A compact chemical miniature of a holoenzyme, coenzyme NADH linked dehydrogenase. Design and synthesis of bridged NADH models and their highly enantioselective reduction

Article abstract of DOI:10.1021/ja992990y

An L-lactate dehydrogenase that requires coenzyme NADH catalyzes the enantioselective reduction of pyruvate to L-lactate in anaerobic glycolysis. As the first homochiral ansa-type NADH models, we designed the bridged NADH models 10a-c having a parapyridinophane structure for strictly mimicking the stereospecificity of hydrogen transfer in the biological asymmetric reduction with NADH. These models were synthesized in several steps from the corresponding bridged nicotinate 5 prepared by our novel pyridine-formation reaction of formyl-substituted (vinylimino)phosphorane 4 with methyl propiolate. The bridged NADH models 10a-c effected excellent biomimetic reduction at various temperatures in the presence of magnesium ion to achieve both the enantioselective and stereospecific reduction of the pyruvate analogues 12u-z into chiral lactate analogues 13u-z with 88-99% ee. The high enantioselectivity was almost completely dependent on the planar chirality of 10a-c but not on the nature of the substituents of their carbamoyl groups. The biomimetic reduction proceeded with retention of the planar chirality, showing that the bridged NADH models are useful for being recycled. An isotope experiment with the deuterated model (±)-10d confirmed the stereospecific hydrogen transfer, which is in good accordance with natural coenzyme characteristics. The model (S)-10c also exhibited good enantioselectivity for the reduction of activated ketones 14k-n into the corresponding chiral alcohols 15k-n with 79-89% ee. The simple bridged NADH model (S)-10c having both a primary carbamoyl group and a shielding bridge feigning an enzyme wall suggests a compact chemical miniature of a holoenzyme, coenzyme NADH linked dehydrogenase, in terms of the unique structure, high enantioselectivity, and recyclability.

Full text of DOI:10.1021/ja992990y

J. Am. Chem. Soc. 2000, 122, 4563-4568
4563
A Compact Chemical Miniature of a Holoenzyme, Coenzyme NADH
Linked Dehydrogenase. Design and Synthesis of Bridged NADH
Models and Their Highly Enantioselective Reduction1
Nobuhiro Kanomata*,†,‡ and Tadashi Nakata§
Contribution from PRESTO, Japan Science and Technology Corporation (JST), and Department of
Industrial Chemistry, School of Science and Technology, Meiji UniVersity, Tama-ku,
Kawasaki 214-8571, Japan, and RIKEN (The Institute of Physical and Chemical Research), Wako,
Saitama 351-0198, Japan
ReceiVed August 17, 1999
Abstract: An L-lactate dehydrogenase that requires coenzyme NADH catalyzes the enantioselective reduction
of pyruvate to L-lactate in anaerobic glycolysis. As the first homochiral ansa-type NADH models, we designed
the bridged NADH models 10a-c having a parapyridinophane structure for strictly mimicking the
stereospecificity of hydrogen transfer in the biological asymmetric reduction with NADH. These models were
synthesized in several steps from the corresponding bridged nicotinate 5 prepared by our novel pyridine-
formation reaction of formyl-substituted (vinylimino)phosphorane 4 with methyl propiolate. The bridged NADH
models 10a-c effected excellent biomimetic reduction at various temperatures in the presence of magnesium
ion to achieve both the enantioselective and stereospecific reduction of the pyruvate analogues 12u-z into
chiral lactate analogues 13u-z with 88-99% ee. The high enantioselectivity was almost completely dependent
on the planar chirality of 10a-c but not on the nature of the substituents of their carbamoyl groups. The
biomimetic reduction proceeded with retention of the planar chirality, showing that the bridged NADH models
are useful for being recycled. An isotope experiment with the deuterated model (()-10d confirmed the
stereospecific hydrogen transfer, which is in good accordance with natural coenzyme characteristics. The model
(S)-10c also exhibited good enantioselectivity for the reduction of activated ketones 14k-n into the
corresponding chiral alcohols 15k-n with 79-89% ee. The simple bridged NADH model (S)-10c having
both a primary carbamoyl group and a shielding bridge feigning an enzyme wall suggests a compact chemical
miniature of a holoenzyme, coenzyme NADH linked dehydrogenase, in terms of the unique structure, high
enantioselectivity, and recyclability.
Introduction
Coenzyme NADH, a cofactor of L-lactate dehydrogenase,
functions as an enantioselective agent that reduces pyruvate to
L-lactate during anaerobic glycolysis. The environment inside
the dehydrogenase achieving the asymmetric reduction satisfies
two important elements: (i) stereoselective transfer of one of
the diastereotopic C-4 hydrogen atoms in NADH to achiral
substrates (HR specific in L-lactate dehydrogenase)2 and (ii)
activation and well-controlled orientation of the substrates with
hydrogen bonds of the amino acid residue3 (Figure 1). The
natural coenzyme NADH contains a simple 1,4-dihydronicotin-
amide moiety as a recyclable redox center for catalytic use,
* To whom correspondence should be addressed. Fax: +81-44-934-
7906. E-mail: kanomata@isc.meiji.ac.jp.
† PRESTO, JST.
