Enantioselective Synthesis of Chiral Amines via Biocatalytic Carbene N-H Insertion

Article abstract of DOI:10.1021/acscatal.0c02794

Optically active amines represent highly valuable building blocks for the synthesis of advanced pharmaceutical intermediates, drug molecules, and biologically active natural products. Hemoproteins have recently emerged as promising biocatalysts for the formation of C-N bonds via carbene transfer, but asymmetric N-H carbene insertion reactions using these or other enzymes have so far been elusive. Here, we report the successful development of a biocatalytic strategy for the asymmetric N-H carbene insertion of aromatic amines with 2-diazopropanoate esters using engineered variants of myoglobin. High activity and stereoinduction in this reaction could be achieved by tuning the chiral environment around the heme cofactor in the metalloprotein in combination with catalyst-matching and tailoring of the diazo reagent. Using this approach, an efficient biocatalytic protocol for the synthesis of a broad range of substituted aryl amines with up to 82% ee was obtained. In addition, a stereocomplementary catalyst useful to access the mirror-image form of the N-H insertion products was identified. This work paves the way to asymmetric amine synthesis via biocatalytic carbene transfer, and the present strategy based on the synergistic combination of protein and diazo reagent engineering is expected to prove useful in the context of these as well as other challenging asymmetric carbene transfer reactions.

Full text of DOI:10.1021/acscatal.0c02794

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Article
Enantioselective Synthesis of Chiral Amines
via Biocatalytic Carbene N—H Insertion
Viktoria Steck, DANIELA MARIA CARMINATI, Nathan Johnson, and Rudi Fasan
ACS Catal., Just Accepted Manuscript • DOI: 10.1021/acscatal.0c02794 • Publication Date (Web): 31 Aug 2020
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Enantioselective Synthesis of Chiral Amines via Biocatalytic
Carbene N—H Insertion
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Viktoria Steck, Daniela M. Carminati, Nathan R. Johnson, and Rudi Fasan*
Department of Chemistry, University of Rochester, 14627 Rochester, New York, USA
* Correspondence should be addressed to R.F. (rfasan@ur.rochester.edu)
Abstract
Optically active amines represent highly valuable building blocks for the synthesis of advanced
pharmaceutical intermediates, drug molecules, and biologically active natural products.
Hemoproteins have recently emerged as promising biocatalysts for the formation of C—N bonds
via carbene transfer, but asymmetric N—H carbene insertion reactions using these or other
enzymes have so far been elusive. Here, we report the successful development of a biocatalytic
strategy for the asymmetric N—H carbene insertion of aromatic amines with 2-diazopropanoate
esters using engineered variants of myoglobin. High activity and stereoinduction in this reaction
could be achieved by tuning the chiral environment around the heme cofactor in the metalloprotein
in combination with catalyst-matching and tailoring of the diazo reagent. Using this approach, an
efficient biocatalytic protocol for the synthesis of a broad range of substituted aryl amines with up
to 82% ee was obtained. In addition, a stereocomplementary catalyst useful to access the mirror-
image form of the N—H insertion products was identified. This work paves the way to asymmetric
amine synthesis via biocatalytic carbene transfer, and the present strategy based on the synergistic
combination of protein and diazo reagent engineering is expected to prove useful in the context of
these as well as other challenging asymmetric carbene transfer reactions.
KEYWORDS: myoglobin, carbene transfer, asymmetric N—H insertion, chiral amines, protein
engineering
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Introduction
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The asymmetric synthesis of chiral amines represents a highly sought-after goal in organic
chemistry due to the ubiquitous presence of nitrogen containing functional groups in biologically
active compounds.1, 2 Nearly half of currently approved pharmaceuticals indeed comprise optically
active amines. Prominent biocatalytic strategies for the enantioselective synthesis of chiral amines
rely on the use of naturally occurring enzyme classes such as amine dehydrogenases,
transaminases, ammonia lyases, aminomutases, and imine reductases.3-8 More recently, engineered
‘nitrene transferases’ for asymmetric amine synthesis via C—H amination have also been
developed.9-11 In this context, the transition-metal catalyzed insertion of carbenoids into N—H
bond represents an attractive and complementary strategy for forging new carbon-nitrogen
bonds.12-15 Hemoproteins have recently emerged as promising biocatalysts for promoting a variety
of abiological carbene transfer reactions,16-28 including N—H carbene insertions.29, 30 In particular,
we and the Arnold group have previously demonstrated that engineered variants of sperm whale
myoglobin (Mb) and cytochrome P450BM3, respectively, can catalyze carbene N—H insertion
reactions involving aromatic amines and ethyl -diazoacetate (EDA).29, 30 More recently, the
substrate scope of the myoglobin-based biocatalysts was extended toward the functionalization of
benzylic and aliphatic amines.31 In addition, artificial metalloenzymes have been also reported to
be able to promote this reaction.32-34 Despite this progress, biocatalytic strategies for asymmetric
carbene N—H insertion have thus far remained elusive. The challenges inherent to realizing this
transformation are further highlighted by studies focused on the development of organometallic
catalysts for asymmetric N—H carbene insertions.14, 15, 35, 36 The difficulty in achieving high
activity and enantioselectivity in these reactions have been attributed to catalyst poisoning by the
nucleophilic amine and facile dissociation of the ylide intermediate from the metal center,
respectively.14, 36 While significant progress was made toward overcoming these challenges, these
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chemocatalytic protocols require the use of precious metals (e.g., Rh, Pd),37-40 synthetically
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challenging chiral ligands and/or co-catalysts,37,
and/or are restricted to -aryl diazo
compounds,39, 40, 44 with only a few exceptions.42, 43 In this context, the development of biocatalytic
alternatives would be therefore highly desirable as it will contribute to the development of
sustainable and environmentally benign approaches for realizing these transformations. Herein,
we report the development of an asymmetric strategy for the preparation of chiral amines via the
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myoglobin-catalyzed N—H insertion of aromatic amines with -diazopropanoate esters. High
activity and stereoinduction in this reaction could be achieved by tuning of the chiral environment
around the heme cofactor in the metalloprotein in combination with catalyst-matching and
tailoring of the diazo reagent. This work provides a new biocatalytic strategy for the synthesis of
optically active -amino acids and it demonstrates the value of a dual protein/substrate engineering
approach for realizing challenging asymmetric carbene transfer reactions using an enzyme.
