Transformation of natural chlorophyll-a into chlorophyll-c analogs possessing the 17-acrylate residue

Article abstract of DOI:10.1246/cl.140798

Chlorophyll(Chl)-a derivatives possessing C18HC17HCH₂CH₂ were transformed into Chl-c analogs possessing C18=C17CH= CH through dehydrogenation to C18=C17, dihydroxylation to C18(OH)C17(OH), and double dehydration. This is the first re

Full text of DOI:10.1246/cl.140798

CL-140798
Received: August 26, 2014 | Accepted: September 6, 2014 | Web Released: September 12, 2014
Transformation of Natural Chlorophyll-a into Chlorophyll-c Analogs
Possessing the 17-Acrylate Residue
Meiyun Xu and Hitoshi Tamiaki*
Graduate School of Life Sciences, Ritsumeikan University, Kusatsu, Shiga 525-8577
(E-mail: tamiaki@fc.ritsumei.ac.jp)
Chlorophyll(Chl)-a derivatives possessing C18H-C17H-
cially available Spirulina (one of the cyanobacteria) powder
according to reported procedures.5 Oxidation of free-base
chlorin H2-1a with 2,3-dichloro-5,6-dicyanobenzoquinone
(DDQ) gave a complex mixture of chlorin products, and the
corresponding porphyrin H2-2a could not be isolated. After zinc
metallation of H2-1a [step (i) in Scheme 2], the resulting Zn-1a
was readily oxidized by DDQ in acetone [step (ii)]6 to give the
desired Zn-2a.7 The facile 17,18-dehydrogenation is ascribable
to a decrease in the oxidation potential by the insertion of zinc at
the central position. Reaction of Zn-2a with osmium tetroxide in
the presence of pyridine, followed by treatment with hydrogen
sulfide [step (iv)], afforded several products possessing
Cβ(OH)-Cβ¤(OH).8 Reverse-phase (RP) HPLC analysis associ-
ated with visible absorption and mass spectral data showed
nine products, 3 singly dihydroxylated isomers and 6 doubly
dihydroxylated isomers: 2,3-, 7,8-, and 17,18-dihydroxychlorins
as well as 2,3,7,8- and 2,3,17,18-tetrahydroxyisobacteriochlorins
CH2-CH2 were transformed into Chl-c analogs possessing
C18=C17-CH=CH through dehydrogenation to C18=C17,
dihydroxylation to C18(OH)-C17(OH), and double dehydration.
This is the first report on the synthesis of the latter porphyrin-
acrylate conjugates by modifying natural chlorin-propionate,
Chl-a.
Chlorophyll(Chl)-c is one of the light-harvesting pigments
in some oxygenic phototrophs, including brown algae and
diatoms.1 Most Chl-c molecules have a fully π-conjugated
porphyrin moiety and a free (unesterified) acrylate residue at
the 17-position, while other Chls possess a partially reduced
porphyrin π-skeleton and a 17-propionate residue esterified with
a lipophilic hydrocarbon chain.2 The unique 17-acrylate residue
is proposed to be enzymatically prepared by dehydrogenation
of the propionate residue in (divinyl-)protochlorophyllide-a
[= (DV-)PChl-aH] as a key intermediate well known for the
biosynthesis of other Chl molecules (Scheme 1),3 but the in vivo
transformation pathway has not yet been identified. Here, we
first report the synthesis of Chl-c analogs consisting of a 17-
acrylate-functionalized porphyrin π-conjugate by modifying
Chl-a. It is noted that Chl-c is divided into some molecular
species characterized by peripheral substituents: typically, Chl-
c1 for 7-methyl-8-ethyl form and Chl-c2 for 7-methyl-8-vinyl
form.4 The present Chl-c analogs were methyl ester forms of
demetallated Chl-c1 derivatives possessing the 3-ethyl and acetyl
groups instead of the 3-vinyl group and lacking the 132-
methoxycarbonyl group.
Methyl mesopyropheophorbide-a (H2-1a, Scheme 2) was
prepared by modifying natural Chl-a extracted from a commer-
Scheme 2. Synthesis of chlorophyll-c analogs H2-4 from
chlorophyll-a derivatives H2-1: (i) Zn(OAc)2¢2H2O/CH3OH-
CH2Cl2; (ii) DDQ/acetone; (iii) aq. HCl/CH2Cl2; (iv) OsO4,
C5H5N/CH2Cl2 and H2S; (v) p-TsOH¢H2O/C6H6, ¦.
Scheme 1. Proposed biosynthetic route of (divinyl-)proto-
chlorophyllide-a [(DV-)PChl-aH] to chlorophylls-c1/c2 (Chls-
c1/c2).
1864 | Chem. Lett. 2014, 43, 1864–1866 | doi:10.1246/cl.140798
© 2014 The Chemical Society of Japan

