New artificial fluoro-cofactor of hydride transfer with novel fluorescence assay for redox biocatalysis

Article abstract of DOI:10.1039/c6cc02002j

A new artificial fluoro-cofactor was developed for the replacement of natural cofactors NAD(P), exhibiting a high hydride transfer ability. More importantly, we established a new and fast screening method for the evaluation of the properties of artificial cofactors based on the fluorescence assay and visible color change.

Full text of DOI:10.1039/c6cc02002j

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New Artificial Fluoro-Cofactor of Hydride Transfer with Novel
Fluorescence assay for Redox Biocatalysis
Received 00th January 20xx,
Accepted 00th January 20xx
a
a
a
b
a
Lei Zhang , Jun Yuan , Yufang Xu *, Y.-H. Percival Zhang *, Xuhong Qian *
DOI: 10.1039/x0xx00000x
www.rsc.org/
New artificial fluoro-cofactor was developed for the
replacement of natural cofactors NAD(P), exhibiting high
hydride transfer ability. More importantly, we established a
new and fast screening method for the evaluation of the
properties of artificial cofactors based on the fluorescence
assay and visible color change.
Nicotinamide adenine dinucleotide (NAD), nicotinamide
adenine dinucleotide phosphate (NADP), and their reduced
form, NADH and NADPH are ubiquitous in all living
because more than 400 oxidoreductases require NAD(P) as
cofactors. Although there are several methods for in situ
1
2
regeneration of NAD(P), including enzymatic, chemical, and
3
electrochemical regeneration, high costs and low-stability of
NAD(P) urge people to find out artificial cofactors which can
4
replace and even surpass NAD(P) . NAD(P) contain two parts,
the nicotinamide moiety acting as hydride donor or accepter
and the adenine dinucleotide moiety playing an important role
Figure 1 Structures of NAD(P)H and reported artificial cofactors
5
in separating between anabolic and catabolic pathways.
7
Although the anabolic and catabolic pathways are necessary resulting in excellent activities and enantioselectivities.
for survival, it is not essential to realize hydride transfer in Moreover, a widely-used artificial cofactor BNAH has been
redox biocatalysis. Hence a number of nicotinamide-containing reported to react with oxidoreductase such as enoate
8
artificial cofactors have been reported (Fig. 1). In chemical reductases for C=C bioreduction, and horse liver alcohol
9
catalysis, Hantzsch ester (HEH) acts as reductant in asymmetric dehydrogenase for chiral synthesis. Most notably, Nigel et al.
6
hydrogenation of benzoxazinones successfully and another reported the cocrystal structures of flavin-containing enzyme
artificial cofactor 9,10-dihydrophenanthridine (DHPD) has ene reductase in complex with NADH and representative
been designed for asymmetric hydrogenation of artificial cofactor, validating that both natural and artificial
benzoxazinones, benzoxazines, quinoxalines and quinolones, cofactors shared similar π-π stacking effect and occupied the
1
0
same region of the active sites.
Inspired by these discoveries, here we reported novel
artificial cofactors based on the 1,4-dihydropyridine skeleton.
These artificial redox coenzymes are inexpensive to synthesize
and stable enough to prolong the lifetime of enzymatic fuel
1
1
cells with lower potential than NADH. Due to the wide
application of fluorine in drug discovery and development, we
also introduced fluorine in our scaffold to expand the
properties and synthetic methodologies which surprisingly
produce a more facile access to a wide range of fluorinated
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Each artificial cofactor has a unique potential, E, that
indicates the ability of the compound to donate or accept
5
electrons. In order to lower its reducing potential, much focus
has been emphasized on the structure modification and
scaffold hopping of 1,4-dihydropyridine skeleton (Fig. 2).
First, we focused on the modification of the C-3 position
with different substituents on the pyridine ring, whose
position is the most important, owing to its role as
coordinating center in the active catalytic pocket of
9
reductase. We replaced -CONH
2
with -CSNH
2
and -COOCH
3
in
C-3 position of the 1,4-dihydropyridine skeleton because they
1
2
have more tightness coordination ability with reductase. In
order to screen skeletons and further optimize hydride
transfer ability of new artificial cofactors, we designed a five
member lactam ring with immobilized intramolecular amide
bond which could strengthen π-π stacking with reductase.
Second, we attempted to introduce methyl on the C-5 position
because methyl could inhibit protonation of the 5,6-double
bond with less negative effect on hydride transfer ability to C4
1
3
14
(
8b vs. 11b) (Table 1). Third, according to known literature,
1
-benzyl substituent on the nitrogen atom at the ring exerted
artificial cofactors. These artificial cofactors allow the a substantial electron-withdrawing effect that could favorably
replacement of NAD(P)H to transfer the hydride to reductase strengthen hydride transfer ability to C4. Hence, we
despiting their apparently minimal structure to the native introduced high electron-negativity group ether side chain and
coenzymes. These artificial cofactors may be applied in sugar- fluorine atom to facilitate transferring hydride. As a kind of
powered biobattery, meanwhile boosting the development of conformational element in vivo, the introduction of F will lead
artificial catalysts in asymmetric synthesis. Moreover, in view an effect on benzyl conformation and influence the binding
1
5
of complicated existing evaluation methodology of artificial mode with the receptor. Notably, the small size of fluorine
cofactors, we chose a substrate that could give remarkable may probably not disturb artificial cofactors entry into pockets.
color change after specific reduction.
The preparation of 1,4-dihydropyridine analogues started
with commercially available substituted nicotinamide, methyl
