Dörnemann, D.

Scheer, Hugo

Senge, M.

Senger, H.

Struck, A.

English

Open Access LMU

Hydroxylation of Chlorinated and Unchlorinated Chlorophylls i n vitro M . Senge*, A . Struck**, D . D ö r n e m a n n * , H . Scheer**, and H . Senger* * Fachbereich Biologie/Botanik, Philipps-Universität. D-355Ü Marburg/Lahn, Bundesrepublik Deutschland ** Botanisches Institut der Universität, Menzinger Straße 67, D-8000 München 19, Bundesrepublik Deutschland Z . Naturforsch. 43c, 515-518 (1988); received March 7, 1988 Chlorophyll Allomerization, Hydroxy-chlorophyll, Thin Layer Chromatography Chlorophyll a is hydroxylated quantitatively in the 13 :-position. when chromatographed on silica gel thin-layer plates. This was shown by using H P L C as a non-destructive method für analyzing photosyntheticpigments. The hydroxy-group of Chi R C I , a 13:-hydroxy-20-chloro-chl o . described by Dörnemann and Senger (1986) and Scheer et cd. (1986). is shown to be artificially introduced during the purification procedure by T L C . Introduction Dur ing the experiments revealing the structure of an unknown Chlorophyll , designated C h i R C I, iso-lated by thin-layer chromatography ( T L C ) from bulk Chlorophylls, a hydroxy-group was detected at the Oppos i t i on [1, 2]. It was already discussed whether this hydroxylation took place i n vivo or was an ar-tefact, induced during the Isolation procedure [1,2] . Such an assumption was suggested by divergent re-sults with H P L C Separation [3], and by preceeding investigations of Chlorophyll alterations on silica gel layers [4, 5]. The reinvestigation of this question was possible by the application of a non-destructive H P L C method for Separation and identification of Chlorophylls, together with the use of synthetic pig-ments. Materials and Methods Chlorophyl l a (Chi a ) was prepared from Scenedes-mus mutant C - 6 E and Scenedesmus obliquus W T -D 3 and prepurified according to Omata and Mura ta [6]. 20-Chloro-pheophytin a and 13 2-hydroxy-20-chloro-pheophytin a were prepared by a modifica-tion of the method of Woodward and Skaric [7] from the respective 20-unsubstituted pigments [2]. Meta la-tion with M g [8] yielded the respective Chlorophylls (2 isomers each). The structures were confirmed by N M R and absorption spectroscopy, interconversions and by demetalation to the pheophytins ( A . Struck and H . Scheer, to be published). Reprint requests to Prof. Dr. H . Senger. Verlag der Zeitschrift für Naturforschung, D-7400 Tübingen 0341-0382/88/0700-0515 $ 01.30/0 For enzymatic chlorination, C h i a was incubated with K C l and H 2 0 2 in K H 2 P 0 4 - b u f f e r (0.3 m, p H 5.0) in the presence of chloroperoxidase ( E C 1.11.1.10, Sigma, M u n i c h , C-0278) for 60 min . The reaction products were extracted with diethylether, dried over M g S 0 4 and subjected to T L C or H P L C analysis. The pigments were identified by absorption spec-troscopy, using an U v i k o n 820 (Kontron) spec-trophotometer. Molecular weights were determined by plasma desorption mass spectrometry ( P D M S ) [Senge and Senger in preparation, 9—11]. T L C was performed on Merck silica gel plates (#5553, M e r c k , Darmstadt, F . R . G . ) , according to D ö r n e m a n n and Senger [1]. H P L C analysis was carried out using a Spheri-sorb S5 RP-18 column with 5 um material (250 mm x 4 mm I D , Kont ron) . The elution System consisted of C H 3 C N / C H 3 O H (75/25; v/v) superim-posed with a multilinear gradient of water content, starting with 10% water. The water content was de-minished to 0% within 40 min , followed by a 10 min phase of regeneration at 0% water. Finally the water content was increased again up to 10% within 10 min [12]. The detection wavelength was 430 nm, the flow rate 1 ml/min. Results and Discussion The reaction product of an enzymatic chlorination of C h i a (comp. I in F ig . 1) was shown to be 13 2-hydroxy-20-chloro-chl a (comp. I V ) after purifica-tion by T L C [9]. This Compound is identical to the C h i R C I described by D ö r n e m a n n and Senger [1]. The purified Compound I V was eluted in the above 516 M . Senge et a l . • Hydroxylation of Chlorophylls in v i t r o J13 'RJ tOOCH3 ^c=o 133 1 3 i I 0 I Phytyl R1 = H R2 = H (I) R1 = H R2 = OH (I) Ri = Cl R 2 r H (I) Ri = Cl R2 = OH (E) Fig. 1. Structural formula of Chi a (I), 132-hydroxy-chl a (II), 20-chloro-chl a (III). 