Scheme 3. Modification of Chls-c1/c2 to H2-4a: (i) aq. HCl/
CH2Cl2; (ii) CH2N2-(C2H5)2O/CH3OH-CH2Cl2; (iii) collidine,
reflux; (iv) H2-PtO2/THF-acetone-C2H5OH.
Scheme 4. Probable dehydrogenation pathways of 17-propio-
nate to 17-acrylate residue.
and 7,8,17,18-tetrahydroxybacteriochlorin (two stereoisomers
for each tetraol) (Scheme S1 and Figure S1 in ESI). No
dihydroxylation occurred at the C12=C13 double bond because
of an electron-withdrawing carbonyl group at the 13-position.
From the reaction mixture, cis-17,18-diol Zn-3a was separated
by RP-HPLC and treated with an aqueous hydrogen chloride
solution [step (iii)] to give H2-3a in 12% isolated yield from
Zn-2a.9 The low yield is primarily ascribed to the low
regioselectivity in the dihydroxylation to the three Cβ=Cβ¤
double bonds of zinc porphyrin Zn-2a, C2=C3, C7=C8, and
C17=C18. It is noted that free-base form H2-2a prepared by
acidic removal of the central zinc of Zn-2a was no longer
transformed into dihydroxylated (bacterio)chlorin compounds
because of its lower oxidizability (vide supra).
Heating a benzene solution of H2-3a and p-toluenesulfonic
acid (p-TsOH) at 50 °C for 6 h [step (v) in Scheme 2]10
consumed all of the starting material and gave a complex
mixture of products which lost water, methanol, or water and
methanol (Scheme S2 and Figure S2 in ESI). As a hydrophobic
fraction with a long retention time, RP-HPLC successfully
afforded the doubly dehydrated product H2-4a in 3% isolated
yield, which was identified by its 1H NMR, MS, and visible
spectra.11 HPLC analysis showed that no more improvement in
the formation of H2-4a could be observed during the reaction.
The coupling constant between the C171- and C172-protons
was 16 Hz, indicating the trans-form in the 17-acrylate residue.
To confirm the molecular structure, H2-4a was alternatively
synthesized by modifying natural Chl-c extracted from com-
mercially available Chaetoceros gracilis cells4 (Scheme 3).
Naturally occurring Chls-c1/c2 were transformed into methyl
pheophorbides-c1/c2 (5c and 5d)12 by an acid and diazomethane.
Pyrolysis of H2-5c and -5d in refluxing collidine gave H2-6c
and -6d, which was hydrogenated on platinum dioxide to afford
3,8-diethyl compound H2-4a after RP-HPLC purification. The
product obtained from Chl-c was identical to the product from
Chl-a aforementioned. In the present hydrogenation, H2-2a
was obtained as a by-product, so the 3-/3,8-(di)vinyl group(s) of
H2-6c and -6d was first reduced and the 17-acrylate residue was
further reduced to the 17-propionate residue.
Similar to the synthesis of 3-ethylated H2-4a, 3-acetylated
H2-4b was produced as follows (Scheme 2). OsO4 oxidation of
Zn-2b13 prepared by DDQ oxidation of Zn-1b gave cis-17,18-
diol Zn-3b with 7,8-diol and 7,8,17,18-tetraol. In the dihydrox-
ylation, neither C2=C3 nor C12=C13 reacted because of the
3- and 13-carbonyl groups. A mixture of the two diols separated
by flash column chromatography on silica gel was treated with
an acid, and RP-HPLC separation afforded H2-3b in 9% isolated
yield from Zn-1b. Acidic dehydration of H2-3b gave H2-4b (5%)
after RP-HPLC separation.14
In summary, methyl pyropheophorbide-a (R3 = CH=CH2
in H2-1),15 one of the Chl-a derivatives possessing the 17-
propionate residue on a chlorin π-skeleton, was converted to
Chl-c analogs H2-4a and -4b possessing the 17-acrylate residue
on a porphyrin π-skeleton. The proposed dehydrogenation
(oxidation and dehydration) mechanism from the 17-CH2CH2
moiety to the CH=CH moiety will be useful for elucidating
the biosynthetic pathway of (DV-)PChl-aH to Chls-c1/c2
(Scheme 4).
We thank Drs. Michio Kunieda and Yusuke Kinoshita of
Ritsumeikan University for preliminary experiments and prep-
aration of the manuscript, respectively. This work was partially
supported by Grants-in-Aid for Scientific Research (A)
(No. 22245030) as well as on Innovative Areas “Artificial
Photosynthesis (AnApple)” (No. 24107002) from the Japan
Society for the Promotion of Science (JSPS).
Supporting Information is available electronically on J-STAGE.
References and Notes
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Chem. Lett. 2014, 43, 1864–1866 | doi:10.1246/cl.140798
© 2014 The Chemical Society of Japan | 1865

