The (4 Z, 10 2, 15Ej-2,3-dihydrobilindione 4, along with the fully unsaturated (E, 2, Z)-analogue
8, has been prepared from the corresponding (Z, Z, Z)-isomer by a variation of Falk's method
(Falk et ul., 1980). The photochemical and acid-catalyzed back-reactions have been studied by UV-vis
and 'H-NMR spectroscopy

Cmiel, Edmund

Kufer, Werner

Rüdiger, W.

Scheer, Hugo

Schneider, Siegfried

Thümmler, F.

English

Universität München: Elektronischen Publikationen

Photocheru. Pkotobiol. Vol. 36. pp. 603 to 607, 1982 
Printed in Great Britain. All rights reserved 
RESEARCH NOTE 
003 143655182, 1 I0603-05$03.00/0 
Copyright 0 1982 Pergamon Press Ltd 
STUDIES ON PLANT BILE PIGMENTS. 11. 
REGIOSELECTIVE PHOTOCHEMICAL AND ACID 
CATALYZED Z,E ISOMERIZATION OF 
DIHYDROBILINDIONE AS PHYTOCHROME MODEL 
w. KUFER*, E. C M I E L t ,  F. THUMMLER*, w. RUDICER*, s. SCHNElDERt and 
H. SCHEER+$ 
*Botanisches Institut der Universitat Munchen, Menzinger Str. 67. 8000 Munchen 19 and 
tInstitut fur Physikalische und Theoretische Chemie, Technische Universitat Munchen, 
D-8046 Garching, W. Germany 
(Rrcercrd 20 April 1982; uccepred 6 J u l y  1982) 
Abstract-The (4 Z, 10 2, 15Ej-2,3-dihydrobilindione 4, along with the fully unsaturated ( E ,  2, Z)-ana- 
logue 8, has been prepared from the corresponding (Z, Z, Z)-isomer by a variation of Falk's method 
(Falk et ul., 1980). The photochemical and acid-catalyzed back-reactions have been studied by UV-vis 
and 'H-NMR spectroscopy. 
The photoconversion of 1, the chromophore of the P, 
form of phytochrome, is the primary photochemical 
reaction of this plant sensory pigment. The structure 
of the stable photoproduct, the P,, chromophore, is 
still unknown. Photooxidation has been indicated 
earlier (Grombein er a/. ,  1975) and model reactions of 
this type have been investigated with the synthetic 
analogue 2 (Krauss and Scheer, 1981) bearing the 
characteristic A-dihydrobilin chromophore system 
(see Scheer, 1981). The characterization of Z,E iso- 
meric bile pigments (Falk and Grubmayr, 1977; Falk 
et a/., 1978, 1980; Gossauer et a/., 1981). isotope 
exchange and spectroscopic studies (Song er a / . ,  1979; 
Sarkar and Song, 1981) and recent studies with P,, 
chromopeptides (Thiimmler et ul., 1981) again raised 
the possibility of a geometric (Z,E)-isomerization as 
the primary reaction of the phytochrome system. Z,E- 
Photoisomerizations have hitherto only been studied 
with the fully unsaturated biliverdin-type pigments 
(Falk and Grubmayr, 1977; Falk et a/ . ,  1978, 1980; 
Gossauer et a/., 1981) and with bilirubins (McDonagh 
et (I/.. 1980; Holzwarth and Schaffner, 1981 ; de Groot 
et a/., 1982; Lightner et d., 1979; Stoll et d., 1979; 
Isobe and Onishi, 1981 ; Falk et a/., 1980). The Z+ E- 
isornerization occurs generally (Falk and Thirring, 
1979, 1980) only with the rubinoid pigments, viz. the 
isomerization of biliverdin proceeds via reversibly 
formed rubinoid addition products (Holzwarth et a/., 
1978; Manitto and Monti, 1979; Falk and Schlederer, 
1978; Falk ei a/., 1980; Kufer and Scheer, 1979, 1982) 
as the substrates proper of the photoreaction (Falk et 
al., 1980), whereas the direct E -+ Z(back-)photoiso- 
:TO whom correspondence should be addressed. 
603 
P A P  36 5--H 
merization is possible with the verdins as well as the 
rubins (Falk and Neufingerl, 1979). 
We are currently investigating the Z, E interconver- 
sions with pigments like 2 bearing the characteristic 
A-dihydrobilindione chromophore of phytochrome. 
They also form reversibly rubinoid addition products 
(Kufer and Scheer. 1979, 1982) but with rather differ- 
ent chromophoric systems in the two 'halves' of the 
molecule. This raises the question of the regioselecti- 
