From the A-dihydrobilindione 3. the pyridinium derivatives 6a,b and analogues thereof have
been obtained by a formal nucleophilic substitution. The reaction is rationalized as a photochemically
assisted oxidation of 3 with a subsequent regioselective addition of pyridine at C-5. By thermolysis.
6a.b yields back the parent bilin 3, together with an oxidation product. The significance of the reaction
with respect to the phytochrome interconversion is discussed

Bubenzer, C.

Krauss, Corinna

Scheer, Hugo

English

Universität München: Elektronischen Publikationen

Phororhumirrr~ und Phrm,hir,loy?. Vol. 30. pp. 473 10 471 
0 Perramon Press Ltd 1979. Prlnted in Great Britain 
0031-X655 79 1001-0473%02.00/0 
STUDIES ON PLANT BILE PIGMENTS-6. 
PHOTOCHEMICALLY ASSISTED REACTION OF 
A MODEL FOR PHYTOCHROME INTERCONVERSION 
A-DIHYDROBILINDIONE WITH NUCLEOPHILES AS 
C. KRAUSS, C. BUBENZER and H. SCHEER* 
Botanisches Institut, Munchen, W. Germany 
(Received 23 Fehruary 1979: accepted I M a y  1979) 
Abstract-From the A-dihydrobilindione 3. the pyridinium derivatives 6a,b and analogues therwf have 
been obtained by a formal nucleophilic substitution. The reaction is rationalized as a photochemically 
assisted oxidation of 3 with a subsequent regioselective addition of pyridine at C-5. By thermolysis. 
6a.b yields back the parent bilin 3, together with an oxidation product. The significance of the reaction 
with respect to the phytochrome interconversion is discussed. 
INTRODUCTION 
Phytochrome, the photomorphogenetic reaction 
center pigment of higher green plants and some algae, 
contains a bile pigment chromophore covalently 
linked to  the protein (Siegelman et a/ . ,  1966; Riidiger, 
1975). The photochromic conversion between the two 
long-lived forms P, and P,, is the primary event of 
the photomorphogenetic reaction chain (Pratt, 1978 ; 
Briggs and Rice, 1972). P, contains the A-dihydrobi- 
lindione chromophore 1 (Klein and Rudiger, 1978. 
Schoch et a/., 1976; Klein et a/., 1977; Grombein e f  
al., 1975; Scheer, 1976). For the chromophore of P,r, 
structure 2 with the B-pyrrolic side chains unaltere'd 
(Klein et a/.,  1977) and a chromophore no longer con- 
taining the A 4,5-double bond (if judged from UV-vis 
spectra, Grombein et a/., 1975) has been proposed. 
This proposal implies a change in the molecular 
structure during the P, e P,, interconversion and 
excludes conformational changes and/or protonation- 
deprotonation events as the sole structural difference 
between the two forms (see Scheer, 1978, for a discus- 
sion). 
In exploiting the synthetic bilin 3 as a model for 
I, an increased reactivity at the A 4.5-double bond 
has been characterized for A-dihydrobilindiones, as 
compared to their fully unsaturated analogues, e.g. 
7 (Scheer and Krauss, 1977; Scheer et al., 1977). 
Specifically, the reversible photodimerization of 3 has 
been proposed as a model type reaction for the phyto- 
chrome interconversions (Scheer and Kraus. 1977). 
Since present evidence supports but one chromophore 
in the monomeric phytochrome (Pratt 1978; Briggs 
and Rice, 1972; and citations therein), the possibility 
of using partners other than a second bilin for addi- 
tion reactions has been studied. Here, we report the 
*Reprint requests should be sent to: Dr. Hugo Scheer. 
Botanisches Institut. Menzinger Str. 67. 8000 Munchen 19. 
W. Germany. 
I 
,Protein 
2 
3 
473 
474 C. KRAUSS, C. BUBENZER and H. SCHEER 
0 7 
- 2 c -  
-ZH+ 
L 4 
Reaction scheme 
reversible, photochemically assisted addition of nuc- 
leophiles of the pyridine type. 
MATERIALS AND METHODS 
The A-dihydrobilindione 3 was prepared by the method 
of Cavaleiro and Smith (1973) from octaethylporphyrin via 
octaethylchlorin (Whitlock et nl., 1969). I 2  and pyridine 
were reagent grade, the latter was dried over basic alumina 
(Woelm-ICN. Eschwege). All solvents were destilled prior 
to use or were reagent grade. Preparative photochemical 
reactions were carried out under nitrogen in pyrex 
cylinders at starting concentrations c : 0.1 mM of com- 
pound 3 with the light of a IOOOW tungsten halogen 
source (13 W/mZ between 550 and 620nm). UV-vis 
absorption spectra were recorded on a DMR 22 spectro- 
photometer (Zeiss, Oberkochen). and are reported in the 
format .I,.,,[nm] (c. Mass spectra were obtained in 
the EI mode with a JMS-D 100 spectrometer (Jeol, Japan). 
and in the FD mode with a model 731 instrument (Varian- 
MAT, Bremen). 'HMR spectra were recorded in the FT 
mode on a HFX 90 instrument (Bruker, Karlsruhe) with 
(CH3),Si as internal standard. Chemical shifts are reported 
in GCppm]. Column chromatography of the reaction mix- 
tures was carried out by an improved HPLC method 
(Schoch et al., 1978) on silica with a slightly alcaline ter- 
nary eluent system. For TLC. silica H (Merck. Darmstadt) 
was used. 