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2.80, 2.02 (each 2H, m, 17-CH2CH2), 2.20 (3H, s, 18-CH3),
1.74 (3H, t, J = 8 Hz, 81-CH3), 1.69 (3H, t, J = 8 Hz, 31-
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11 To a solution of H2-3a (8 mg, 14 ¯mol) in benzene (15 mL)
was added p-TsOH¢H2O (4.5 mg, 24 ¯mol), and the solution
was stirred at 50 °C under N2 for 6 h. After cooling down, the
reaction mixture was diluted with CH2Cl2 (20 mL), then
washed with H2O, 4% aq. NaHCO3, and H2O, dried over
Na2SO4, and evaporated. The residue was purified by HPLC
(Cosmosil 5C18-AR-II 10 º © 250 mm, MeOH/MeCN =
1/1, 2.5 mL min¹1, retention time: 38 min) to give H2-4a
(3%): vis (CH2Cl2) -max = 649 (relative absorbance, 0.01),
6
7
1
591 (0.07), 575 (0.07), 529 (0.07), 436 nm (1.00); H NMR
(CDCl3): ¤ 10.03 (1H, s, 10-H), 9.88 (1H, s, 5-H), 9.87
(1H, s, 20-H), 9.18 (1H, d, J = 16 Hz, 17-CH), 6.88 (1H, d,
J = 16 Hz, 171-CH), 5.81 (2H, s, 131-CH2), 4.08 (2H, q,
J = 8 Hz, 8-CH2), 4.07 (3H, s, 12-CH3), 3.96 (2H, q,
J = 8 Hz, 3-CH2), 3.86 (3H, s, 172-COOCH3), 3.72 (3H, s,
7-CH3), 3.63 (3H, s, 12-CH3), 3.51 (3H, s, 18-CH3), 1.88
(3H, t, J = 8 Hz, 81-CH3), 1.84 (3H, t, J = 8 Hz, 31-CH3),
¹2.24, ¹3.16 (each 1H, br, NH © 2); HRMS (APCI) found:
m/z = 547.2704, calcd for C34H35N4O3: MH+, 547.2704.
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were added pyridine (400 ¯l) and OsO4 (30 mg, 0.12 mmol),
and the solution was stirred at room temperature overnight
under N2. MeOH (10 mL) was added and into the solution
was bubbled H2S for 15 min, then the mixture was filtered
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(Cosmosil 5C18-AR-II 10 º © 250 mm, MeOH/H2O =
87/13, 2.0 mL min¹1, retention time; 28 min) to give Zn-3a.
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1
597 (0.06), 579 (0.07), 535 (0.05), 439 nm (1.00); H NMR
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s, 20-H), 8.54 (1H, d, J = 15 Hz, 17-CH), 6.62 (1H, d,
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3.70 (3H, s, 172-COOCH3), 3.63 (3H, s, 18-CH3), 3.38 (3H,
s, 7-CH3), 3.33 (3H, s, 3-COCH3), 1.84 (3H, t, J = 8 Hz, 81-
CH3) [two NH signals were too broad to be visible.]; HRMS
(APCI) found: m/z = 561.2496, calcd for C34H33N4O4:
MH+, 561.2496.
1
407 nm (1.00); H NMR (CDCl3): ¤ 9.46 (1H, s, 5-H), 9.29
(1H, s, 10-H), 8.70 (1H, s, 20-H), 5.43, 5.24 (each 1H, d,
J = 20 Hz, 131-CH2), 3.86 (2H, q, J = 8 Hz, 8-CH2), 3.68
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1866 | Chem. Lett. 2014, 43, 1864–1866 | doi:10.1246/cl.140798
© 2014 The Chemical Society of Japan