‡ Meiji University.
§ RIKEN.
(1) A part of this work has previously been published as a communica-
tion: Kanomata, N.; Nakata, T. Angew. Chem., Int. Ed. Engl. 1997, 36,
1207-1211.
(2) (a) Loewus, F. A.; Ofner, P.; Fisher, H. F.; Westheimer, F. H.;
Vennesland, B. J. Biol. Chem. 1953, 202, 699-704. (b) You, K.-s. CRC
Crit. ReV. Biochem. 1984, 17, 313-451.
(3) (a) Holbrook, J. J.; Liljas, A.; Steindel, S. J.; Rossmann, M. G. In
The Enzymes, 3rd ed.; Boyer, P. D., Ed.; Academic Press: New York, 1975;
Vol. XI, pp 191-292. (b) Bra¨nde´n, C.-I.; Eklund, H. In Dehydrogenases
Requiring Nicotinamide Coenzymes; Jeffery, J., Ed.; Birkhauser Verlag:
Basel, 1980; pp 40-84.
Figure 1. Schematic description of biological reduction with coenzyme
NADH in L-lactate dehydrogenase.
which has both prochiral unsubstituted hydrogens at its C-4
position and a primary carbamoyl group, and the corresponding
10.1021/ja992990y CCC: $19.00 © 2000 American Chemical Society
Published on Web 04/29/2000

4564 J. Am. Chem. Soc., Vol. 122, No. 19, 2000
Kanomata and Nakata
apoenzyme provides a chiral environment for its biological
reduction to induce high enantioselectivities toward its sub-
strates. This asymmetric induction has attracted the attention
of organic and bioorganic chemists to explore the features of
such a stereospecific reduction by challenging its mimicry,4-10
or the related asymmetric reduction of activated ketones,8a,c,10b,11
imines,11b or enamines6e in organic media. However, the
conventional strategy to design an artificial NADH is the
syntheses of the 1,4-dihydronicotinamides or their analogues
having a chiral center at their C-4 positions5 and/or chiral
sidearms on their carbamoyl groups.6-10 Therefore, the former
suffers from loss of chirality at C-4 during the course of the
model reactions, and the latter generally requires significant
modification of the dihydronicotinamide unit in such a way to
introduce a fused ring system, additional chiral auxiliaries, a
chiral sulfoxide group, etc. To strictly mimic the stereoselective
transfer of the hydrogen atom in recyclable artificial systems,
we have designed the novel bridged NADH models 10a-c,
which incorporate the oligomethylene bridge feigning an
“enzyme wall” to regulate the stereoselective approach of
pyruvate analogues for accomplishment of their biomimetic
reduction with high enantioselectivity. Especially, the model
(S)-10c incorporating not only the ansa bridge but also a primary
carbamoyl group is worth being tested as a compact chemical
Scheme 1a
a E ) an ester group.
miniature of lactate dehydrogenase containing coenzyme NADH
to probe whether such a simple model compound efficiently
works in artificial systems. We now report the detailed results
of the synthesis of the bridged NADH models 10a-c, the first
homochiral ansa-type NADH models,12 and their highly enan-
tioselective reduction of pyruvate analogues as well as some
activated ketones with good selectivity to demonstrate that
stereoselective shielding of a dihydronicotinamide ring is the
absolute and the minimally sufficient factor for artificial NADH
systems with high enantioselectivity.
(4) Recent review articles: (a) Kanomata, N. J. Synth. Org. Chem., Jpn.
1999, 57, 512-522. (b) Murakami, Y.; Kikuchi, J.; Hisaeda, Y.; Hayashida,
O. Chem. ReV. 1996, 96, 721-758. (c) Dupas, G.; Levacher, V.; Bourgui-
gnon, J.; Que´guiner, G. Heterocycles 1994, 39, 405-429. (d) Burgess, V.
A.; Davies, S. G.; Skerlj, R. T. Tetrahedron: Asymmetry 1991, 2, 299-
328.
(5) (a) Ohno, A.; Ikeuchi, M.; Kimura, T.; Oka, S. J. Am. Chem. Soc.
1979, 101, 7036-7040. (b) Mikata, Y.; Hayashi, K.; Mizukami, K.;
Matsumoto, S.; Yano, S.; Yamazaki, N.; Ohno, A. Tetrahedron Lett. 2000,
41, 1035-1038. Ohno, A.; Kashiwagi, M.; Ishihara, Y. Tetrahedron 1986,
42, 961-973. (c) de Kok, P. M. T.; Bastiaansen, L. A. M.; van Lier, P. M.;
Vekemans, J. A. J. M.; Buck, H. M. J. Org. Chem. 1989, 54, 1313-1320.