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Results and Discussion
Target reaction and activity enhancement via diazo reagent optimization. The insertion of -
diazopropanoate esters into the N—H bond of aniline (1) was chosen as the target reaction. This
reaction provides an attractive approach to the synthesis of chiral -amino acids45 as well as the
preparation of chiral -methyl-amine motifs occurring in various drug candidates.46, 47 Using ethyl
2-diazopropanoate (EDP, 2a) as the carbene donor, we initially tested the performance of
myoglobin variant Mb(H64V,V68A), which was previously identified as an excellent biocatalyst
for catalyzing the N—H insertion of ethyl 2-diazoacetate (EDA) with aniline and other
arylamines.29 In stark contrast to the reaction with EDA (6,150 turnovers or TON),29
Mb(H64V,V68A) was found to exhibit a drastically reduced catalytic activity in the
functionalization of aniline (1) with EDP (2a) to give the N—H insertion product 2b (130 TON;
Figure 1, Entry 2) under identical reaction conditions. The strikingly diminished activity of the
hemoprotein in the reaction with EDP vs. EDA can be attributed to an increase in steric congestion
at the level of the iron-carbenoid intermediate48, 49 and/or a reduction of the electrophilicity due to
the presence of the methyl group (vs. -H) in alpha to the carbene atom, resulting in overall reduced
reactivity toward attack by the amine nucleophile. A similar trend was indeed observed in the
context of Mb-catalyzed S—H insertion.18
To overcome this limitation, we decided to test the N—H insertion reactivity of
Mb(H64V,V68A) in the presence of other 2-diazopropanoate esters such as isopropyl (3a), tert-
butyl (4a), and benzyl derivatives (5a). While low TON values were observed for the reactions
with 3a and 4a (13-27 TON), Mb(H64V,V68A) exhibited a significantly increased catalytic
efficiency (835 TON) in the presence of benzyl 2-diazopropanoate (BnDP, 5a) to give the N—H
insertion product 5b. Interestingly, a similar trend was observed using wild-type Mb as the catalyst.
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Encouraged by these results, we examined a broader panel of myoglobin variants previously found
to be active toward N—H insertion with EDA29 in the aniline reaction with the differently
substituted -diazopropanoates 2a-5a. As shown in Figure 1, all of these other myoglobin variants
showed significantly higher catalytic turnovers in the N—H insertion reaction with benzyl 2-
diazopropanoate 5a (2,650 - 4,830 TON) compared to the corresponding reactions in the presence
of the other diazopropanoate esters 2a-4a (13 - 1,360 TON). Among them, Mb(H64V),
Mb(L29A,H64V), and Mb(F43V,V68F) emerged as the most promising biocatalysts for the
desired reaction, supporting 4,430 - 4,830 TON for conversion of aniline to 5b in the presence of
BnDP (5a). The beneficial effect of the benzyl substituent in the diazo substrate was apparent also
for other Mb variants such as Mb(F43V) and Mb(V68A), which exhibited 4,090 and 3,095 TON,
respectively, toward the synthesis of 5b as opposed to their negligible activity toward formation
of 2b-4b.
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Figure 1. Catalytic activity (TON) of wild-type Mb, Mb(H64V,V68A), and other engineered Mb
variants toward the N—H insertion of aniline with the 2-diazopropanoate esters 2a-5a. Reaction
conditions: 10 mM aniline 1, 10 mM 2a-5a, 1 M Mb catalyst, 10 mM Na2S2O4, 50 mM phosphate
buffer (pH 8). See Table S1 for further details.
Optimization of enantioselectivity via protein engineering. Having identified BnDP (5a) as a
well-matched carbene donor reagent for achieving high activity in the desired N—H insertion
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reaction with an -diazopropanoate substrate, we assessed the enantioselectivity of the
aforementioned Mb-catalyzed transformations (Table 1). Despite their high catalytic activity,
none of these Mb variants was found to induce significant levels of enantiomeric excess (ee) in the
N—H insertion reaction (2 - 4% ee, Table 1, Entries 1 and 14; Supporting Information Table S2).
Disappointingly, further mutagenesis of Mb(L29A,H64V), which was one of the most active
biocatalysts identified from the initial screening (Figure 1), did not yield variants with improved
enantioselectivity (Supporting Information, Table S2). Based on these results, we decided to
screen a broader collection of Mb-based ‘carbene transferases’, which were previously developed
for various asymmetric carbene transfer reactions,17-20, 24, 25, 28 including asymmetric S—H
insertion reactions.18 Despite differing by multiple mutations at the level of their ‘active site’
residues (L29, F43, H64, V68, and I107), none of these metalloproteins show noticeable levels of
enantioselectivity toward the N—H insertion reaction with aniline (1) and 5a (-3 - 4% ee,
Supporting Information, Table S2). These results underscored the fundamental challenges of
achieving high enantioselectivity in an asymmetric carbene N—H insertions as anticipated at the
incipit of this work.
