Expanding the chemical diversity in artificial imine reductases based on the biotin-streptavidin technology

Article abstract of DOI:10.1002/cctc.201300825

We report on the optimization of an artificial imine reductase based on the biotin-streptavidin technology. With the aim of rapidly generating chemical diversity, a novel strategy for the formation and evaluation of biotinylated complexes is disclosed. Tethering the biotin-anchor to the Cp* moiety leaves three free coordination sites on a d⁶ metal for the introduction of chemical diversity by coordination of a variety of ligands. To test the concept, 34 bidentate ligands were screened and a selection of the 6 best was tested in the presence of 21 streptavidin (Sav) isoforms for the asymmetric imine reduction by the resulting three legged piano stool complexes. Enantiopure α-amino amides were identified as promising bidentate ligands: up to 63 % ee and 190 turnovers were obtained in the formation of 1-phenyl-1,2,3,4-tetrahydroisoquinoline with [IrCp*^biotin(L-ThrNH₂)Cl]?SavWT as a catalyst. Biotinspired! A new strategy for the generation of chemical diversity in artificial transfer hydrogenases (ATHases) based on the biotin-streptavidin technology is disclosed. By combining a biotinylated MCp* fragment with 34 commercially available ligands in the presence of wild-type streptavidin, promising candidates for the asymmetric reduction of imines are identified. Selected ligands are screened against 21 streptavidin isoforms and the performance of the resulting constructs is evaluated.

Full text of DOI:10.1002/cctc.201300825

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DOI: 10.1002/cctc.201300825
Expanding the Chemical Diversity in Artificial Imine
Reductases Based on the Biotin–Streptavidin Technology
Tommaso Quinto, Fabian Schwizer, Jeremy M. Zimbron, Albert Morina, Valentin Kçhler, and
Thomas R. Ward*[a]
We report on the optimization of an artificial imine reductase
based on the biotin-streptavidin technology. With the aim of
rapidly generating chemical diversity, a novel strategy for the
formation and evaluation of biotinylated complexes is dis-
closed. Tethering the biotin-anchor to the Cp* moiety leaves
three free coordination sites on a d6 metal for the introduction
of chemical diversity by coordination of a variety of ligands. To
test the concept, 34 bidentate ligands were screened and a se-
lection of the 6 best was tested in the presence of 21 strepta-
vidin (Sav) isoforms for the asymmetric imine reduction by the
resulting three legged piano stool complexes. Enantiopure a-
amino amides were identified as promising bidentate ligands:
up to 63 % ee and 190 turnovers were obtained in the forma-
tion of 1-phenyl-1,2,3,4-tetrahydroisoquinoline with [IrCp*biotin
-
(L-ThrNH2)Cl]ꢀSavWT as a catalyst.
Introduction
Artificial metalloenzymes result from the incorporation of an
organometallic catalyst or a catalyst precursor within a macro-
molecular scaffold (protein or oligonucleotide).[1–19] The result-
ing hybrid catalyst can be optimized by using either genetic or
chemical methods.[1–8,18–21] In the context of artificial metalloen-
zymes based on the biotin-streptavidin technology, we and
others have relied on synthesizing various biotinylated ligands
to provide chemical diversity.[1,4,15,20,21] To generate larger artifi-
cial cofactor libraries quickly, we reasoned that separating the
necessary biotin anchor moiety and variable ligand
elements would enable us to screen commercially
available ligands in the presence of streptavidin (Sav;
Scheme 1). To test the validity of this strategy, we se-
lected the asymmetric transfer hydrogenation of pro-
chiral imines catalyzed with biotinylated moieties
(M=Rh, Ir).
metal for the reaction to proceed.[24–27] This reaction is thus ide-
ally suited to test the concept outlined in Scheme 1b.
