f o r t . l . c . The author re-examined t h i s problem by c a r r y i n g out a sma ll s c a le o x i d a t i o n th at showed a s i n g l e product spot on t . l . c . a n a l y s i s . I n c r e a s i n g the s c a le o f the o x i d a t i o n and p u r i f y i n g t h i s product by p r e p a r a t i v e t . l . c . enabled a ' H NMR spectrum to be o b t a in e d . T h i s spectrum, shown in F i g . 3 .14 , shows a s i n g l e t methyl peak at 2 . 1 p .p.m . and f o r the o l e f i n i c

hydrogens, two s i n g l e t s at 4.9 and 5.2 p .p.m . The la r g e peak at 5.3 p.p.m. i s due to contamination w i t h dic hlo rom e th a nc . The A , ! ^ q u a r t e t t y p i c a l o f p a r a - d i s u b s t i t u t e d a r o m a tic compounds shows as two d ou b le ts c e n t r e d on 6.65 and 7.2 p .p.m . T h is i n t e r p r e t a t i o n o f the spectrum showed that the product was the phenol produced by

p a r a - h y d r o x y l a t i o n of the a r o m a t i c ring. T h i s c o n c l u s i o n w a s

supported by the u . v . spectrum which showed a ^ o f 258 nm. A d d i t i o n o f one drop o f 5 M NaOH r e s u l t e d in a movement o f t h i s peak to 282 nm and an i n c r e a s e in i t s e x t i n c t i o n c o e f f i c i e n t . A d d i t i o n o f I M MCI caused the peak t o move back t o i t s o r i g i n a l p o s i t i o n o f 258 nm w it h a c o r re s p o n d in g r e d u c t io n in peak h e i g h t . T his type o f behav iou r i s t y p i c a l o f ph e n o ls , tr eatm en t w i t h a base caus ing a b s t r a c t i o n o f the p h e n o lic hydrogen ( F i g . 3 . 7 ) . The r e s u l t a n t anion is s t a b i l i s e d by c o n ju g a t io n w ith the a r o m a tic r i n g g i v i n g a l a r g e r chromophorc w ith concomitant in c r e a s e in i t s

\

. A c i d i f i c a t i o n r e - p r o t o n a t e s t h i s anion w ith

max r e g e n e r a t i o n o f the phenol.

Attempts at f u r t h e r c h a r a c t e r i s a t i o n o f the product o f enzymic o x i d a t i o n o f a -m e t h y ls t y r c n e by e l e c t r o n impact mass s p e c tr o m e tr y were not e n t i r e l y s u c c e s s fu l due, p r o b a b ly , to s e l f - p r o t o n a t i o n o f the phenol to g i v e an apparent m o le c u la r ion at m/z o f 145 in s te ad o f 144.

The f a c t th at the major i s o l a b l e product was the p ar a- d i s u b s t i t u t e d arene suggeste d th at the o x i d i s i n g ag ent used by

M. capaulaLuo

i s e l e c t r o p h i l i c in n a tu r e, l i k e the s p e c ie s b e l i e v e d to p a r t i c i p a t e in cytochrome P-450 o x i d a t i o n s .

98

To see i f t h i s r e s u l t was r ep e a te d w ith o t h e r ar om at ic compounds we i n i t i a l l y chose e t h y lb e n z e n e as a s u b s t r a t e . The reason f o r t h i s c h o ic e was th at a l l p o s s i b l e products from mono- h y d r o x y l a t i o n o f the ar om ati c r i n g , i . e . , 2 - , 3- and 4 - e t h y l p h e n o l were r e a d i l y a v a i l a b l e , as were th e p o t e n t i a l products from o x i d a t i o n o f the s id e c h a in , i . e . , 1- and 2 - p h e n y l e t h a n o l . A s m a l l - s c a l e o x i d a t i o n was c a r r i e d out and the products were an alys ed by t . l . c . This showed two sp ots which were con fir m ed t o be p ro d ucts o f mono­ oxygenase a c t i v i t y by a p p r o p r i a t e c o n t r o l e xp erim ent s ( s e e M a t e r i a l s and Methods s e c t i o n ) . One o f thes e spots co-chromatographed w ith A -e th y lp h e n o l and the o t h e r w ith I - p h e n y l e t h a n o l . I n c r e a s i n g the s c a l e and a n a ly s in g the products by *H NMR s p e c tr o s c o p y supported th es e d e d u ct io n s . One product showed the A2B2 c o u p l i n g p a t t e r n i n th e ar om ati c