RESULTS AND DISCUSSION 
Irradiation of 3 in benzene/pyridine (4:l) in the 
presence of I2 or other one-electron oxidants leads 
t o  a rapid colour change from blue to  violet, similar 
t o  the change occurring during dimerization in the 
absence of pyridine (Scheer and Krauss, 1977) (Fig. 1). 
Chromatography of the product mixture yields only 
traces of the dimers, however. Two main products 
can be isolated, which migrate o n  silica only at high 
methanol concentrations, and which are irreversibly 
adsorbed upon prolonged exposure to  silica. TLC: 
Rf = 0.52 and 0.62, respectively, in dichloromethane/ 
methanol = 9:1, containing 1% aqueous ammonia; 
Phytochrome interconversion model 
A 
[a,..] 
475 
I 
nT 
- A [nm] 
Figure 1. UV-vis spectra of the photochemical reaction of 3 ( 4 0 p M )  in benzene containing pyridine 
(2.47 M )  and I2 (19.0 pM). Each trace corresponds to 1 min irradiation time, for the light conditions, 
see text. The arrows denote the change of absorption during the reaction. 
HPLC: V, = 106 and 182m(, respectively, on 4 x 
300 mm silica with dichloromethane/methanol = 
1:l containing 0.04% aqueous ammonia as eluent. 
The two products are interconvertible, e.g. by acid, 
and thus assigned as isomers. From the slower migra- 
ting zone, the isomer 6a of 2,3-dihydro-5(1-pyri- 
dinium)-octaathyl-l,19(21,24H)-bilindione has been 
isolated, the faster migration zone contains 6b. 
. UV-uis (see also Fig. 2): Cation (corresponding to 
the chromophore free base): 545 (10.27). 326 (19.9); 
dication (corresponding to the chromophore monoca- 
tion): 603 (25.5), 327 (2 1.4); zwitterion (corresponding 
to the chromophore anion): 585 (9.7); zinc complex: 
630 ( I  7.5). 585 (9.7). 330 (25.5). 
8.21 (Pyr-H-B,B‘, ‘d’), 8.49 (Pyr-H-A, 7’). 9.04 
(Pyr-H-C,C’, ‘d’). 6b: 6.76 (H-10. s) 5.96 (H-15, s) 8.29 
(Pyr-H-B,B’, *d’). 8.70 (Pyr-H-A, ‘t’) 9.38 (Pyr-H-C, C’. 
‘d’). (C2H3),CO: 6a: 7.07 (H-I0 s), 6.21 (H-15, s). 8.42 
(Pyr-H-B,B’, ‘d’), 8.92 Pyr-H-A, ‘t’), 9.59 (Pyr-H-C,C. 
W). 6b: 7.04 (H-10. s). 6.18 (H-15, s), 8.50 (Pyr-H-B,B’. 
W), 8.94 (Pyr-H-A. ‘t’), 9.57 (Pyr-H-C,C‘ ‘d’). 
CF,C002H: 6a: 7.71 (H-10, s), 6.60 (H-15, s), 8.35 
(Pyr-H-B,B, ‘d’), 8.70 (Pyr-H-A, ‘t’), 9.1 1 (Pyr-H-C,C, 
‘HMR: C’HClS: 6a: 6.72 (H-10, s), 5.92 (H-15. s), 
Figure 2. UV-vis spectra of the pyridinium salt 6s in methanol (-), its protonated form obtained 
after addition of 0.25% of 35% HCI (. . . . .). its deprotonated form obtained after addition of 0.25”,, 
methanolic KOH (-.-,-), and its Zn-complex obtained after addition of 0.25”; saturated methanolic 
Zn-acetate (-----). Extinction coefficients are based on the assumption of OH- as counterion after 
silica chromatography. 
416 C. KRAUSS, C. BUBENZER and H. SCHEER 
‘d‘). 6b: 7.68 (H-10, s), 6.48 (H-15, s), 8.43 (Pyr-H-B,B, 
‘d), 8.80 (Pyr-H-A, ‘t’), 9.29 (Pyr-H-C,C, ‘d’). 
MS:EI-Mode:  556 (loo%), 554 (6673, 527 (46%), 
432 (45%), 421 (24%), 418 (26%). FD-Mode: 634 
(loo%), 556 (6%). 
Structure 6 of the pyridinium adducts has been 
established by the following arguments: ( I )  The 
‘HMR spectrum shows the ABB’CC’ pattern (two 
‘doublets’ and one ‘triplet’ (J.,PPdrc,,,  z 7 Hz) in the 
ratio 2:2:1) and the chemical shifts typical for N-sub- 
stituted pyridinium ions (Abramovitch. 1974; Diner 
and Lown, 1971); (2) The F D  mass spectrum has a 
peak at the highest mass of 634rnle (base peak), the 
only major fragment occurs at 556/e. In the E l  spec- 
trum, there are no reproducible peaks above the basis 
peak at 556 m/r; (3) The chromatographic properties 
indicate an ionic compound: (4) The site of substitu- 
tion is indicated from the ‘HMR spectrum. Of the 
three methine resonances in 3 assigned to H-5 
(5.44 pprn), H-10 (6.56 ppm) and H-15 (5.97 ppm) 
(Cavaleiro and Smith, 1973), the ones of H-15 and 
H-10 are slightly shifted, but still present in 6a and 
6b while the high-field signal characteristic for H-5 
is lacking; (5) The UV-vis spectra (Fig. 2) of the 
monocation. dication, zwitterion and zinc complex 
are in the wavelength ranges typical of the free bases, 
cations, anions and zinc complexes, respectively, of 
‘biliviolins’ containing the tripyrrinone chromophore 
8*. Thus, the A 4,5-double-bond, although still 
present, is spectroscopically no longer evident. This 
effect of large meso-substituents has been observed 
earlier by Bonfiglio et a/. (1977) and related to the 
uncoupling of the adjacent rings by a pronounced 
twist between them; (6) The hydrogenated ring A is 
still present, as evidenced by the characteristic ’ HMR 
multiplet (Scheer et a/., 1977) at 6 = 1.5-1.8 ppm 
(2’,3’-CH2). by the 556m/e peak in the mass spectra. 