132-hydroxy-20-chloro-chl a (IV). A l l Compounds exist as epimers at C-13 2. "i 1 1 r 20 30 40 50 60 Retention time [min] Fig. 2. H P L C elution profiles for the reaction products of an enzymatic chlorination of 132-hydroxy-chl (A) and of Chi a (B). I - Chi a. II - 132-HO-chl a% III - 20-CI-chl a . IV - 132-HO-20-Cl-chl a , 5 - chl a\ 6 - 20-CI-chl a'. described H P L C System after a retention time of 33 min . In order to circumvent the laborious T L C Separa-t ion, the reaction products of the enzymatic chlorina-tion of C h i a were extracted from the reaction mix-ture and directly subjected to H P L C analysis. The elution profile (Fig. 2 B ) showed major peaks at re-tention times of 32 and 37 min. M i n o r peaks derived from epimerization of Chlorophylls at C-13 2 , general-ly occurring in polar solvents and during extraction in Chlorophylls containing an enolizable ß -ke toes te r . B y using C h i a, purified by H P L C as internal and external Standard, the peak at 32 min was identified as C h i a (I). It showed the typical absorption spec-trum with maxima at 661 nm and 428 nm in acetone, and P D M S revealed its molecular weight to be 893 (Table I). The Compound eluting after 37 min ex-hibited absorption maxima at 668 nm and 432 nm in acetone. The absorption spectrum was identical to the one obtained for C h i R C I ( I V ) , but while C h i R C I has a molecular weight of 943 [1], this Com-pound gave a M + - p e a k at 926 ± 1 in the PDMS-spec -trum. It follows that different products are obtained, when either T L C or H P L C are used for Separation of the reaction products of an enzymatic chlorination of C h i a. Similar discrepancies were observed, when C h i a was purified by H P L C or T L C . When C h i a was analyzed by H P L C before and after being subjected to T L C it showed different retention times. C h i a purified by H P L C was eluted after 32 min , while C h i a purified by T L C eluted in the H P L C System after 29 min . A spectral analysis showed that both Com-pounds exhibited the same absorption spectrum, with maxima at 661 nm and 428 nm in acetone. P D M S confirmed that only the H P L C purified C h i a had a molecular weight of 893, in conformity with the Table I. Absorption maxima in acetone. retention times in H P L C , and plasma desorption mass spectrometry data of Chi a (I), 132-hydroxy-chl a (II). 20-chloro-chl a (III), and 132-hydroxy-20-chloro-chl a (IV). Compound Retention Absorption Mass No. maxima of M +-peak time [min] [nm] I 32 661 428 893 II 29 661 428 908 III 37 668 432 927 IV 33 668 432 943 M . Senge et al. • Hydroxylation of Chlorophylls in v i t r o 517 weight expected from the formula. The C h i a sub-jected to T L C gave a PDMS-spec t rum with the M + -peak at 908 mass units. Hence , C h i a before and after T L C showed the same mass difference of 16 mass units as enzymatically chlorinated C h i a , when purified either by T L C or H P L C . When C h i purified by T L C , i.e. the Compound eluting at 29 min , was incubated with chloroperoxid-ase and the extracted reaction products were ana-lyzed by H P L C , an elution profile as given in Fig . 2 A was obtained. It shows major peaks at 29 min . i.e. not converted starting material and at 33 min . i.e. Compound I V . Since hydroxylation of Chlorophyll is a very com-mon allomerization reaction of porphyrins [13—16], and the mass difference of 16 mass units before and after T L C would be in good accordance with the introduction of a hydroxyl-group, chemically synthe-sized Compounds of known structure were used for comparison. 13 2-hydroxy-20-chloro-chl a ( IV) was E c o 00 O c ö JQ i_ O l/) -Q < J A » T 1 — i 1 •M B 0 10 20 30 40 50 60 Retention time [min] Fig. 3. H P L C elution profiles for the reaction products of a chemical synthesis of 132-hydroxy-20-chloro-chl a (A) and a mixture of chl a (I) and 20-chloro-chl a (III) (B). For iden-tification of the peaks see Fig. 2. The epimer mixture of IV is not separated under the conditions of the Separation. eluted after a retention time of 33 min (Fig. 3 A ) , and 20-chloro-chl a (III) was eluted after 37 min (Fig . 