2-Ethyl-3-methylmaleimide in Tokyo Bay sediments providing the first evidence for its formation from chlorophylls in the present photic and oxygenic zone

Article abstract of DOI:10.1271/bbb.66.1844

Tokyo Bay bottom sediments were analyzed for 2-ethyl-3-methylmaleimide, a degradation product of chlorophylls, which has been detected in ancient sediments. It was found in all sediments examined in concentrations of about 1 to 15 nmol/g- of dried sediment, and it was shown to be preserved for 100 years in the sediments. Its depth distribution agreed with that of the reported total organic carbon content of the sediments, reflecting a change in primary productivity. We concluded that this maleimide was produced under photic and oxygenic conditions in nature before the incorporation of photosynthesizing organisms into sediments.

Full text of DOI:10.1271/bbb.66.1844

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2-Ethyl-3-methylmaleimide in Tokyo Bay Sediments
Providing the First Evidence for its Formation from
Chlorophylls in the Present Photic and Oxygenic
Zone
Masaki KOZONOa, Shinya NOMOTOa, Hajime MITAa, Ryoshi ISHIWATARIb & Akira
SHIMOYAMAa
a Department of Chemistry, University of Tsukuba Tsukuba 305-8571, Japan
b Department of Chemistry, Tokyo Metropolitan University 1-1 Minami-ohsawa, Hachioji,
Tokyo 192-0397, Japan
Published online: 22 May 2014.
To cite this article: Masaki KOZONO, Shinya NOMOTO, Hajime MITA, Ryoshi ISHIWATARI & Akira SHIMOYAMA (2002) 2-
Ethyl-3-methylmaleimide in Tokyo Bay Sediments Providing the First Evidence for its Formation from Chlorophylls in
the Present Photic and Oxygenic Zone, Bioscience, Biotechnology, and Biochemistry, 66:9, 1844-1847, DOI: 10.1271/
bbb.66.1844
To link to this article: http://dx.doi.org/10.1271/bbb.66.1844
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Biosci. Biotechnol. Biochem., 66 (9), 1844–1847, 2002
2-Ethyl-3-methylmaleimide in Tokyo Bay Sediments Providing the
First Evidence for its Formation from Chlorophylls in the
Present Photic and Oxygenic Zone
Masaki KOZONO,1 Shinya NOMOTO † Hajime MITA,1 Ryoshi ISHIWATARI
, ,
and Akira SHIMOYAMA
1,
2,††
1,
†
1Department of Chemistry, University of Tsukuba, Tsukuba 305-8571, Japan
2Department of Chemistry, Tokyo Metropolitan University, 1-1 Minami-ohsawa, Hachioji,
Tokyo 192-0397, Japan
Received February 19, 2002; Accepted May 10, 2002
Tokyo Bay bottom sediments were analyzed for
2-ethyl-3-methylmaleimide, a degradation product of
chlorophylls, which has been detected in ancient sedi-
ments. It was found in all sediments examined in con-
cient sediments derived from chlorophylls or pho-
tosynthetic organisms.
Since the discovery of porphyrins in sediments by
Treibs in 1934,4) these compounds have been widely
studied as biomarkers for photosynthetic organisms.
