Identification of tetrapyrrole compounds excreted by Rhodobacter sphaeroides and sources of the methyl hydrogens of bacteriochlorophyll a biosynthesized by R. sphaeroides, based on13C-NMR spectral analysis of coproporphyrin III tetramethyl ester

Article abstract of DOI:10.1248/cpb.55.1067

Red-fluorescent tetrapyrrole compounds excreted by Rhodobacter sphaeroides into the culture broth were concluded to be coproporphyrinogen (Copro'gen) III and uroporphyrinogen (Uro'gen) I, based on the ¹³C-NMR spectral identification of coproporphyrin (Copro) III tetramethyl ester and uroproporphyrin (Uro) I octamethyl ester. The sources of the methyl hydrogens of bacteriochlorophyll a were established by analysis of the ¹³C-NMR spectra of ²H,¹³C-Copro III tetramethyl ester chemically derived from ²H,¹³C-Copro'gen III biosynthesized through the feeding of δ-amino[2-¹³C]levulinic acid (ALA) to R. sphaeroides in medium containing 50% ²H₂O. We confirmed the previous finding that one of the methyl hydrogens was derived from water in the medium during decarboxylation of four acetyl side chains of Uro'gen III to generate Copro'gen III. It was further shown that the other hydrogen atoms, previously reported to be derived from methylene hydrogens at C-2 of ALA, had been exchanged with hydrogen of water in the medium in the biosynthetic pathways leading from ALA to Copro'gen III.

Full text of DOI:10.1248/cpb.55.1067

July 2007
Notes
Chem. Pharm. Bull. 55(7) 1067—1069 (2007)
1067
Identification of Tetrapyrrole Compounds Excreted by Rhodobacter
sphaeroides and Sources of the Methyl Hydrogens of Bacteriochlorophyll a
Biosynthesized by R. sphaeroides, Based on 13C-NMR Spectral Analysis of
Coproporphyrin III Tetramethyl Ester
Katsumi IIDA,* Masayuki NAKAMURA, Hiroshi HANAMITSU, and Masahiro KAJIWARA*
Department of Medicinal Chemistry, Meiji Pharmaceutical University; 2–522–1 Noshio, Kiyose, Tokyo 204–8588, Japan.
Received October 13, 2006; accepted April 16, 2007; published online April 20, 2007
Red-fluorescent tetrapyrrole compounds excreted by Rhodobacter sphaeroides into the culture broth were
concluded to be coproporphyrinogen (Copro’gen) III and uroporphyrinogen (Uro’gen) I, based on the 13C-NMR
spectral identification of coproporphyrin (Copro) III tetramethyl ester and uroproporphyrin (Uro) I octamethyl
ester. The sources of the methyl hydrogens of bacteriochlorophyll a were established by analysis of the 13C-NMR
spectra of 2H,13C-Copro III tetramethyl ester chemically derived from 2H,13C-Copro’gen III biosynthesized
through the feeding of d-amino[2-13C]levulinic acid (ALA) to R. sphaeroides in medium containing 50% 2H2O. We
confirmed the previous finding that one of the methyl hydrogens was derived from water in the medium during
decarboxylation of four acetyl side chains of Uro’gen III to generate Copro’gen III. It was further shown that the
other hydrogen atoms, previously reported to be derived from methylene hydrogens at C-2 of ALA, had been ex-
changed with hydrogen of water in the medium in the biosynthetic pathways leading from ALA to Copro’gen III.
Key words Rhodobacter sphaeroides; bacteriochlorophyll a; coproporphyrin III; 13C-NMR; d-amino[2-13C]levulinic acid (ALA);
2H2O
Analysis of the 13C-NMR and broad-band deuterium and
Therefore, we tried to identify the compounds having red
proton-decoupled 13C-NMR (13C-{1H}{2H}NMR) spectra of fluorescence in the culture broth, and utilized them to re-
2H,13C-bacteriochlorophyll a (after conversion to methyl investigate the biosynthetic sources of the methyl hydrogens
2H,13C-bacteriopheophorbide a with CH3OH/H2SO4) derived of bacteriochlorophyll a by repeating the [2-13C]ALA feed-
from our previous two feeding experiments with d-amino[2- ing experiment in medium containing 50% 2H2O in R.