3 B ) . Hence, the reaction product of the en-zymatic chlorination of C h i a is 20-chloro-chl a (III) and the hydroxylgroup of C h i R C I is introduced artificially during thin layer chromatography. Further support for this conclusion was obtained by the following experiments. Pure Ch i a (I) was converted with chloroperoxidase to chlorinated Chlorophyll (III) [9], and then subjected to T L C Sep-aration. The whole T L C plate was scraped off, and the pigments were extracted exhaustively with acetone. When this sample was analyzed by H P L C an elution profile similar to F ig . 2 A was obtained (cf. F ig . 5). The hydroxylation of 20-chloro-chl a (III) to Com-pound ( IV) could be followed during Separation on the T L C plate. When a sample of the chemically synthesized Compounds (III) and ( IV) were applied to a T L C plate and developed after the method of D ö r n e m a n n and Senger [1], the band of Compound (III) migrated initially ahead of the band of Com-pound ( I V ) . Dur ing the development the spatial difference between both bands became smaller, and at the end of the T L C procedure the band of Com-pound (III) comigrated with the band of ( I V ) . The Compound ( IV) deriving from Compound (III), was scraped off, extracted and subjected to H P L C analy-sis. The elution profile is given in F ig . 4. The major peak has a retention time of 33 min , hence Com-pound (III) has been converted to Compound ( IV) during the T L C procedure. 0) ü c o n o < E 1) I V 1 I T 1 1 1 0 10 20 30 40 50 60 Retention time [min] Fig. 4. H P L C elution profile of chemically synthesized 20-chloro-chl a (see Fig. 2B) after development on Merck T L C plates. 518 M . Senge et al. • Hydroxylation of Chlorophylls in v i t r o Cht a + CPO ^ - 20-Cl-Chta 132-H0-Chl a * CPO HPLC - 132-H0-20-Ü-Chl a (1) (N) Fig. 5. Scheine of the connections between pure Chi a and the products obtained in enzymatic chlorination reactions, using either T L C or H P L C for purification of the reaction prod-ucts. CPO = chloroperoxidase and cosubstrates, see [9]. The scheme of F ig . 5 summarizes the different ex-periments. The results clearly demonstrate that hy-droxylation of Chlorophylls on silica gel plates is a nearly quantitative reaction. This Supports the vvarn-ing of Strain et al. [4] not to use silica gel materials in Chlorophyll chromatography. The results reported here have to be considered during further investiga-tions on the nature of C h i R C I. A ckn o xvledgem en ts The work was supported by grants from the Deutsche Forschungsgemeinschaft to H . Senger and to H . Scheer (Forschergruppe "Pflanzliche Tetra-pyrrole") . M . S . was supported by the Studienstif-tung des deutschen Volkes . [1] D . Dörnemann and H . Senger, Photochem. Photo-bio!. 43, 573-581 (1986). [2] H . Scheer, E . Groß, B . Nitsche. E . Cmiel, S. Schneider, W. Schäfer, H . - M . Schiebe!, and H. -R. Schulten, Photochem. Photobiol. 43, 559-571 (1986). [3] T. Watanabe, M . Kobayashi, M . Nakazato, I. Ikegami, and T. Hiyama, in: Progr. in Photosynth. Res., Vol. I, pp. 491-498 (J. Biggens, ed.), Martinus Nijhoff Publ., Dordrecht 1987. [4] H . H . Strain, J . Sherma, and M . Grandolfo, Anal . Chem. 39, 926-932 (1967). [5] M . F. Bacon, Biochem. J. 101, 3 4 C - 3 6 C (1966). [6] T. Omata and N . Murata, Plant Cell Physiol. 24, 1093-1100 (1983). [7] R. B . Woodward and V . Skaric. J. A m . Chem. Soc. 83, 4676-4677 (1961). [8] H.-P. Isenring, E . Zass, K . Smith, H . Falk, J . -L. Luisier, and A . Eschenmoser. Helv. Chim. Acta 58, 2357-2367 (1975). [9] H . Senger, D . Dörnemann, K . Kotzabasis. M . Senge. W. Schmidt, and M . Wissenbach, in: Progr. in Photo-synth. Res., Vol. IV, pp. 491-498 (J. Biggins, ed.), Martinus Nijhoff Pub!., Dordrecht 1987. [10] H . Jungclas, H . Danigel, and H . Schmidt, Org. Mass Spectrom. 17, 86-90 (1982). [11] R. D . Macfarlane and D. F. Torgerson, Science 191, 920-925 (1976). [12] K . Humbeck. S. Römer, and H . Senger, submitted to Botanica Acta. [13] A . Stoll and E . Wiedemann, Fortschr. d. Chem. org. Naturst. I, 159-253 (1938). [14] P. H . Hynninen and S. Assandri, Acta Chem. Scand. 27, 1478-1486 (1973). [15] F. C. Pennington, H . H . Strain, W. A . Svec, and J. J. Katz, J . A m . Chem. Soc. 89, 3875-3881 (1967). [1.6] P. M . Schaber, J. E . Hunt, R. Fries, and J. J . Katz. J. Chromatogr. 316, 25-41 (1984). 

