A novel NADH model: Design, synthesis, and its chiral reduction and fluorescent emission

Article abstract of DOI:10.1002/adsc.200900610

A novel chiral nicotinamide adenine dinucleotide hydrogen (NADH) model with C₃ symmetry was designed and synthesized. Hydrogens at the C-4 position of all dihydropyridine rings in the inner part of the bowl could transfer to the substrate with powerful enantioselectivity. This novel C ₃ symmetrical NADH model is capable of fluorescence emission at 455 nm when excited at 390 nm.

Full text of DOI:10.1002/adsc.200900610

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DOI: 10.1002/adsc.200900610
A Novel NADH Model: Design, Synthesis, and its Chiral
Reduction and Fluorescent Emission
Nai-Xing Wanga,b,* and Jia Zhaoa,b
a
Technical Institute of Physics and Chemistry, Chinese Academy of Sciences, Beijing 100190, Peopleꢀs Republic of China
College of Chemistry and Chemical Engineering, Graduate University of Chinese Academy of Sciences, Beijing 100049,
b
Peopleꢀs Republic of China
Fax : (+86)-10-6255-4670; e-mail: nxwang@mail.ipc.ac.cn
Received: September 4, 2009; Revised: November 12, 2009; Published online: December 8, 2009
Supporting information for this article is available on the WWW under
http://dx.doi.org/10.1002/adsc.200900610.
Abstract: A novel chiral nicotinamide adenine di-
nucleotide hydrogen (NADH) model with C3 sym-
metry was designed and synthesized. Hydrogens at
the C-4 position of all dihydropyridine rings in the
inner part of the bowl could transfer to the sub-
strate with powerful enantioselectivity. This novel
C3 symmetrical NADH model is capable of fluores-
cence emission at 455 nm when excited at 390 nm.
Keywords: chiral NADH model; enantioselectivity;
fluorescence; magnesium(II); synthesis
second aspect was to incorporate a substituent at the
reaction center: the C-4 position of the dihydropyri-
dine ring.[3] The third one was to design the specific
conformation to obtain high stereoselectivity.[4] How-
ever, in the first situation the dihydronicotinamide
The coenzyme nicotinamide adenine dinucleotide hy- unit needs to be modified significantly by introducing
drogen (NADH) is an important molecule in nature. big chiral auxiliaries, a complicated ring or by chang-
Over the past several decades, NADH mimics have ing the amide group to a chiral sulfinyl group. The
become one of the highlights of biochemistry and or- second one suffered from loss of chirality at the C-4
ganic chemistry.[1] To the best of our knowledge, the position during the course of the model reduction re-
dihydropyridine amido group is the key structure for action. The third one was particular and by far fewer
NADH models.
Kanomata[1f] designed and synthesized a bridged NADH model compound 1 with C3-symmetry was de-
NADH model with an [n](2,5)pyridinophane (para- signed and synthesized.
pyridinophane) skeleton as a sterically-demanding Our model 1 has six chiral carbon centers. (1R,2R)-
models have been studied. Herein, a novel chiral
AHCTUNGTRENNUNG
side chain that induces high enantioselectivity. Kano- Diaminocyclohexane is introduced as chiral source to
mata believed that the oligomethylene chain bridging connect three identical pyridine-3,5-dicarbonyl groups
the 2- and 5-positions of a dihydronicotinoyl ring into a large ring. Then three identical 1,4-dihydropyri-
would behave as an “enzyme wall” that allows the dine units are connected by the phenyl-1,3,5-trimethy-
substrates to approach from the opposite side exclu- lene group to form model 1. Molecular modeling via
sively. In the asymmetric reduction of methyl benzoyl- molecular dynamics followed by energy minimization
formate, mandelate was obtained with a high enantio- with Gaussian 03 shows that the bowl-shaped confor-
meric excess when a 1 molar ratio of magnesium was mation shown in Figure 1 is the most stable one. The
used.
phenyl-1,3,5-trimethylene group is the “bottom” of
Generally, chemists had formerly designed NADH the “bowl” and the rigidly defined concave cavity of
models according to three aspects: the first one was the bowl can hold a metal ion in a fixed position rela-
to control the stereoselectivity by changing the substi- tive to the 1,4-dihydropyridine. As a result, regulation
tutent of the dihydronicotinamide with a view to form of the stereoselective approach of the substrates for
a remote sterically-demanding side chain.[2] The accomplishment of their biomimetic reduction with
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Nai-Xing Wang and Jia Zhao
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Figure 1. The structure of model 1 and its conformation.