and by the reversion experiments; (7) The product 
is formulated as the tautomer 6 rather than 5 for 
the lack of a third meso proton signal in the ‘HMR 
spectrum, and for the generally higher basicity of N 
as compared to C. 
Due to its ready interconversion, compound 6a 
contained 19% 6b. In addition to the thermal dispro- 
portionation (see below). this is another reason for 
us not being able to  obtain a defined melting point. 
The isomer mixture 6a.b can be analyzed by ‘HMR. 
The signals of the methine protons and of the pyri- 
dinium ring protons listed above are most suitable 
due to their large separation for the respective 
isomers, but doubling of some signals can even be 
*The term ‘violin’ is used here as a phenomenological 
description designating linear oligopyrroles with a single 
visible absorption band of the free base around 560nm. 
Formula 8 with R, .  R,. R3 # 0 is typical of these com- 
pounds. Historically, the term has been associated with 
4.5-dihydro- I ,  I9 (2 I ,  24 H) bilindiones. 
?The central methine bridge-(C-10) is the principal point 
of nucleophilic attack in the (unoxidized) free bilins both 
for bilindiones (Falk and Schlederer, 1978; Holzwarth et 
a/., 1978) and dihydrobilindiones (Kufer and Scheer, 1979). 
observed in the methyl- and methylene region. After 
equilibration in CF,COO’H, the isomer ratio 
6a:6b = 2:1 has been obtained, as compared to 4.5:I 
in the reaction mixture. 
The stereochemical origin for 6 to occur as isomers 
can be related to steric hindrance of the meso-pyri- 
dinium and the P-pyrrolic substituents. By this means, 
rotation around the C-5, C-6 bond is hindered, lead- 
ing to atropisomers. In conjunction with the asym- 
metric centers at ring A (transoid configuration of 
the ethyl groups), this gives rise to two diastereomeric 
pairs of enantiomers, even in case of the rings B, C 
and D being coplanar. Based on this model, the 
5-nitrobilindione obtained from an achiral bilindione 
(Bonfiglio et al., 1973) possibly is a racemate. 
Reaction 3- 6 is formally a nucleophilic substitu- 
tion at C-5. It can be rationalized by the sequence 
shown in Scheme 1. Electron abstraction by the oxi- 
dant and subsequent deprotonation by the base 
present leads to a radical, and by repetition to the 
hypothetical dehydrobilindione 4, either one of which 
is attacked by pyridine at  C-5 to yield 6 after tauto- 
merization. Accordingly, the reaction is facilitated 
with the 2,4, 2,6 and 3,5-lutidines due to the inductive 
effect of the alkyl groups, and by increasing concen- 
trations of the oxidant, 12. Nucleophilic attack on 
cation radicals or dications of fully unsaturated bilin- 
diones have been reported to occur at both the outer 
methine bridges (Eivazi et al., 1977). Although we 
have not yet identified all by-products of the reaction 
of 3, the predominant formation of 6a,b supports 
again (Scheer, 1978). that hydrogenation of ring A 
activates selectively the adjacent methine bridge 
under oxidizing conditions.? Formation of 6a,b is 
photochemically assisted, but occurs principally in the 
dark, too. An anaerobic solution of 3 (40 p M )  in ben- 
zene containing I, (7.75 p M )  and pyridine (2.47 M) 
has been quenched after 2 h standing in the dark by 
filtration over Na,S20, into glycine buffer (pH 2.7). 
After workup, it contained 5% of the isomers 6a,b 
(in addition to minor amounts of a dimeric violin), 
besides unaltered 3. The dark reaction is accompanied 
by a slight blue-shift of the absorption spectrum, 
which indicates the formation of an intermediate. The 
educt 3 can be recovered in high yield from this inter- 
mediate, but the latter is an activated form of 3 which 
forms rapidly 5-oxobilindiones and formyltripyr- 
rionones (Scheer el al., 1977) upon addition of Zn’+ 
and admission of air. A fresh solution of 3 with pyri- 
dine and I,, in which the spectral changes are not 
yet visible, reacts at least an order of magnitude 
slower under otherwise identical conditions. At the 
present state, it is impossible to assign a specific struc- 
ture (isomer, complex, or a more or less oxidized 
species) to this intermediate formed in the dark. As 
the photochemical reaction occurs rapidly even in 
freshly prepared solutions, the latter appears to  be 
independent from the dark intermediate. 
To account for the long-wavelength absorption of 
P,.,, Grombein et a/. (1975) have invoked the chromo- 
Phytochrome interconversion model 477 
Table I .  Apparent pK-values 
for protonation (pK,)  and 
deprotonation (pK,) of bile 
pigments. The values are deter- 
mined by titration of the bilins 
(c 2 IO-’pM) with HCI and 
KOH. respectively, in a I : I  
mixtuye of a saturated solution 
of guanidinium chloride in 
water and methanol (Scheer. 
