Biomimetic systems involving sequential redox reactions in glycolysis-the sulfur effect

Article abstract of DOI:10.1039/d0cc05185c

Magnesium hemithioacetates were used as model cysteine compounds to mimic natural hemithioacetals, and their biomimetic oxidation reactions using a model NAD+ compound were investigated. Cyclic hemithioacetate was found to be the best substrate for the reaction with the model NAD+ compound, which gave the corresponding NADH analog in excellent yield. This journal is

Full text of DOI:10.1039/d0cc05185c

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Biomimetic systems involving sequential redox
reactions in glycolysis – the sulfur effect†
Cite this:DOI: 10.1039/d0cc05185c
Narihito Ogawa, *a Sei Furukawa,b Yuya Kosugi,a Takayuki Takazawaa and
b
Received 30th July 2020,
Accepted 17th September 2020
Nobuhiro Kanomata
*
DOI: 10.1039/d0cc05185c
rsc.li/chemcomm
Magnesium hemithioacetates were used as model cysteine compounds biomimetic oxidation of a model NAD+ compound using a
to mimic natural hemithioacetals, and their biomimetic oxidation series of hemithioacetal derivatives via a biomimetic system
reactions using a model NAD+ compound were investigated. Cyclic involving sequential redox reactions.
hemithioacetate was found to be the best substrate for the reaction
with the model NAD+ compound, which gave the corresponding in situ by the reaction of the magnesium thiolate derived
An active acyclic hemithioacetal derivative 2 was prepared
NADH analog in excellent yield.
from ethanethiol (1) and acetaldehyde in THF (Table 1). We
initially investigated the biomimetic oxidation reaction
Coenzyme NAD+ and its reduced form (NADH) function as using compound 2 and N-benzyl pyridinium salt 3a, a model
redox agents in a number of biological reactions including the NAD+ compound. A regioselective hydrogen transfer reaction
glycolysis process observed during carbohydrate metabolism.1 occurred from 2 to 3a to exclusively give 1,4-dihydropyridine
NAD+ oxidizes glyceraldehyde-3-phosphate to bisphospho- 4a3a,4 in up to 23% yield (entry 1). Efforts to increase the
glyceric acid in D-glyceraldehyde-3-phosphate dehydrogenase product yield by the addition of a polar aprotic co-solvent such
(GAPDH), while NADH paired with the oxidation step reduces as DMF or the use of an excess amount of 2 were unsuccessful
pyruvate to L-lactate with high enantioselectivity in lactate (entries 2 and 3). Performing the reaction at higher temperature
dehydrogenase (LDH) during anaerobic glycolysis (Scheme 1). was inefficient (entry 4) and indicated acyclic hemithioacetal
Consequently, there have been a large number of artificial derivative 2 was not very stable upon heating. Although our
NADH model compounds reported for LDH-type asymmetric
reduction reactions,2 but only a few examples have been
studied in the GAPDH-type biomimetic oxidation reaction.3
We have previously reported that regioselective hydrogen trans-
fer from a 1,1-diolate, a model substrate mimicking the biolo-
gical system, proceeds very efficiently to form a series of model
NAD+ compounds during the GAPDH-type reaction without the
use of a transition metal catalyst.3a However, natural biological
oxidation during glycolysis is known to proceed via a hemi-
thioacetal intermediate produced via the addition of a cysteine
residue to an aldehyde substrate in GAPDH. To develop a process
similar to that found in biological systems, we have designed an
active hemithioacetal derivative representing a new class of model
compounds generated from D-glyceraldehyde-3-phosphate and
the cysteine residue in GAPDH. In this paper, we report the
a Department of Applied Chemistry, Meiji University, Kawasaki,
Kanagawa 214-8571, Japan. E-mail: narihito@meiji.ac.jp
b Department of Chemistry and Biochemistry, Waseda University,
Shinjuku-ku, Tokyo 169-8555, Japan. E-mail: kanomata@waseda.jp
Scheme 1 GAPDH and LDH holoenzymatic redox during anaerobic
† Electronic supplementary information (ESI) available. See DOI: 10.1039/
glycolysis.
d0cc05185c
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Table 1 Biomimetic oxidation using acyclic hemithioasetal derivative 2
Table 2 Biomimetic oxidation using cyclic hemithioacetal derivative 10
10
Temp. Time
Ratio
Entry (equiv.)
3
(1C)
(h)
Solvents
4a (%) (4 : 5)b
1
2
3
4
5
6
7
8
5
2
2
2
2
2
5
5
5
2
2
3a 90
3a 90
3a 90
3a 90
3a 90
3a 90
3a 50
3a 50
3a 50
3b 90
3b 50
1
1
1
1
1
1
1
3
6
1
3
THF
THF
61
31
499 : 1
499 : 1
499 : 1
499 : 1
499 : 1
—
Entry 2 (equiv.) Temp. (1C) Solvents
4a (%)a Ratio (4a : 5a)b
THF/DMF = 6 : 1 72
THF/DMF = 4 : 1 80
THF/HMPA = 6 : 1
THF/DMSO = 6 : 1 Trace
THF/DMF = 4 : 1 Trace
THF/DMF = 4 : 1 47
THF/DMF = 4 : 1 37
THF/DMF = 4 : 1 74
THF/DMF = 4 : 1 39
1
2
3
4
1
1
3
1
50
50
50
90
THF
23
499 : 1
499 : 1
499 : 1
499 : 1
6
THF/DMF = 5 : 1 25
THF/DMF = 5 : 1 23
THF
—
12
499 : 1
499 : 1
499 : 1
499 : 1
a
b
Isolated yield. Determined by 1H NMR.