high enantioselectivity could be realized. Besides, to obtain 8 by treatment of 7 with excess (1R,2R)-di-
three identical dihydropyridine units are arranged in
a large ring system in such way that all of the three
ACHTUNGTRENNUNG
AHCTUNGTRENNUNG
hydrogens in the C3 symmetrical model can transfer lution of 8 and the resulting reaction mixture was
to the substrate. Our NADH model should be inter- heated at 858C, 9 was obtained and used directly in
esting for the research on biosimulation. This C3 sym- the next procedure without further purification.
metrical NADH model should also be a new fluores- Crude 9 was reduced by sodium dithionite to afford
cent probe in the course of the signal transduction crude product 1. After separation on Sephadex LH-20
and for some research on the reaction mechanism.
with methanol as the eluent, the pure model com-
We originally attempt to synthesize model 1 by the pound 1 was obtained.
tri-directional synthetic method outlined in Scheme 1.
It is interesting that there is an obvious difference
Since three identical 3,5-diethoxycarbonylpyridine of the chemical shifts between the two prochiral C-4
1
units were fixed by a phenyl-1,3,5-trimethylene group, protons in the H NMR spectrum of model 1 (Dd=
the most difficult ring-closing reaction in this route 0.4 ppm). As shown in Figure 1, this significant fea-
would be more feasible.
ture suggests that the carbonyl groups in the C-3 and
It seems that this synthetic route should have been C-5 positions of the dihydropyridine rings are greatly
available.[5–7] Unfortunately, we found that our inter- restricted by the rigidly defined concave cavity and
mediate 5 was very sensitive to temperature, light and have an intilted orientation. Thereby the prochiral C-
air. Compound 5 was very difficult to keep its stability 4 protons are highly diastereotopically distinguished
in the following procedures. Thus the route shown in by the special bowl-shaped conformation. Three hy-
Scheme 1 failed.
drogens at the C-4 positions of all dihydropyridine
A practical and efficient synthetic route is outlined rings in the inner part of the bowl could transfer to
in Scheme 2. The key method in Scheme 2 was the re- the substrate in the reduction reactions of our novel
duction reaction which occurred in the last step. At NADH model with powerful enantioselectivity.
first, the large heterocycle compound 8 was expected
The C3 symmetrical NADH model 1 was found to
to be obtained by the reaction of 3,5-dichlororocarbo- effect a good biomimetic reduction simply, and this is
nylpyridine with (1R,2R)-diaminocyclohexane.[8] How- not in prevailing organocatalyzed asymmetric reduc-
ever, we found that the products were very complicat- tion. The reaction of 1 with methyl benzoylformate
ed and some linear polymer also was formed. It was carried out in the presence of magnesium per-
seemed that the acyl chloride was too active to make chlorate in acetonitrile (Table 1).
the reaction controllable. Then we tried to look for a
Enantioselective reduction is one of the key reac-
suitable reagent which was a little bit less active than tions to produce chiral compounds in academia and in
the acyl chloride. Pentafluorophenol was found to be the chemical industry.[9] Recently the Hantzsch dihy-
a good reagent and the pentafluorophenoxy moiety dropyridine (not a chiral hydride source) has been
was a good leaving group. Compound 7 was prepared used as a transfer-hydrogenation reagent for organo-
by reaction of 2 with pentafluorophenol. It was easy catalytic asymmetric reactions.[10–13] However, enantio-
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A Novel NADH Model: Design, Synthesis, and its Chiral Reduction
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Scheme 1. Attempted synthesis of model 1. (a) C2H5OH, H2SO4, reflux, 69%; (b) 1,3,5-tris(bromomethyl)benzene, CH3OH,
reflux, 45%; (c) Na2S2O4, Na2CO3, H2O, room temperature, 26%; (d) 1. NaOH, H2O, room temperature, 5 h; 2. SOCl2,
reflux, 8 h; (e) (1R,2R)-diaminocyclohexane, THF, room temperature, 12 h.
Table 1. Asymmetric reduction reactions of methyl benzoylformate with 1. (The ratio of NADH model to methyl benzoylfor-
mate is 1:1).
Entry
T [oC]
t
Ratio Mg(ClO4)2/model 1
10
ee [%][a,b]
Configuration
Yield [%][c]
1
2
r.t.