Results and Discussion
The synthesis of the biotinylated catalyst precursors
[MCp*biotinCl2]2 (M=Ir, Rh) is described elsewhere.[12,19,28,29] The
dimeric precursor was reacted in situ with a selection of com-
mercially available bidentate ligands (Scheme 2). To identify
Considering the robustness of the MCp*
moiety,[22,23] we reasoned that for d6 transition metals
tethering the biotin anchor to the Cp* unit would
leave three free coordination sites around the metal
Scheme 1. Artificial metalloenzymes consist of an organometallic catalyst or a catalyst
precursor incorporated within a protein scaffold. The optimization of their catalytic per-
formance is achieved either by mutating the protein (*) or by varying the bidentate
ligand (A–A’). a) The recognition element (3) and the variable bidentate ligand are cova-
lently linked. b) The recognition element and the bidentate ligand (A–A’) are distinct,
thus allowing for screening of commercially available ligands.
for further functionalization. It is widely accepted
that three-legged piano stool complex-catalyzed
asymmetric transfer hydrogenation proceeds via an
outer sphere hydride transfer mechanism, which re-
quires only one free coordination site around the
suitable coordination conditions, the chiral ligand l-ProNH2
was complexed in situ with the dinuclear catalyst precursors
[MCp*biotinCl2]2 (M=Ir, Rh) at different pH values within the
buffer range of 3-(N-morpholino)propanesulfonic acid (MOPS)
or 2-(N-morpholino)ethanesulfonic acid. As the best conversion
for the asymmetric transfer hydrogenation of prochiral imine
[a] T. Quinto, F. Schwizer, Dr. J. M. Zimbron, A. Morina, Dr. V. Kçhler,
Prof. Dr. T. R. Ward
Department of Chemistry
University of Basel
Spitalstrasse 51, CH-4056 Basel (Switzerland)
E-mail: thomas.ward@unibas.ch
Homepage: http://www.chemie.unibas.ch/~ward/index.html
1a was observed at pH 7.8, this value was selected for all sub-
sequent studies.
Supporting information for this article is available on the WWW under
http://dx.doi.org/10.1002/cctc.201300825.
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and in the presence of Sav,
whereas [RhCp*biotinCl2]2 demon-
strates significant activity only in
the absence of Sav (Table 1,
entry 1).
2) Although most ligands
tested afford >60 turnovers
(>30% yield) if combined with
[RhCp*biotinCl2]2, only four ligands
give rise to >60 turnovers with
[IrCp*biotinCl2]2.
Notably,
the
widely used amino sulfonamide
ligand scaffold (ligand 9) per-
forms only moderately in com-
parison to the best ligands
under the experimental condi-
tions adopted for this screening.
With high-throughput screening
in mind, no effort was made to
improve
of individual
combinations.
the
performance
metal–ligand
3) Incorporation within Sav
leads to a significant decrease in
conversion in all but two metal–
ligand combinations. Both gly-
cine amide 3 and bisoxazoline
10 combined with [IrCp*biotinCl2]2
afforded more than 180 turn-
Scheme 2. Initial selection of bidentate ligands (3–10) for the creation of artificial imine reductases based on the
biotin–streptavidin technology.
A preliminary screening was performed with eight commer-
cially available bidentate ligands using [MCp*biotinCl2]2 (M=Ir,
Rh) for the reduction of 1-phenyl-3,4-dihydroisoquinoline 1a
with formate as a hydride source. The results are presented in
Table 1.
overs for the reduction of cyclic imine 1a. Owing to the enan-
tiopure environment provided by the Sav host, the corre-
sponding amine 2a is produced in enantioenriched form: (S)-
2a in 43% ee with 3 and (S)-2a in 14% ee with 10 (Table 1, en-
tries 2 and 9 with [IrCp*biotinCl2]2). The ATHase derived from gly-
cine 4 performs poorly in the presence of Sav compared with
glycine amide 3 (Table 1, entries 2 and 3 with [IrCp*biotinCl2]2).
After identifying the a-amino amide scaffold as a promising
activating ligand[30–36] in the ATHase assembly for the reduction
of imine 1a, 28 commercially available amino amides were
tested in conjunction with [IrCp*biotinCl2]2 in the presence of
wild-type Sav (Sav-WT; Figure 1 and Table 2).