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Pursuing an alternative approach toward this goal, we turned our attention to modification
of the highly conserved50 proximal heme-coordinating histidine residue (His93; Figure S1) in the
Mb(H64V,V68A) background. Accordingly, we tested the performance in the N—H insertion
reaction of a series of Mb(H64V,V68A)-based variants in which the His93 residue is substituted
for both metal-coordinating (i.e., Cys, Asp, Ser, Tyr) and non-nucleophilic residues (i.e. Phe, Ala).
Gratifyingly, these experiments revealed a pronounced effect of the axial ligand substitution on
the enantioselectivity of the N—H insertion reaction, especially in the presence of Ser, Ala, or Tyr
in place of His93 (Table 1, Entries 2-7). From these analyses, Mb(H64V,V68A,H93Y) was
identified as the most enantioselective catalyst for this reaction, producing 5b with an enantiomeric
excess of 20% while maintaining good activity (1,180 TON). We also tested the effect of
introducing non-canonical amino acids such as N-methyl histidine (NMH), -(3-thienyl)-alanine
(3ThA), (3-(3’-pyridyl)-alanine (3PyA) and p-aminophenylalanine (pAmF) at the His93 position
of Mb(H64V,V68A) (Table 1, Entries 9-11). While these axial ligand modifications have
produced functional carbene transferases,51 none of these mutations had a beneficial impact on the
enantioselectivity of the present reaction.
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From these studies, we selected Mb(H64V,V68A,H93Y) as the most promising candidate
for further optimization of its enantioselectivity via protein engineering. To facilitate these efforts,
we established that these Mb-catalyzed transformations can be carried out using E. coli whole cells
expressing the Mb(H64V,V68A,H93Y) biocatalyst. Interestingly, the whole-cell reactions brought
about a slight improvement in enantioselectivity compared to the in vitro reactions with purified
protein (20→28% ee; Table 1, Entries 1 vs. 8). Using this approach, Mb(H64V,V68A,H93Y) was
subjected to site-saturation mutagenesis at the active site positions corresponding to Leu29 and
Phe43, followed by screening of the resulting variants as whole cells arrayed in multi-well plates.
While the large majority of these quadrupole mutants showed reduced enantioselectivity compared
to the parent enzyme (see Table S2 for representative examples), an improved variant carrying a
Leu29Met substitution could be identified. Mb(L29M,H64V,V68A,H93Y) catalyzes the
formation of 5b with an improved enantioselectivity of 53% ee compared to 28% ee for
Mb(H64V,V68A,H93Y) (Table 1, Entries 12 vs. 8; see also Figure 2). Furthermore, the
Leu29Met mutation was found to be beneficial also toward increasing the efficiency of the
reaction, resulting in an improved yield of 94% compared to 39% for the parent enzyme. Site-
saturation mutagenesis of the yet unaltered active site position 107 in
Mb(L29M,H64V,V68A,H93Y) failed to give further improvements in enantioselectivity.
Combined with the results from the screening efforts mentioned earlier, these protein engineering
studies revealed both a strong cooperativity of the active site mutations in dictating and controlling
protein-induced stereoinduction and a highly rugged fitness landscape52 with respect to this
property. Indeed, while resulting in folded and functional variants, other substitutions at position
29 (e.g., L29H, L29K) and both isosteric (e.g., F43H) and non-isosteric (e.g., F43C, F43I)
substitutions at position 43 were found to lead to nearly complete loss of stereoselectivity (1-14%
ee; Supporting Information Table S3, Entries 2-9). At position 107, only -branched residues
structurally related to the wild-type isoleucine residue (i.e., Thr and Val) were tolerated without a
complete loss of enantioselectivity (38-42% ee vs. 53% ee; Supporting Information Table S3,
Entries 10 and 11).
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Table 1. Catalytic activity and enantioselectivity of selected engineered myoglobin variants in
the N—H insertion reaction with aniline 1 and benzyl 2-diazopropanoate 5a.a
O
O
H
NH2
0.02 mol% Mb variant
Na2S2O4, KPi pH 8
N
+
*
O
O
N2
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1
5a
Catalyst
5b
Entry
Yieldb
TONb
% eec
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Mb(H64V,V68A)
41%
20%
19%
21%
14%
15%
24%
39%
6%
2,030
980
960
1,040
700
750
1,180
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310
1,195
830
1,300
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13
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28
6
4
-6
3
Mb(H64V,V68A,H93A)
Mb(H64V,V68A,H93C)
Mb(H64V,V68A,H93D)
Mb(H64V,V68A,H93F)
Mb(H64V,V68A,H93S)
Mb(H64V,V68A,H93Y)
Mb(H64V,V68A,H93Y)e
Mb(H64V,V68A,H93(NMH))
Mb(H64V,V68A,H93(3ThA))
Mb(H64V,V68A,H93(3PyA))
24%
17%
Mb(H64V,V68A,H93(pAmF))
Mb(L29M,H64V,V68A,H93Y)e
Mb(F43V,V68F)
26%
94%
40%
39%
64%
36%
34%
33%
53
4
1,990
1,960
-
Mb(F43V,H64A,V68F)
Mb(F43V,H64A,V68F)e
Mb(F43V,H64C,V68F)e
Mb(F43V,H64G,V68F)
Mb(F43V,H64T,V68F)e
-43
-56
42
-17
48
-
1,700
-
aReaction conditions: 5 mM 1, 5 mM 5a, 1 M Mb catalyst, 50 mM phosphate buffer (pH 8), room temperature.
bYield and TON determined based on HPLC conversion using calibration curves with isolated 5b. Enantiomeric
c
excess determined by chiral SFC. eReaction conditions: 5 mM 1, 5 mM 5a, E. coli whole cells expressing Mb catalyst
(OD=20), 50 mM phosphate buffer (pH 7.2), room temperature.