Hydroxylation of chlorinated and unchlorinated chlorophylls in vitro

Universität München: Elektronischen Publikationen

Hydroxylation of Chlorinated and Unchlorinated Chlorophylls i n vitro 
M . Senge*, A . Struck**, D . D ö r n e m a n n * , H . Scheer**, and H . Senger* 
* Fachbereich Biologie/Botanik, Philipps-Universität. D-355Ü Marburg/Lahn, 
Bundesrepublik Deutschland 
** Botanisches Institut der Universität, Menzinger Straße 67, D-8000 München 19, 
Bundesrepublik Deutschland 
Z . Naturforsch. 43c, 515-518 (1988); received March 7, 1988 
Chlorophyll Allomerization, Hydroxy-chlorophyll, Thin Layer Chromatography 
Chlorophyll a is hydroxylated quantitatively in the 13 :-position. when chromatographed on silica 
gel thin-layer plates. This was shown by using H P L C as a non-destructive method für analyzing 
photosyntheticpigments. The hydroxy-group of Chi R C I , a 13:-hydroxy-20-chloro-chl o . described 
by Dörnemann and Senger (1986) and Scheer et cd. (1986). is shown to be artificially introduced 
during the purification procedure by T L C . 
Introduction 
Dur ing the experiments revealing the structure of 
an unknown Chlorophyll , designated C h i R C I, iso-
lated by thin-layer chromatography ( T L C ) from bulk 
Chlorophylls, a hydroxy-group was detected at the 
Oppos i t i on [1, 2]. It was already discussed whether 
this hydroxylation took place i n vivo or was an ar-
tefact, induced during the Isolation procedure [1,2] . 
Such an assumption was suggested by divergent re-
sults with H P L C Separation [3], and by preceeding 
investigations of Chlorophyll alterations on silica gel 
layers [4, 5]. The reinvestigation of this question was 
possible by the application of a non-destructive 
H P L C method for Separation and identification of 
Chlorophylls, together with the use of synthetic pig-
ments. 
Materials and Methods 
Chlorophyl l a (Chi a ) was prepared from Scenedes-
mus mutant C - 6 E and Scenedesmus obliquus W T -
D 3 and prepurified according to Omata and Mura ta 
[6]. 
20-Chloro-pheophytin a and 13 2-hydroxy-20-
chloro-pheophytin a were prepared by a modifica-
tion of the method of Woodward and Skaric [7] from 
the respective 20-unsubstituted pigments [2]. Meta la-
tion with M g [8] yielded the respective Chlorophylls 
(2 isomers each). The structures were confirmed by 
N M R and absorption spectroscopy, interconversions 
and by demetalation to the pheophytins ( A . Struck 
and H . Scheer, to be published). 
Reprint requests to Prof. Dr. H . Senger. 
Verlag der Zeitschrift für Naturforschung, D-7400 Tübingen 
0341-0382/88/0700-0515 $ 01.30/0 
For enzymatic chlorination, C h i a was incubated 
with K C l and H 2 0 2 in K H 2 P 0 4 - b u f f e r (0.3 m, p H 
5.0) in the presence of chloroperoxidase ( E C 
1.11.1.10, Sigma, M u n i c h , C-0278) for 60 min . The 
reaction products were extracted with diethylether, 
dried over M g S 0 4 and subjected to T L C or H P L C 
analysis. 
The pigments were identified by absorption spec-
troscopy, using an U v i k o n 820 (Kontron) spec-
trophotometer. Molecular weights were determined 
by plasma desorption mass spectrometry ( P D M S ) 
[Senge and Senger in preparation, 9—11]. 
T L C was performed on Merck silica gel plates 
(#5553, M e r c k , Darmstadt, F . R . G . ) , according to 
D ö r n e m a n n and Senger [1]. 