(d) Meyers, A. I.; Oppenlaender, T. J. Am. Chem. Soc. 1986, 108, 1989-
1996. Meyers, A. I.; Brown, J. D. J. Am. Chem. Soc. 1987, 109, 3155-
3156.
(6) (a) Combret, Y.; Torche´, J. J.; Ple´, N.; Duflos, J.; Dupas, G.;
Bourguignon, J.; Que´guiner, G. Tetrahedron 1991, 47, 9369-9382. (b)
Combret, Y.; Torche´, J. J.; Binay, P.; Dupas, G.; Bourguignon, J.; Que´guiner,
G. Chem. Lett. 1991, 125-128. (c) Combret, Y.; Duflos, J.; Dupas, G.;
Bourguignon, J.; Que´guiner, G. Tetrahedron 1993, 49, 5237-5246. (d)
Be´dat, J.; Levacher, V.; Dupas, G.; Que´guiner, G.; Bourguignon, J. Chem.
Lett. 1995, 327-328. Be´dat, J.; Levacher, V.; Dupas, G.; Que´guiner, G.;
Bourguignon, J. Chem. Lett. 1996, 359-360. (e) Leroy, C.; Levacher, V.;
Dupas, G.; Que´guiner, G.; Bourguignon, J. Tetrahedron: Asymmetry 1997,
8, 3309-3318.
(7) Burgess, V. A.; Davies, S. G.; Skerlj, R. T.; Whittaker, M.
Tetrahedron: Asymmetry 1992, 3, 871-901. Burgess, V. A.; Davies, S.
G.; Skerlj, R. T. J. Chem. Soc., Chem. Commun. 1990, 1759-1762. Davies,
S. G.; Skerlj, R. T.; Whittaker, M. Tetrahedron: Asymmetry 1990, 1, 725-
728.
(8) (a) Seki, M.; Baba, N.; Oda, J.; Inouye, Y. J. Am. Chem. Soc. 1981,
103, 4613-4615. (b) Hoshide, F.; Ohi, S.; Baba, N.; Oda, J.; Inouye, Y.
Agric. Biol. Chem. 1982, 46, 2173-2175. (c) Seki, M.; Baba, N.; Oda, J.;
Inouye, Y. J. Org. Chem. 1983, 48, 1370-1373. (d) Skog, K.; Wennerstro¨m,
O. Tetrahedron Lett. 1992, 33, 1751-1754.
(9) de Vries, J. G.; Kellogg, R. M. J. Am. Chem. Soc. 1979, 101, 2759-
2761. Jouin, P.; Troostwijk, C. B.; Kellogg, R. M. J. Am. Chem. Soc. 1981,
103, 2091-2093. Talma, A. G.; Jouin, P.; de Vries, J. G.; Troostwijk, C.
B.; Werumeus Buning, G. H.; Waninge, J. K.; Visscher, J.; Kellogg, R. M.
J. Am. Chem. Soc. 1985, 107, 3981-3997.
(10) Chiral sulfoxide models: (a) Imanishi, T.; Hamano, Y.; Yoshikawa,
H.; Iwata, C. J. Chem. Soc., Chem. Commun. 1988, 473-475. (b) Obika,
S.; Nishiyama, T.; Tatematsu, S.; Miyashita, K.; Iwata, C.; Imanishi, T.
Tetrahedron 1997, 53, 593-602. (c) Obika, S.; Nishiyama, T.; Tatematsu,
S.; Miyashita, K.; Imanishi, T. Chem. Lett. 1996, 853-854. Obika, S.;
Nishiyama, T.; Tatematsu, S.; Miyashita, K.; Imanishi, T. Tetrahedron 1997,
53, 3073-3082. (d) Obika, S.; Nishiyama, T.; Tatematsu, S.; Nishimoto,
M.; Miyashita, K.; Imanishi, T. Heterocycles 1998, 49, 261-267.
(11) (a) Vasse, J. L.; Charpentier, P.; Levacher, V.; Dupas, G.; Que´guiner,
G.; Bourguignon, J. Synlett 1998, 1144-1146. (b) Versleijen, J. P.; Sanders-
Hovens, M. S.; Vanhommerig, S. A.; Vekemans, J. A.; Meijer, E. M.
Tetrahedron 1993, 49, 7793-7802.
Results
Synthesis of Bridged Nicotinate. Scheme 1 outlines our
synthetic strategy for bridged nicotinate, a crucial synthetic
intermediate for the NADH model compounds. We employed
the aza-Wittig reaction as the key step since it seems useful for
the syntheses of nicotinates13 and pyridinophane derivatives.12b,14
The retrosynthetic cleavage of the CdN double bond in the
pyridinophane skeleton reveals the dienal intermediate A as the
precursor, which is a valence isomer of the corresponding
cyclobutene intermediate B. The enamine-type [2+2] cycload-
dition disassembles B to the formyl-substituted (vinylimino)-
phosphorane and methyl propiolate.