Based on these results, we decided to explore an alternative evolutionary trajectory starting
from Mb(F43V,V68F), which was one of the most active biocatalysts for the N—H insertion
reaction with BnDP as identified from the screening of the initial panel of Mb variants (>5,000
TON, Figure 1). In this case, site-saturation mutagenesis was directed to the distal His64 residue,
as mutation of this position was also determined to be beneficial to improve catalytic activity in
this reaction (i.e., Mb(H64A), Figure 1). Screening of this library enabled the identification of two
variants, Mb(F43V,H64C,V68F)
and
Mb(F43V,H64T,V68F),
featuring
improved
enantioselectivity (4% → 42% and 48% ee, respectively) for the synthesis of the same enantiomer
of 5b generated using Mb(L29M,H64V,V68A,H93Y) (Figure 2). The latter remained a superior
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biocatalyst for this transformation, however, in reasons of its slightly higher enantioselectivity and
better yield (93% vs. 33-36%). Interestingly, two Mb variants with inverted enantioselectivity were
also identified from this Mb(F43V,V68F)-based library, bearing a His64→Gly or His64→Ala
mutation (Table 1, Entries 18 and 15). Mb(F43V,H64A,V68F), in particular, was determined to
be the most promising biocatalyst for achieving enantiocomplementarity in this reaction, enabling
the synthesis of 5b in -56% ee and 64% yield using whole-cells and with identical
enantiopreference and -43% ee using purified protein (Table 1, Entries 16 vs. 15; see also Figure
2). Further mutagenesis of position 29 and 107 in Mb(F43V,H64A,V68F) did not yield further
improvements in enantioselectivity (see Table S3 for selected examples). As noted earlier for
Mb(L29M,H64V,V68A,H93Y), a strong synergy among the active site mutations in influencing
enantioselectivity became apparent also for the stereocomplementary counterpart
Mb(F43V,H64A,V68F), as judged by the low enantioselectivity (0-4% ee) of both
Mb(F43V,V68F) and the single-site variant Mb(H64A) (Supporting Information, Table S2). In
addition, the high sensitivity of this property to subtle modifications of Mb active site configuration
was evident from the complete switch in enantiopreference (48% ee vs. -56% ee) observed for
Mb(F43V,H64T,V68F) vs. Mb(F43V,H64A,V68F), which is induced by a small change at
position 64 (Thr vs. Ala).
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(L29M,H64V,V68A,H93Y) + 12a
100
(82% ee)
(H64V,V68A,H93Y) + 12a
(75% ee)
80
(L29M,H64V,V68A,H93Y)
60
40
(53% ee)
(H64V,V68A,H93Y)
(F43V,H64T,V68F)
(28% ee)
(48% ee)
20
(H64V,V68A)
(2% ee)
WT Mb
(0% ee)
0
(F43V,V68F)
(4% ee)
-20
-40
-60
-80
-100
(F43V,H64A,V68F)
(-56% ee)
(F43V,H64A,V68F) + 10a
(-63% ee)
Figure 2. Optimization of enantioselectivity of Mb-catalyzed N—H insertion reaction with aniline
and -diazopropanoate esters via protein and substrate engineering. When not specified, the diazo
reagent is BnDP (5a).
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Optimization of enantioselectivity via tailoring of the diazo substrate. From the studies
summarized above, Mb(L29M,H64V,V68A,H93Y) and Mb(F43V,H64A,V68F) emerged as the
most selective biocatalysts for the synthesis of 5b with complementary enantioselectivity (Figure
2). Given our results with the different diazopropanoate esters (Figure 1), we envisioned that
modification of the carbene donor reagent could offer an additional strategy, complementary to
protein engineering, to further fine-tune the enantioselectivity of these Mb-catalyzed
transformations. Specifically, we surmised that modification of the benzyl group in BnDP, which
was critical for imparting high activity in this reaction (Figure 1), could provide a means to
influence also stereoinduction, possibly through affecting the facial selectivity of amine attack to
the heme-bound carbenoid and/or via other mechanisms. Accordingly, we investigated the
reactivity of Mb(L29M,H64V,V68A,H93Y) toward the aniline functionalization reaction in the
presence of a panel of benzyl 2-diazopropanoate derivatives in which a methyl substituent was
added in ortho- (6a), meta- (7a), and para- (8a) position of the aromatic ring of the benzyl group
(Figure 3). Gratifyingly, these experiments revealed a major effect of the methyl substitution
pattern on the enantioselectivity of the reaction, with the use of 4-methylbenzyl 2-diazopropanoate
8a enabling the largest increase in enantiomeric excess of the corresponding N—H insertion
product 9b compared to the reaction in the presence of 5a (53→68% ee). In comparison, the
reactions with 7a and 6a resulted in a more modest improvement (62% ee) and a reduction (25%
ee), respectively, of enantioinduction under identical experimental conditions, revealing a
correlation between this property and the increasing distance of the steric bulk derived from the
methyl group (i.e., para > meta > ortho) from the -carbon atom of the diazo reagent, and thus of