9
10
11
initial attempts at modeling the biooxidation reaction were
unsatisfactory, it is still worth noting that none of the 1,6-
dihydropyridine isomer 5a was formed in the reaction upon
a
b
Isolated yield. Determined by 1H NMR.
careful examination of the crude products by 1H NMR place at 90 1C to give 4a in 72 and 80% yield, respectively
spectroscopy.3a,4c
(entries 3 and 4). The reaction was performed using HMPA and
Based on our assumption that the thioethyl group is easily DMSO as the reaction cosolvent, but the product yield was low
removed from 2 at high temperature required for breaking the (entries 5 and 6). The biomimetic reaction performed at 50 1C
aromaticity of the pyridinium moiety, we subsequently afforded NADH model compound 4a in trace amounts (entry 7).
designed cyclic hemithioacetate derivative 10 as a new class However, extending the reaction time gave 4a in 47% yield
of model substrate for the GAPDH-type oxidation reaction (entry 8). Furthermore, the reactivity in the biomimetic oxida-
(Scheme 2). The synthesis of 10 is shown in Scheme 2. Phtha- tion did not improve when conducting the reaction for more
lide (6) was reacted with benzyl mercaptan in DMF to give than 3 h (entry 9). In entries 10 and 11, we performed a
carboxylic acid 7 in quantitative yield. A subsequent cyclization biomimetic oxidation using NAD+ model 3b. As a result, the
reaction of 7 in the presence of trifluoroacetic anhydride yield was similar to that when 3a was used (vs. entries 4 and 8).
afforded thiolactone 8 in 97% yield.5,6 After the reduction of 1,6-Dihydropyridine 5a, b was formed in the reaction, but in
8 using Red-Al, the resulting cyclic hemithioacetal 9 was treated trace amounts in all entries. The regioselectivity of NADH
with methylmagnesium bromide to give hemithioacetal deriva- models 4 and 5 was improved compared to our previous results
tive 10, which was used in the biomimetic oxidation reaction of the biomimetic oxidation using 1,1-diolate.3a The use of
without isolation.
DMF as a co-solvent is useful to improve both the reactivity and
The reaction of model NAD+ compound 3 with 5 equiv. of the regioselectivity affording the desired a 1,4-dihydropyridine
10 in THF at 90 1C afforded 1,4-dihydropyridine 4a in 61% yield motif as an NADH model.4c
(Table 2, entry 1). In this reaction, thiolactone 8 was isolated in
The reaction using cyclic hemiacetal derivative 11 was
54% yield. The reaction using 2 equiv. of 10 under similar performed in order to compare its reactivity and selectivity
reaction conditions also proceeded to give 4a, albeit in 31% with those of compound 10 (Scheme 3).
yield (entry 2). Subsequently, we carried out the reaction in the
The reaction using model NAD+ compound 3 and 11
presence of different amounts of DMF as an aprotic co-solvent performed at 90 1C in THF also gave compound 4a, but the
and the regioselective hydrogen transfer reaction of 9 took product yield was low (6%), which was in remarkable contrast
to the result obtained using sulfur analog 10 (Table 2, entry 1).
Scheme 2 Synthesis of cyclic hemithioacetal derivative 10.
Scheme 3 Biomimetic oxidation using hemiacetal derivative 11.
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isomer (the ring-opened aldehyde was not observed by
1H NMR spectroscopy). Finally, hemithioacetal derivative 17
was synthesized in situ upon reaction with methylmagnesium
bromide. Magnesium hemithiacetate 20 was generated from g-
butylothiolactone (18) via hemithioacetal 19. The ring-opened
aldehyde formed from cyclic hemithioacetal 19 was not
observed using 1H NMR spectroscopy, being consistent with
the other hemthioacetals, 9 and 16.
We carried out the biomimetic reaction using magnesium
hemithioacetate 17 and 20 (Scheme 6). The reduction using
hemithioacetate 17 did not take place under the optimized
reaction conditions shown in Table 2. On the other hand, the
reaction using hemithioacetate 20 gave 1,4-dihydropyridine 4
in 46% yield. These results indicate that the reactivity of 10,
which has a dihydrobenzo[c]thiophenol structure, was remark-
ably higher than that observed for the isothiochromanol (17)
and tetrahydrothiophenol (20) derivatives in the biomimetic
oxidation reaction. Hydrogen, a vital component in the stereo-
selective biomimetic reduction, acquires the highest reactivity
at a benzylic position of 10, followed by that of aliphatic 20. The
sulfur atom conjugated with the benzene ring deprives the
hydrogen atom of its reactivity, as is exemplified in 17.