The actual synthesis of the bridged nicotinate 5 is summarized
in Scheme 2. The Vilsmeier-Haack formylation15 of the readily
available cyclododecanone afforded cis-2-chloro-1-cyclododecene-
carbaldehyde (cis-1) and its isomer, trans-1, in 78% and 17%
yields, respectively. Compound cis-1 was treated with sodium
azide and a catalytic amount of lithium chloride to give trans-
2-azido-1-cyclododecenecarbaldehyde (trans-2) and the formyl-
substituted cyclododec[b]azirinecarbaldehyde (3) in a 39/61
(12) Pyridinophane coenzyme models were reported. For NAD+/NADH
models: (a) Kuroda, Y.; Seshimo, H.; Kondo, T.; Shiba, M.; Ogoshi, H.
Tetrahedron Lett. 1997, 38, 3939-3942. (b) Oikawa, T.; Kanomata, N.;
Tada, M. J. Org. Chem. 1993, 58, 2046-2051. (c) de Kok, P. M. T.; Buck,
H. M. J. Chem. Soc., Chem. Commun. 1985, 1009-1010. de Kok, P. M.
T.; Donkersloot, M. C. A.; van Lier, P. M.; Meulendijks, G. H. W. M.;
Bastiaansen, L. A. M.; van Hooff, H. J. G.; Kanters, J. A.; Buck, H. M.
Tetrahedron 1986, 42, 941-959. (d) Hasselbach, H.-J.; Krieger, C.; Decker,
M.; Staab, H. A. Liebigs Ann. Chem. 1986, 765-776. (e) Murakami, Y.;
Aoyama, Y.; Kikuchi, J.; Nishida, K. J. Am. Chem. Soc. 1982, 104, 5189-
5197. (f) See refs 8d and 9. For vitamin B6 models: (g) Koh, J. T.; Delaude,
L.; Breslow, R. J. Am. Chem. Soc. 1994, 116, 11234-11240. (h) Kuzuhara,
H.; Iwata, M.; Emoto, S. J. Am. Chem. Soc. 1977, 99, 4173-4175. (i)
Tachibana, Y.; Ando, M.; Kuzuhara, H. Bull. Chem. Soc. Jpn. 1983, 56,
3652-3656. Tachibana, Y.; Ando, M.; Kuzuhara, H. Chem. Lett. 1982,
1765-1768.
(13) Kanomata, N.; Nakata, T. Heterocycles 1998, 48, 2551-2558.
(14) (a) Kanomata, N.; Nitta, M. Tetrahedron Lett. 1988, 29, 5957-
5960. Kanomata, N.; Nitta, M. J. Chem. Soc., Perkin Trans. 1 1990, 1119-
1126. (c) Nitta, M.; Akie, T.; Iino, Y. J. Org. Chem. 1994, 59, 1309-
1314.
(15) (a) Marson, C. M.; Giles, P. R. Synthesis Using Vilsmeier Reagents;
CRC Press: Boca Raton, 1994. (b) Virgilio, J. A.; Heiweil, E. Org. Prep.
Proced. Int. 1982, 14, 9-20. (c) Ziegenbein, W.; Lang, W. Chem. Ber.
1960, 93, 2743-2749.

Design and Synthesis of Bridged NADH Models
J. Am. Chem. Soc., Vol. 122, No. 19, 2000 4565
Scheme 2a
amide formation with (S)-valinol gave the bridged nicotinamides
(S,S)-8a and (R,S)-8b. For the synthesis of the primary amide
(S)-8c via the chiral bridged nicotinic acid (S)-7, the racemic
acid (()-7 was converted into the homochiral imide derivatives,
(S,S)-11a and (R,S)-11b. The compounds 8a,b and 11a,b are
quite stable molecules so that flipping of the oligomethylene
bridges is frozen and does not disturb the separation from each
other at room temperature. This observation is in contrast with
the case of 2,8-dithia[10](2,5)pyridinophane racemizing itself.12h
Heating 8a,b and 11a,b triggered the flipping that is useful to
control the planar chirality. The interconversion reached an
equilibrium in refluxing toluene after 8 h to give a nearly 1/1
mixture of (S,S)-8a and (R,S)-8b and, more interestingly, a
mixture of (S,S)-11a and (R,S)-11b in a ca. 3/2 ratio. The
selective preparation of homochiral 8a,b and 11a,b was achieved
since repeated “separation and interconversion” could stop the
flipping down into either side of the desired planar chirality.
This concept is exemplified by preferential synthesis of pure
(S,S)-11a, the yield of which increased to 83% (93%, theoreti-
cally) after two repeated separations and interconversions.
