Transfer hydrogenations catalyzed by streptavidin-hosted secondary amine organocatalysts

Article abstract of DOI:10.1039/d0cc08142f

Here, the streptavidin-biotin technology was applied to enable organocatalytic transfer hydrogenation. By introducing a biotin-tethered pyrrolidine (1) to the tetrameric streptavidin (T-Sav), the resulting hybrid catalyst was able to mediate hydride transfer from dihydro-benzylnicotinamide (BNAH) to α,β-unsaturated aldehydes. Hydrogenation of cinnamaldehyde and some of its aryl-substituted analogues was found to be nearly quantitative. Kinetic measurements revealed that the T-Sav:1 assembly possesses enzyme-like behavior, whereas isotope effect analysis, performed by QM/MM simulations, illustrated that the step of hydride transfer is at least partially rate-limiting. These results have proven the concept thatT-Savcan be used to host secondary amine-catalyzed transfer hydrogenations.

Full text of DOI:10.1039/d0cc08142f

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Transfer hydrogenations catalyzed
by streptavidin-hosted secondary
amine organocatalysts†
Cite this: Chem. Commun., 2021,
57, 1919
Received 15th December 2020,
Accepted 14th January 2021
a
ab
c
c
´
Nicolo Santi,
`
Louis Y. P. Luk
Louis C. Morrill,
*
Katarzyna Swiderek, Vicent Moliner
and
ab
DOI: 10.1039/d0cc08142f
rsc.li/chemcomm
Here, the streptavidin–biotin technology was applied to enable hydroxylases,14 cytochrome P450,15 2-hydroxybiphenyl 3-monooxy-
organocatalytic transfer hydrogenation. By introducing a biotin- genase (HbpA),16 Old Yellow Enzyme (TsOYE),17 and glucose dehy-
tethered pyrrolidine (1) to the tetrameric streptavidin (T-Sav), the drogenases (GDH).18 Here, we aim to expand this collection by
resulting hybrid catalyst was able to mediate hydride transfer from proving the concept that organocatalytic artificial enzymes can
dihydro-benzylnicotinamide (BNAH) to a,b-unsaturated aldehydes. catalyze transfer hydrogenation using BNAH as a hydride source.19
Hydrogenation of cinnamaldehyde and some of its aryl-substituted
Artificial enzymes can be created by docking chemical
analogues was found to be nearly quantitative. Kinetic measure- catalysts into a designated protein scaffold, and the resulting
ments revealed that the T-Sav:1 assembly possesses enzyme-like complex can potentially use hydride donors for reactions.7
behavior, whereas isotope effect analysis, performed by QM/MM Different approaches such as computational design, genetic
simulations, illustrated that the step of hydride transfer is at least code expansion, supramolecular approaches and amino acid
partially rate-limiting. These results have proven the concept that modification have been used to accommodate chemical cata-
T-Sav can be used to host secondary amine-catalyzed transfer lytic systems for bioorthogonal reactions.19–25 Previously, the
hydrogenations.
streptavidin–biotin technology has been applied to host Ru and
Ir-mediated transfer hydrogenation with the use of NAD(P)H
In biological systems, nicotinamide is frequently used in the and formic acid as hydride donors.7,26–28 Recently, streptavidin
transfer of electrons and hydrogen atoms1,2 and is typically found has also been used to host organocatalysis, including secondary
in the form of NAD(P)H attached to an adenosine dinucleotide amine, anion–p and DMAP catalysis;19,20,23,29,30 however,
appendix.3 While the adenosine dinucleotide motif is crucial for the reaction profile of these organocatalytic systems remains
molecular recognition and metabolic regulation in cells,4 this largely unexplored, and their possibility of mediating organo-
appendix is a large and complex molecular burden that needs to catalytic transfer hydrogenation reaction has not been tested.