50
3 d
18 h
3.0
3.0
88
82
R
R
96
92
[a]
Determined by HPLC.
No 2-propanol present.
Isolated yields.
[b]
[c]
selective hydrogenation with Hantzsch dihydropyri- chiral hydride sources, thus, asymmetric reduction
dine has to use costly chiral organocatalysts. To the with Hantzsch dihydropyridine needs chiral organoca-
best of our knowledge, most of the Hantzsch dihydro- talysts. NADH is a coenzyme molecule in nature,
pyridine compounds are neither chiral molecules nor Hantzsch ester compounds would not be subject to
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Nai-Xing Wang and Jia Zhao
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Scheme 2. Successful synthesis of model 1. Reagents and conditions: (a) pentafluorophenol, DMF, room temperature, 6 h,
92%; (b) (1R,2R)-diaminocyclohexane, THF, room temperature, 4 h, 21%; (c) 1,3,5-tris(bromomethyl)benzene, DMF, 858C,
12 h, 35%; (d) Na2S2O4, Na2CO3, H2O, room temperature, 10 h, 42%.
NADH models.[1e] Researches on NADH models are displays fluorescent pH-sensing activity in the physio-
mainly useful for some of the biosimulating and life logical pH range.
sciences in the future.
On the basis of the results described, we believe
Nicotinamide adenine dinucleotide hydrogen that the C3 symmetrical NADH model 1 should be a
(NADH) in nature is capable of fluorescent emission new pH-sensing fluorescent probe, which should have
at 430–445 nm when excited at 340 nm (lexc =340 nm, some good potential as a small probe molecule for
lem =430–445 nm), while the oxidized forms (NAD+) the research on atherosclerosis in the course of signal
are not.[14a] Blomquist reported that the fluorescence transduction.[15a] Plenio reported the use of fluores-
emission intensity at 460 nm of reduced NADH in so- cence signal molecules to follow the course of a
lution is increased in the presence of deuterium oxide Suzuki coupling reaction, and the fluorescence signal
over that observed in water.[14b] We found that our changes at each stage of the reaction.[15b]
model 1 is capable of fluorescent emission at 455 nm
In summary, we have designed and synthesized the
when excited at 390 nm (lexc =390 nm, lem =455 nm). first C3 symmetrical NADH model with a special
The dihydropyridine rings in the model 1 could act bowl-shaped conformation. Three identical dihydro-
similarly as pH-sensitive fluorophores, this is a unex- pyridine units were connected to form a rigidly de-
pected new finding in our model 1. Figure 2 shows the fined concave cavity which could encase and fix some
pH dependence on the emission spectrum of 1 that substrates to accomplish the biomimetic reduction
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A Novel NADH Model: Design, Synthesis, and its Chiral Reduction
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Figure 2. Emission spectra of 1 in methanol as a function of pH. Spectra were recorded with excitation at 390 nm at the dif-
ferent pH values.
ture of the bis(pentafluorophenyl) ester of 3,5-pyridinedicar-
with high enantioselectivity. Meanwhile, all three hy-
boxylic acid 7 (5.0 g, 10.0 mmol) in THF at 08C (ice bath),
drogens at the C-4 positions of three dihydropyridine
The resulting reaction mixture was stirred at room tempera-
rings in the inner part of the bowl could transfer to
ture for 4 h. The solvent was removed to give the crude
the substrate in the reduction reactions of NADH
product. The product was purified by column chromatogra-
model 1 with powerful enantioselectivity. The C3 sym-
metrical NADH model should also be a new fluores-
cent probe for the research on atherosclerosis in the
course of signal transduction[15a] and some reaction
mechanisms.[15b]
phy, ethanol and ethyl acetate (1: 9) was used as eluent to
give the white solid 8; yield: 0.5 g (21%); 1H NMR
[400 MHz, (CD3)2SO]: d=1.32 (m, 6H, CHH’CH2CHNH),
1.52–1.55 (m, 6H, CHH’CH2CHNH), 1.76–1.78 (m, 6H,
CHH’CHNH), 1.88–1.92 (m, 6H, CHH’CHNH), 3.93 (m,
6H, CHNH), 8.38 (s, 3H, pyr-4-CH), 8.66 (d, J=7.56 Hz,
6H, NH), 8.87 (s, 6H, pyr-2,6-CH); 13C NMR [100 MHz,
(CD3)2SO]: d=24.9 (CH2CH2CHNH), 31.5 (CH2CHNH),
53.5 (CHNH), 130.3 (pyr-3,5-C),134.8 (pyr-4-CH), 150.1
(pyr-2,6-CH), 165.0, 165.1 (C=O); IR (KBr): v=3252, 3065,
2938, 2860, 1635, 1541, 1298, 705 cmÀ1; ESI-MS m/z=736.5
[M+H]+, 774.5 [M+K]+, 734.3 [MÀH]À, 770.4 [M+Cl]À;
[a]D: À177.48 (c 0.002, DMSO).