The structure of (S,S)-8a, colorless plates recrystallized from
ethyl acetate, was unequivocally solved by X-ray crystal-
lographic analysis (Figure 2). The interesting feature of the
structure is that the oligomethylene bridge significantly blocks
the face of the pyridine moiety, and this result agrees with our
design that the bridge would function as a bulky shielding wall
against substrate approach. The absolute structure of (S,S)-11a
was established after hydrolysis; the reaction with lithium
hydroperoxide in THF-H2O (3/1) gave nicotinic acid (S)-7 and
nicotinamide (S,S)-8a in 55% and 24% yields, respectively, the
latter of which was identical to the authentic specimen. The
hydrolysis in DMF-THF-H2O (3/3/1) allowed the clean
removal of the chiral auxiliary19 to give (S)-7 exclusively in
84% yield. The chiral acid (S)-7 was converted into the
corresponding primary amide (S)-8c in a conventional manner.
The N-alkylation of 8a-c with methyl iodide afforded NAD+
models 9a-c (X ) I), in quantitative yields, and the following
regioselective 1,4-reduction with aqueous sodium dithionite
resulted in the formation of the bridged NADH models 10a-
c.20
Biomimetic Reduction with Bridged NADH Models. The
bridged NADH models 10a-c achieved excellent biomimetic
reduction of the pyruvate analogues 12u-z to produce the
optically active lactate analogues 13u-z in the presence of
magnesium perchlorate in acetonitrile (Table 1). The model
(S,S)-10a accomplished the enantioselective hydrogen transfer
to methyl benzoylformate (12u) to afford (R)-methyl mandelate
[(R)-13u (R1 ) Ph)] with 99% ee at room temperature (entry
1). It is noteworthy that the rate of the reaction was remarkably
enhanced as the reaction temperature increased without a
significant decrease in enantioselectiVity. The reactions were
complete within 24 h at 50 °C and within 6 h at 75 °C to give
(R)-13u with 99% ee and 98% ee, respectively (entries 2 and
3). The planar chirality of the bridged NADH models plays a
decisive role in determining the stereochemistry of the products.
The diastereomer model (R,S)-10b having the opposite planar
chirality behaved as the pseudoenantiomer of (S,S)-10a, the
reduction with which resulted in (S)-13u with 97% ee at room
temperature and 98% ee at 75 °C (entries 4 and 5). It is of
particular importance to mention that, in each reaction, the
homochiral N-methylpyridinium perchlorate 9a,b (X ) ClO4)
a Reagents: (a) NaN3, LiCl, THF-H2O, rt, 5 h. (b) hν (Pyrex),
CHCl3, rt, 8 h (83% yield for two steps). (c) PPh3, toluene, reflux, 3 h
(91% yield, 3/1 trans- and cis-4). (d) Methyl propiolate, toluene, 140
°C, 12 h (21% and 7% yields for 5 and 6).
ratio.16 The following photoirradiation of the mixture in
chloroform accomplished the conversion of trans-2 into azirine
3 (83% yield from cis-1). The thermal ring-opening reaction of
3 with triphenylphosphine proceeded in refluxing toluene to give
the iminophosphorane 4 in 91% yield. The 1H NMR spectrum
showed that compound 4 is a mixture of trans- and cis-isomers
in a ratio of ca. 3/1 according to their aldehyde signals at δ
9.87 ppm (0.75H) and δ 10.66 ppm17 (0.25H), respectively. The
1H NMR spectra of 1, 2, and 4 were clearly informative for
assigning the configuration of CdC double bonds. For the cis-
isomers, the aliphatic signals of each geminal pair agreed with
their symmetric structure, while those of the trans-isomers
independently appeared according to their substituent groups
restricting the free dynamic motion of the decamethylene bridge
beyond the CdC double bonds. 3-Methoxycarbonyl[10](2,5)-
pyridinophane (5) was successfully synthesized from 4 by the
novel pyridine-formation reaction with methyl propiolate. The
desired bridged nicotinate 5 was obtained in 21% yield as well
as its ortho-bridged isomer 6 in 7% yield.18 The structures of
1
these compounds were deduced on the basis of their H and
13C NMR spectra and mass spectra. The H NMR spectrum
1
showed that one of the oligomethylene protons of 5 that
appeared at δ 0.13 ppm was located in a shielding region above
the pyridine ring. Furthermore, every bridge proton is located
in a different environment since all the aliphatic signals
independently appeared. These findings indicate that compound
5 has a racemic parapyridinophane skeleton and the flipping
(jump-rope rotation) of the oligomethylene bridge is frozen on
the NMR time scale at room temperature. The structure of 6
was easily assigned since the signals of the oligomethylene
protons are half as many as those of 5 due to its symmetric
structure.
Synthesis of Bridged NADH Models. Scheme 3 illustrates
the synthetic pathways for the bridged NADH models 10a-c.