the heme-carbenoid intermediate.48 Based on these findings, we tested the para-trifluoromethyl-
and para-methoxy-substituted diazo reagents 9a and 10a, respectively, which led to the formation
of the corresponding products 9b and 10b in comparable enantiomeric excess (67% ee) as
observed for the para-methyl-substituted counterpart 8b (Figure 3). These results indicated that
substrate-dependent steric effects are primarily responsible for affecting the enzyme’s
enantioselectivity compared to electronic factors. Based on that and given the higher
enantioselectivity levels obtained for 8b and 7b over 6b, we chose to further increase the steric
bulk in the para and meta position of the aryl ring in the diazo compound by using the 3,4-
dimethyl- (11a), 4-isopropyl (12a) and 4-tert-butyl (13a) substituted diazo reagents. Gratifyingly,
these modifications resulted in a further improvement of enantioselectivity, with the best results
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being obtained with 12a to give the N—H insertion product 12b in 82% ee and 51% yield (Figure
3). Beside their beneficial effect on stereoselectivity, it is worth noting that all of the
diazopropanoate reagents were readily accepted by Mb(L29M,H64V,V68A,H93Y) without a
significant drop in the yield of these reactions (50-87% vs. 93%; Figure 3), thus denoting a good
tolerance of the biocatalyst also in the presence of relatively bulky reagents such as 13b. To
examine the generality of this substrate engineering approach, we also tested the panel of 2-
diazopropanoate esters in the presence of Mb(H64V,V68A,H93Y) as the catalyst. Insightfully, a
very similar structure-enantioselectivity trend was obtained for this biocatalyst (i.e., 4-iPr ≈ 4-tBu
≥ 4-CF3 > 4-MeO ≈ 3,4-dimethyl = 4-Me >> 3-Me >> 2-Me; Scheme S1) compared to that
observed for Mb(L29M,H64V,V68A,H93Y) (i.e., 4-iPr > 4-tBu > 3,4-dimethyl ≥ 4-CF3= 4-Me =
4-MeO > 3-Me >> 2-Me). Furthermore, an even larger improvement in enantioselectivity was
observed in the case of the former enzyme (i.e., 28%→75% ee vs. 53→82% ee) upon re-
engineering of the diazo reagent (Scheme S1).
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Similar experiments were then conducted using the enantio-complementary biocatalyst
Mb(F43V,H64A,V68F). Also in this case, substitution of the benzyl group was found to affect the
enantioselectivity of the biocatalyst (Figure 3), further supporting the generality of this strategy.
At the same time, an overall different structure-activity profile was obtained for this Mb variant
compared to Mb(L29M,H64V,V68A,H93Y) and Mb(H64V,V68A,H93Y), which is not surprising
since the benzyl group in the corresponding heme-carbenoid intermediates is expected to interact
with different regions of the active site in the two enantiodivergent catalysts. For
Mb(F43V,H64A,V68F), most of the other para substitutions were tolerated without significant
change in the performance of the biocatalyst, whereas both a 2-methyl (6b) and 4-tBu substitution
in the diazo compound (13b) reduced enantioselectivity (-30% ee and -37% ee) compared to the
reaction with BnDP (-56% ee). In contrast, an improvement in both enantioselectivity (-56% → -
63% ee) and yield (64%→ 94%) was achieved using the 4-methoxy substituted diazopropanoate
derivative 10a, resulting in the formation of 10b in -63% ee and 94% yield. Importantly, for all of
the Mb variants tested, the performance of these biocatalysts could be further optimized via re-
engineering of the diazo compound, demonstrating the value of this strategy as a complement to
protein engineering.
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Figure 3. Substrate scope (left box) and comparison of enantioselectivity (right graph) for
Mb(L29M,H64V,V68A,H93Y) (blue) and Mb(F43V,H64A,-V68F) (orange) in the N—H
insertion reaction with aniline 1 and substituted benzyl 2-diazopropanoates 6a-13a. Reaction
conditions: 5 mM aniline, 5 mM 6a-13a, E. coli whole cells expressing Mb catalyst (OD=20), 50
mM phosphate buffer (pH 7.2), room temperature. Yield determined based on HPLC conversion
using calibration curves with isolated 6b-13b. c Enantiomeric excess determined by chiral SFC.
b
Optimization of the Mb(L29M,H64V,V68A,H93Y)-catalyzed reaction. Having identified 12a
as the best matched carbene donor reagent for the Mb(L29M,H64V,V68A,H93Y)-catalyzed N—
H insertion reaction, the latter was subjected to further optimization and characterization (Table
2). Using a 1:2 aniline:diazo compound ratio, the yield of 12b from the whole cell reaction with
this enzyme could be increased from 51% to 82% while maintaining high enantioselectivity (79%
ee; Table 2, Entry 3 vs. 1). Under these conditions, nearly quantitative yields (94-98%) for this
reaction could be obtained using a higher cell density (OD600) of 40-60 (vs. 20), albeit with a
reduction in enantiopurity (74% ee; Table 2, Entry 5-6).