Experimental Section
Preparation of Compound 7
1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochlo-
ride (EDC) (6.4 g, 33.2 mmol) was added to a solution of
3,5-pyridinedicarboxylic acid (2.8 g, 16.8 mmol) and penta-
fluorophenol (6.2 g, 33.2 mmol) in 150 mL of dry DMF. The
resulting solution was stirred at room temperature for 6 h,
water (200 mL) was added and a white precipitate formed.
The product was collected by filtration and purified by flash
column chromatography (silica gel, CH2Cl2) to give the
Preparation of Compound 9
Compound 8 (0.4 g, 0.54 mmol) and 1,3,5-tris(bromome-
thyl)benzene (0.2 g, 0.56 mmol) were added to dry DMF
(5 mL), and the resulting solution was stirred under nitrogen
at 858C for 12 h, then cooled and ether was added. The pre-
cipitate was collected by filtration and washed with ether to
give the crude product as a white solid (yield: 0.53 g, 89%)
which was used without further work-up.
1
white solid 7; yield: 7.80 g (93%); mp 154–1578C; H NMR
(300 MHz, CDCl3): d=9.20 (s, 1H, pyr-4-CH), 9.68 (s, 2H,
pyr-2,6-CH); IR (KBr): v=3061, 2670, 2465, 1773, 1655,
1602, 1516, 1471, 1430, 1321, 1294, 1223, 1154, 1095, 996,
731 cmÀ1; EI-MS: m/z (%)=316 (100), 260 (23.8), 183 (6.6),
105 (10.8).
Preparation of Compound 1
To an aqueous solution of compound 9 (0.5 g, 0.46 mmol),
an aqueous solution of sodium dithionite (1.9 g, 10.9 mmol)
and sodium carbonate (0.73 g, 6.5 mmol) were added drop-
wise at room temperature under nitrogen. The mixture was
Preparation of Compound 8
A
solution
of
(1R,2R)-diaminocyclohexane
(1.3 g,
11.4 mmol) in THF (30 mL) was added dropwise to a mix-
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Nai-Xing Wang and Jia Zhao
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left to stir overnight at room temperature, during the time a
yellow precipitate formed. The product was collected by fil-
tration and purified by gel chromatography (Sephadex LH-
20, CH3OH) to give 1 as a yellow solid; yield: 0.16 g (yield
42%); 1H NMR [400 MHz, (CD3)2SO]: d=1.20 (m, 12H,
CH2CH2CHNH), 1.52–1.54 (m, 6H, CH2CHH’CHNH), 1.78
(m, 6H, CH2CHH’CHNH), 2.64 (d, J=18.3 Hz, 3H, pyr-4-
CHH’), 3.03 (d, J=18.3 Hz, 3H; pyr-4-CHH’), 3.63 (m, 3H,
CHNH), 3.78–3.80 (m, 3H, CHNH), 3.95 (d, J=14.1 Hz,
3H, ArCHH’), 4.56 (d, J=14.1, 3H, ArCHH’), 6.44 (s, 3H,
C6H3), 6.92 (d, J=8.4 Hz, 3H, CHNH), 7.06 (d, J=7.6 Hz,
3H, CHNH), 7.16 (s, 3H, pyr-2,6-CHCH’), 7.21 (s, 3H, pyr-
2,6-CHCH’); 13C NMR [150 MHz, (CD3)2SO]: d=25.2
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(CH2CH2CHNH),
25.6
(C’H2CH2CHNH),
32.0
(CH2CHNH), 32.9 (C’H2CHNH), 40.5 (pyr-4-CH2), 52.0
(CHNH), 53.0 (C’HNH), 57.3 (ArCH2), 107.3 (pyr-3,5-CC’),
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Acknowledgements
Support for this research was provided by the Natural Science
Foundation of China (20472090 and 10576034).
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