Hydrolysis of the nicotinate 5 with lithium hydroxide afforded
the corresponding nicotinic acid (()-7, and the subsequent
(16) The 1H NMR spectrum measured immediately after the reaction at
0 °C showed the formation of cis-2-azido-1-cyclododecenecarbaldehyde (cis-
2): 1H NMR (CDCl3) δ 1.79 (m, 2H), 2.24 (t, J ) 7.5 Hz, 2H), 2.62 (t, J
) 7.7 Hz, 2H), 10.1 (s, 1H); the other signals (14H) were overlapped with
those of trans-2. Warming the mixture of cis- and trans-2 caused the rapid
transformation of cis-2 into azirine 3 even at room temperature.
(17) The aldehyde signal of cis-4 has a chemical shift similar to that of
the known cyclohexene analogue with exclusive cis-configuration (δ 10.66
in DMSO-d6). We also prepared this compound and confirmed that the
chemical shift in CDCl3 is δ 10.72 ppm: Tabyaoui, B.; Aubert, T.; Farnier,
M.; Guilard, R. Synth. Commun. 1988, 18, 1475-1482.
(18) Our previous communication discusses the reaction pathways for
the formation of 5 and 6. See ref 1. For further synthetic application of
formyl-substituted (vinylimino)phosphoranes to highly functional pyridines,
see ref 13.
(19) Jacobi, P. A.; Zheng, W. Tetrahedron Lett. 1993, 34, 2585-2588.
(20) The dihydropyridines 10a-c were used in the next step immediately
without further purification. These compounds are apparently sensitive to
oxygen.

4566 J. Am. Chem. Soc., Vol. 122, No. 19, 2000
Kanomata and Nakata
Scheme 3a
a Reagents: (a) LiOH, MeOH-H2O, rt, 12 h (81% yield). (b) (COCl)2, rt, 1 h; (S)-valinol, Et3N, CHCl3, rt, 2 h (48% each). (c) MeI, MeCN, 50
°C, 24 h (quant). (d) Na2S2O4, Na2CO3, H2O-CH2Cl2, rt, 12 h. (e) Toluene, reflux, 8 h (quant). (f) (COCl)2, rt, 1 h; NaH, (S)-4-isopropyloxazolidinone,
CH2Cl2, rt, 2 h [49% and 48% yields for (S,S)-11a and (R,S)-11b]. (g) LiOH, H2O2, DMF-THF-H2O (3/3/1), 0 °C, 2 h (84% yield). (h) (COCl)2,
rt, 1 h; NH3 (gas), CHCl3, rt, 1 h (96% yield). (i) Na2S2O4, Na2CO3, H2O-MeOH-CH2Cl2, rt, 14 h (quant).
in the transition state of the reduction for good asymmetric
induction in artificial systems (vide infra).1 However, it is so
surprising that the model (S)-10c having an unsubstituted
carbamoyl group reduced 12u with remarkably high ee’s and
achieved the almost identical optical yields of 13u with slightly
higher chemical yields as compared to those of 10a,b.
Scheme 4 summarizes the isotope and related experiments
showing the selectivity and the reactivity of the C-4 hydrogens
Figure 2. X-ray crystallographic structure of (S,S)-8a.
in the bridged NADH model systems. The reduction of (()-9c
(X ) I) with sodium dithionite in deuterated solvent underwent
was recovered by chromatography on silica gel in 60-81%
yields and that, therefore, the bridged NAD+/NADH model
systems were shown to retain their planar chirality throughout
the entire redox processes and were recyclable. The primary
amide model (S)-10c also allowed the excellent enantioselec-
tivity to give (R)-13u with 98% ee at room temperature and
with 97% ee at 75 °C (entries 6 and 7). Pyruvate esters such as
n-butyl and benzyl pyruvate, 12v,w (R1 ) Me), and the related
R-ketoesters such as 12x,y (R1 ) Et and n-Bu) were also
efficiently reduced with (S)-10c to afford the corresponding
lactates and their analogues (R)-13v-y with 96-98% ee’s
(entries 8-15). The other analogue 12z having a slightly bulky
alkyl group (R1 ) i-Bu) gave (R)-13z with 82-92% ee’s (entries
16 and 17). In each reaction, the (S)- or (R)-planar chirality of
10a-c resulted in the formation of the (R)- or (S)-lactate
analogues, respectively, with high stereospecificity. The corre-
sponding NAD+ model (S)-9c (X ) ClO4) was also obtained
in 68-83% isolated yields. These results indicate that the high
enantioselectivity with the bridged NADH models 10a-c is
fully dependent on their planar chirality of the parapyridinophane
moieties, and not on the nature of the carbamoyl sidearms
(entries 1-7). When we initially designed the chiral models
10a,b, the chelation sidearms of the hydroxyl groups were
introduced to expect the formation of tight ternary complexes
stereospecific labeling12c of the outer C-4 hydrogen of (()-10d;
1
a signal resonating at δ 2.8021 disappeared in the H NMR
spectrum, and the deuterated signal appeared at δ 2.78 in the
2H NMR spectrum. The deuterium located at the less hindered
side of (()-10d was almost exclusively transferred in the
successive biomimetic reduction to give the labeled mandelate
(()-13u-d, with 99% deuterium incorporation. The recovered
(()-9c (X ) ClO4) was found not to contain a detectable amount
of deuterium. These findings are unambiguous evidence that
only one side of the dihydronicotinamide ring is the active face
in this redox system. The ternary complex22 illustrated in Figure
3 rationalizes the enantioselectivity, where magnesium ion
exclusively chelates to methyl benzoylformate beneath the
bridged NADH model since its top side is protected by the
oligomethylene bridge. For further investigation of the reactivity
of an inner hydrogen at C-4, we synthesized the 4-methyl-
substituted model (()-10e, the hydrogen of which is sandwiched
between the methyl group and the bridge (Scheme 4). The
(21) Geminal C-4 protons of 10c (δ 2.80 and δ 2.89) were unequivocally
assigned by the COSY, HMQC, HMBC, and NOE differencial spectra. The
NOE enhancement (3%) was observed between the inner proton (δ 2.89)
and one of the oligomethylene protons (δ 1.60).