13C]levulinic acid (ALA) (ALA, a biosynthetic intermediate sphaeroides.
2
of tetrapyrrole) in medium containing 50% H2O and [2,2-
Experimental
2H2,2-13C]ALA to Rhodobacter sphaeroides led us to pro-
Chemicals and Instruments [2-13C]ALA was synthesized by our
pose a mechanism involving decarboxylation of the four
acetyl side chains of uroporphyrinogen (Uro’gen) III in the
biosynthesis of coproporphyrinogen (Copro’gen) III (Fig.
1).1) As regards the sources of the methyl hydrogens of bacte-
riochlorophyll a, we concluded that two of the hydrogens of
each methyl group were retained from ALA and the other
one was derived from water in the medium. However, in a
[2,2-2H2,2-13C]ALA feeding experiment, signals influenced
by none and by only one deuterium atom, which could not be
interpreted by the proposed decarboxylation mechanism,
were observed in the 13C-NMR spectra of 2H,13C-methyl bac-
teriopheophorbide a. These signals could be assigned to the
method2) from sodium acetate (99 atom% 13C), which was purchased from
2
2
Cambridge Isotope Laboratories. H2O (98 atom% H) was purchased from
1
Shoko Co., Ltd. All other chemicals were of analytical grade. All H-NMR
(300 MHz) spectra were recorded on a Varian Gemini-300 spectrometer, and
the signal of TMS (0 ppm) was used as an internal standard. All 13C-NMR
(150 MHz) spectra were recorded on a JEOL JNM-ECA 600 spectrometer
with a cold probe for a solution of 2H,13C-Copro III tetramethyl ester in
C2HCl3; the signal of C2HCl3 (77.0 ppm) was used as an internal standard.
All UV spectra were recorded on a Jasco UVIDEC-610C spectrometer. All
FAB-MS spectra were recorded on a JEOL DX302 with the aid of 3-ni-
trobenzyl alcohol (3-NOBA).
Isolation of Compounds Having Red Fluorescence from the Culture
Broth of R. sphaeroides The culture of R. sphaeroides IFO 12203 was
carried out by a modification of the method described in our previous
paper.1) The cultures were anaerobically grown under illumination
(2400 lux) in seed culture medium (60 ml), which consisted of yeast extract
(2.0 g·lꢀ1), DL-malic acid (2.7 g·lꢀ1), KH2PO4 (0.5 g·lꢀ1), K2HPO4
(0.5 g·lꢀ1), (NH4)2HPO4 (0.8 g·lꢀ1), MgSO4 (0.2 g·lꢀ1), EDTA
2
four methyl carbons of H,13C-methyl bacteriopheophorbide
a derived from [2-13C]ALA and [2-2H,2-13C]ALA contained
in [2,2-2H2,2-13C]ALA synthesized by our method.1,2) These
results suggested that the deuterium atom, which was derived
(2.5 mg·lꢀ1),
ZnCl2
(2.5 mg·lꢀ1),
FeSO4·7H2O
(1.0 mg·lꢀ1),
from [2,2-2H2,2-13C]ALA, had been exchanged with hydro- MnCl2·4H2O (0.5 mg·lꢀ1), CuCl2·2H2O (0.1 mg·lꢀ1), CoCl2·6H2O
(0.5 mg·lꢀ1) and H3BO4 (0.02 mg·lꢀ1) at pH 6.8 (adjusted with saturated
NaHCO3 solution), in a 60 ml test tube at 27 °C. Seed culture (60 ml) incu-
bated for 7 d and a sterilized solution of ALA (60 mg) in H2O (10 ml), which
had been filtered through a membrane filter (0.2 mm), were added to fermen-
tation culture medium (1 l), which had the same composition as the seed cul-
ture medium, in a 1 l fermentation bottle. The cultures of R. sphaeroides in
two 1 l fermentation bottle were continuously grown photosynthetically
(2400 lux) at 27 °C for 7 d. Sephadex DEAE A-25 (3.0 g) was added to the
supernatant obtained by centrifugation of the culture broth for 20 min at
12300 g, and the mixture was stirred. After 30 min, the Sephadex was
gen of water in the medium in the biosynthetic steps from
ALA to bacteriochlorophyll a in R. sphaeroides. The [2-
13C]ALA feeding experiment in medium containing 50%
2H2O resulted in low incorporation of labeled compounds
2
under the influence of H2O, and the signals of the four
methyl carbons influenced by at least two deuterium atoms
could not be clearly observed in 13C-NMR spectra of 2H,13C-
methyl bacteriopheophorbide a. However, we observed a red
fluorescence in the culture broth of R. sphaeroides, which
produces bacteriochlorophylls intracellularly.