1
6
nicotinate, thionicotinamide and I2
two steps, substituted nicotinamides were alkylated by benzyl
. Through straightforward
17
bromide in THF or CH CN to obtain bromide salts and
3
reduced by Na S O to yield 1,4-dihydropyridines (2b-16b
2
)
2 4
1
8
(Scheme 1).
Substitution with F at the para- position of the 1-benzyl
yielding 12b, which displays lower potential than 11b due to
a
Potential of aitificial cofactors and NADH recorded at a glassy
−1
carbon electrode. The voltage scan rate was 100 mV s .
b
Solubility of biomimetic cofactors conducted by UV-visable
spectrophotometer in 0.1 M PBS buffer, pH 7.4.
2
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electron-withdrawing effect as we expected. (3b vs 2b
, 6b vs
5b
,
9b vs 8b, 15b vs 14b) (Table 1, Fig. S1, ESI). Besides, sugar-
powered biobatteries usually run on the aqueous buffer so we
determine the water solubility of artificial cofactors. Though
the introduction of F may sacrifice solubility to some extent,
the solubility of 15b is just a bit lower than BNAH, without
impeding the use of this artificial cofactors.
To appraise the hydride transfer ability of our artificial
cofactors, we utilized the hydrogenation of α,β-epoxy ketones
to β-hydroxy ketones that mediated through a catalytic
amount of artificial cofactors in chemical system to evaluate
1
9
the hydride transfer ability of our artificial cofactors. In this
enzyme-free reaction, Na S O was used as the reducing agent
2
2 4
+
to regenerate BNAH from BNA Br , with H O as hydride source,
-
2
and the best optimized condition is CH CN/H O (1:1, v/v) at 25
3
2
o
C, giving complete conversion with high isolated yields.
As shown in the Table 2, all artificial cofactors could
transfer hydride to form β-hydroxy ketones, and the isolated These phenomena were in consistent with the results of cyclic
yields of the best promising compounds such as 9b and 12b voltammetry, indicating the advantage of the introduction of
are up to 85%, higher than positive control BNAH (63%, fluorine.
respectively) which is outperform than natural coenzymes
0
through steady-state-kinetics.
Furthermore, we designed a new assay to evaluate
1
The reaction rates of artificial cofactors that could react with reductase. This new
compounds 5b-7b are faster than other compounds, strongly assay was better than current assays, such as UV-visible
suggesting better σ-donor S in this position could shorten absorption spectra, GC analyses, HPLC, steady-state kinetics.
reaction time indeed. We were pleased to find that all artificial We selected a flavin-containing enzyme nitroreductase (NTR)
cofactors could realize hydrogenation of α,β-epoxy ketones to from Escherichia coli and a fluorescence quenched substrate
form β-hydroxy ketones in this optimized reaction conditions, that will give fluorescence signals observed by naked eyes if
which means that our artificial cofactors could act as hydride reduced. Through the optical signal, we can judge whether the
donor in this chemistry system. Besides, among these five artificial cofactor can react with nitroreductase for the hydride
series cofactors, fluoro-cofactors displayed higher isolated transfer (Fig. 3).
yields than the cofactors substituted by H and -OCH CH OCH .
2
NTR could effectively reduce nitroaromatic compounds to
the corresponding amines in the presence of NADH as an
electron donor by transferring the hydride. NTR exhibits equal
capability of using either NADH or NADPH as a cofactor.
Richard et al. demonstrated that the adenine, dinucleotide
moiety, was not necessary and NTR could recognize simple
2
3
1
,4-dihydropyridine compounds as effective as NAD(P)H in its
2
0
ability to transfer hydride.
Our group previously has reported semi-CyHP could be
used as a selective off-on fluorescent probe which could detect
NTR. Until reduced by NADH catalyzed by NTR, the amino
group of semi-CyHF reconstructed the electronic push–pull
2
1
system and a strong fluorescence was observed. Without
NADH or replacing NADH with other biological reductants such
as glutathione (GSH), homocysteine (Hcy), dithiothreitol (DTT),
cysteine (Cys), remarkable enhancement couldnot be obtained.
This result demonstrated the importance of NADH in this
system for transferring the hydride to NTR. These results
suggested that fluorescence spectrosopy response could be
used to evaluate the effects of our artificial cofactors.
The assay of artificial cofactors toward the reduction of
semi-CyHP was performed in the phosphate buffered saline
o
PBS) buffer at 37 C. The fluorescence of semi-CyHP solution
(
-
5
(
10 M) was undetectable when excited at around 490 nm.
-1
-4
After addition of 2.5 μg mL of NTR and 5×10 M NADH, a
strong fluorescence enhancement at around 575 nm was
observed. The reduction of the probe semi-CyHP was realized
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Notes and references
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Figure 4 Fluorescence response of probe semi-CyHP (10 M)
-
-4
after adding artificial cofactors (5×10 M) and NTR (2.5 μg mL
1
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intervals at around 575 nm as indicated in the figure with
excitation at 490 nm. Silt: 10, 10 nm.
1
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selectively responds to NADH, we could replace NADH with
our artificial cofactors (2b-16b) (Fig. S2, ESI) to appraise the
effects of artificial cofactors based on the fluorescence
9
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1
1
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,
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5
(
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1
1
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1
1
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,
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This work is financially supported by the National Natural
Science Foundation of China (Grants 21236002).
4
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