vity of the isomerization in pigments like 2 which is 
relevant to the structure of the P,,-chromophore. 
The isomerization of the A-dihydrobilindione 2-4 
was performed by a modification of Falk et a/.'s 
(1980) general procedure. Compound 2 (8.3 mg) was 
treated with 2-mercaptoethanol (10 m/) in dimethyl- 
sulfoxide (30mP) to yield the addition product 3 
(%,,,dx = 421 nm, see Kufer and Scheer, 1982). It was 
irradiated for 10min under N,  with light from a 
1000 W halogen lamp, filtered through 5 cm each of 
an aqueous solution of NaNO, (2.1 7 M )  and a sol- 
ution of CuSO, (0.035 M )  in 2.7 A4 aqueous ammonia 
(transmission maximum: 426 nm, cut off; 0.1% trans- 
mission 390 nm; modified from Calvert and Pitts, 
1966). After photolysis, the solution was partitioned 
between chloroform and water, thoroughly washed 
with water, and chromatographed on silica H (Merck, 
Darmstadt) plates with chloroform/methanol = 20: 1 
(Falk et a/ . ,  1978). Due to the ready back-isomerisa- 
tion of 4 to 2, the work-up and further handling of 4 
was performed either in complete darkness or, when 
necessary, with red safety lights. The (15 E)-isomer 4 
(typical yield 4%) was isolated from the slower mov- 
ing zone, in addition to the educt 2 (37%) as the major 
colored component, and a few oxidation products. It 
was rechromatographed on silica plates with carbon 
Figiirc 1 .  Photoisomerization of (4 Z, 10 Z .  15 €)-2.3- 
dihydrobilindione 4. Visible absorption spectrum of 4 in 
acetone (-1 and or the same solution after 3 min ir-  
radiation w i t h  white light (1400 W.rn- '  in the range 
between 460 and 600 nm) (---). According to TLC analysis 
the product of the photoreaction was the corresponding 
(Z,Z,Z)-isomer 2. 
tetrachloride acetone = 10:l. The structure of 4 was 
established as follows: (i) the UV-vis spectra of the 
free base (;.,,,, = 520nm in acetone) and the cation 
(in,,?, = 570nm in acetone. 14, concentrated HCI) 
were red shifted by 76 and 8 5 n m  with respect to the 
corresponding forms of the ( 1 5  Z)-isomer 2. This 
matches with the data given by Blacha-Puller (1979) 
for the (1 5 E)-isomer of a dihydrobilindione prepared 
by total synthesis [,'-,,, = 533 (1  5 E )  vs. : 605 nm 
CO Protein coo 
(15 Z) in CHCI,. 605 (15 15) and 676 nm (15  ZI in 
CHCl,/HCI]. The shift is much largcr than that 
expected for the 4 E isomer 5. in which onl! the 
4-double bond is uncoupled. ( i i )  In the N M R  spec- 
trum, the signals of the 5 H (5.38 ppm) and I0 H 
(6.93 ppm) remain unchanged. whcreas the 15 H 
(6.16 ppm) signal is shifted by 0.23 ppni to loner field. 
A shift of this order is typical for methine-protons at 
the site of isomerization (Gossauer o r  ( I / . .  1981 : Falk 
er L I L ,  1978). In particular. the data are similar to thc 
ones given by Blacha-Puller ( I  979) for ;I ( I  5 E )  I5 H 
at  6.2 I ppm) and ( I  5 Z)-isomer ( 15 H at 5.97 ppml of a 
dihydrobilindione. (iii) Irradiation of 4 o r  its cation 
with white light yielded back the isomcr 2 ;IS the sole 
product (Fig. 1). The reaction \+.as also studied b! 
NMR spectroscopy. Under non-exchangins con- 
ditions. the 15 H signal was shifted back to its orip- 
inal position at  5.93 ppm in 2. whereas the other sip- 
nals remained unchanged. 
To study the influence o f  the reduced ring A on the 
behavior of 2. the isomerization \ \as also studied with 
the bilindione 6 (octaethylbilindione) with ring A 
unsaturated. The conditions for the reaction sequence 
were less critical than with 2. The addition product 7 
was already formed quantitatively in the presence of 
7.5% of the thiol, and the complete removal of 3-mer- 
captoethanol was also facilitated by the stability of 6 
towards bases which allows washing with dilute 
KOH. The final yield of 8 (10",), however. WLLS similar 