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Table 2. Mb(L29M,H64V,V68A,H93Y)-catalyzed enantioselective N—H insertion reaction with
aniline 1 and substituted benzyl 2-diazopropanoate (6a-13a) under different conditions.a
O
O
H
NH2
Mb(L29M,H64V,V68A,H93Y)
50 mM KPi pH 7.2
N
*
O
O
+
N2
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1
12a
12b
Entry
Catalyst loading
Aniline (1)
Diazo (12a)
Yieldb
TONb
% eec
1
2
3
4
5
OD=20
OD=20
OD=20
OD=10
OD=40
OD=60
20 M
5 M
5 mM
10 mM
5 mM
5 mM
5 mM
5 mM
5 mM
5 mM
5 mM
2.5 mM
5 mM
5 mM
51%
61%
82%
80%
94%
98%
54%
55%
47%
80%
n.d.
151
202
509
119
79
82
80
79
70
74
74
36
30
28
30
10 mM
10 mM
10 mM
10 mM
10 mM
10 mM
10 mM
10 mM
6
7
137
571
2,470
399
8
9
1 M
5 M
10
a Reaction conditions: 1 and 12a at the indicated concentration, Mb(L29M,H64V,V68A,H93Y) catalyst in whole cells
or as purified protein at the indicated OD600 or concentration, respectively, in 50 mM phosphate buffer (pH 7.2), room
temperature. b Yield and TON determined based on HPLC conversion using calibration curves with isolated 12b. n.d.
= not determined. c Enantiomeric excess determined by chiral SFC.
Further characterization of Mb(L29M,H64V,V68A,H93Y) in purified form showed that it
supports up to 2,470 total turnovers for the formation of 12b (Table 2, Entry 9), with an initial rate
of 100 turnovers/min. The reaction reaches completion within 60 minutes.29 Interestingly, both
yield and enantioselectivity of the N—H insertion reactions with purified
Mb(L29M,H64V,V68A,H93Y) were generally inferior than those obtained from the whole-cell
reactions with cells expressing this Mb variant (Table 2, Entries 3-6 vs. 7-10), indicating a
beneficial effect of the intracellular environment on the performance of the biocatalyst possibly
due to macromolecular crowding or other factors. In addition, also Mb(H64V,V68A,H93Y) and
Mb(F43V,H64A,V68F) exhibit this trend (Table 1, Entries 7 vs. 8, 15 vs. 16). This phenomenon
has been observed before for enzymatic reactions,53 including certain biocatalytic carbene transfer
reactions.54 We further noticed that a comparable yield of 50-60% was obtained in the presence of
5 mM aniline across variable catalyst loading conditions (i.e., between 0.02 and 0.4 mol%),
whereas a higher yield could be achieved by reducing the substrate concentration (55% → 80%;
Table 2, Entries 8 vs. 10). These results suggest the presence of product inhibition. Supporting
this conclusion, controls experiment showed that a Mb(L29M,H64V,V68A,H93Y) reaction with
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4-bromoaniline as the substrate (vide infra) gave the expected N—H insertion product in the
absence but not in the presence of 12b spiked into to the reaction mixture. Interestingly, no signs
of product inhibition were observed for the whole-cell reaction, adding to the benefits of the latter
approach for performing these transformations. Although the reasons for this phenomenon were
not investigated, we hypothesize that it could derive from a favorable effect of macromolecular
crowding toward destabilizing the product/enzyme complex and/or from efficient diffusion (or
export) of the N—H insertion product outside of the cell, both of which are expected to relieve the
biocatalyst from product inhibition effects.
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Analysis of the Amine Substrate Scope. Using the reaction conditions optimized above, we next
investigated the performance of Mb(L29M,H64V,V68A,H93Y) toward enabling the asymmetric
insertion of diazopropanoate 12a across different aromatic amines (Scheme 1). Notably, a broad
range of aniline derivatives (16a-i) could be efficiently transformed by the enzyme to give the
desired N—H insertion products in good to excellent yields (50-99%) and good enantioselectivity
(57-81% ee). Specifically, Mb(L29M,H64V,V68A,H93Y) was found to tolerate well both
electron-withdrawing (16e-f) and electron-donating (16c-d) substituents in the para position of the
aniline substrate, exhibiting a generally higher enantioselectivity toward functionalization of the
latter (70-81% vs. 57-60% ee, respectively). Similar results were obtained for meta-substituted
aniline derivatives 16b and 16gwhich could be converted into 17b and 17g in 74-88% yield and
74-78% ee. The ortho-substituted aniline derivative 17a was obtained in quantitative yield (99%)
but with a lower enantioselectivity of 31% ee, indicating that meta and para substitution are better
tolerated than ortho substitutions with respect to enantioselectivity as judged based on the 17a-c
regioisomer series. A sterically encumbered substrate such as 4-tert-butyl aniline (16i) was also
accepted by the catalyst, although an increase in steric bulk at the para position affected both yield
and enantioselectivity, as derived from comparison of the results with 16i with those for the
isopropyl- (16h) and methyl-substituted (16c) counterparts. 2-Naphthylamine was also tested but
found not to participate in this reaction (data not shown), likely due to the poor solubility of this
compound in the aqueous reaction media.