collected by filtration, lyophilized and suspended in
a mixture of
CH3OH/H2SO4 (95 : 5, v/v). The mixture was left for 24 h at room tempera-
∗ To whom correspondence should be addressed. e-mail: iida@my-pharm.ac.jp; kajiwara@my-pharm.ac.jp
© 2007 Pharmaceutical Society of Japan

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Vol. 55, No. 7
Fig. 1. Biosynthetic Pathways to Bacteriochlorophyll a from ALA via Uroporphyrinogen (Uro’gen) III and Coproporphyrinogen (Copro’gen) III in R.
sphaeroides, and Structures of Chemically Derived Methyl Bacteriopheophorbide a and Coproporphyrin (Copro) III Tetramethyl Ester
ture. Methanol-insoluble material was removed, then the solution was neu-
tralized with saturated NaHCO3 solution and evaporated. The residue was
dissolved in ion-exchanged water and the solution was extracted three times
with CH2Cl2. The combined organic layer was washed with water. The or-
the culture broth of R. sphaeroides were identified as
Copro’gen III and Uro’gen I on the basis of the identifi-
cation of their derivatives Copro III tetramethyl ester and
Uro I octamethyl ester by analysis of the FAB-MS, UV and
1H-NMR spectra and comparison of our previous studies.3—6)
Copro III tetramethyl ester derived from Copro’gen III, a
biosynthetic intermediate of bacteriochlorophyll a, is more
stable than bacteriochlorophyll a. Therefore, Copro III tetra-
methyl ester isolated from the culture broth of R. sphaeroides
can be used to determine the sources of the methyl hydrogens
of bacteriochlorophyll a biosynthesized by R. sphaeroides.
2H,13C-Copro III tetramethyl ester was isolated from
the supernatant obtained by centrifugation of the culture
ganic layer was dried over anhydrous sulfate and evaporated. The resulting
mixture was purified by column chromatography on silica gel with ben-
zene/ethyl acetate (10 : 1—8 : 1, v/v) to give Copro III tetramethyl ester
(11 mg), and with benzene/ethyl acetate (2 : 1, v/v)–CHCl3 to give Uro I oc-
tamethyl ester (24 mg); Copro III tetramethyl ester; FAB-MS (3-NOBA):
Calcd for C40H46O8N4; 710.83, Found; 711 (MHꢁ), UV lmax (CHCl3) nm:
401.2, 500.0, 532.4, 569.6, 623.2, 1H-NMR (300 MHz, CDCl3) d: ꢀ3.81
(2H, br, ꢂNH), 3.28 (8H, m, –CH2CH2CO2–), 3.61, 3.63, 3.64, 3.65 (12H,
4ꢃs, –CH2CH2CO2CH3), 3.67, 3.68, 3.69. 3.70 (12H, 4ꢃs, –CH3), 4.40
(8H, m, –CH2CH2CO–), 10.06, 10.07, 10.08, 10.09 (4H, 4ꢃs, meso proton),
Uro I octamethylester; FAB-MS (3-NOBA): Calcd for C48H54O16N4; 942.98,
Found; 943 (MHꢁ), UV lmax (CHCl3) nm: 405.6, 501.6, 536.4, 573.2, 626.0,
1H-NMR (300 MHz, CDCl3) d: ꢀ3.63 (2H, br, ꢂNH), 3.38 (8H, t,
Jꢂ7.7 Hz, –CH2CH2CO2–), 3.40 (12H, s, –CH2CH2CO2CH3), 3.79 (12H, s,
–CH2CO2CH3), 4.47 (8H, t, Jꢂ7.7 Hz, –CH2CH2CO2–), 5.15 (8H, s,
–CH2CO2–), 10.21 (4H, s, meso protons).