to the best yields obtained for the isomerization of 2. 
Data of 8:  UV-vis: free base (CHCI,): = 585. 
/J 
2 I 
CH~OH 
3 4 
Research Note 605 
dH 
7 
6 
CO-Prdcin COO- 
9 I0 
368 nm; cation (CHCl,/trifluoroacetic acid): A,, = 
598, 362 nm; 'H-NMR in CDCI,: 6.86 ppm (s, 10 H), 
6.20 (s, 15 EH), 5.90 (s, 5 ZH), 2.2-2.8 (m, -CH2-), 
0.8-1.4 (m, --CH3); in CDCl,/d-trifluoroacetic acid: 
7.28 (s, IOH), 6.52 (s, 15 EH), 6.25 (s, 5 ZH), 2.3-3.0 
(m, -CH2-), 1.e1.5 (m, -CH3). These data are 
comparable to the ones reported by Falk et ai. (1978) 
and Gossauer et al. (1981) for E,Z,Z-bilindiones of 
the verdin series, and in particular the assignment of 
the methine NMR signals has been made in analogy. 
The (E)-isomer reverts photochemically to the 
(Z)-educt 6. Like other (E)-isomers (Falk and Grub- 
mayr, 1979), it also reverts in acidic solution in a dark 
reaction. Both the photochemical and the acid-cata- 
lyzed back reactions give isosbestic points (642, 460, 
335, 31 1 nm in CHCl,/trifluoroacetic acid) when fol- 
lowed by UV-vis spectroscopy, and yield the (Z,Z,Z)- 
isomer 6 as the sole product. The mechanism of the 
acid catalyzed reaction has been studied by 'H-NMR 
in the presence of deuterated trifluoroacetic acid. 
There is apparently no simultaneous H/D exchange at 
C-15 with the isomerization (Fig. 2), indicating that 
no protonation-deprotonation mechanism at this 
position is operative for the isomerization. The iso- 
merization is then likely to involve protonation at 
other sites and/or tautomerization similar to the 
recently suggested processes in the primary steps of 
phytochrome phototransformation (Sarkar and Song, 
1981). The negligible proton exchange at C-15 also 
indicates that the electron density at the isomerized 
methine bridges is neither strongly increased in the 
(E,Z, Z)-isomer nor during the isomerization, as com- 
pared to the (Z,Z,Z)-isomer. The C-5 in 2 is selec- 
tively attacked by electrophilic reagents and the 5 H 
is readily exchanged with deuterated trifluoroacetic 
acid (Krauss, 1980). This is supported by M O  calcu- 
606 
Figure 2. 'H-NMR spectra of the acid catalyzed thermal isomerization of the ( E .  Z. Z)-hilindione 8 to 
the (2. 2. Z)-isomer 6. Only the range between 6 = 6-8 ppm is shown, which contains the signals of the 
methine protons (indicated). as well as the signal of residual CHCI, at 6 = 7.26 ppm. The first spectrum 
(lower left) was scanned 30 min after the addltion of trifluoroacetic acid to a solution of 8 in CDCI,. The 
subsequent spectra mere scanned at intervals of IS min. 
lations indicating an  increased electron densit j  at  the 
methine bridge next to  the reduced ring (Scheer et d.. 
1982). 
The  results obtained with 2 show that the Z.E- 
(photo)isomerizations of A-dihydrobilindiones occur 
along the lines previously established for biliverdins 
with an unsaturated ring A (see Fdlk et ul., 1980). A 
particular feature. however. of the A-dihydrobilin- 
diones is their asymmetry. T w o  rather different 
products (9. lo) can principally be formed by isomeri- 
ration at either c-5 or C-15. respectively. a point 
which is obviously important to  the phytochrome 
problem. The  adduct 3 as  the educt proper for photoi- 
somerization has two absorption maxima (421 and 
315 nm in the reaction mixture*) which are assigned 
to the dipqrromethenone chromophore comprising 
rings C and D, and  to  the vinylpyrrole chromophore 
comprising rings A and  B. respectively (Kufer and 
Scheer. 1983). The  irradiation of 3 has been carried 
out  with filtered light absorbed preferentially by the 
dipyrromethenone chromophore. leading to  an  over- 
all regioselective transformation of 2 to  its (4 Z.  10 Z,  
15 E )  isomer 4. The  ready direct photoisomerization 