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Scheme 1. Substrate scope of Mb(L29M,H64V,V68A,H93Y) (A) and Mb(F43V,H64A,V68F) (B)
in the N—H insertion reaction with -alkyl diazoesters and substituted anilines 16a-i.a
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7
8
9
E. coli whole cells expressing
O
O
A
Mb(L29M,H64V,V68A,H93Y) ( )
H
NH2
R1
N
B
or Mb(F43V,H64A,V68F) ( )
*
O
O
R
+
R
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N2
R1
iPr
iPr
OD = 20
50 mM KPi pH 7.2
1 16a-i
17a-m
,
12a
14a
15a
(R= Me)
(R= Et)
(R= n-Pr)
yieldb (% ee)c
O
O
H
O
H
H
N
N
*
N
*
*
O
O
O
iPr
iPr
iPr
17a
17b
17c
A
B
Ad
Ad
: 99% (31% ee)
: 99% (-9% ee)
: 88% (78% ee)
: 75% (70% ee)
B
: 49% (-64% ee)
B
: 62% (-53% ee)
O
H
O
H
O
H
N
*
N
*
N
*
O
O
O
MeO
iPr Cl
iPr
Br
iPr
17d
17e
17f
A
B
A
B
A
B
: 54% (81% ee)
: 65% (-43% ee)
: 76% (60% ee)
: 59% (-20% ee)
: 94% (57% ee)
: 93% (-31% ee)
O
H
O
H
O
H
Cl
F
N
*
N
*
N
*
O
O
O
iPr
iPr
iPr
17g
17h
17i
A
B
Ad
A
B
: 74% (74% ee)
: 64% (-5% ee)
: 74% (63% ee)
: 50% (44% ee)
: 78% (-38% ee)
B
: 56% (-40% ee)
O
O
H
H
N
N
*
*
O
O
Et
Et
iPr
MeO
iPr
17k
17j
A
B
A
B
: 77% (8% ee)
: 43% (-41% ee)
: 84% (22% ee)
: 60% (-29% ee)
O
O
H
H
N
N
*
*
O
O
nPr
nPr
iPr
MeO
iPr
17l
17m
A
B
: 51% (15% ee)
: 44% (-5% ee)
A
B
: 65% (13% ee)
: 25% (-6% ee)
a Reaction conditions: 5 mM aniline, 10 mM 12a or 14a or 15a, E. coli whole cells expressing Mb catalyst (OD=20),
50 mM phosphate buffer (pH 7.2), room temperature. b Yield determined based on HPLC conversion using calibration
curves with isolated product. c Enantiomeric excess determined by chiral SFC. d Reaction performed using OD=60.
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Having established the broad substrate scope of Mb(L29M,H64V,V68A,H93Y), we were
interested in comparing and contrasting the performance of the enantiocomplementary catalyst
Mb(F43V,H64A,V68F) across the same set of substrates (Scheme 1). In all cases,
Mb(F43V,H64A,V68F) was able to process the aryl amine derivative, furnishing the
corresponding N—H insertion product 17a-m with a yield ranging from 49% to 99%. In addition,
for all substrates, this biocatalyst furnished the opposite enantiomer with respect to
Mb(L29M,H64V,V68A,H93Y), thus exhibiting a consistent enantiodivergent selectivity
compared to the latter. While sharing with Mb(L29M,H64V,V68A,H93Y) a comparably broad
substrate scope, Mb(F43V,H64A,V68F) was found to display a much more pronounced substrate-
dependent effect on enantioselectivity, which varied from -5% to -64% ee (Scheme 1). The
substituent effect on stereoselectivity also differed between the two biocatalysts. For example,
while in both cases substitutions in para and meta of the aniline substrate are better tolerated than
ortho substitutions (e.g., 17b and 17c vs. 17a), the results with 17i, 17h, and 17c indicate that the
enantioselectivity of Mb(F43V,H64A,V68F) is significantly less affected by increasing steric bulk
at the para position (Me > iPr = tBu) when compared with Mb(L29M,H64V,V68A,H93Y) (Me >
iPr > tBu). Mb(F43V,H64A,V68F)-dependent stereoinduction is also significantly more sensitive
to the electronic properties of the substituent (e.g., 17c vs. 17e). We attribute the differential
behavior of two catalysts as emerging from these and the studies with the different
diazopropanoate esters (Figure 3) to different mode of interaction with the diazo reagent and
aniline substrate, and further studies are warranted to elucidate these aspects in more detail.
For both catalysts, we then tested their performance in the asymmetric N—H insertion with
aniline (1) and a representative aniline derivative (i.e., p-methoxy-aniline, 16d) in the presence of
other -alkyl substituted diazoesters, namely the -diazobutanoate reagent 14a and -
diazopentanoate 15a (Scheme 1). Notably, both catalysts were able to readily accept these carbene
donor reagents to give the desired products 17k-17m in good to moderate yields (51-84% for
(Mb(L29M,H64V,V68A,H93Y) and 25-60% for Mb(F43V,H64A,V68F; Scheme 1). In addition,
the two biocatalysts maintained enantiodivergent selectivity in these reactions. Not surprisingly,
however, the enantioselectivity of these reactions was significantly diminished compared to the
reactions with the -diazopropanoate reagent 12a (see 12b in Figure 3 and 17d in Scheme 1),
supporting the importance of the diazo reagent/catalyst match for optimal chiral induction. As an
exemption, the Mb(F43V,H64A,V68F) catalyst was found to tolerate a larger substituent (Et) at
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the alpha position of the diazo reagent, producing 17k and 17j with enantiomeric excess values
comparable to those for the -methyl substituted counterparts (-41% ee vs. -50% ee and -29% ee
vs. -41% ee, respectively).