2-Ethyl-3-methylmaleimide (1) was recently found in
Permian and Triassic marine sediments by Grice
centrations of about 1 to 15 nmol g- of dried sediment,
W
and it was shown to be preserved for 100 years in the
sediments. Its depth distribution agreed with that of the
reported total organic carbon content of the sediments,
re‰ecting a change in primary productivity. We con-
cluded that this maleimide was produced under photic
and oxygenic conditions in nature before the incorpora-
tion of photosynthesizing organisms into sediments.
et al.,5–6) and in Neogene and Cretaceous Tertiary
W
sediments by us,7–8) together with other alkylmalei-
mides. The compound is a novel biomarker for chlo-
rophylls, because many kinds of chlorophylls contain
a 3-ethyl-4-methylpyrrole moiety. No other precur-
sors in nature have been known to aŠord the 2-ethyl-
3-methylmaleimide structure, as far as we know.
Higher plants and phytoplankton are the major
contributors to terrestrial and marine sedimentary
organic matter, respectively, and the sedimentary
maleimide, therefore, is presumably derived from
chlorophylls of photosynthetic organisms. An advan-
tage of the use of this new biomarker compound is
that it can be extracted more easily from sediments
and identiˆed by GC-MS than porphyrins can be.
The transformation of maleimides from pyrrole
moieties of chlorophylls occurs under photic or
oxygenic conditions or both. Therefore, the occur-
rence of the maleimide in ancient sediments raised
Key words: maleimide; porphyrin; chlorophyll; sedi-
ment
Portions of biomolecules that have ceased to play
their roles in living organisms are deposited in sedi-
ments. Some of these molecules are decomposed to
simple molecules, mainly by the action of microor-
ganisms in the sediments, but others can remain in
sediments for geological periods, undergoing minor
or major modiˆcation by chemical transformation.
Consequently, these molecules become one of the ob-
jects in the ˆeld of organic geochemistry. It is im-
portant to understand the transformation processes
of biomolecules in sediments, to provide information
on the histories of the molecules and their sedimenta-
ry environments. For instance, from knowledge of
molecular transformations, such as racemization of
L
-amino acids to
D
and decarboxylation of n-fatty
acids to -alkanes in sediments, evolved the concepts
n
of the molecular clock and the carbon preference in-
dex, respectively.1–3) The preserved biomolecules that
have undergone only limited modiˆcation and can be
unambiguously linked with precursor compounds or
further with speciˆc organisms are called biomar-
kers; examples are porphyrins in crude oils and an-
Scheme 1.
†
To whom correspondance should be addressed.
Present adress: 4-24-4 Eifuku, Suginami-ku, Tokyo 168-0064, Japan
††