of 4 back to 2 compares well with that of denatured 
or peptic digested P,, (Thiimmler et al.. 1981). which 
would be in support of an  E.Z,Z isomer like 4 a s  a 
P,-,-model. rather than pigments derived from pho- 
tooxidative reactions (see Scheer, 1981). The  absorp- 
tion maximum of the E , Z . Z - 4  cation (570nm) is, 
however. considerably shorter than that of denatured 
*The absorption maxima of bilirubins are strongly sol- 
vent dependent (see Kuenzle. 1970: Holzwarth et d.. 1978. 
1981). 
or peptic digested P,, in acidic solvents (615 nm), even 
if the influence of the 18-vinyl-group is taken into 
account. It is, therefore, desirable to have at  hand. 
too, the (4 E ,  10 2, 15 Z)-isomer 5 of 2. Unsuccessful 
attempts for a total synthesis of ;I pigment hearing 
this chromophore (Blacha-Puller, 1981) indicate an  
even decreased stability as  compared to the (4 Z.  10 Z.  
15 E)-isomer. The  effects of irradiation into the vinyl- 
pyrrole chromophore of 3 comprising rings A and B 
(Kufer and  Scheer, 1982) are currently being studied. 
Ackiioic Irdyeriicw-This work mas supported b) the 
Deutsche Forschungsgemeinschaft. Bonn. 
REFERENCES 
Blacha-Puller, M. (1979) Dissertation. Technische Uni- 
versitiit. Braunschweig. 
Calvert. J .  G. and J. N. Pitts (1966) Phorochcriiisrr.y. 
p. 737. Wiley, New York. 
de Groot. J. A., R. van der Steen. R. Fokkens and J .  Lug- 
tenburg (1982) R e d .  Trrir. Chirn. Puys-Brrs 101, 
Falk, H.  and K.  Grubmayr (1977) Anqew. Chrrii. 89. 
Fnlk, H. and K. Grubmayr (1977) Arryrw. Chm.  1r11.  
Ed. Eiiyi. 16, 470. 
Fxlk, H. and I(. Grubmayr (1979) Moizuisii. C'liem 110, 
1237-1242. 
Falk, H., K.  Grubmayr, E. Haslinger. T. Schlederer and 
K. Thirring (1978) Monursli. Chrni. 109, 1451-1473. 
Falk, H., N. Miiller and T. Schlederer (1980) Moriutsh. 
Chem. 111, 159-175. 
Falk, H. and F. Neutingerl (1979) !tloriutsh. Chrrn. 110, 
1243-1 255. 
Falk, H .  and T. Schlederer (1978) Moriurush. Chrii. 109, 
101 3-1 0 15. 
35-40, 
487-488. 
Research Note 607 
Falk, H. and K. Thirring (1979) Z. Naturforsch. 34b, 
Falk, H. and K. Thirring (1980) Z .  Nutrtrjbrsch. 35b, 
376380. 
Gossauer, A,, M. Blacha-Puller, R. Zeisberg and V. 
Wfay (1981) Liehigs. Atiii .  Chem. 342-346. 
Grombein, S., W. Riidiger and H. Zimmermann (1975) 
Hoppe-Seyler’s Z. Physiol. Chern. 356, 1709-1 7 14. 
Holzwarth, A. R., H.  Lehner, S. E. Braslavsky and 
K. Schaffner (1978) Liebiys Ann. Chern. 2002-2017. 
Holzwarth, A. R.  and K. Schaffner (1981) Photochem. 
Photobiol. 33, 635-639. 
Isobe. K. and S. Onishi (1981) Biorhrrn. J .  193, 
Krauss, C. (1980) Dissertation, Universitat Miinchen, 
Krauss, C. and H.  Scheer (1981) Photochern. Phorohiol. 
Kuenzle, C. C. (1970) Biochcm. J .  119, 395-409. 
Kufer. W. and H. Scheer (1979) Hoppe-Seyler’s Z .  Phy- 
1448-1453. 
1029-103 1. 
Munchen. 
34, 385-391. 
siol. C ~ ~ J I I .  360, 935-956. 
Kufer. W. and H. Scheer (1982) 2. Naturforsch. 37c, 
Lightner, D. A., T. A. Wooldridge and A. F. McDonagh 
Manitto, P. and D. Monti (1979) Eupericnria 35, 
McDonagh, A. F., L. A. Palma and D. A. Light- 
Sarkar. H. K. and P.-S. Song (1981) Biochemistry 20, 
Scheer, H. (1981) Anyew. Cheni. 93, 230-250. 
Scheer H. (1981) AJXJW. Chrm. I l i t .  Ed .  Eny l .  20, 
Scheer, H., H. Formanek and S. Schneider (1982) Pho- 
Song. P.-S., Q. Chae and J. D. Gardner (1979) Biochirn. 
Stoll. M. S., E. A. Zenone, J. D. Ostrow and J. E. Zarem- 
Thiimmler, F., T. Brandlmeier and W. Riidi- 
I 79- 192. 
(1979) 
141 8-1420. 
ner (1980) Science 208. 145:151. 
43 15-4320. 
Biochem. Biophys. Res. Comnturi. 86, 235-243. 
241 -26 I .  
rocheni. Photohiol. 36, 259-272. 
Biophys. Actu 576, 479-496. 
bo (1979) Biocheni. J .  183, 139-146. 
ger (1981) 2. Narurfosc/i. 36c, 440-449. 