be addressed when it comes to synthetic applications.5 Conse-
Here, we demonstrate that secondary amine hosted by
quently, various cofactor recycling systems have been designed.6,7 streptavidin can be used to catalyze transfer hydrogenation
In an alternative and potentially simpler approach, small organic reactions (Fig. 1). Hybrid catalysts have been created by introducing
molecules can be used as hydride donors,8,9 such as the Hantzsch biotinylated secondary amines (1 and 2) to the tetrameric strep-
ester,10 dihydro-benzylnicotinamide (BNAH) and their deri- tavidin (T-Sav) or the monomeric counterpart (M-Sav). Upon
vatives.11,12 Nevertheless, there are only a handful of protein- optimization, conversion of cinnamaldehyde (3) to its reduced
based catalysts reported to use simple hydride donors for reac- counterpart (5) was found to be 490% (Table 1). This work lays
tions, including ene-reductases (ERs),6,13 salicylate (SalH), the basis for enabling organocatalytic transfer hydrogenation in
para-hydroxybenzoate (PHBH) and hydroxybenzoate (3HB6H) biological contexts.
In previous studies, we have demonstrated that biotinylated
a School of Chemistry, Main Building, Cardiff University, Cardiff CF10 3AT, UK.
E-mail: lukly@cardiff.ac.uk; Tel: +44 (0)29 2251 0161
catalysts (1 and 2) were able to mediate Michael addition and
aldol condensation when introduced to the tetrameric strep-
tavidin (T-Sav).31 In this work, the reduction of cinnamaldehyde
3 by dihydrobenzyl nicotinamide (BNAH, 4) in KPi buffer
(10 mM, 10% methanol, pH 7.0) at room temperature serves
as the model transfer hydrogenation reaction and was assessed
by 1H NMR spectroscopy (Table 1). In the absence of protein or
b Cardiff Catalysis Institute, School of Chemistry, Main Building, Cardiff University,
Cardiff CF10 3AT, UK
c
´
´
´
´
Departament de Quımica Fısica i Analıtica, Universitat Jaume I, Castello 12071,
Spain
† Electronic supplementary information (ESI) available. See DOI: 10.1039/
d0cc08142f
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Table 1 Screening reaction for transfer hydrogenation from BNAH to
cinnamaldehydeabc
Hydrogen
donor
Loading
(mol%)
% Estimated
conversiona
Entry
Host
Guest
1
2
3
4
5
6
7
8
N.A.
N.A.
N.A.
N.A.
1
2
1
1
1
1
2
1
2
1
BNAH (4)
BNAH (4)
BNAH (4)
BNAH (4)
BNAH (4)
BNAH (4)
BNAH (4)
BNAH (4)
BNAH (4)
BNAH (4)
BNAH (4)
BNAH (4)
NADH (5)
Hantzsch (6)
N.A.
1
1
1
1
1
2
1
1
1
1
1
1
3
5
4
T-Sav
M-Sav
N.A.
26
25
77
95
94d
53e, f
51
21
10
5
N.A.
T-Sav
T-Sav
T-Sav
T-Sav
T-Sav
M-Sav
M-Sav
T-Sav
T-Sav
9
10
11
12
13
14
1
1
0
a
Estimated conversion was determined by the ratio of the corres-
ponding 1H NMR peak integration (see ESI). Reactions performed in
b
a mixture 90 : 10 KPi 10 mM : MeOH at pH 7.0 using 1 equivalent of
c
aldehyde (6.6 mM) and 2 equivalents of 4 (13.2 mM). In the absence of
catalyst, partial oxidation of BNAH was observed, but the oxidation rate
is negligible when compared to the protein-hosted organocatalytic
d
reaction. Reactions performed in a mixture 75 : 25 KPi 10 mM : MeOH
at pH 7.0 using 1 equivalent of aldehyde (6.6 mM) and 5 equivalents of
e
f
4 (33 mM). 0 rpm, side-product observed. ppt observed. 1 mol% of
T-Sav/M-Sav corresponds to 66 nmol catalytic sites. N.A. indicates not
added.