1976) monitored with a glass 
electrode 
Compound pK, pK, 
3 5.6* ICllt 
7 5.l* I O - l l t  
Dimer of 3 1.4 1 1  
6a.b 4.2 9.2 
*From Scheer (1976). 
tDestruction at pH > 10 
during titration. 
phore to be present in a deprotonated form. Based 
on this suggestion, a model for the PI, chromophore 
should not only have a violin-type absorption spec- 
trum, but also a sufficiently low pK-value to  be 
deprotonated under physiological conditions. Violins 
generally have too high pK-values in this respect 
(Falk and Leodolter, 1978). and this is true, too, for 
the dimeric violins. By contrast, the pK of 6a,b is 
considerably lower. Acid-base titrations of 6a,b in a 
mixed solvent system (Scheer, 1976) gave a poorly 
defined pK = 9.1 (Table 1) for the reversible depro- 
tonation to the zwitterion (spectrum in Fig. 2). The 
pK decrease is rationalized by coulomb interactions, 
similar to, e.g. the pK decrease of the carboxyl group 
in amino acids. 
Like the dimers described earlier (Scheer and 
Krauss 1977), compound 6a,b reverts thermally to the 
educt 3. The reversion occurs already at lower tem- 
peratures, e.g. in the FD source of the mass spec- 
trometer at 76”. If carried out  on a micropreparative 
scale, 3 is accompanied by the bilindione 7 and 
another unidentified oxidation product of the violin 
type. In contrast to the cleavage of the dimers, the 
reversion reaction of 6a,b in the absence of an added 
reductant can only be accomplished by a dispropor- 
tion reaction. This type of reaction has recently been 
indicated for the bilindione series, too (Eivazi et al., 
1977). It should be noted in this context that the in 
uitro dark reversion of Pf, to P, is accelerated by 
reducing agents (Mumford and Jenner, 1971 ; Kufer 
and Scheer, 1979). 
If the principle of a photochemically assisted nuc- 
leophilic addition is applied to the phytochrome in- 
terconversion. one would expect the protein side 
chains of cysteine, tyrosine, histidine or tryptophane 
as the most likely reaction partners. Preliminary 
results with the respective N- and C-protected amino 
acids have indicated that the latter three can add to 
compound 3, thus supporting this model. 
Ackrioevledgemertts-This work was supported by a grant 
from the Deutsche Forschungsgemeinschaft Bonn-Bad 
Godesberg. We are indebted to the Munchner Univer- 
sitatsgesellschaft for financial aid. We thank Prof. W. 
Riidiger for helpful discussions and continuing support. 
Prof. H. Budzikiewicz (University. Koln) for providing the 
FD-mass spectra. and G. Schild for measurement of the 
‘HMR spectra. 
Note  added $‘n pro05 In an attempt to identify intermediates 
of the oxidative reaction, a long-lived n-cation radical pro- 
duced from the zinc complexes of 3 and 7 has recently been 
characterized (Krauss, C. and H. Scheer (1979) Tetrahedron 
Lett., in press). 
REFERENCES 
Abramovitch. A. (1974) Pyridine and its Derirarrs. Vol. 14. Suppl. pt I. Interscience. New York. 
Bonfiglio. J. V.. R. Bonnett, M. B. Hursthouse, K. M. A. Malik and S. C. Naithani (1977) J .  Chrnl. 
Brigs, W. R. and H. V. Rice (1972) Ann. Reis. Plant Physiol. 23, 293-334. 
Cavaleiro. J. A. S. and K. M. Smith (1973) J. Chent. SOC. Prrkin Trans. I 2149-2155. 
Diner, U. E. and E. W. Lown (1971) Can. J. Chrm. 49, 403-415. 
Eivazi, F.. W. M. Lewis and K. M. Smith (1977) Tetraltedron Lett .  3083-3086. 
Falk, H. and T. Schlederer (1978) Monatsh. Cheni. 109. 1013-1015. 
Falk, H. and A. Leodolter (1978) Monarsh. Chem. 109, 883-897. 
Grombein. S.. W. Riidiger and H. Zimmermann (1975) Hoppr Srylrr’s Z. Phj.sio/. Ch~rll. 346. 
Holzwarth. A. R.. H. Lehner. S. E. Braslavsky and K. Schaffner (1978) Lirhiys Ann.  Chrn7. 2002-2017. 
Klein, G., S. Grombein and W. Rudiger (1977) Hoppe Seylrr’s 2. Physiol. Chem. 358, 1077-1079. 
Klein, G. and W. Riidiger (1978) Liebigs Ann. Chem. 267-279. 
Kufer, W. and H. Scheer (1979) Hoppe Seyler’s Z. Physiol. Chem. 360, 935-956. 
Mumford, F. E. and E. L. Jenner (1971) 
Pratt, L. H. (1978) Photochem. Photobiol. 27, 81-105. 
Riidiger, W. (1975) Ber. Dt.  Bor. Ges. 88, 125-139. 
Scheer, H. (1976) Z. Nururforsch. C 31, 413-417. 
Scheer, H. and C. Krauss (1977) Photochem. Photohiol. 25. 31 1-314. 
Scheer, H., U. Linsenmeier and C. Krauss (1977) Hoppe Seylrr’s 2. Physiol. Chrn~. 358. 185-196. 
Scheer, H. 