STUDIES ON PLANT BILE PIGMENTS.

Open Access LMU

Photocheru. Pkotobiol. Vol. 36. pp. 603 to 607, 1982 Printed in Great Britain. All rights reserved RESEARCH NOTE 003 143655182, 1 I0603-05$03.00/0 Copyright 0 1982 Pergamon Press Ltd STUDIES ON PLANT BILE PIGMENTS. 11. REGIOSELECTIVE PHOTOCHEMICAL AND ACID CATALYZED Z,E ISOMERIZATION OF DIHYDROBILINDIONE AS PHYTOCHROME MODEL w. KUFER*, E. C M I E L t ,  F. THUMMLER*, w. RUDICER*, s. SCHNElDERt and H. SCHEER+$ *Botanisches Institut der Universitat Munchen, Menzinger Str. 67. 8000 Munchen 19 and tInstitut fur Physikalische und Theoretische Chemie, Technische Universitat Munchen, D-8046 Garching, W. Germany (Rrcercrd 20 April 1982; uccepred 6 J u l y  1982) Abstract-The (4 Z, 10 2, 15Ej-2,3-dihydrobilindione 4, along with the fully unsaturated ( E ,  2, Z)-ana- logue 8, has been prepared from the corresponding (Z, Z, Z)-isomer by a variation of Falk's method (Falk et ul., 1980). The photochemical and acid-catalyzed back-reactions have been studied by UV-vis and 'H-NMR spectroscopy. The photoconversion of 1, the chromophore of the P, form of phytochrome, is the primary photochemical reaction of this plant sensory pigment. The structure of the stable photoproduct, the P,, chromophore, is still unknown. Photooxidation has been indicated earlier (Grombein er a/. ,  1975) and model reactions of this type have been investigated with the synthetic analogue 2 (Krauss and Scheer, 1981) bearing the characteristic A-dihydrobilin chromophore system (see Scheer, 1981). The characterization of Z,E iso- meric bile pigments (Falk and Grubmayr, 1977; Falk et a/., 1978, 1980; Gossauer et a/., 1981). isotope exchange and spectroscopic studies (Song er a / . ,  1979; Sarkar and Song, 1981) and recent studies with P,, chromopeptides (Thiimmler et ul., 1981) again raised the possibility of a geometric (Z,E)-isomerization as the primary reaction of the phytochrome system. Z,E- Photoisomerizations have hitherto only been studied with the fully unsaturated biliverdin-type pigments (Falk and Grubmayr, 1977; Falk et a/ . ,  1978, 1980; Gossauer et a/., 1981) and with bilirubins (McDonagh et (I/.. 1980; Holzwarth and Schaffner, 1981 ; de Groot et a/., 1982; Lightner et d., 1979; Stoll et d., 1979; Isobe and Onishi, 1981 ; Falk et a/., 1980). The Z+ E- isornerization occurs generally (Falk and Thirring, 1979, 1980) only with the rubinoid pigments, viz. the isomerization of biliverdin proceeds via reversibly formed rubinoid addition products (Holzwarth et a/., 1978; Manitto and Monti, 1979; Falk and Schlederer, 1978; Falk ei a/., 1980; Kufer and Scheer, 1979, 1982) as the substrates proper of the photoreaction (Falk et al., 1980), whereas the direct E -+ Z(back-)photoiso- :TO whom correspondence should be addressed. 603 P A P  36 5--H merization is possible with the verdins as well as the rubins (Falk and Neufingerl, 1979). We are currently investigating the Z, E interconver- sions with pigments like 2 bearing the characteristic A-dihydrobilindione chromophore of phytochrome. They also form reversibly rubinoid addition products (Kufer and Scheer. 