Fig. 1 Combining the tetrameric streptavidin (T-Sav) with biotinylated
organocatalysts 1 or 2 to generate organocatalytic artificial enzymes
(T-Sav:1, PDB: 6GH7, 1.08 Å) for transfer hydrogenation reactions.
reactions.33 M-Sav was thus tested as an alternative host for
the organocatalytic transfer hydrogenation reaction. Neverthe-
less, in this case the conversion dropped to 21% and 10% by
catalyst 1 and 2 in M-Sav, respectively (entries 11 and 12). This
catalysts, only 3% of dihydrogenated product was observed observation suggests that secondary amine-catalyzed transfer
after 24 hour incubation when two equivalents of BNAH were hydrogenation prefers a more shielded protein scaffold. To see
used (entry 1). Similarly, T-Sav (and its monomeric counterpart how a change in hydride donor affects the conversion rate,
M-Sav32,33) has negligible effects in catalyzing hydride transfer alternative hydride donors including Hantzsch ester (5) and
(entries 2 and 3). When 1 mol% of catalyst 1 or 2 is used, the NADH (6) were tested, but their use resulted in significantly
estimated reaction conversion increased to about 25% (entries lower conversion (entry 13 and 14). The negligible conversion
4 and 5). When both T-Sav and catalyst 1 are included (1 : 1.2 observed with NADH (5) could be caused by its structural
ratio of catalyst to protein), the estimated conversion increased complexity, whereas the low solvent solubility of Hantzsch ester
to 77 and 95% at 1 and 2 mol% respectively (entries 6 and 7). (6) in aqueous buffer can influence its ability to transfer
These results are in line with the findings that organocatalysis hydride within the T-Sav scaffold.
is favored when executed in an environment that has low
Having the optimal conditions determined (Table 1, entry 8),
dielectric constant, such as organic solvents and protein surface various aromatic a,b-unsaturated aldehydes were tested as
or interior.19 Increasing the amount of BNAH to 5 equivalents in a alternative substrates. The T-Sav:1 assembly showed significant
75 : 25 KPi : MeOH mixture enhances the conversion up to 94% substrate promiscuity; cinnamaldehyde analogues with chloro,
with only 1 mol% of T-Sav:1 (entry 8). Interestingly, when no fluoro or nitro substituent added at the para position can be
shaking was applied to the reaction mixture, the product conver- converted into the corresponding dihydro-products (Table 2),
sion decreases to 53% (entry 9). Lastly, in agreement with the while the background reactions remain negligible. Supported
previous studies which illustrated that catalyst 2 is more flexible by NMR analysis, modest conversion was also observed for the
and solvent-exposed when bound to T-Sav,20 the conversion was methyl, bromo and methoxy analogues (60–76%). Together,
lower when catalyst 2 was used (entry 10).
In most streptavidin-based artificial enzymes, the catalytic is largely affected by electrostatic properties of the substituents.
centers are surrounded by residues at the intersubunit Kinetic properties of the streptavidin-based transfer hydrogena-
these observations suggest that the efficiency of hydride transfer
interface.24,34 It however has recently been demonstrated that tion system were evaluated. The protein-based catalyst demon-
such a shielded environment might not be ideal for some of strates enzyme-like kinetic behaviours with catalytic efficiency
the chemical catalytic reactions; indeed, the monomeric variant (kcat/KMcinnamaldehyde) estimated to be 8.50 Æ 0.5 MÀ1ÁsÀ1 at 25 1C
M-Sav can be a superior host to T-Sav for Rh-catalyzed (Fig. 2). The corresponding kcat/KM for flavoprotein was reported to
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Table 2 Substrate scope for transfer hydrogenation from BNAH to aro-
matic a,b-unsaturated aldehydes mediated by T-Sav:1 assemblya
Aldehyde
% Estimated conversionb
Cinnamaldehyde (3)
p-Cl (8)
p-F (9)
94
94
499
60
p-Br (10)
p-OCH3 (11)
p-NO2 (12)
p-CH3 (13)
69
Fig. 3 Detail of the transition state for the hydride transfer from BNAH to
cinnamaldehyde in the active site of T-Sav:1 optimized at the M06-2X/MM
level. Average distances in Å.