Schoch, S., G. Klein, U. Linsenmeier and W. Riidiger (1976) Liebigs Ann. Chem. 549-558. 
Schoch, S., U. Lempert, H. Wieschhoff and H. Scheer (1978) J. Chromatogr. 157, 357-364. 
Siegelman, H. W., B. C. Turner and S. B. Hendricks (1966) Plant Physiol. 41, 1289-1292. 
Whitlock, H. W.. Jr., R. Hanauer, M. Y. Oester and B. K. Bower (1969) J. Ant. C/t f~ t .  SOC. 91. 
SOC. Chem. Cornmun. 829-830. 
1709-1 7 14. 
Biochemistry 10, 98-101. 
(1978) Proceedings of the European Meetiny on Photomorphogenesis. Aarhus. 21-52. 
7485-7489. 


STUDIES ON PLANT BILE PIGMENTS-6. PHOTOCHEMICALLY ASSISTED REACTION OF A-DIHYDROBILINDIONE WITH NUCLEOPHILES AS A MODEL FOR PHYTOCHROME INTERCONVERSION

Open Access LMU ( Ludwig-Maximilians-Univ. München)

Phororhumirrr~ und Phrm,hir,loy?. Vol. 30. pp. 473 10 471 0 Perramon Press Ltd 1979. Prlnted in Great Britain 0031-X655 79 1001-0473%02.00/0 STUDIES ON PLANT BILE PIGMENTS-6. PHOTOCHEMICALLY ASSISTED REACTION OF A MODEL FOR PHYTOCHROME INTERCONVERSION A-DIHYDROBILINDIONE WITH NUCLEOPHILES AS C. KRAUSS, C. BUBENZER and H. SCHEER* Botanisches Institut, Munchen, W. Germany (Received 23 Fehruary 1979: accepted I M a y  1979) Abstract-From the A-dihydrobilindione 3. the pyridinium derivatives 6a,b and analogues therwf have been obtained by a formal nucleophilic substitution. The reaction is rationalized as a photochemically assisted oxidation of 3 with a subsequent regioselective addition of pyridine at C-5. By thermolysis. 6a.b yields back the parent bilin 3, together with an oxidation product. The significance of the reaction with respect to the phytochrome interconversion is discussed. INTRODUCTION Phytochrome, the photomorphogenetic reaction center pigment of higher green plants and some algae, contains a bile pigment chromophore covalently linked to  the protein (Siegelman et a/ . ,  1966; Riidiger, 1975). The photochromic conversion between the two long-lived forms P, and P,, is the primary event of the photomorphogenetic reaction chain (Pratt, 1978 ; Briggs and Rice, 1972). P, contains the A-dihydrobi- lindione chromophore 1 (Klein and Rudiger, 1978. Schoch et a/., 1976; Klein et a/., 1977; Grombein e f  al., 1975; Scheer, 1976). For the chromophore of P,r, structure 2 with the B-pyrrolic side chains unaltere'd (Klein et a/.,  1977) and a chromophore no longer con- taining the A 4,5-double bond (if judged from UV-vis spectra, Grombein et a/., 1975) has been proposed. This proposal implies a change in the molecular structure during the P, e P,, interconversion and excludes conformational changes and/or protonation- deprotonation events as the sole structural difference between the two forms (see Scheer, 1978, for a discus- sion). In exploiting the synthetic bilin 3 as a model for I, an increased reactivity at the A 4.5-double bond has been characterized for A-dihydrobilindiones, as compared to their fully unsaturated analogues, e.g. 7 (Scheer and Krauss, 1977; Scheer et al., 1977). Specifically, the reversible photodimerization of 3 has been proposed as a model type reaction for the phyto- chrome interconversions (Scheer and Kraus. 1977). Since present evidence supports but one chromophore in the monomeric phytochrome (Pratt 1978; Briggs and Rice, 1972; and citations therein), the possibility of using partners other than a second bilin for addi- tion reactions has been studied. Here, we report the *Reprint requests should be sent to: Dr. Hugo Scheer. Botanisches Institut. Menzinger Str. 67. 8000 Munchen 19. W. Germany. I ,Protein 2 3 473 474 C. KRAUSS, C. BUBENZER and H. SCHEER 0 7 - 2 c -  -ZH+ L 4 Reaction scheme reversible, photochemically assisted addition of nuc- leophiles of the pyridine type. MATERIALS AND METHODS The A-dihydrobilindione 3 was prepared by the method of Cavaleiro and Smith (1973) from octaethylporphyrin via octaethylchlorin (Whitlock et nl., 1969). I 2  and pyridine were reagent grade, the latter was dried over basic alumina (Woelm-ICN. Eschwege). All solvents were destilled prior to use or were reagent grade. Preparative photochemical reactions were carried out under nitrogen in pyrex cylinders at starting concentrations c : 0.1 mM of com- pound 3 with the light of a IOOOW tungsten halogen source (13 W/mZ between 550 and 620nm). UV-vis absorption spectra were recorded on a DMR 22 spectro- photometer (Zeiss, Oberkochen). and are reported in the format .I,.,,[nm] (c. Mass spectra were obtained in the EI mode with a JMS-D 100 spectrometer (Jeol, Japan). and in the FD mode with a model 731 instrument (Varian- MAT, Bremen). 