6. Dhclus: algoritmo de clustering jer´ arquico divisivo
6.5. An´ alisis de la complejidad
Un less o t h e r w i s e s t a t e d , a l l s o l v e n t s and c h em ica ls used were o f a n a l y t i c gra d e o r were p u r i f i e d by d i s t i l l a t i o n . Anhydrous s o l v e n t s were p re p ared as f o l l o w s .
D i e t h y l E th e r
AR d i e t h y l e t h e r was heated to r e f l u x o v e r , and f r a c t i o n a l l y d i s t i l l e d from L i A l l l ^ , under a stream o f n i t r o g e n , b . p . 35°C.
P y r i d i n e
AR p y r i d i n e was heated t o r e f l u x w it h KOH p e l l e t s f o r
1 hour, f r a c t i o n a l d i s t i l l a t i o n gave anhydrous p y r i d i n e ( b . p .
115-116 ° C ) , s t o r e d o v e r KOH.
Dichloromet liane
Dichlo romethane was d i s t i l l e d from calc ium h y d r id e b . p .
4C°C.
Di met Ity tsn 1 pliox i de
Heated to r e f l u x o v e r Call, f o r 2 hours f o l l o w e d by f r a c t i o n a l d i s t i l l a t i o n b . p . 85-87°C.
Petrol Et lier
1 4 6 - -
bonate and ag ain w a t e r , d r i e d w it h calc iu m c h l o r i d e and then f r a c t i o n a l l y d i s t i l l e d .
C hlorof orm
E t h a n o l - f r e e c h lo r o fo r m was pre p are d by p as s in g AR c h lo r o f o r m down a s h o rt alumina column.
NADH
NADU was p u r i f i e d by d i s s o l v i n g in water to g i v e the r e q u ir e d c o n c e n t r a t i o n . This was then e x t r a c t e d w i t h d i e t h y l e t h e r to remove s t a b i l i s i n g e t h a n o l , and the r e s u l t a n t s o l u t i o n p la c e d on a r o t a r y e v a p o r a to r t o remove r e s i d u a l d i e t h y l e t h e r .
Water
In an attempt to remove the t r a c e s o f acetone mentioned p r e v i o u s l y (Chapter 2, c y c lo p r o p a n e s ) and any o t h e r t r a c e i m p u r i t i e s , doubly d i s t i l l e d water was heated a t r e f l u x temper ature w it h a l k a l i n e KMnO^ f o r 24 ho ur s. The water was then f r a c t i o n a l l y d i s t i l l e d t w i c e , but g . l . c . a n a l y s i s s t i l l showed the presenc e o f a t r a c e im pu ri ty that co-chromatographed w ith a c e to n e .
P r e p a r a t i v e t . l . c . p l a t e s
As mentioned in the t e x t ( e . g . page 9 6 ), the *H n . m . r . s p e c t r o s c o p i c an aly s e s o f the products o f enzymic o x i d a t i o n s were o f t e n c o m p lic a t e d by resonances from t r a c e i m p u r i t i e s . To minimise th es e problems, e i t h e r AR grade s o l v e n t s were used o r the s o l v e n t s were p u r i f i e d as d e s c r ib e d e a r l i e r in t h i s Chapter (p ag e 140). The
p r e p a r a t i v e t . l . c . p l a t e s used f o r p u r i f i c a t i o n o f th e prod ucts were f i r s t e l u t e d w i t h I0Z MeOH in CH2C12 , d r i e d and r e a c t i v a t e d
b e f o r e use.
Enzymic O xid atio n s
Enzymic o x i d a t i o n s were c a r r i e d out on two d i f f e r e n t s c a l e s , the s m a ll e r s c a l e o x i d a t i o n b e in g used as an a n a l y t i c run t o see i f o x i d a t i o n had o c c u r r e d . A number o f c o n t r o l s were used i n c lu d in g the i n c l u s i o n o f a c e t y l e n e (a known i n h i b i t o r o f the M.C. monooxygenase) and cya nid e which i n h i b i t s the a l c o h o l dehydrogena se but not the monooxygenase.
Exp erim ental
For o x i d a t i o n s o f s o l i d o r l i q u i d compounds f i v e 5 cm3 c o n i c a l f l a s k s were used and the c o n te n ts o f each i s l i s t e d below. F la s k I F la sk 2 Flas k 3 Fla s k 4 Phosphate b u f f e r , 20 mnolar, pH 7, (0 . 4 cm ) , - 3 s u b s tra te (5 x 10 Phosphate b u f f e r , 20 mmolar, pH 7, (0 . 4 cm ) , - 3 s u b s tra te (5 x 10 Phosphate b u f f e r , 20 mmolar, pH 7, (0 .4 cm )
3
Phosphate b u f f e r , 20 nm olar, pH 7, (0 . 4 cm ) , (5 x 10"39) , KCN s o l u t i o n , 0.5 im o l a r s u b s tr a teF la sk 5 Phosphate b u f f e r , 20 mmolar, pH 7, (0 . 4 cm3) , -3
s u b s t r a t e (5 x 10
g)
3 The f l a s k s were then suba s e a le d and t o f l a s k 5 one cm o f a c e t y l e n e was added from a s y r in g e d i s p l a c i n g an e q u iv a le n t amount o f a i r . The f l a s k s were then warmed to 45°C on a shaking water
-3
3
bath and M.C. e x t r a c t (100 x 10 cm ) was added by s y r i n g e , alo ng -3 3
w it h NAD11 s o l u t i o n 0.1 molar (50 x 10 cm ) . No NADH s o l u t i o n was added t o f l a s k 2. The f l a s k s were then r etu rn e d t o the shaking water
3
bath f o r 60 minutes a f t e r which they were e x t r a c t e d w it h ( 1 cm ) which
was removed, d r i e d (MgSO^), f i l t e r e d and c o n c e n t r a t e d to
oa.