499c
76
a
Estimated conversion was determined by ratio of the corresponding
1H NMR peak integration (see ESI). Reactions performed in a mixture
b
transfer. The resulting KIE was found to be 3.89 Æ 0.15 when
the ground state of BNAH in aqueous solution was used, and
this parameter further increased to 4.28 Æ 0.18 when the
equilibrium between the Michaelis complex and the hydride
transfer transition state were used for calculation (see ESI,† for
details). These values are similar to those previously obtained
for hydride transfer processes catalyzed by NADH-dependent
enzymes,35,36 including L-lactate dehydrogenase (in the range
between 3.36 and 2.80)37 and morphinone reductase (8.4 Æ 1.6),38
but significantly higher than that of the experimental counterpart
measured in the present study. Interestingly, regarding the
hydride donor–acceptor distance and the relative orientation of
the different involved moieties, the nature of the optimized
transition state can be considered as equivalent to those located
in previous studies (Fig. 3).35–38 It therefore suggests that the
hydride transfer step in the T-Sav artificial enzyme is at least
partially rate-limiting.
In summary, we have described the use of streptavidin as a
host for organocatalytic transfer hydrogenations. At 1 mol%,
the T-Sav:1 assembly was able to mediate the hydrogenation of
various aromatic a,b-unsaturated aldehydes in excellent con-
versions, and the resulting products have been used in the
synthesis of pharmaceutical compounds.39 Moreover, T-Sav:1
possesses Michaelis Menten kinetic properties for the organo-
catalytic transfer hydrogenation, presenting as a great starting
point for artificial enzyme engineering. Studies focusing on the
reaction mechanisms, the use of prochiral aldehydes and
potential applications in cascade reactions are currently
ongoing.
75 : 25 KPi 10 mM : MeOH at pH 7.0 using 1 equivalent of aldehyde
(6.6 mM) and 5 equivalents of 4 (33 mM) with 1 mol% of T-Sav:1.
c
Reaction performed in a mixture of 9 : 1 KPi 10 mM : MeOH at pH
7.0 using 1 equivalent of aldehyde (6.6 mM) and 2 equivalents of 4
(13.2 mM). 1 mol% of T-Sav/M-Sav correspond to 66 nmol catalytic sites.
be 5900 MÀ1ÁsÀ1. Furthermore, BNAH has been used for aromatic
hydroxylation and the reduction of the a,b-unsaturated carbonyl
substrate; their biomolecular reaction rate constants were measured
to be 1.2Á104 and 1.9Á106 MÀ1ÁsÀ1, respectively.13,14 Although these
values are significantly higher than that of the streptavidin system,
the presented bottom-up approach has not yet been engineered and
offers significant flexibility in design.
QM/MM molecular dynamics (MD) simulations were con-
ducted to obtain a detailed understanding of the hydride
transfer step in the T-Sav:1 system. Exploration of the free
energy surface at M06-2X:RM1/MM level enabled identification
of the isolated structures of the reactants (in solution as
solvated species and in the active site of the protein scaffold
as the ‘‘Michaelis complex’’) and the hydride transfer transition
state (see ESI,† for details). Hence, the magnitude of the
[1,2-2H/1H] KIE was computationally assessed, including quan-
tum tunneling corrections required for chemical reactions
involving the transfer of a light particle such as hydride
This work was supported by Cardiff University through the
start-up fund provided by the Cardiff School of Chemistry, the
Leverhulme Trust through a grant to L. Y. P. L. (RPG-2017-195),
the Royal Society through a grant to L. C. M. (RG150466), and the
UK’s Wellcome Trust through grants to L. Y. P. L. (202056/Z/16/Z).
Conflicts of interest
Fig. 2 Kinetic evaluation of the T-Sav:1 complex for transfer hydrogena-
tion from BNAH to cinnamaldehyde.
There are no conflicts to declare.
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