H P L C analysis was carried out using a Spheri-
sorb S5 RP-18 column with 5 um material 
(250 mm x 4 mm I D , Kont ron) . The elution System 
consisted of C H 3 C N / C H 3 O H (75/25; v/v) superim-
posed with a multilinear gradient of water content, 
starting with 10% water. The water content was de-
minished to 0% within 40 min , followed by a 10 min 
phase of regeneration at 0% water. Finally the water 
content was increased again up to 10% within 10 min 
[12]. The detection wavelength was 430 nm, the flow 
rate 1 ml/min. 
Results and Discussion 
The reaction product of an enzymatic chlorination 
of C h i a (comp. I in F ig . 1) was shown to be 13 2-
hydroxy-20-chloro-chl a (comp. I V ) after purifica-
tion by T L C [9]. This Compound is identical to the 
C h i R C I described by D ö r n e m a n n and Senger [1]. 
The purified Compound I V was eluted in the above 
516 M . Senge et a l . • Hydroxylation of Chlorophylls in v i t r o 
J13 
'RJ tOOCH3 
^c=o 133 1 3 i 
I 
0 
I 
Phytyl 
R1 = H R2 = H (I) 
R1 = H R2 = OH (I) 
Ri = Cl R 2 r H (I) 
Ri = Cl R2 = OH (E) 
Fig. 1. Structural formula of Chi a (I), 132-hydroxy-chl a 
(II), 20-chloro-chl a (III). 132-hydroxy-20-chloro-chl a 
(IV). A l l Compounds exist as epimers at C-13 2. 
"i 1 1 r 
20 30 40 50 60 
Retention time [min] 
Fig. 2. H P L C elution profiles for the reaction products of 
an enzymatic chlorination of 132-hydroxy-chl (A) and of 
Chi a (B). I - Chi a. II - 132-HO-chl a% III - 20-CI-chl a . 
IV - 132-HO-20-Cl-chl a , 5 - chl a\ 6 - 20-CI-chl a'. 
described H P L C System after a retention time of 
33 min . 
In order to circumvent the laborious T L C Separa-
t ion, the reaction products of the enzymatic chlorina-
tion of C h i a were extracted from the reaction mix-
ture and directly subjected to H P L C analysis. The 
elution profile (Fig. 2 B ) showed major peaks at re-
tention times of 32 and 37 min. M i n o r peaks derived 
from epimerization of Chlorophylls at C-13 2 , general-
ly occurring in polar solvents and during extraction in 
Chlorophylls containing an enolizable ß -ke toes te r . 
B y using C h i a, purified by H P L C as internal and 
external Standard, the peak at 32 min was identified 
as C h i a (I). It showed the typical absorption spec-
trum with maxima at 661 nm and 428 nm in acetone, 
and P D M S revealed its molecular weight to be 893 
(Table I). The Compound eluting after 37 min ex-
hibited absorption maxima at 668 nm and 432 nm in 
acetone. The absorption spectrum was identical to 
the one obtained for C h i R C I ( I V ) , but while C h i 
R C I has a molecular weight of 943 [1], this Com-
pound gave a M + - p e a k at 926 ± 1 in the PDMS-spec -
trum. It follows that different products are obtained, 
when either T L C or H P L C are used for Separation of 
the reaction products of an enzymatic chlorination of 
C h i a. 
Similar discrepancies were observed, when C h i a 
was purified by H P L C or T L C . When C h i a was 
analyzed by H P L C before and after being subjected 
to T L C it showed different retention times. C h i a 
purified by H P L C was eluted after 32 min , while C h i 
a purified by T L C eluted in the H P L C System after 
29 min . A spectral analysis showed that both Com-
pounds exhibited the same absorption spectrum, 
with maxima at 661 nm and 428 nm in acetone. 
P D M S confirmed that only the H P L C purified C h i a 