'HMR spectra were recorded in the FT mode on a HFX 90 instrument (Bruker, Karlsruhe) with (CH3),Si as internal standard. Chemical shifts are reported in GCppm]. Column chromatography of the reaction mix- tures was carried out by an improved HPLC method (Schoch et al., 1978) on silica with a slightly alcaline ter- nary eluent system. For TLC. silica H (Merck. Darmstadt) was used. RESULTS AND DISCUSSION Irradiation of 3 in benzene/pyridine (4:l) in the presence of I2 or other one-electron oxidants leads t o  a rapid colour change from blue to  violet, similar t o  the change occurring during dimerization in the absence of pyridine (Scheer and Krauss, 1977) (Fig. 1). Chromatography of the product mixture yields only traces of the dimers, however. Two main products can be isolated, which migrate o n  silica only at high methanol concentrations, and which are irreversibly adsorbed upon prolonged exposure to  silica. TLC: Rf = 0.52 and 0.62, respectively, in dichloromethane/ methanol = 9:1, containing 1% aqueous ammonia; Phytochrome interconversion model A [a,..] 475 I nT - A [nm] Figure 1. UV-vis spectra of the photochemical reaction of 3 ( 4 0 p M )  in benzene containing pyridine (2.47 M )  and I2 (19.0 pM). Each trace corresponds to 1 min irradiation time, for the light conditions, see text. The arrows denote the change of absorption during the reaction. HPLC: V, = 106 and 182m(, respectively, on 4 x 300 mm silica with dichloromethane/methanol = 1:l containing 0.04% aqueous ammonia as eluent. The two products are interconvertible, e.g. by acid, and thus assigned as isomers. From the slower migra- ting zone, the isomer 6a of 2,3-dihydro-5(1-pyri- dinium)-octaathyl-l,19(21,24H)-bilindione has been isolated, the faster migration zone contains 6b. . UV-uis (see also Fig. 2): Cation (corresponding to the chromophore free base): 545 (10.27). 326 (19.9); dication (corresponding to the chromophore monoca- tion): 603 (25.5), 327 (2 1.4); zwitterion (corresponding to the chromophore anion): 585 (9.7); zinc complex: 630 ( I  7.5). 585 (9.7). 330 (25.5). 8.21 (Pyr-H-B,B‘, ‘d’), 8.49 (Pyr-H-A, 7’). 9.04 (Pyr-H-C,C’, ‘d’). 6b: 6.76 (H-10. s) 5.96 (H-15, s) 8.29 (Pyr-H-B,B’, *d’). 8.70 (Pyr-H-A, ‘t’) 9.38 (Pyr-H-C, C’. ‘d’). (C2H3),CO: 6a: 7.07 (H-I0 s), 6.21 (H-15, s). 8.42 (Pyr-H-B,B’, ‘d’), 8.92 Pyr-H-A, ‘t’), 9.59 (Pyr-H-C,C. W). 6b: 7.04 (H-10. s). 6.18 (H-15, s), 8.50 (Pyr-H-B,B’. W), 8.94 (Pyr-H-A. ‘t’), 9.57 (Pyr-H-C,C‘ ‘d’). CF,C002H: 6a: 7.71 (H-10, s), 6.60 (H-15, s), 8.35 (Pyr-H-B,B, ‘d’), 8.70 (Pyr-H-A, ‘t’), 9.1 1 (Pyr-H-C,C, ‘HMR: C’HClS: 6a: 6.72 (H-10, s), 5.92 (H-15. s), Figure 2. UV-vis spectra of the pyridinium salt 6s in methanol (-), its protonated form obtained after addition of 0.25% of 35% HCI (. . . . .). its deprotonated form obtained after addition of 0.25”,, methanolic KOH (-.-,-), and its Zn-complex obtained after addition of 0.25”; saturated methanolic Zn-acetate (-----). Extinction coefficients are based on the assumption of OH- as counterion after silica chromatography. 416 C. KRAUSS, C. BUBENZER and H. SCHEER ‘d‘). 6b: 7.68 (H-10, s), 6.48 (H-15, s), 8.43 (Pyr-H-B,B, ‘d), 8.80 (Pyr-H-A, ‘t’), 9.29 (Pyr-H-C,C, ‘d’). MS:EI-Mode:  556 (loo%), 554 (6673, 527 (46%), 432 (45%), 421 (24%), 418 (26%). FD-Mode: 634 (loo%), 556 (6%). Structure 6 of the pyridinium adducts has been established by the following arguments: ( I )  The ‘HMR spectrum shows the ABB’CC’ pattern (two ‘doublets’ and one ‘triplet’ (J.,PPdrc,,,  z 7 Hz) in the ratio 2:2:1) and the chemical shifts typical for N-sub- stituted pyridinium ions (Abramovitch. 1974; Diner and Lown, 1971); (2) The F D  mass spectrum has a peak at the highest mass of 634rnle (base peak), the only major fragment occurs at 556/e. In the E l  spec- trum, there are no reproducible peaks above the basis peak at 556 m/r; (3) The chromatographic properties indicate an ionic compound: (4) The site of substitu- tion is indicated from the ‘HMR spectrum. Of the three methine resonances in 3 assigned to H-5 (5.44 pprn), H-10 (6.56 ppm) and H-15 (5.97 ppm) (Cavaleiro and Smith, 1973), the ones of H-15 and H-10 are slightly shifted, but still present in 6a and 6b while the high-field signal characteristic for H-5 is lacking; (5) The UV-vis spectra (Fig. 2) of the monocation. dication, zwitterion and zinc complex are in the wavelength ranges typical of the free bases, cations, anions and zinc complexes, respectively, of ‘biliviolins’ containing the tripyrrinone chromophore 8*. Thus, the A 4,5-double-bond, although still present, is spectroscopically no longer evident. This effect of large meso-substituents has been observed earlier by Bonfiglio et a/. (1977) and related to the uncoupling of the adjacent rings by a pronounced twist between them; (6) The hydrogenated ring A is still present, as evidenced by the characteristic ’ HMR multiplet (Scheer et a/., 1977) at 6 = 1.5-1.8 ppm (2’,3’-CH2). by the 556m/e peak in the mass spectra. and by the reversion experiments; (7) The product is formulated as the tautomer 6 rather than 5 for the lack of a third meso