1979, 1982) but with rather differ- ent chromophoric systems in the two 'halves' of the molecule. This raises the question of the regioselecti- vity of the isomerization in pigments like 2 which is relevant to the structure of the P,,-chromophore. The isomerization of the A-dihydrobilindione 2-4 was performed by a modification of Falk et a/.'s (1980) general procedure. Compound 2 (8.3 mg) was treated with 2-mercaptoethanol (10 m/) in dimethyl- sulfoxide (30mP) to yield the addition product 3 (%,,,dx = 421 nm, see Kufer and Scheer, 1982). It was irradiated for 10min under N,  with light from a 1000 W halogen lamp, filtered through 5 cm each of an aqueous solution of NaNO, (2.1 7 M )  and a sol- ution of CuSO, (0.035 M )  in 2.7 A4 aqueous ammonia (transmission maximum: 426 nm, cut off; 0.1% trans- mission 390 nm; modified from Calvert and Pitts, 1966). After photolysis, the solution was partitioned between chloroform and water, thoroughly washed with water, and chromatographed on silica H (Merck, Darmstadt) plates with chloroform/methanol = 20: 1 (Falk et a/ . ,  1978). Due to the ready back-isomerisa- tion of 4 to 2, the work-up and further handling of 4 was performed either in complete darkness or, when necessary, with red safety lights. The (15 E)-isomer 4 (typical yield 4%) was isolated from the slower mov- ing zone, in addition to the educt 2 (37%) as the major colored component, and a few oxidation products. It was rechromatographed on silica plates with carbon Figiirc 1 .  Photoisomerization of (4 Z, 10 Z .  15 €)-2.3- dihydrobilindione 4. Visible absorption spectrum of 4 in acetone (-1 and or the same solution after 3 min ir-  radiation w i t h  white light (1400 W.rn- '  in the range between 460 and 600 nm) (---). According to TLC analysis the product of the photoreaction was the corresponding (Z,Z,Z)-isomer 2. tetrachloride acetone = 10:l. The structure of 4 was established as follows: (i) the UV-vis spectra of the free base (;.,,,, = 520nm in acetone) and the cation (in,,?, = 570nm in acetone. 14, concentrated HCI) were red shifted by 76 and 8 5 n m  with respect to the corresponding forms of the ( 1 5  Z)-isomer 2. This matches with the data given by Blacha-Puller (1979) for the (1 5 E)-isomer of a dihydrobilindione prepared by total synthesis [,'-,,, = 533 (1  5 E )  vs. : 605 nm CO Protein coo (15 Z) in CHCI,. 605 (15 15) and 676 nm (15  ZI in CHCl,/HCI]. The shift is much largcr than that expected for the 4 E isomer 5. in which onl! the 4-double bond is uncoupled. ( i i )  In the N M R  spec- trum, the signals of the 5 H (5.38 ppm) and I0 H (6.93 ppm) remain unchanged. whcreas the 15 H (6.16 ppm) signal is shifted by 0.23 ppni to loner field. A shift of this order is typical for methine-protons at the site of isomerization (Gossauer o r  ( I / . .  1981 : Falk er L I L ,  1978). In particular. the data are similar to thc ones given by Blacha-Puller ( I  979) for ;I ( I  5 E )  I5 H at  6.2 I ppm) and ( I  5 Z)-isomer ( 15 H at 5.97 ppml of a dihydrobilindione. (iii) Irradiation of 4 o r  its cation with white light yielded back the isomcr 2 ;IS the sole product (Fig. 1). The reaction \+.as also studied b! NMR spectroscopy. Under non-exchangins con- ditions. the 15 H signal was shifted back to its orip- inal position at  5.93 ppm in 2. whereas the other sip- nals remained unchanged. To study the influence o f  the reduced ring A on the behavior of 2. the isomerization \ \as also studied with the bilindione 6 (octaethylbilindione) with ring A unsaturated. The conditions for the reaction sequence were less critical than with 2. The addition product 7 was already formed quantitatively in the presence of 7.5% of the thiol, and the complete removal of 3-mer- captoethanol was also facilitated by the stability of 6 towards bases which allows washing with dilute KOH. The final yield of 8 (10",), however. WLLS similar to the best yields obtained for the isomerization of 2. Data of 8:  UV-vis: free base (CHCI,): = 585. /J 2 I CH~OH 3 4 Research Note 605 dH 7 6 CO-Prdcin COO- 9 I0 368 nm; cation (CHCl,/trifluoroacetic acid): A,, = 598, 362 nm; 'H-NMR in CDCI,: 6.86 ppm (s, 10 H), 6.20 (s, 15 EH), 