had a molecular weight of 893, in conformity with the 
Table I. Absorption maxima in acetone. retention times in 
H P L C , and plasma desorption mass spectrometry data of 
Chi a (I), 132-hydroxy-chl a (II). 20-chloro-chl a (III), and 
132-hydroxy-20-chloro-chl a (IV). 
Compound Retention Absorption Mass No. 
maxima of M +-peak 
time [min] [nm] 
I 32 661 428 893 
II 29 661 428 908 
III 37 668 432 927 
IV 33 668 432 943 
M . Senge et al. • Hydroxylation of Chlorophylls in v i t r o 517 
weight expected from the formula. The C h i a sub-
jected to T L C gave a PDMS-spec t rum with the M + -
peak at 908 mass units. Hence , C h i a before and after 
T L C showed the same mass difference of 16 mass units 
as enzymatically chlorinated C h i a , when purified 
either by T L C or H P L C . 
When C h i purified by T L C , i.e. the Compound 
eluting at 29 min , was incubated with chloroperoxid-
ase and the extracted reaction products were ana-
lyzed by H P L C , an elution profile as given in 
Fig . 2 A was obtained. It shows major peaks at 
29 min . i.e. not converted starting material and at 
33 min . i.e. Compound I V . 
Since hydroxylation of Chlorophyll is a very com-
mon allomerization reaction of porphyrins [13—16], 
and the mass difference of 16 mass units before and 
after T L C would be in good accordance with the 
introduction of a hydroxyl-group, chemically synthe-
sized Compounds of known structure were used for 
comparison. 13 2-hydroxy-20-chloro-chl a ( IV) was 
E 
c 
o 
00 
O 
c 
ö 
JQ 
i_ 
O 
l/) 
-Q 
< 
J 
A 
» T 1 — i 1 
•M B 
0 10 20 30 40 50 60 
Retention time [min] 
Fig. 3. H P L C elution profiles for the reaction products of a 
chemical synthesis of 132-hydroxy-20-chloro-chl a (A) and a 
mixture of chl a (I) and 20-chloro-chl a (III) (B). For iden-
tification of the peaks see Fig. 2. The epimer mixture of IV 
is not separated under the conditions of the Separation. 
eluted after a retention time of 33 min (Fig. 3 A ) , 
and 20-chloro-chl a (III) was eluted after 37 min 
(Fig . 3 B ) . Hence, the reaction product of the en-
zymatic chlorination of C h i a is 20-chloro-chl a (III) 
and the hydroxylgroup of C h i R C I is introduced 
artificially during thin layer chromatography. 
Further support for this conclusion was obtained 
by the following experiments. Pure Ch i a (I) was 
converted with chloroperoxidase to chlorinated 
Chlorophyll (III) [9], and then subjected to T L C Sep-
aration. The whole T L C plate was scraped off, and 
the pigments were extracted exhaustively with 
acetone. When this sample was analyzed by H P L C 
an elution profile similar to F ig . 2 A was obtained 
(cf. F ig . 5). 
The hydroxylation of 20-chloro-chl a (III) to Com-
pound ( IV) could be followed during Separation on 
the T L C plate. When a sample of the chemically 
synthesized Compounds (III) and ( IV) were applied 
to a T L C plate and developed after the method of 
D ö r n e m a n n and Senger [1], the band of Compound 
(III) migrated initially ahead of the band of Com-
pound ( I V ) . Dur ing the development the spatial 
difference between both bands became smaller, and 
at the end of the T L C procedure the band of Com-
pound (III) comigrated with the band of ( I V ) . The 
Compound ( IV) deriving from Compound (III), was 
scraped off, extracted and subjected to H P L C analy-