2-Ethyl-3-methylmaleimide in Tokyo Bay Sediments
1845
the issue of the conditions and timing of the oxida-
tion of tetrapyrrole pigments, because ancient sedi-
ments do not provide such oxidative conditions. In
this study, on the assumption that the maleimide is
formed in photic or oxygenic environments and
preserved in the sediments, we undertook a search
for maleimides in the sediments of Tokyo Bay, the
depositional environment of which is apparently ox-
idative.
Materials and Methods
General procedure for analysis of maleimide in
Tokyo Bay sediments. The Tokyo Bay bottom sedi-
ments that we analyzed were portions of a 100-cm
core collected at about 5 km o‹ng from the in‰ux of
the Tama River in 1986. The core was cut every
2.5 cm from the surface to 30 cm in depth and every
5.0 cm from 30 to 100 cm in depth. These layers were
lyophilized, pulverized, and kept in a freezer until
analysis. The samples (0.4 g) were extracted by soni-
Fig. 1. Total Ion Chromatogram (a) and Mass Fragmentogram
(b) of an Extract of Tokyo Bay Sediment and Total Ion Chro-
matogram of Authentic 2-Ethyl-3-methylmaleimide (c).
maleimide (1). Diethyl 2-ethyl-3-methylmaleate was
prepared by a similar method to that reported for the
stereoselective synthesis of diethyl 2-butyl-3-
methylmaleate,9) from diethyl 1-ethoxycarbonyl-
propane-1-phosphonate and ethyl pyruvate as a
cation with n-hexane, a 7:3 mixture of n-hexane and
CH2Cl2, CH2Cl2, a 4:1 mixture of CH2Cl2-methanol
and methanol (2 ml each) 6 times each, and each ex-
tract was put of a silica gel column (175
with -hexane, CH2Cl2, CH2Cl2-acetone (9:1), and
methanol (12 ml each) as eluents. The eluent was con-
centrated to 50 l under a stream of nitrogen. One
l solution was analyzed by GC-
×
10 mm i.d.)
colorless oil in the yield of 56
z
. 1H-NMR (CDCl3)
d
:
＝
＝
n
1.08 (3H, t,
J
J
7.5 Hz, 2-CH2CH3), 1.30, 1.32 (total
6.5 Hz, OCH2CH3), 1.96 (3H, s,
×
6H, t 2,
m
3-CH3), 2.38 (2H, q,
J
＝
7.5 Hz, 2-CH2CH3), 4.20,
＝
6.5 Hz, OCH2CH3).
×
4.22 (total 4H, q 2, J
microliter of a 50-
m
MS.
The maleate thus obtained (2.1 g, 10 mmol) in
ethanol (5 ml) was treated with 2 NaOH (5 ml) at
40 C for 3 h. The solution was acidiˆed with 2 HCl
at 0 C and extracted with ether. The extract was con-
centrated and the residual oil was reacted with urea
(2.0 g) at 100 C for 1 h. The resulting solid was ex-
For analysis of dihydrohematinic acid (3) or
hematinic acid (4), the fraction obtained from 1 g of
surface sediment was concentrated to dryness and
M
9
M
9
treated with 15
z BF3-methanol (1 ml) at 809C for
20 min. The reaction mixture was then concentrated
to dryness, dissolved in CH2Cl2, and analyzed by
GC-MS.
9
tracted with chloroform and the extract was puriˆed
by silica gel column chromatography with benzene-
ethyl acetate as the eluent to give the desired
The samples were analyzed with a Hewlett Packard
G1800A GCD system. The GC was equipped with an
maleimide (1) as needles (0.59 g, 43
z
), mp 67–8
: 1.15 (3H, t,
7.6 Hz, 2-CH2CH3), 1.98 (3H, s, 3-CH3), 2.42 (2H,
9C
1
FFAP bonded FS-WCOT capillary column (60 m
0.25 mm i.d.). The oven temperature was pro-
grammed to give 50 C for 2 min, to increase from
50 C to 150 C at the rate of 10
C min„1, then from
150 C to 230 C at the rate of 3
C min„1, and to hold
×
(lit. 68
9
C). H-NMR (CDCl3)
d
J
＝
＝
7.6 Hz, 2-CH2CH3), 8.30 (1H, broad s, NH).
9
q,
J
9
9
9
z
Anal. Found: C, 60.46; H, 6.55; N, 9.97 . Calcd.
9
9
9
for C7H9O2N: C, 60.42; H, 6.52; N, 10.07z.
at the ˆnal temperature for 50 min. Helium was used
as a carrier gas with a ‰ow rate of 1 ml min„1. Elec-
tron impact (70 eV) mass spectra were obtained by
Results and Discussion
scanning from m z 45 to 425 every 0.5 s.
Each of 16 samples of the divided core was extract-
ed successively by sonication with -hexane,
hexane-CH2Cl2 (7:3), CH2Cl2, CH2Cl2-methanol
(4:1), and methanol. The extracts were each separat-
ed by silica gel column chromatography, each frac-
tion being analyzed by GC-MS.
2-Ethyl-3-methylmaleimide (1) was detected in the
CH2Cl2-acetone fractions of the CH2Cl2-methanol
extracts of all the samples analyzed. Typical GC-MS
data of the extract are shown in Figs. 1 and 2,
together with those of an authentic sample, which
W
Identiˆcation of 2-ethyl-3-methylmaleimide was
made by comparison of the retention time on the GC
and of the MS fragment patterns with those of the
standard compound, which was synthesized through
a new easier path as stated below. The abundance of
maleimide was estimated by comparison of the peak
area on a mass fragmentogram of molecular ion to
that of the standard compound.
n
n-
Preparation of authentic 2-ethyl-3-methyl-

