with anhydrous ferne Chloride in glacial acetic acid (95 -100% yield). The ...... Anaspec Research Laboratories Ltd., Pearl House, Bartholomew St., Newbury, ...
Biological Techniques Series
J. E. TREHERNE
P. H. RUBERY
D e p a r t m e n t of B i o c h e m i s t r y U n i v e r s i t y of C a m b r i d g e England
Department o f Zoology U n i v e r s i t y of C a m b r i d g e England
Ion-sensitive Intracellular Microelectrodes, R. C. Thomas, 1978 Time-lapse Cinemicroscopy, P . N . R i d d l e , 1979 Immunochemical Methods in the Biological Sciences: Enzymes and Proteins, R. J . M a y e r and J . H . W a l k e r ,
1980
Microclimate Measurement for Ecologists, D . M . U n w i n , 1980 Whole-body Autoradiography, C. G. C u r t i s , S. A . M . C r o s s , R. J . M c C u l l o c h and
G. M . P o w e l l ,
1981
Microelectrode Methods for Intracellular Recording and Ionophoresis, R. D . P u r v e s ,
1981
Red Cell Membrances—A Methodological Approach, J . C. J . D . Young,
1982
A. L .Moore,
1983
Ellory
Techniques of Flavonoid Identification, K . R. M a r k h a m , 1982 Techniques of Calcium Research, M . V. Thomas, 1982 Isolation of Membranes and Organelles from Plant Cells, J . L . Intracellular Staining of Mammalian Neurones, A . R. E . W. Fyffe,
1984
Hall
G. B r o w n a n d
and and
Techniques in Photomorphogenesis Edited by
Harry Smith Department of Botany University of Leicester Leicester, England
M. G. Holmes
Smithsonian Environmental Research Rockville, Maryland
Center
1984 ACADEMIC PRESS
(Harcourt Brace Jovanovich, Publishers)
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Department der Universum • j tünchen =
Bereich 8
Z), a n d these p i g m e n t s are t h e n g e n e r a l l y o n l y isolated i n the Z , Z , Z f o r m (Falk et a l , 1979). The 1 0 , 2 3 - d i h y d r o b i l i n d i o n e s ( b i l i r u b i n s ) a r e c a p a b l e , b y c o n t r a s t , o f photoisomerisations i n both d i r e c t i o n s a n d t h u s f o r m , u p o n i r r a d i a t i o n w i t h v i s i b l e light, p h o t o s t a t i o n a r y m i x t u r e s c o n t a i n i n g m o d e r a t e a m o u n t s of the E , Z , Z and Z , Z , E i s o m e r s a n d a m i n o r a m o u n t o f t h e E , Z , E i s o m e r (MacDonagh et a l , 1982). Falk et a l (1980) h a v e d e v i s e d a g e n e r a l m e t h o d w h i c h is based o n t h i s fact. The b i l i v e r d i n is first c o n v e r t e d to a r u b i n o i d p i g m e n t b y addition o f a n u c l e o p h i l e t o C-10, w h i c h is t h e n s u b j e c t e d to p h o t o i s o m e r i s a t i o n . Workup u n d e r conditions w h i c h r e m o v e t h e n u c l e o p h i l e l e a d t h e n t o a m i x t u r e o f the corresponding Z ^ - i s o m e r i c b i l i v e r d i n s . The p r e p a r a t i o n of t h e i s ^ Z - b i l i n d i o n e l b g i v e n b e l o w (Kufer et a l , 1982a) f o l l o w s essentially t h e o r i g i n a l p r o c e d u r e o f Falk et a l (1979). Owing to the s y m m e t r y of t h e Z,Z,Z-adduct l a , only one E , Z , Z i s o m e r ( l b ) can be formed. Although t h e m e t h o d is p r i n c i p a l l y a p p l i c a b l e t o all b i l i v e r d i n s , practical Problems m a y arise f r o m u n s y m m e t r i c p r o d u c t s ( S e p a r a t i o n , regioselect i v i t y ) , f r o m labile s u b s t i t u e n t s (e.g., i r r e v e r s i b l e a d d i t i o n o f t h e n u c l e o p h i l e ) a n d / o r f r o m b i l i v e r d i n s w h i c h d o n o t r e a d i l y a d d n u c l e o p h i l e s at C-10. These problems a r e of c o n s i d e r a b l e i n t e r e s t w i t h r e g a r d t o t h e photoisomeris a t i o n of t h e n a t i v e p h y t o c h r o m e chromophore. The g e n e r a l p r o c e d u r e m a y then h a v e t o b e m o d i f i e d . For example, t h e u n s y m m e t r i c a n d m o r e unstable d i h y d r o b i l i n d i o n e 4a r e q u i r e s a l a r g e r a m o u n t of the thiol to form the r u b i n o i d a d d i t i o n p r o d u c t , a n d forms o n l y o n e ( 1 5 E ) (4b) of the t w o possible E i s o m e r s (4b,c), w h i c h is also m o r e labile u n d e r t h e w o r k u p condi-
delocalisation
tions (Kufer et al, 1982a).