2
broth containing 50% H2O of R. sphaeroides in the pres-
ence of [2-13C]ALA. The magnified 13C-NMR and 13C-
2
{1H}{2H}NMR spectra for the methyl region of H,13C-
Copro III tetramethyl ester are shown in Figs. 2a and b, re-
spectively. In Fig. 2a, four singlet signals (11.5325, 11.5708,
11.6259, 11.6642 ppm) and four triplet signals (11.2860,
11.3219, 11.3745, 11.4152 ppm) can be observed. In Fig. 2b,
there are three groups of four singlet signals. The chemical
shifts of the four most downfield singlet signals were
11.5301, 11.5708, 11.6259 and 11.6642 ppm. The chemical
shifts of the four singlet signals of the center group were
11.2836, 11.3219, 11.3745 and 11.4176 ppm, and those of
the four most upfield singlet signals were 11.0346, 11.0682,
11.1208 and 11.1663 ppm.
Feeding of [2-13C]ALA to R. sphaeroides in Medium Containing 50%
(v/v) 2H2O The above-mentioned seed culture (60 ml) and a sterilized
2
solution of [2-13C]ALA (60 mg, 4.5 mmol·lꢀ1) in 50% (v/v) H2O (10 ml),
which had been filtered through a membrane filter (0.2 mm), were added to
50% (v/v) 2H2O fermentation culture medium (1 l), which had the same
composition as the seed culture medium, in a 1 l fermentation bottle. The
cultures of R. sphaeroides were continuously grown photosynthetically
2
(2400 lux) at 27 °C for 7 d. H,13C-Copro III tetramethyl ester (5 mg) was
isolated from the supernatant obtained by centrifugation of the culture broth
as described above.
Results and Discussion
Two tetrapyrrole compounds having red fluorescence in
By comparison of Fig. 2a with Fig. 2b, the four most

July 2007
1069
(11.2860, 11.3219, 11.3745, 11.4152 ppm) in Fig. 2a are
transformed to the four singlet signals (11.2836, 11.3219,
11.3745, 11.4176 ppm) in Fig. 2b by additional deuterium
decoupling. Therefore, these four triplet signals were derived
from the four singlet signals, which were split into triplets
2
owing to H–13C spin coupling of 19.6 Hz (J2H–13C) and
shifted upfield by 37.8 Hz owing to one deuterium a-isotope
effect, so they can be assigned to four 13C-methyl carbons
(C-21, C-71, C-121 and C-191, respectively) bearing one deu-
terium atom. Thus, these results confirmed that one hydrogen
atom in these 13C-methyl groups was introduced from deu-
2
terium of H-water in the medium by the previous proposed
decarboxylation mechanism when the four methyl groups of
Copro’gen III were generated from the four acetyl side
chains of Uro’gen III in R. sphaeroides. The small singlet
signals (11.0346, 11.0682, 11.1208, 11.1663 ppm), which did
not appear in Fig. 2a, appeared after additional deuterium de-
coupling (Fig. 2b). These four singlet signals are a-isotope-
shifted (75.6 Hz) by two deuterium atoms, indicating that two
hydrogen atoms of the 13C-methyl hydrogens at C-21, C-71,
C-121 and C-191 were exchanged with deuterium of 2H-water
in the medium (one deuterium atom was introduced into the
methyl group during decarboxylation, as mentioned above).
These results show that the hydrogen atoms derived from the
methylene hydrogens at C-2 of ALA were exchanged with
2
the deuterium of H-water in the medium during the biosyn-
thetic steps from ALA to Copro’gen III in R. sphaeroides.
Acknowledgment We wish to thank Mrs. Nemoto N. and Asakura K. of
JEOL Co. for measurement of 13C-NMR spectra.
References
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(1995).
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Fig. 2. Magnified (a) 13C-NMR Spectrum and (b) 13C-{1H}{2H}NMR
Spectrum of 2H,13C-Coproporphyrin (Copro) III Tetramethyl Ester in the
Methyl Region
downfield singlet signals of Figs. 2a and b can be assigned to
four 13C-methyl carbons (C-21, C-71, C-121 and C-191),
which were derived from the 13C-carbon of [2-13C]ALA, in
2H,13C-Copro III tetramethyl ester. The four triplet signals
6) Kajiwara K., Hara K., Takatori K., Chem. Pharm. Bull., 42, 817—820
(1994).