Scheme 4 Existence ratio of hemithioacetal 9 and hemiacetal 13.
The difference observed between the ‘‘oxygen’’ and ‘‘sulfur’’
reactions was apparently dependent on the abundance of the
acetal and thioacetal formed under the reaction conditions
(Scheme 4). The 1H NMR spectrum of hemithioacetal 9 showed
no significant peak beyond d 8.0 ppm, indicating that the
formation of aldehyde 12 was negligible in its equilibrium
state. On the other hand, hemiacetal 13 exists as an equili-
brium mixture with aldehyde 14 in a 66 : 34 ratio, as determined
1
by H NMR spectroscopy (see ESI†). This sulfur effect favoring
We have designed a one-pot sequential biomimetic oxida-
tion and reduction process mediated by an NAD+/NADH sys-
tem, which can be used as a mimic of anaerobic glycolysis. In
addition, we recognized that a stoichiometric amount of Mg2+
is released as an essential activator for the biomimetic
reduction of the pyruvate analog when using the synthetic
NADH analog (Scheme 7).3a,9
the ring-closed form of the hemithioacetal is directly connected
to higher efficiency observed in the hydrogen transfer step from
10 compared to 11. However, there must be a more hidden
sulfur effect: this is because the yield of 4a with reactive
hemithioacetate 10 was ca. 10 times larger than that observed
with 11, which exceeds the value estimated from their abun-
dances determined using 1H NMR spectroscopy. The superb
reactivity exhibited during the hydrogen transfer process from
10 depends on the higher electron-donating effect of sulfur
compared to oxygen.7 This interpretation is also closely related
to GADPH oxidation observed in nature, which employs a
cysteine residue to activate glyceraldehyde-3-phosphate. A deep
and rigid pocket in the enzyme binding site of the substrate
may also prevent the entropic loss observed in the system.
Further biomimetic reactions were investigated using iso-
meric 17 and aliphatic 20. Thiolactone 15 was prepared from
commercially available benzo[b]thiophen-2-boronic acid using
a literature procedure (Scheme 5).8 During the reduction of 15
using DIBAL, hemithioacetal 16 was produced as the sole
The reaction using deuterated cysteine derivative 10-d1
resulted in the formation of 4a and the C-4 deuterated 1,4-
dihydropyridines 4a-d1 and 4a-d2, which was attributed to the
deuterium–hydrogen scrambling catalyzed by 3a.3a After
removal of the reaction solvent (THF), a LDH-type reaction
using methyl benzoylformate (21) in CH3CN was carried out
without any further addition of magnesium salts. After stirring
at room temperature for 4 d, the desired product, methyl
mandelate (22), was obtained and the hydrogen–deuterium
ratio was 70 : 30 by 1H NMR spectroscopy. We then investigated
the reduction of 21 using the mono-deuterated NADH analog
4a-d1, which was prepared independently from 3 and sodium
dithionite in D2O in the presence of magnesium perchlorate.3a,10
Partially deuterated 22 was obtained with an identical H/D ratio
of 70: 30.11 This value was in good agreement with that obtained
in the relay model reaction when considering the ratio of 4a,
4a-d1, and 4a-d2 formed in the preceding reaction. These findings
clearly show that the preferential deuterium–hydrogen relay
Scheme 6 Biomimetic oxidation using hemithioacetal derivative 17 and
20.
Scheme 5 Synthesis of magnesium hemithioacetate 17 and 20.
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reaction as well as its application to a model catalytic system is
now actively underway in our laboratories.
Conflicts of interest
There are no conflicts to declare.
Notes and references
¨
´
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3 GAPDH-type biomimetic oxidation: (a) N. Kanomata, M. Suzuki,
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Scheme 7 Synthesis of deuterated hemithioacetal derivative 10-d1, and
the sequential biomimetic oxidation and reduction system.
4 (a) V. Carelli, F. Liberatore, L. Scipione, B. D. Rienzo and S. Tortorella,
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transfer reaction from 10-d1 to 22 was mediated by the redox
cycle of the model NAD+/NADH compounds as well as the real
coenzyme system during glycolysis.
In summary, we have accomplished a GAPDH-type oxidation
reaction using a series of hemithioacetal derivatives in a model
NAD+/NADH system and demonstrated its application in
GAPDH- and LDH-type relay model reactions. When we carried
out the biomimetic oxidation reaction using cyclic hemithio-
acetate 10 bearing a five-membered ring, the hydrogen transfer
reaction proceeded under mild conditions using a THF/DMF
6 D. R. Boyd, N. D. Sharma, S. D. Shepherd, S. G. Allenmark and
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9 The reaction of NAD+ model compound 3 with cyclic hemithioacetal
10 is expected to generate one equivalent of MgBr2.
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11 The NADH model compound 4 did not reduce 21 to 22 in the
10 in the biomimetic oxidation reaction was better than that
observed for compounds 2, 11, 17, and 20. A further detailed
absence of Mg2+. Therefore, the preceding GAPDH-type model
reaction must provide an activator such as MgBr2 to the following
mechanistic study exploring the scope and limitation of the
LDH-type model reaction.
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