sis. The elution profile is given in F ig . 4. The major 
peak has a retention time of 33 min , hence Com-
pound (III) has been converted to Compound ( IV) 
during the T L C procedure. 
0) 
ü 
c 
o 
n 
o 
< 
E 
1) 
I V 
1 I T 1 1 1 
0 10 20 30 40 50 60 
Retention time [min] 
Fig. 4. H P L C elution profile of chemically synthesized 20-
chloro-chl a (see Fig. 2B) after development on Merck 
T L C plates. 
518 M . Senge et al. • Hydroxylation of Chlorophylls in v i t r o 
Cht a + CPO ^ - 20-Cl-Chta 
132-H0-Chl a * CPO HPLC - 132-H0-20-Ü-Chl a 
(1) (N) 
Fig. 5. Scheine of the connections between pure Chi a and the 
products obtained in enzymatic chlorination reactions, using 
either T L C or H P L C for purification of the reaction prod-
ucts. CPO = chloroperoxidase and cosubstrates, see [9]. 
The scheme of F ig . 5 summarizes the different ex-
periments. The results clearly demonstrate that hy-
droxylation of Chlorophylls on silica gel plates is a 
nearly quantitative reaction. This Supports the vvarn-
ing of Strain et al. [4] not to use silica gel materials in 
Chlorophyll chromatography. The results reported 
here have to be considered during further investiga-
tions on the nature of C h i R C I. 
A ckn o xvledgem en ts 
The work was supported by grants from the 
Deutsche Forschungsgemeinschaft to H . Senger and 
to H . Scheer (Forschergruppe "Pflanzliche Tetra-
pyrrole") . M . S . was supported by the Studienstif-
tung des deutschen Volkes . 
[1] D . Dörnemann and H . Senger, Photochem. Photo-
bio!. 43, 573-581 (1986). 
[2] H . Scheer, E . Groß, B . Nitsche. E . Cmiel, S. 
Schneider, W. Schäfer, H . - M . Schiebe!, and H. -R. 
Schulten, Photochem. Photobiol. 43, 559-571 (1986). 
[3] T. Watanabe, M . Kobayashi, M . Nakazato, I. 
Ikegami, and T. Hiyama, in: Progr. in Photosynth. 
Res., Vol. I, pp. 491-498 (J. Biggens, ed.), Martinus 
Nijhoff Publ., Dordrecht 1987. 
[4] H . H . Strain, J . Sherma, and M . Grandolfo, Anal . 
Chem. 39, 926-932 (1967). 
[5] M . F. Bacon, Biochem. J. 101, 3 4 C - 3 6 C (1966). 
[6] T. Omata and N . Murata, Plant Cell Physiol. 24, 
1093-1100 (1983). 
[7] R. B . Woodward and V . Skaric. J. A m . Chem. Soc. 
83, 4676-4677 (1961). 
[8] H.-P. Isenring, E . Zass, K . Smith, H . Falk, J . -L. 
Luisier, and A . Eschenmoser. Helv. Chim. Acta 58, 
2357-2367 (1975). 
[9] H . Senger, D . Dörnemann, K . Kotzabasis. M . Senge. 
W. Schmidt, and M . Wissenbach, in: Progr. in Photo-
synth. Res., Vol. IV, pp. 491-498 (J. Biggins, ed.), 
Martinus Nijhoff Pub!., Dordrecht 1987. 
[10] H . Jungclas, H . Danigel, and H . Schmidt, Org. Mass 
Spectrom. 17, 86-90 (1982). 
[11] R. D . Macfarlane and D. F. Torgerson, Science 191, 
920-925 (1976). 
[12] K . Humbeck. S. Römer, and H . Senger, submitted to 
Botanica Acta. 
[13] A . Stoll and E . Wiedemann, Fortschr. d. Chem. org. 
Naturst. I, 159-253 (1938). 
[14] P. H . Hynninen and S. Assandri, Acta Chem. Scand. 
27, 1478-1486 (1973). 
[15] F. C. Pennington, H . H . Strain, W. A . Svec, and J. J. 
Katz, J . A m . Chem. Soc. 89, 3875-3881 (1967). 
[1.6] P. M . Schaber, J. E . Hunt, R. Fries, and J. J . Katz. J. 
Chromatogr. 316, 25-41 (1984). 


https://epub.ub.uni-muenchen.de/2296/1/2296.pdf

Hydroxylation of chlorinated and unchlorinated chlorophylls in vitro

Abstract

Similar works

Full text

Available Versions

Open Access LMU

Universität München: Elektronischen Publikationen