proton signal in the ‘HMR spectrum, and for the generally higher basicity of N as compared to C. Due to its ready interconversion, compound 6a contained 19% 6b. In addition to the thermal dispro- portionation (see below). this is another reason for us not being able to  obtain a defined melting point. The isomer mixture 6a.b can be analyzed by ‘HMR. The signals of the methine protons and of the pyri- dinium ring protons listed above are most suitable due to their large separation for the respective isomers, but doubling of some signals can even be *The term ‘violin’ is used here as a phenomenological description designating linear oligopyrroles with a single visible absorption band of the free base around 560nm. Formula 8 with R, .  R,. R3 # 0 is typical of these com- pounds. Historically, the term has been associated with 4.5-dihydro- I ,  I9 (2 I ,  24 H) bilindiones. ?The central methine bridge-(C-10) is the principal point of nucleophilic attack in the (unoxidized) free bilins both for bilindiones (Falk and Schlederer, 1978; Holzwarth et a/., 1978) and dihydrobilindiones (Kufer and Scheer, 1979). observed in the methyl- and methylene region. After equilibration in CF,COO’H, the isomer ratio 6a:6b = 2:1 has been obtained, as compared to 4.5:I in the reaction mixture. The stereochemical origin for 6 to occur as isomers can be related to steric hindrance of the meso-pyri- dinium and the P-pyrrolic substituents. By this means, rotation around the C-5, C-6 bond is hindered, lead- ing to atropisomers. In conjunction with the asym- metric centers at ring A (transoid configuration of the ethyl groups), this gives rise to two diastereomeric pairs of enantiomers, even in case of the rings B, C and D being coplanar. Based on this model, the 5-nitrobilindione obtained from an achiral bilindione (Bonfiglio et al., 1973) possibly is a racemate. Reaction 3- 6 is formally a nucleophilic substitu- tion at C-5. It can be rationalized by the sequence shown in Scheme 1. Electron abstraction by the oxi- dant and subsequent deprotonation by the base present leads to a radical, and by repetition to the hypothetical dehydrobilindione 4, either one of which is attacked by pyridine at  C-5 to yield 6 after tauto- merization. Accordingly, the reaction is facilitated with the 2,4, 2,6 and 3,5-lutidines due to the inductive effect of the alkyl groups, and by increasing concen- trations of the oxidant, 12. Nucleophilic attack on cation radicals or dications of fully unsaturated bilin- diones have been reported to occur at both the outer methine bridges (Eivazi et al., 1977). Although we have not yet identified all by-products of the reaction of 3, the predominant formation of 6a,b supports again (Scheer, 1978). that hydrogenation of ring A activates selectively the adjacent methine bridge under oxidizing conditions.? Formation of 6a,b is photochemically assisted, but occurs principally in the dark, too. An anaerobic solution of 3 (40 p M )  in ben- zene containing I, (7.75 p M )  and pyridine (2.47 M) has been quenched after 2 h standing in the dark by filtration over Na,S20, into glycine buffer (pH 2.7). After workup, it contained 5% of the isomers 6a,b (in addition to minor amounts of a dimeric violin), besides unaltered 3. The dark reaction is accompanied by a slight blue-shift of the absorption spectrum, which indicates the formation of an intermediate. The educt 3 can be recovered in high yield from this inter- mediate, but the latter is an activated form of 3 which forms rapidly 5-oxobilindiones and formyltripyr- rionones (Scheer el al., 1977) upon addition of Zn’+ and admission of air. A fresh solution of 3 with pyri- dine and I,, in which the spectral changes are not yet visible, reacts at least an order of magnitude slower under otherwise identical conditions. At the present state, it is impossible to assign a specific struc- ture (isomer, complex, or a more or less oxidized species) to this intermediate formed in the dark. As the photochemical reaction occurs rapidly even in freshly prepared solutions, the latter appears to  be independent from the dark intermediate. To account for the long-wavelength absorption of P,.,, Grombein et a/. (1975) have invoked the chromo- Phytochrome interconversion model 477 Table I .  Apparent pK-values for protonation (pK,)  and deprotonation (pK,) of bile pigments. The values are deter- mined by titration of the bilins (c 2 IO-’pM) with HCI and KOH. respectively, in a I : I  mixtuye of a saturated solution of guanidinium chloride in water and methanol (Scheer. 