The Z,Z, Z - b i l i n d i o n e l a is t r e a t e d w i t h 2 - m e r c a p t o e t h a n o l i n d i m e t h y l s u l p h o x i d e (7.5% v / v ) t o y i e l d a y e l l o w S o l u t i o n o f t h e a d d i t i o n p r o d u c t . It is i r r a d i a t e d f o r 10 m i n w i t h b l u e l i g h t u n d e r n i t r o g e n . All t h e f o l l o w i n g Steps m u s t b e carried o u t u n d e r a d i m g r e e n safelight o r , w h e r e p o s s i b l e , i n d a r k ness. The S o l u t i o n is first p o u r e d i n t o C h l o r o f o r m , a n d t h e 2 - m e r c a p t o e t h anol is e x t r a c t e d w i t h d i l u t e a q u e o u s p o t a s s i u m h y d r o x i d e . The r e s u l t i n g g r e e n C h l o r o f o r m S o l u t i o n is w o r k e d u p , a n d the r e s i d u e is c h r o m a t o g r a p h e d o n silica H plates w i t h C h l o r o f o r m - m e t h a n o l (20:1). The y i e l d o f t h e E , Z, Z i s o m e r l b is 10%.
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H U G O SCHEER
D. 21iV,24A^Methyleneetiobiliverdin-IVy One of the problems in bile pigment chemistry is their large conformational freedom. 21N,24N-Methyleneetiobilindione-IV}> (6) (Falk and Thirring, 1981) is restricted to cyclic conformations. Based on substantiated molecular orbital calculations and spectroscopic data, a predominantly cyclic helical conformation has been assigned to biliverdin 9 and many other free bilindiones (see Scheer, 1981, for leading references). Another conformationally restricted bilindione, the "extended" isophorcabilin 19 is described in Section III. I. Compound 6 has been prepared by Falk and Thirring (1981) from its open-chain parent, etiobiliverdin-IVy, by insertion of a methylene group. The procedure should be applicable to all octaalkylbilindiones. The reaction is (probably for steric reasons) regioselective, and only small amounts of the isomers bridged between the neighbouring N-21 and N-22 ( = N-24 and N-23) or N-22 and N-23 atoms are formed. The anion of etiobiliverdin-IVy is treated in dimethyl sulphoxidepotassium hydroxide with diiodomethane under argon at 100°C. Compound 6 is purified and separated from its isomers by chromatography on silica G with C h l o r o f o r m - m e t h a n o l (50:1). The yield is 20%, the two isomers are isolated in 1 and 2% yield, respectively. III. SEMISYNTHETIC BILE PIGMENTS A. Biliverdins from bilirubins — example: biliverdin-IXa Biliverdin-IXa (7) is prepared from commercial bilirubin (=bilirubin-IXa:) (8) by oxidation with high potential quinones. Several other oxidants have been described in the literature (see MacDonagh, 1979), but quinones are now most widely used