The following trends emerged from these results:
1) The dinuclear catalyst precursor [IrCp*biotinCl2]2, devoid of
any additional ligands, demonstrates only low detectable artifi-
cial transfer hydrogenase (ATHase) activity both in the absence
Table 1. Identification of the most suitable ligand for activating a biotiny-
lated piano stool catalyst precursor for the reduction of imine 1a.[a]
The following trends emerged from these results:
1) In the absence of Sav, the enantiopure biotinylated piano
stool complexes demonstrate up to 29% ee for the reduction
of imine 1a. The distant enantiopure biotin anchor does not
significantly affect the enantioselectivity either in the presence
of achiral ligands (Table 1) or in the presence of mirror-image
amino amides. Accordingly (in the absence of Sav), l-phenyla-
lanine amide and d-phenylalanine amide afford the amine (S)-
2a in 17% ee and (R)-2a in 16% ee, respectively (Table 2, en-
tries 10 and 11).
Entry
Ligand
Yield (ee)[d] [%]
Sav-WT[b] No protein[c]
No protein[b]
Sav-WT[c]
1
2
3
4
5
6
7
8
9
–
3
4
5
6
7
8
9
6 (À1)
93 (0)
8 (À3)
0 (0)
4 (À2)
31 (0)
44 (0)
16 (À1)
99 (0)
3 (38)
94 (43)
2 (21)
0 (0)
81 (0)
60 (0)
51 (0)
34 (0)
16 (À1)
35 (À1)
6 (À1)
36 (0)
37 (0)
5 (À5)
5 (7)
4 (1)
3 (À1)
3 (À1)
3 (À1)
2 (À6)
3 (1)
0 (0)
13 (16)
0 (0)
6 (13)
51 (14)
2) In most of the cases, the transfer hydrogenation activities
in the absence and in the presence of Sav are comparable.
However, incorporation within Sav leads to a significant in-
crease in ee values for several combinations (Table 2, entries 1,
2, 4, 6, 8, 12, 18, and 20) or to an inversion of the preferred
product enantiomer (Table 2, entries 5, 7, 9, 11, and 17). In gen-
10
4 (0)
[a] The best results are highlighted in boldface. For full experimental de-
tails, see the Supporting Information; [b] Metal catalyst precursor=
[IrCp*biotinCl2]2; [c] Metal catalyst precursor=[RhCp*biotinCl2]2; [d] A positive
ee value refers to the S enantiomer; a negative value refers to the R enan-
tiomer of amine 2a.
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eral, incorporation in Sav-WT shifts the stereoselectivity in favor
3) Upon incorporation within Sav, >60% ee of (S)-2a and
ꢁ170 turnovers are obtained with l-LeuNH2, d-ArgNH2, l-
IleNH2, or l-ThrNH2 (Table 2, entries 6, 9, 18, and 20). The a-
amino amides bearing a softer donor side chain (e.g., l-me-
thionine amide and l-histidine amide; Table 2, entries 19 and
25) completely inhibit the catalysis. We hypothesize that the
binding mode of these potentially tridentate ligands may differ
from that of the other amino amide ligands tested.[34]
of the S enantiomer.
4) The absolute configuration of the amino amide has
a modest effect on the enantioselectivity of the corresponding
artificial metalloenzyme. In the isolated cases tested, Dee
values for the matched versus mismatched combinations differ
by ꢂ20% in the protein (Table 2, entries 4 and 5, entries 6 and
7, entries 8 and 9, entries 10 and 11, entries 16 and 17), except
in proline (Dee=75%; entries 2 and 3).
After identifying promising amino amide ligands, we pro-
ceeded to the genetic optimization of the ATHase activity. For
this purpose, six amino amide ligands (l-ProNH2, l-ValNH2, l-
LeuNH2, l-IleNH2, l-ThrNH2, and GlyNH2) were selected and
screened in the presence of 21 Sav isoforms for the reduction
of cyclic imine 1a. The corresponding results are presented as
a fingerprint in Figure 2.
Figure 1. Selection of amino amide ligands screened for the enantioselective
reduction of imine 1a.
