Reaction mechanism of the macrophomate synthase: Experimental evidence on intermediacy of a bicyclic compound

Article abstract of DOI:10.1016/S0040-4039(99)02311-4

In the reaction of methyl coumalate 4 and oxalacetate with the macrophomate synthase, an aberrant product 5 was formed instead of the expected benzoate 6. The structure of 5 strongly indicates intermediacy of a bicyclic compound 10 in the extraordinary five-step transformation catalyzed by the macrophomate synthase. (C) 2000 Elsevier Science Ltd.

Full text of DOI:10.1016/S0040-4039(99)02311-4

TETRAHEDRON
LETTERS
Pergamon
Tetrahedron Letters 41 (2000) 1443–1446
Reaction mechanism of the macrophomate synthase:
experimental evidence on intermediacy of a bicyclic compound
Kenji Watanabe, Takashi Mie, Akitami Ichihara, Hideaki Oikawa ∗ and Mamoru Honma
Department of Applied Bioscience, Graduate School of Agriculture, Hokkaido University, Sapporo 060-8589, Japan
Received 15 November 1999; accepted 3 December 1999
Abstract
In the reaction of methyl coumalate 4 and oxalacetate with the macrophomate synthase, an aberrant product 5
was formed instead of the expected benzoate 6. The structure of 5 strongly indicates intermediacy of a bicyclic
compound 10 in the extraordinary five-step transformation catalyzed by the macrophomate synthase. © 2000
Elsevier Science Ltd. All rights reserved.
Keywords: enzymes; enzyme reaction; mechanisms.
Recently, we have purified the macrophomate synthase1 which catalyzes extraordinary multistep
transformation from oxalacetate and 2-pyrone 2 to macrophomic acid 1 as shown in Scheme 1. This
enzyme accepted various 2-pyrones which were converted to the corresponding benzoates.2 To our
knowledge, dehydroquinate synthase3 and mechanistically related 2-deoxy-scyllo-inosose synthase4 are
only examples on catalyzing different types of chemical reactions. This type of enzymatic reaction is of
great interest to not only enzymology but also bio-organic chemistry. Previously, we proposed that the
2-pyrone 2 was first transformed into a bicyclic intermediate 3 on the basis of observation that bicyclic
compounds mimicking the plausible intermediate 3 effectively inhibited the enzymatic reaction.1 In this
report, we describe experimental evidence that supports our proposed reaction mechanism shown in
Scheme 1.
Scheme 1. Transformation catalyzed by the macrophomate synthase
∗
Corresponding author.
0040-4039/00/$ - see front matter © 2000 Elsevier Science Ltd. All rights reserved.
PII: S0040-4039(99)02311-4
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During the study on the substrate selectivity of the macrophomate synthase,2 we found that a simple
2-pyrone, methyl coumalate 4, was consumed very rapidly to afford an aberrant product 55 which is
totally different from the expected benzoate 6 (Scheme 2). Under standard incubation conditions (10
min, 30°C), 10% of 2 was converted to the corresponding 1 while 4 was completely consumed within 10
min. Among 14 substrates tested,2 4 revealed the highest conversion rate.
Scheme 2. Enzymatic reaction with methyl coumalate 4 and oxalacetate
Based on the molecular formula C10H10O7 obtained from HR-MS, it was suggested that 5 is an adduct
of 4 and oxalacetate with concomitant loss of carbon dioxide. Since the aberrant product 5 was relatively
unstable, its structure was elucidated after conversion to the corresponding dimethyl ester 76 (Scheme
2). The NMR data including 1H, 13C NMR, COSY and HSQC spectra allowed us to deduce the partial
structure of 7 and then the gross structure was determined by HMBC analysis as shown in Fig. 1.
Fig. 1.
In the NOED experiment with 7, no NOE was detected between C2-H and either of unequivalent
methylene protons C6-H7 while the large coupling constant (9.4 Hz) between C2-H and C3-H was
1
observed in the H NMR spectrum of the trimethyl ester 8 derived from 7 by methanolysis of the
lactone moiety (Scheme 3). These data unambiguously confirmed the relative stereochemistry of 5,
7 and 8. In order to determine the absolute configuration of 8, we chose the method8 developed by
Fukushi due to the steric hindrance of the secondary alcohol at C3. Thus, trimethyl ester 8 was converted
to the corresponding (aR)- and (aS)-20-methoxy-10,10-binaphthyl-2-carboxylic acid (MBNC) esters 9a
and 9b, respectively. The calculated ∆δ values (∆δ=δ(aS)−δ(aR)) in Fig. 2 established the absolute
configuration of 8 as shown in Scheme 3.
Formation of the aberrant product 5 in the reaction of 4 would be reasonably explained by conversion
from the plausible intermediate 10 via lactone ring opening with concomitant allylic rearrangement and
the subsequent lactonization with C-8 carboxylate as shown in Scheme 4. These conversions might
occur in the active site of the enzyme because no quenched product of the corresponding cation was
detected. Relatively large differences (6.6 kcal/mol) on the steric energy between 5 and 10 supports