1846
M. KOZONO et al.
Fig. 2. Mass Spectra of Maleimide in the Extract of Tokyo Bay
Sediment (a) and Authentic 2-Ethyl-3-methylmaleimide (b).
certiˆed the identiˆcation of 1. A search for other ex-
pected oxidation products of chlorophylls, 2-methyl-
3-vinylmaleimide (2) and dihydrohematinic acid (3),
or hematinic acid (4), was also done by GC-MS, and
among them, only 4 was found as traces in the
methanol fraction of the CH2Cl2-methanol extract
after esteriˆcation. The source of 4 seems ambigu-
ous, as it can be formed both by oxidation of chlo-
rophylls accompanying dehydrogenation and by oxi-
dation of heme.
Fig. 3. Depth Distributions of 2-Ethyl-3-methylmaleimide and
Total Amount of Chlorophyll
a and Related Compounds (a)
and Total Organic Carbon Contents13) (b) in the Tokyo Bay
Sediments.
pounds will be reported elsewhere. The linear
sedimentation rate had been assumed to be
Jen and Mackinney reported the formation of 2-
ethyl-3-methylmaleimide (1) by sunlight irradiation
1.5 cm year from the surface to 25 cm in depth and
W
of chlorophyll
a
in organic solvents, the pho-
1.0 cm year below 25 cm in depth.13)
W
todecomposition accompanying oxygen uptake of
3 moles or more per chlorophyll molecule.10) They
found no other maleimides or related compounds,
such as 2, 3, or 4, in the reaction mixture. On the
other hand, the enzymatic breakdown pathway of
chlorophylls has recently become clearer, and an
oxygenolytic opening of porphyrin rings was found
to occur in senescent leaves between C-4 and C-5
of chlorophylls (see Scheme 1).11–12) However, no
reports have appeared about the enzymatic produc-
tion of maleimides from chlorophylls. Therefore,
oxygen-assisted photodecomposition of chlorophylls
or their enzymatic catabolites might contribute to the
formation of the 2-ethyl-3-methylmaleimide (1) that
we detected in Tokyo Bay sediments. This means that
maleimide generation probably occurs in dead organ-
isms before they are buried in the deposits of the
Tokyo Bay bottom. The practical absence of 2, 3, or
4 in the sediments is consistent with the previous
report.
The sediments below 30 cm in depth were found to
contain maleimide 1 in almost equal concentration
(about 2 nmol g), and a rather steep increase of the
W
maleimide content (from 2 to 13 nmol g) was ob-
W
served from the 20-cm- to the 30-cm-depth sample.
The reported TOC increase from the bottom to the
30-cm-depth sample has been attributed to the in-
crease in organic pollutants corresponding to popula-
tion growth in the metropolitan area. The higher and
almost uniform TOCs of the upper sediments had
been considered to re‰ect both the pollutant in‰ux
and the increased propagation of phytoplankton
caused by advanced eutrophication, the latter having
operated about 10 years later than the former. The
increase in the maleimide content being delayed be-
hind that of TOC suggests that maleimide 1 found in
the sediments originates from phytoplankton rather
than from terrestrial plants.
This analytical study provides the ˆrst evidence for
formation of 2-ethyl-3-methylmaleimide from chlo-
rophylls under photic and oxygenic conditions in
nature. This maleimide was found to be preserved for
about 100 years in the Tokyo Bay sediments, and its
depth distribution seemed to re‰ect a change in
primary productivity. In our previous study,
To examine the preservation of 1 in the sediments,
we analyzed the depth distribution of the quantity of
1 throughout the core, as shown in Fig. 3, together
with the depth proˆle of the total organic carbon
content (TOC) of the sediments.13) Included in Fig. 3
for comparison is the depth distribution of the total
Cretaceous Tertiary sediments proved to contain no
W
amount of chlorophyll
a
and related compounds
, pyropheophorbide a, and pyro-
); the details of analysis for these com-
2-methylmaleimide in spite of the existence of
methylpyrrole-containing porphyrins in appreciable
amounts.8) This fact may rule out the possibility of
(pheophytin
pheophytin
a
a

2-Ethyl-3-methylmaleimide in Tokyo Bay Sediments
1847
(Lower Rhine Basin, northwest Germany) inferred
from molecular and isotopic compositions of biomar-
ker components. Org. Geochem., 26, 677–690 (1997).
7) Kozono, M., Nomoto, S., Mita, H., and Shimoyama,
A., Detection of maleimides and their characteristics
in Neogene sediments of the Shinjo basin, Japan.
Geochem. J., 35, 225–236 (2001).
diagenetic generation of maleimides from porphyrins
in sediments. The maleimide detected in the ancient
sediments could have been produced before the in-
corporation of dead bodies of photosynthesizing
organisms into sediments, rather than from sedimen-
tary porphyrins by the action of diagenesis in sedi-
ments.
8) Shimoyama, A., Kozono, M., Mita, H., and
Nomoto, S., Maleimides in the Cretaceous Tertiary
W
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