Table 2. Selected results obtained for the screening of amino amide li-
gands for the reduction of imine 1a with [IrCp*biotinCl2]2 (with or without
Sav-WT) as a complex precursor.[a]
Entry
Ligand
Yield (ee)[b] [%]
No protein
Sav-WT
1
2
3
4
5
6
7
8
GlyNH2
93 (0)
94 (43)
47 (67)
28 (À8)
70 (63)
66 (45)
96 (63)
96 (51)
71 (52)
85 (61)
98 (33)
96 (13)
98 (52)
94 (30)
5 (44)
17 (58)
87 (34)
88 (36)
89 (65)
0 (0)
96 (63)
91 (56)
86 (39)
90 (36)
89 (26)
0 (0)
l-ProNH2
d-ProNH2
l-ValNH2
99 (17)
99 (À29)
99 (20)
99 (À12)
97 (9)
99 (À13)
98 (7)
71 (À12)
97 (17)
99 (À16)
99 (1)
99 (11)
83 (13)
94 (24)
57 (6)
56 (À6)
98 (5)
d-ValNH2
l-LeuNH2
d-LeuNH2
l-ArgNH2
d-ArgNH2
l-PheNH2
d-PheNH2
l-SerNH2
l-GluNH2
l-Pro-NHethyl
l-3,4-Dehydro-ProNH2
l-AlaNH2
d-AlaNH2
l-IleNH2
l-MetNH2
l-ThrNH2
l-AsnNH2
l-GlnNH2
l-TyrNH2
l-LysNH2
l-HisNH2
l-AspNH2
l-TrpNH2
b-GlyNH2
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
0 (0)
86 (6)
85 (12)
45 (12)
70 (16)
99 (1)
0 (0)
66 (13)
65 (20)
7 (2)
94 (43)
95 (37)
3 (28)
[a] The best results are highlighted in boldface. Reaction conditions:
35 mm substrate, 0.25 mol% [IrCp*biotinCl2]2, 0.55 mol% amino amide,
7.4 mgmLÀ1 Sav-WT, 0.56m MOPS, 1.4m formate, pH 7.8, incubation for
18 h at 508C. All listed results are the average of two runs; for full details,
see the Supporting Information; [b] A positive ee value refers to the S
enantiomer; a negative value refers to the R enantiomer of amine 2a.
Figure 2. Results of the chemogenetic optimization of ATHases for the asym-
metric reduction of imine 1a, which are presented in the bubble-chart
format. The size of the bubbles is proportional to the conversion (between 4
and 99%), and the color codes the enantiomeric excess. See Table 2 for ex-
perimental details. The numerical values (conversion and ee) are listed in
Table S1.
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From the results presented in Figure 2, the following conclu-
sions could be drawn:
Variation of the substrate led to the following observations
and trends:
1) The best results in terms of enantioselectivity for any
ligand are obtained with Sav-WT.
1) Conversion of bulky substrate 1a generally led to the high-
est observed enantioselectivities in favor of the S product.
2) Reducing the size of the substituent at position 1 of the di-
hydroisoquinoline moiety from phenyl to methyl resulted
in a considerable decrease in enantioselectivity.
3) Introduction of methoxy substituents at positions 6 and 7
of the dihydroisoquinoline moiety only moderately affected
the selectivity.
2) Mutating single residues at position S112 or K121, or mu-
tating both residues simultaneously, leads to a decrease in
enantioselectivity for all ligands tested. The S112Y mutant led,
in combination with l-LeuNH2, to an inversion of enantioselec-
tivity compared to Sav-WT (63% ee of (S)-2a in Sav-WT and
20% ee of (R)-2a in the presence of S112Y).
3) All mutants at position K121 typically lead to high con-
versions with all amino amide ligands tested. In the presence
of either l-ProNH2 or l-ValNH2, significantly improved conver-
sions compared to Sav-WT were observed with K121N, K121A,
K121H, and K121Y mutants.
4) With 2-substituted 1-pyrrolines, conversions were generally
good to excellent.
5) The highest ee value for 1-pyrrolines was observed for the
bulky substrate carrying a cyclohexyl substituent in combi-
nation with l-ThrNH2. The ee values for the benzyl- and
phenyl-substituted substrates 1e and 1 f, respectively, were
generally low.
4) In the presence of the L124K, L124Y, or L124F mutant,
a marked decrease in enantioselectivity compared with that in
the presence of Sav-WT is observed for all ligands except l-
ProNH2. Conversions were generally good to excellent with the
L124Y or L124F mutant.
5) Good to excellent conversions were observed for all
double mutant–ligand combinations, which were accompanied
by a decrease in enantioselectivity (except l-ProNH2).