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Scheme 3.
Fig. 2.
that these reactions are favorable. The reason why the reaction with 4 provided the lactone 5 instead
of the formation of normal product 6 might be ascribed to flexibility of the plausible intermediate 10
in the active site of the enzyme. Since the bicyclic intermediate 10 has only one substituent compared
with another intermediate 3 which possesses three substituents, this conformational freedom allowed
10 to escape the usual dehydrative decarboxylation process. The C–C bond formations described in
Scheme 4 are possibly regarded as a Michael addition–aldol reaction although a possibility involving a
Diels–Alder reaction cannot be rigorously ruled out. Currently, we are working on elucidating the full
reaction sequence in this extraordinary enzymatic transformation.
Scheme 4. The formation of an aberrant product 5 in the enzymatic reaction with 4. The steric energies (SE) were calculated by
MM2

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Acknowledgements
We are grateful to Mr. K. Watanabe and Dr. E. Fukushi in our department for measuring MS and NMR
spectra, and to Dr. Y. Fukushi in our department for providing the useful reagent MBNCs. This work was
supported by a Grant-in-Aid from the Ministry of Education, Science and Culture of Japan. K.W. thanks
the JSPS fellowship for Japanese Junior Scientists.
References
1. Oikawa, H.; Watanabe, K.; Yagi, K.; Ohashi, S.; Mie, T.; Ichihara, A.; Honma, M. Tetrahedron Lett. 1999, 40, 6983–6986.
2. Watanabe, K.; Mie, T.; Ichihara, A.; Oikawa, H.; Honma, M. Biosci. Biotechnol. Biochem. in press.
3. Widlanski, T. S.; Bender, S. L.; Knowles, J. R. J. Am. Chem. Soc. 1987, 109, 1873–1875; Widlanski, T. S.; Bender, S. L.;
Knowles, J. R. J. Am. Chem. Soc. 1989, 111, 2299–2300.
4. Iwase, N.; Kudo, F.; Yamauchi, N.; Kakinuma, K. Biosci., Biotechnol. Biochem. 1998, 62, 2396–2407.
5. Compound 5: [α]22D=+25.5 (c 0.17, MeOH); IR (CHCl3) νmax 2924, 1790, 1716, 1645, 1441, 1261, 1047, 955, 801 cm−1
;
1H NMR (270 MHz, CDCl3) δ 7.14 (m, 1H, C5-H), 5.65 (dd, J=5.9 and 1.5 Hz, 1H, C3-H), 3.82 (s, 3H, OCH3), 3.72 (d,
J=5.9 Hz, 1H, C2-H), 3.11 (dd, J=20.2 and 3.0 Hz, 1H, C6-H), 2.69 (dd, J=20.2 and 2.7 Hz, 1H, C6-H); 13C NMR (125 MHz,
CDCl3) δ 176.3 (C8), 171.5 (C1), 164.1 (C9), 142.6 (C5), 131.9 (C4), 73.2 (C7), 70.9 (C3), 52.3 (C2), 52.2 (C10), 34.2 (C6);
MS (FD+) m/z (relative intensity) 243 (M++H, 100); FDHRMS for C10H11O7 (M++H) calcd: 243.0505; found: 243.0487.
6. Compound 7: [α]22D=+18.9 (c 0.80, CHCl3); IR (CHCl3) νmax 2956, 2332, 1733, 1645, 1436, 1258, 966, 916, 732 cm−1
;
1H NMR (270 MHz, CDCl3) δ 7.04 (m, 1H, C5-H), 5.56 (dd, J=5.4 and 1.8 Hz, 1H, C3-H), 3.75 (s, 3H, OCH3), 3.66 (s,
3H, OCH3), 3.57 (d, J=5.6 Hz, C2-H), 3.01 (dd, J=20.2 and 3.3 Hz, 1H, C6-H), 2.59 (dd, J=20.2 and 4.0 Hz, 1H, C6-H);
13C NMR (125 MHz, CDCl3) δ 175.8 (C8), 166.9 (C1), 163.7 (C10), 142.3 (C5), 131.7 (C4), 73.3 (C3), 71.1 (C7), 52.6 (C2),
52.6 (OCH3), 52.5 (OCH3), 34.5 (C6); MS (FD+) m/z (relative intensity) 257 (M++H, 100); FDHRMS for C11H13O7 (M++H)
calcd: 257.0661; found: 257.0652.
3
7. The relative stereochemistry of 7 was also confirmed by relative intensities of the cross peaks originated from J (C,H)
correlation in the HMBC spectrum optimized for 3J (C,H)=7 Hz. See: Bax, A.; Summers, M. F. J. Am. Chem. Soc. 1986, 108,
2093–2094.
8. Fukushi, Y.; Yajima, C.; Mizutani, J. Tetrahedron Lett. 1994, 35, 599–602.