1976) monitored with a glass electrode Compound pK, pK, 3 5.6* ICllt 7 5.l* I O - l l t  Dimer of 3 1.4 1 1  6a.b 4.2 9.2 *From Scheer (1976). tDestruction at pH > 10 during titration. phore to be present in a deprotonated form. Based on this suggestion, a model for the PI, chromophore should not only have a violin-type absorption spec- trum, but also a sufficiently low pK-value to  be deprotonated under physiological conditions. Violins generally have too high pK-values in this respect (Falk and Leodolter, 1978). and this is true, too, for the dimeric violins. By contrast, the pK of 6a,b is considerably lower. Acid-base titrations of 6a,b in a mixed solvent system (Scheer, 1976) gave a poorly defined pK = 9.1 (Table 1) for the reversible depro- tonation to the zwitterion (spectrum in Fig. 2). The pK decrease is rationalized by coulomb interactions, similar to, e.g. the pK decrease of the carboxyl group in amino acids. Like the dimers described earlier (Scheer and Krauss 1977), compound 6a,b reverts thermally to the educt 3. The reversion occurs already at lower tem- peratures, e.g. in the FD source of the mass spec- trometer at 76”. If carried out  on a micropreparative scale, 3 is accompanied by the bilindione 7 and another unidentified oxidation product of the violin type. In contrast to the cleavage of the dimers, the reversion reaction of 6a,b in the absence of an added reductant can only be accomplished by a dispropor- tion reaction. This type of reaction has recently been indicated for the bilindione series, too (Eivazi et al., 1977). It should be noted in this context that the in uitro dark reversion of Pf, to P, is accelerated by reducing agents (Mumford and Jenner, 1971 ; Kufer and Scheer, 1979). If the principle of a photochemically assisted nuc- leophilic addition is applied to the phytochrome in- terconversion. one would expect the protein side chains of cysteine, tyrosine, histidine or tryptophane as the most likely reaction partners. Preliminary results with the respective N- and C-protected amino acids have indicated that the latter three can add to compound 3, thus supporting this model. Ackrioevledgemertts-This work was supported by a grant from the Deutsche Forschungsgemeinschaft Bonn-Bad Godesberg. We are indebted to the Munchner Univer- sitatsgesellschaft for financial aid. We thank Prof. W. Riidiger for helpful discussions and continuing support. Prof. H. Budzikiewicz (University. Koln) for providing the FD-mass spectra. and G. Schild for measurement of the ‘HMR spectra. Note  added $‘n pro05 In an attempt to identify intermediates of the oxidative reaction, a long-lived n-cation radical pro- duced from the zinc complexes of 3 and 7 has recently been characterized (Krauss, C. and H. Scheer (1979) Tetrahedron Lett., in press). REFERENCES Abramovitch. A. (1974) Pyridine and its Derirarrs. Vol. 14. Suppl. pt I. Interscience. New York. Bonfiglio. J. V.. R. Bonnett, M. B. Hursthouse, K. M. A. Malik and S. C. Naithani (1977) J .  Chrnl. Brigs, W. R. and H. V. Rice (1972) Ann. Reis. Plant Physiol. 23, 293-334. Cavaleiro. J. A. S. and K. M. Smith (1973) J. Chent. SOC. Prrkin Trans. I 2149-2155. Diner, U. E. and E. W. Lown (1971) Can. J. Chrm. 49, 403-415. Eivazi, F.. W. M. Lewis and K. M. Smith (1977) Tetraltedron Lett .  3083-3086. Falk, H. and T. Schlederer (1978) Monatsh. Cheni. 109. 1013-1015. Falk, H. and A. Leodolter (1978) Monarsh. Chem. 109, 883-897. Grombein. S.. W. Riidiger and H. Zimmermann (1975) Hoppr Srylrr’s Z. Phj.sio/. Ch~rll. 346. Holzwarth. A. R.. H. Lehner. S. E. Braslavsky and K. Schaffner (1978) Lirhiys Ann.  Chrn7. 2002-2017. Klein, G., S. Grombein and W. Rudiger (1977) Hoppe Seylrr’s 2. Physiol. Chem. 358, 1077-1079. Klein, G. and W. Riidiger (1978) Liebigs Ann. Chem. 267-279. Kufer, W. and H. Scheer (1979) Hoppe Seyler’s Z. Physiol. Chem. 360, 935-956. Mumford, F. E. and E. L. Jenner (1971) Pratt, L. H. (1978) Photochem. Photobiol. 27, 81-105. Riidiger, W. (1975) Ber. Dt.  Bor. Ges. 88, 125-139. Scheer, H. (1976) Z. Nururforsch. C 31, 413-417. Scheer, H. and C. Krauss (1977) Photochem. Photohiol. 25. 31 1-314. Scheer, H., U. Linsenmeier and C. Krauss (1977) Hoppe Seylrr’s 2. Physiol. Chrn~. 358. 185-196. Scheer, H. Schoch, S., G. Klein, U. Linsenmeier and W. Riidiger (1976) Liebigs Ann. Chem. 549-558. Schoch, S., U. Lempert, H. Wieschhoff and H. Scheer (1978) J. Chromatogr. 157, 357-364. Siegelman, H. W., B. C. Turner and S. B. Hendricks (1966) Plant Physiol. 41, 1289-1292. Whitlock, H. W.. Jr., R. Hanauer, M. Y. Oester and B. K. Bower (1969) J. Ant. C/t f~ t .  SOC. 91. SOC. Chem. Cornmun. 829-830. 1709-1 7 14. Biochemistry 10, 98-101. (1978) Proceedings of the European Meetiny on Photomorphogenesis. Aarhus. 21-52. 7485-7489. 

Hoppe Seylrr’s 2.

Hoppr Srylrr’s Z.

Pyridine and its Derirarrs.

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STUDIES ON PLANT BILE PIGMENTS-6. PHOTOCHEMICALLY ASSISTED REACTION OF A-DIHYDROBILINDIONE WITH NUCLEOPHILES AS A MODEL FOR PHYTOCHROME INTERCONVERSION

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