5.90 (s, 5 ZH), 2.2-2.8 (m, -CH2-), 0.8-1.4 (m, --CH3); in CDCl,/d-trifluoroacetic acid: 7.28 (s, IOH), 6.52 (s, 15 EH), 6.25 (s, 5 ZH), 2.3-3.0 (m, -CH2-), 1.e1.5 (m, -CH3). These data are comparable to the ones reported by Falk et ai. (1978) and Gossauer et al. (1981) for E,Z,Z-bilindiones of the verdin series, and in particular the assignment of the methine NMR signals has been made in analogy. The (E)-isomer reverts photochemically to the (Z)-educt 6. Like other (E)-isomers (Falk and Grub- mayr, 1979), it also reverts in acidic solution in a dark reaction. Both the photochemical and the acid-cata- lyzed back reactions give isosbestic points (642, 460, 335, 31 1 nm in CHCl,/trifluoroacetic acid) when fol- lowed by UV-vis spectroscopy, and yield the (Z,Z,Z)- isomer 6 as the sole product. The mechanism of the acid catalyzed reaction has been studied by 'H-NMR in the presence of deuterated trifluoroacetic acid. There is apparently no simultaneous H/D exchange at C-15 with the isomerization (Fig. 2), indicating that no protonation-deprotonation mechanism at this position is operative for the isomerization. The iso- merization is then likely to involve protonation at other sites and/or tautomerization similar to the recently suggested processes in the primary steps of phytochrome phototransformation (Sarkar and Song, 1981). The negligible proton exchange at C-15 also indicates that the electron density at the isomerized methine bridges is neither strongly increased in the (E,Z, Z)-isomer nor during the isomerization, as com- pared to the (Z,Z,Z)-isomer. The C-5 in 2 is selec- tively attacked by electrophilic reagents and the 5 H is readily exchanged with deuterated trifluoroacetic acid (Krauss, 1980). This is supported by M O  calcu- 606 Figure 2. 'H-NMR spectra of the acid catalyzed thermal isomerization of the ( E .  Z. Z)-hilindione 8 to the (2. 2. Z)-isomer 6. Only the range between 6 = 6-8 ppm is shown, which contains the signals of the methine protons (indicated). as well as the signal of residual CHCI, at 6 = 7.26 ppm. The first spectrum (lower left) was scanned 30 min after the addltion of trifluoroacetic acid to a solution of 8 in CDCI,. The subsequent spectra mere scanned at intervals of IS min. lations indicating an  increased electron densit j  at  the methine bridge next to  the reduced ring (Scheer et d.. 1982). The  results obtained with 2 show that the Z.E- (photo)isomerizations of A-dihydrobilindiones occur along the lines previously established for biliverdins with an unsaturated ring A (see Fdlk et ul., 1980). A particular feature. however. of the A-dihydrobilin- diones is their asymmetry. T w o  rather different products (9. lo) can principally be formed by isomeri- ration at either c-5 or C-15. respectively. a point which is obviously important to  the phytochrome problem. The  adduct 3 as  the educt proper for photoi- somerization has two absorption maxima (421 and 315 nm in the reaction mixture*) which are assigned to the dipqrromethenone chromophore comprising rings C and D, and  to  the vinylpyrrole chromophore comprising rings A and  B. respectively (Kufer and Scheer. 1983). The  irradiation of 3 has been carried out  with filtered light absorbed preferentially by the dipyrromethenone chromophore. leading to  an  over- all regioselective transformation of 2 to  its (4 Z.  10 Z,  15 E )  isomer 4. The  ready direct photoisomerization of 4 back to 2 compares well with that of denatured or peptic digested P,, (Thiimmler et al.. 1981). which would be in support of an  E.Z,Z isomer like 4 a s  a P,-,-model. rather than pigments derived from pho- tooxidative reactions (see Scheer, 1981). The  absorp- tion maximum of the E , Z . Z - 4  cation (570nm) is, however. considerably shorter than that of denatured *The absorption maxima of bilirubins are strongly sol- vent dependent (see Kuenzle. 