(22) Toyooka, Y.; Matsuzawa, T.; Eguchi, T.; Kakinuma, K. Tetrahedron
1995, 51, 6459-6474.

Design and Synthesis of Bridged NADH Models
J. Am. Chem. Soc., Vol. 122, No. 19, 2000 4567
Table 1. Biomimetic Reduction of Pyruvate Analogues 12u-z with Bridged NADH Models 10a-c
13u-z
9a-c
entry
10a-c
12u-z
R1
Ph
Ph
Ph
Ph
Ph
Ph
Ph
Me
Me
Me
Me
Et
R2
T
time
yield (%)a
config.
ee (%)b
yield (%)a
1
2
3
4
5
6
7
a
a
a
b
b
c
c
c
c
c
c
c
c
c
c
c
c
u
u
u
u
u
u
u
v
v
w
w
x
x
y
y
z
Me
Me
Me
Me
Me
Me
Me
n-Bu
n-Bu
Bn
Bn
Bn
Bn
Bn
rt
5 d
24 h
6 h
4 d
6 h
2 d
6 h
4 d
6 h
5 d
6 h
2 d
3 h
3 d
5 h
3 d
6 h
64
70
63
64
63
84
78
81d
78d
81
76
80
71
85
74
42
44
R
R
R
S
99
99
98
97
98
98
97
98
97
97
96
98
98
96
96
88
92
66
81
65
60
71
69
72
50 °C
75 °C
rt
75 °C
rt
75 °C
rt
75 °C
rt
75 °C
rt
75 °C
rt
75 °C
rt
75 °C
S
R
R
R
R
R
R
R
R
R
R
R
R
8c
9c
10
11
12
13
14
15
16
17
72
>99d
83
68
80
74
77
70
81
71
Et
n-Bu
n-Bu
i-Bu
i-Bu
Bn
Bn
Bn
z
a Isolated yields otherwise specified. b Determined by HPLC. c The reduction was carried out in CD3CN. d Measured by 1H NMR spectra using
cyclohexanecarboxaldehyde as an internal standard.
Scheme 4a
Table 2. Asymmetric Reduction of 14k-n with (S)-10c
a Obtained on the basis of the formation of (S)-9c. b Determined by
HPLC. c Isolated yields.
compounds having a trifluoromethyl or 2-pyridyl group with a
bridged NADH model compound. As well as the above-
mentioned reduction of a series of R-ketoesters including
pyruvates, the model (S)-10c reduced activated ketones such
as 14k-n with reasonably good enantioselectivity in the
presence of magnesium ion to give the corresponding chiral
alcohols (R)-15k-n with 79-89% ee. The introduction of the
same (R)-configuration as in the case of the biomimetic
reduction with (S)-10c suggests that those reactions also proceed
through the ternary complex similar to that in Figure 3, in which
the coordinating ester group is replaced with either a trifluo-
romethyl or a 2-pyridyl group.
a Reagents: (a) (i) Na2S2O4, Na2CO3, D2O-MeOD-CH2Cl2, rt, 11
h. (b) MeMgBr, THF, -20 °C and then rt, 45 min (36% yield and
32% for its regioisomer).
Figure 3. Proposed transition state for the reduction of methyl
benzoylformate with NADH models 10c.
Discussion
compound (()-10e was fully inert toward 12u in the presence
of magnesium ion even upon heating in acetonitrile at 75 °C
for 6 h. This result suggests that the reacting C-4 hydrogen needs
to reach its substrate by way of chelation with magnesium ion
to form a ternary complex. In the case of (()-10e, a remote
interaction between the NADH model and the substrate is
apparently insufficient to trigger the reaction.