100 x“ 3 3 . «
10 cm w ith a stream o f n i t r o g e n . The c o n te n ts o f the f l a s k s were then c h r o m a t o g r a p h ic a lly a n a ly s e d .
F or gaseous o r v o l a t i l e s u b s t r a t e s the same procedure was used
3
w it h the e x c e p t i o n th a t the s u b s t r a t e ( 1 cm o f v ap ou r) was added by
s y r i n g e a f t e r the f l a s k s were suba s e a l e d . A ls o the used to e x t r a c t the pro duc ts was added by s y r i n g e b e f o r e the suba s e a l was removed.
L a r g e r s c a l e ox id a tio n s d esig ne d to g i v e s u f f i c i e n t product f o r s p e c t r o s c o p i c a n a l y s i s (wz. 27, c o n v e r s i o n ) were c a r r i e d out as f o l l o w s .
A 250 cm3 c o n i c a l f l a s k was charged w i t h phosphate b u f f e r ,
20 mmolar, pH 7 ( 8 cm3) and 0.1 g o f s u b s t r a t e . The f l a s k was then
suba s e a le d and warmed to 45°C on a shaking w a t e r bath. NADH s o l u t i o n ,
- 3
0 . 1 molar ( I cm ) was then added a lo n g w i t h crude e x t r a c t ( 2 cm )
and the f l a s k was r etu rn e d t o the water bath f o r 1 hour. The con te nts o f the f l a s k were then e x t r a c t e d w ith (3 x 20 cm3) with
c e n t r i f u g a t i o n , where n e c e s s a r y , t o a s s i s t s e p a r a t i o n o f the l a y e r s . The CH,,C12 a l i q u o t s were then d r i e d (MgSO^), f i l t e r e d and the s o lv e n t
removed by r o t a r y e v a p o r a t o r . The product was then p u r i f i e d by chromatography. I f the s u b s t r a t e s were v o l a t i l e o r gaseous
1 4 9 * - '
Q <1
the r e a c t i o n was c a r r i e d out in a 50 cm c o n ic a l f l a s k and 6 cmJ
o f the vapour was added by s y r i n g e . Dichloromethane was added to e x t r a c t the products b e f o r e the suba s e a l was removed.
E xc e p ti on s t o the above work up procedure were as f o l l o w s : in the case o f the o x i d a t i o n o f methylcyclopropane
3
con tin uo us e x t r a c t i o n by d i e t h y l e t h e r ( 1 0 0 cm ) was r eq u ire d
2
f o r a p e r i o d o f 48 hours; a f t e r th e o x i d a t i o n o f C l - H]pro pene,
3
benzene ( I cm ) was added, the p ro d u c t was e x t r a c t e d and the benzene
I 2
la y e r s e p a r a t e d t d r i e d (MgSO,) , f i l t e r e d and analysed by H and H NMR d i r e c t l y .
I t i s p o s s i b l e that p ro d u c ts from the enzymic o x i d a t i o n s undergo f u r t h e r o x i d a t i o n or some o t h e r type o f r e a c t i o n . For
cytochrome P4 5 0 i t has been argu ed * th at a second o x i d a t i o n i s u n l i k e l y .
This i s because once the product has been r e le a s e d from the a c t i v e s i t e i t would be p re s e n t in a r e l a t i v e l y la r g e pool o f s u b s t r a t e and hence s t a t i s t i c a l l y u n l i k e l y t o encou nter the enzyme a second tim e. A l s o , i t has been argued th at i n c o r p o r a t i o n o f an oxygen atom i n t o the s u b s t r a t e w i l l i n c r e a s e i t s p o l a r i t y and conse qu en tly d e cr ease i t s a f f i n i t y f o r the hydrophobic a c t i v e s i t e . The f i r s t argument i s r e l e v a n t to the M.C. enzyme, p a r t i c u l a r l y as the c o n ve r s io n s o f s u b s t r a t e t o product ar e g e n e r a l l y low (ca. 1%). No products were e v e r seen on t . l . c . a n a l y s i s t h a t could be a s c r ib e d to f u r t h e r o x i d a t i o n .
Where p o s s i b l e the enzymic products were a l s o an alysed by mass s p e c tr o s c o p y and the r e s u l t a n t s p ec tr a compared with those ob ta in e d from a u t h e n tic samples o f the compounds. T h i s , ho we ver, was o f l i m i t e d use due t o the i m p u r it ie s in the products g i v i n g r i s e t o anomalous peaks i n the s p e c tr a .