1970: Holzwarth et d.. 1978. 1981). or peptic digested P,, in acidic solvents (615 nm), even if the influence of the 18-vinyl-group is taken into account. It is, therefore, desirable to have at  hand. too, the (4 E ,  10 2, 15 Z)-isomer 5 of 2. Unsuccessful attempts for a total synthesis of ;I pigment hearing this chromophore (Blacha-Puller, 1981) indicate an  even decreased stability as  compared to the (4 Z.  10 Z.  15 E)-isomer. The  effects of irradiation into the vinyl- pyrrole chromophore of 3 comprising rings A and B (Kufer and  Scheer, 1982) are currently being studied. Ackiioic Irdyeriicw-This work mas supported b) the Deutsche Forschungsgemeinschaft. Bonn. REFERENCES Blacha-Puller, M. (1979) Dissertation. Technische Uni- versitiit. Braunschweig. Calvert. J .  G. and J. N. Pitts (1966) Phorochcriiisrr.y. p. 737. Wiley, New York. de Groot. J. A., R. van der Steen. R. Fokkens and J .  Lug- tenburg (1982) R e d .  Trrir. Chirn. Puys-Brrs 101, Falk, H.  and K.  Grubmayr (1977) Anqew. Chrrii. 89. Fnlk, H. and K. Grubmayr (1977) Arryrw. Chm.  1r11.  Ed. Eiiyi. 16, 470. Fxlk, H. and I(. Grubmayr (1979) Moizuisii. C'liem 110, 1237-1242. Falk, H., K.  Grubmayr, E. Haslinger. T. Schlederer and K. Thirring (1978) Monursli. Chrni. 109, 1451-1473. Falk, H., N. Miiller and T. Schlederer (1980) Moriutsh. Chem. 111, 159-175. Falk, H. and F. Neutingerl (1979) !tloriutsh. Chrrn. 110, 1243-1 255. Falk, H .  and T. Schlederer (1978) Moriurush. Chrii. 109, 101 3-1 0 15. 35-40, 487-488. Research Note 607 Falk, H. and K. Thirring (1979) Z. Naturforsch. 34b, Falk, H. and K. Thirring (1980) Z .  Nutrtrjbrsch. 35b, 376380. Gossauer, A,, M. Blacha-Puller, R. Zeisberg and V. Wfay (1981) Liehigs. Atiii .  Chem. 342-346. Grombein, S., W. Riidiger and H. Zimmermann (1975) Hoppe-Seyler’s Z. Physiol. Chern. 356, 1709-1 7 14. Holzwarth, A. R., H.  Lehner, S. E. Braslavsky and K. Schaffner (1978) Liebiys Ann. Chern. 2002-2017. Holzwarth, A. R.  and K. Schaffner (1981) Photochem. Photobiol. 33, 635-639. Isobe. K. and S. Onishi (1981) Biorhrrn. J .  193, Krauss, C. (1980) Dissertation, Universitat Miinchen, Krauss, C. and H.  Scheer (1981) Photochern. Phorohiol. Kuenzle, C. C. (1970) Biochcm. J .  119, 395-409. Kufer. W. and H. Scheer (1979) Hoppe-Seyler’s Z .  Phy- 1448-1453. 1029-103 1. Munchen. 34, 385-391. siol. C ~ ~ J I I .  360, 935-956. Kufer. W. and H. Scheer (1982) 2. Naturforsch. 37c, Lightner, D. A., T. A. Wooldridge and A. F. McDonagh Manitto, P. and D. Monti (1979) Eupericnria 35, McDonagh, A. F., L. A. Palma and D. A. Light- Sarkar. H. K. and P.-S. Song (1981) Biochemistry 20, Scheer, H. (1981) Anyew. Cheni. 93, 230-250. Scheer H. (1981) AJXJW. Chrm. I l i t .  Ed .  Eny l .  20, Scheer, H., H. Formanek and S. Schneider (1982) Pho- Song. P.-S., Q. Chae and J. D. Gardner (1979) Biochirn. Stoll. M. S., E. A. Zenone, J. D. Ostrow and J. E. Zarem- Thiimmler, F., T. Brandlmeier and W. Riidi- I 79- 192. (1979) 141 8-1420. ner (1980) Science 208. 145:151. 43 15-4320. Biochem. Biophys. Res. Comnturi. 86, 235-243. 241 -26 I .  rocheni. Photohiol. 36, 259-272. Biophys. Actu 576, 479-496. bo (1979) Biocheni. J .  183, 139-146. ger (1981) 2. Narurfosc/i. 36c, 440-449. 

Dissertation. Technische Universitiit.

Hoppe-Seyler’s Z.

Liebiys Ann.

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STUDIES ON PLANT BILE PIGMENTS.

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