r e g i o n e xp e c te d from 4 - e t h y l p h o n o l . The second product showed a s i n g l e t at 6 7.25 p.p.m. and a q u a r t e t at A . 85 p.p.m . These s i g n a l s corre spon d

to the resonances in the *H NMR spectrum o f a u t h e n tic I - p h e n y l e t h a n o l , from i t s ar om atic and s i d e - c h a i n m eth ine, r e s p e c t i v e l y . The resonance from the methyl group was masked by i m p u r i t i e s , but th e c o u p l i n g c on stant o f the methine hydrogen t o t h i s methyl group was o b s e rv e d to be

ap p ro xim ate ly 6 Hz. A u . v . spectrum o f the 4 - e t h y l p h e n o l showed a

\

a t 280 nm th at on tr eatm en t w it h 5 M NaOH moved t o 288 nm and r eturn e d t o max

280 nm on tr eatm en t with I M HC1. As a f i n a l method o f c h a r a c t e r i s a t i o n g . l . c . a n a l y s i s was used. With a column o f SPI000 on Carbopac P i t was p o s s i b l e to s e p a r a t e the t h r e e e t h y lp h e n o l isom ers . From the product o f enzymic o x i d a t i o n o f e t h y l b e n z e n e , two predominant peaks were obs erved c o r re s p o n d in g to A - e t h y lp h e n o l and I - p h e n y l e t h a n o l . No s i g n i f i c a n t peak cou ld be seen th a t would be c o n s i s t e n t w it h the presence o f 2 -p h e n y le t h a n o l. A l s o , no peaks were seen t o c o ­ chromatograph w ith a u t h e n t ic 2- and 3 - e t h y l p h e n o l s . The acc ur ac y o f the an aly s e s f o r these phenols was r e l a t i v e l y poor because t h e i r r e t e n t i o n times were long and peaks were r a t h e r broad. However, i t

was p o s s i b l e to deduce that ÿ

27.

2- and $ 1% 3 - e t h y lp h e n o l had be en formed (

7.

o f the 4 - e t h y l p h e n o l isomer f o r m e d ).

The o b s e rv e d p a r a - h y d r o x y l a t i o n s imply an e l e c t r o p h i l i c c h a r a c t e r f o r the enzymic o x i d i s i n g a g e n t . The low y i e l d or absence o f m o ta -h y d r o x y la t e d produc t i s not s u r p r i s i n g . However, the non­ f o r m a tio n o f o r t h o - h y d r o x y l a t e d product needs some e x p l a n a t i o n . T h is i s p r o b a b ly a consequence o f the way the a c t i v e s i t e o f the M.C. mono-oxygenase binds the s u b s t r a t e . I f the a c t i v e s i t e i s a na rr ow d e p r e s s io n or c l e f t in the enzym e's s u r fa c e and the a r om ati c s u b s t r a t e binds in one o f the c o n f i g u r a t i o n s shown in F i g . 3 . 8 , then the s i t e s o f o x i d a t i o n would be determ ined p r i m a r i l y by t h e i r r e l a t i v e p r o x i m i t y to the o x i d i s i n g a g e n t .

I f the b in d in g co n fo rm a tio n were the o n l y c r i t e r i a then one would

e x p e c t , from the model shown, th at e t h y lb e n z e n e would g i v e 2_{- p h e n y le t h a n o l}

in p r e f e r e n c e to I - p h o n y l e t h a n o l . That t h i s does not occur may i n d i c a t e the need f o r the s u b s t r a t e to be a b l e t o s t a b i l i s e some t r a n s i t o r y charge in the o x i d a t i o n p r o c e s s . T h is p o i n t w i l l be d is cu ss e d l a t e r in th in Chapter and a l s o in Chapter 5.

O x i d a t i o n o f to lu e n e gave two produc t sp ots on t . l . c . a n a l y s i s that co-chromatographed w it h a u t h e n t i c p a r a - c r e s o l and b e n z y l a l c o h o l . Attempts t o s e p a ra t e the 2 - , 3- and 4 - c r e s o l isomers o r t h e i r 3 , 5 - d i n i t r o b e n z o a t e s by t . l . c . was u n s u c c e s s fu l. I n c r e a s i n g

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