Carbamoyl substituents of NADH model compounds are well-
known to affect the stereochemistry of the products in coenzyme-
mimetic reactions. A series of NADH models having hydroxyl
groups exhibited typical sidearm effects;4c,6,7,22 the model 16
provided 85-94% ee for (R)-mandelate, whereas a similar
model, 17,6b without a hydroxyl group had only 20% ee. The
absolute configuration of the hydroxyl branch is still important
for NADH models with competitive chiral auxiliaries. Davies
reported NADH models having both a chiral carbamoyl group
Asymmetric Reduction of Activated Ketones. Table 2
indicates asymmetric reduction of some activated carbonyl

4568 J. Am. Chem. Soc., Vol. 122, No. 19, 2000
Kanomata and Nakata
and a chiral iron moiety designed for selective shielding of their
dihydropyridine rings.7 However, there seems to be both
matched and mismatched combinations between the two aux-
iliary groups, and although the former achieved 97-98% ee
for optically active mandelate, the latter gave only 15-16%
ee. On the other hand, all the bridged models 10a-c induced
high enantioselectivity, which is strictly subject to the absolute
structure of their solid parapyridinophane moieties. Thus, the
carbamoyl group derived from (S)-valinol does not behave as a
“chirality-inducing group” in our model systems, and its
presence or absence, including the issue of absolute configu-
ration, has almost negligible effects on the selectivity.
Recyclable NADH models achieving temperature independency
of high enantioselectivity should be seriously considered in
directing our efforts to catalyze the redox reactions. These
classes of NAD+/NADH models would give us significant
discretion to design the reversed course of model reactions
(NAD+ f NADH type) at various temperatures to challenge
the unprecedented catalytic asymmetric model reactions. The
bridged NAD+/NADH model systems could also correlate with
our recent findings of glycolysis-type reactions including the
biomimetic oxidation and reduction in aprotic organic media.23
Conclusion
The bridged models 10a-c, designed as the first homochiral
ansa-type NADH models, successfully functioned in controlling
both the direction and the orientation of the substrate approach
to the model compounds for inducing high enantioselectivity
in their biomimetic and related reactions. In particular, the (S)-
10c model having both a primary carbamoyl group and a
shielding bridge feigning an enzyme wall provides a compact
chemical miniature of a holoenzyme, coenzyme NADH linked
dehydrogenase, in terms of its unique and simple structure, high
enantioselectivity, and usefulness for being recycled. In other
words, the combination of the stereoselective shielding of a
dihydronicotinamide core and the substrate control with mag-
nesium ion satisfies the requirements for the highly enantiose-
lective reduction of pyruvate analogues in the small model
system of 10a-c, which is comparable to the chiral environment
of a holoenzyme during biological reduction.
Another unique property of 10a-c is the temperature
independency of the enantioselectivity during the reduction of
12u-y. Previous studies on the temperature effect of NADH
model reactions showed that higher temperatures decreased more
or less the enantioselectivity (5-29% decrease of ee’s).5c,6c,8a
The compounds 10a-c, however, demonstrated a temperature
independency of ee’s though an additional chelation sidearm is
missing in the case of 10c. This means that, in the presence of
magnesium ion, the stereoselective blocking of a dihydronico-
tinamide core is paramount for strictly mimicking the asym-
metric reduction with coenzyme NADH and the principle is
applicable even at higher temperatures. A striking advantage
of bridged NADH models over C-4-substituted models is
substantial recyclability, whereas the models such as 185a and
195c suffer from a loss of chirality at C-4 in each model reaction.
Acknowledgment. N.K. gratefully acknowledges the finan-
cial support by a Grant-in-aid for Science Research from the
Ministry of Education, Science, and Culture (07740514) and
Special Grant for Promotion of Research from RIKEN (The
Institute of Physical and Chemical Research). We thank Ms.
Kimiko Kobayashi for the X-ray crystallographic analysis and
Ms. Kumiko Harata for operation of the FAB mass spectrometer.
Supporting Information Available: Complete experimental
details, including characterization data for cis-1, trans-1, trans-
2, 3-7, 8a-c, 9a-c (X ) I and ClO4), 10a-e, and 11a,b, the
details of the crystal structure data for (S,S)-8a, and copies of
1H NMR spectra of 10d and 1H and 13C NMR spectra of trans-
1, trans-2, 3, 8b, 10a-c, 10e, and 11a,b (PDF, CIF). This
material is available free of charge via the Internet at
http://pubs.acs.org.
(23) Kanomata, N.; Suzuki, M.; Yoshida, M.; Nakata, T. Angew. Chem.,
Int. Ed. 1998, 37, 1410-1412.
JA992990Y