Finally, the substrate scope of the artificial imine reductases
was evaluated (Figure 3). For this purpose, six prochiral cyclic
Conclusions
To readily access large chemical diversity, a new artificial metal-
loenzyme design based on the biotin–streptavidin technology
has been presented. Relying on three-legged piano stool com-
plexes and tethering the biotin anchor on the Cp* moiety al-
lowed us to screen various bidentate ligands for the asymmet-
ric reduction of cyclic imines. An initial screening led to the
identification of amino amides as versatile bidentate ligands in
conjunction with the {IrCp*biotin} moiety. Genetic diversity was
introduced by site-directed mutagenesis. Both chemical diver-
sity and genetic diversity were shown to have a significant
effect on the activity and selectivity of the resulting artificial
metalloenzyme.
By taking into account the versatility of the {Cp*MLn} moiety
in catalysis, we reasoned that the strategy disclosed herein will
find wide application for the chemical optimization of artificial
metalloenzymes. Current efforts are aimed at the structural
and kinetic characterization of such hybrid catalysts.
Figure 3. Selection of substrates for the asymmetric transfer hydrogenation
of cyclic imines.
imines were tested in the presence of artificial metalloenzymes
[IrCp*biotin(amino amide)Cl]ꢀSav-WT (amino amide=l-ProNH2,
l-ValNH2, l-LeuNH2, l-IleNH2, l-ThrNH2, GlyNH2). The results of
this screening are summarized in Table 3.
Experimental Section
General method for the asymmetric transfer hydrogenation
Buffer A (100 mL, 0.6m in MOPS in Milli-Q H2O, pH 7.8) was placed
in a polypropylene (PP) tube, followed by the addition of the bio-
tinylated metal complex [MCp*biotinCl2]2 stock solution (3.75 mL,
5.0 mm in DMSO) and the ligand stock solution (3.75 mL, 11 mm in
Milli-Q H2O or DMSO, depending on the ligand). The mixture was
agitated for 30 min at 308C and 600 rpm in a thermo mixer for
precomplexation. The corresponding lyophilized Sav mutant
(1.6 mg) was dissolved in buffer B (100 mL, 0.6m in MOPS, 3.0m in
HCOONa in Milli-Q H2O, pH 7.8). Then, Sav-mixture (100 mL) was
added to the PP tube containing the metal complex and agitation
was continued for 15 min at 308C and 600 rpm to ensure binding
of the biotinylated complex to Sav. Finally, the substrate stock solu-
tion was added (7.5 mL, 1m in DMSO) and the mixture was agitated
at 508C for 18 h. Subsequently, NaOH(aq) (60 mL, 5m solution) was
added to the reaction mixture, followed by the addition of CH2Cl2
Table 3. Screening of different substrates for the [IrCp*biotin(amino ami-
de)Cl]ꢀSav-WT-mediated transfer hydrogenation.
Entry Ligand
Yield (ee)[a] [%]
1a
1b
1c
1d
1e
1 f
1
2
3
4
5
6
l-ProNH2 47 (67) 99 (5) 100 (6) 69 (À32) 60 (À11) 100 (18)
l-ValNH2 70 (63) 100 (17) 100 (17) 84 (À44) 80 (À22) 86 (16)
l-LeuNH2 96 (63) 100 (3) 100 (18) 95 (À45) 96 (À31) 100 (20)
l-IleNH2 89 (65) 100 (20) 100 (43) 83 (À32) 79 (À22) 10 (18)
l-ThrNH2 96 (63) 100 (25) 100 (41) 98 (À57) 95 (À35) 91 (20)
GlyNH2
94 (43) 100 (4) 100 (18) 96 (À46) 94 (À9)
79 (2)
[a] Positive and negative ee values correspond to S and R enantiomers, re-
spectively. The absolute configuration of the amines 2d–f was not deter-
mined. The best results are highlighted in boldface.
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Acknowledgements
We acknowledge the financial support from the European Union
Seventh Framework Programme (FP7/2007-2013) under grant
agreement No. FP7-PEOPLE-ITN-2008-238531. Additional funding
was provided by the Swiss National Science Foundation (200020_
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Keywords: asymmetric
technology · imine reduction · metalloenzymes · transfer
hydrogenation
catalysis
·
biotin–streptavidin
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Received: September 27, 2013
Published online on January 16, 2014
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