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Conclusions
In conclusion, we have reported the first example of a biocatalytic strategy for the
asymmetric synthesis of chiral amines via carbene N—H insertion. Specifically, an engineered
myoglobin catalyst capable of promoting the insertion of 2-diazopropanoates into the N—H bonds
of a broad range of aniline derivatives with up to 99% yield and up to 82% ee was developed. In
addition, a stereodivergent biocatalyst that offers up to -64% ee for the same reaction was obtained,
thus providing access to both enantiomeric forms of the desired N—H insertion product. These
reactions can be carried out in whole cell systems, which simplify their application for organic
synthesis. Our protein engineering studies revealed a highly cooperative effect of beneficial active
site mutations in inducing and controlling stereoselectivity in this reaction, but also highlighted
the challenge of achieving high enantiocontrol through this strategy alone. As demonstrated by the
present studies, tailoring and catalyst-matching of the diazo reagent provided an effective strategy
for both enhancing the catalytic efficiency (Figure 1) and optimizing the enantioselectivity
(Figures 2 and 3) of this metalloprotein-catalyzed reaction. To the best of our knowledge, this is
the first example of the use of diazo substrate engineering for fine-tuning the stereoselectivity of
an enzyme-catalyzed carbene transfer reaction. This work lays the foundation for future
development of other asymmetric N—H insertions and it is anticipated that the present strategy
based on the synergistic combination of protein and diazo reagent engineering could prove
valuable toward developing biocatalytic strategies for these and other challenging asymmetric
carbene-mediated transformations.
Experimental Details
Reagents and Analytical Methods. All chemicals and reagents were purchased from commercial
suppliers (Sigma-Aldrich, TCI Chemicals) and used without any further purification, unless
otherwise stated. The diazo compounds isopropyl 2-diazopropanoate (3a), tert-butyl 2-
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diazopropanoate (4a) and benzyl 2-diazopropanoate (5a) were prepared according to previously
reported procedures.1 All moisture- or oxygen-sensitive reactions were carried out under argon
atmosphere in oven-dried glassware with magnetic stirring using standard gas-tight syringes,
cannulae and septa. 1H, 19F and 13C NMR spectra were measured on a Bruker DPX-400 instrument
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(operating at 400 MHz for H, 376 MHz for F and 100 MHz for C) or a Bruker DPX-500
instrument (operating at 500 MHz for 1H and 125 MHz for 13C). Tetramethylsilane (TMS) served
as the internal standard (0 ppm) for 1H NMR, CDCl3 was used as the internal standard (77.0 ppm)
for 13C NMR. Silica gel chromatography purifications were carried out using AMD Silica Gel 60
Å 230-400 mesh. Thin Layer Chromatography (TLC) was carried out using Merck Millipore TLC
silica gel 60 F254 glass plates. UV-Vis measurements were performed on a Shimadzu UV-2401PC
UV-Vis spectrometer. HPLC analyses were performed on a Shimadzu LC-2010A-HT equipped
with a VisionHT C18 column and a UV-Vis detector. Stereoisomer resolution was performed by
Supercritical Fluid Chromatography (SFC) analysis, using a JASCO Analytical and Semi-
Preparative SFC instrument.
Cloning and Mutagenesis. pET22b(+) (Novagen) was used as the recipient plasmid vector for
cloning of all of the myoglobin variants. The plasmids encoding for the selected engineered variant
of sperm whale myoglobin were prepared as described previously.2 The Mb(H64V,V68A,H93Y)
and Mb(F43V,H64A,V68F)-derived single-site saturation libraries were prepared by using "small
intelligent mutagenesis"3 as described previously.4 Using the corresponding gene encoding the
parent protein as template, small-intelligent mutagenesis libraries for each of the four target active
site positions (i.e., 29, 43, 64, and 107) were prepared using a mixture of four primers containing
the codon NDT, VMA, ATG and TGG at the target position in a 12 : 6 : 1 : 1 ratio. After gene
assembly via SOE PCR, the corresponding library was cloned into the Nde I / Xho I cassette of
pET22b(+), followed by transformation into E. coli DH5 cells. As an example, for preparing the
site-saturation mutagenesis library at position 64, a 3’-terminal fragment of the target gene was
prepared by PCR (NEB Phusion Polymerase) using a 12 : 6 : 1 : 1 mixture of forward primers #18
through #21, the reverse super primer #2, and vector pET22b_Mb(F43V,V68F) as template. In a
separate PCR reaction, the 5’-terminal gene fragment was prepared using forward super primer
#1, reverse primer #22, and vector pET22b_Mb(F43V,V68F) as template. The two fragments were
then fused together using stitching with overlap extension PCR (SOE PCR) and super primers #1
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and #2 to yield the target gene encoding for Mb(F43V,H64X,V68F) where X is the randomized
position. The plasmid pET22b(+) and the SOE product were digested with Nde I and Xho I for 2
hr at 37 °C. The gene insert and plasmid were then ligated with T4 DNA ligase. The ligation mix
was transformed into chemically competent E. coli DH5α cells. At least 60 colonies per library
were pooled, sequenced, and transformed into chemically competent E. coli C41(DE3) cells for
expression. The sequences of the oligonucleotide primers used in this project are given in Table
S4.
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Protein expression and purification. Wild-type Mb and engineered Mb variants were cloned and
expressed in E. coli C41(DE3) cells as described previously.2 Briefly, cells were grown in TB
medium (ampicillin, 100 mg L−1) at 37 °C (180 rpm) until OD600 reached 0.8-1.0. Cells were then
induced with 0.25 mM β-D-1-thiogalactopyranoside (IPTG) and 0.3 mM δ-aminolevulinic acid
(ALA). After induction, cultures were shaken at 27 °C (180 rpm), harvested after 20 h by
centrifugation (4,000 rpm, 20 min, 4 °C) and resuspended in Ni-NTA Lysis Buffer (50 mM KPi,
250 mM NaCl, 10 mM histidine, pH 8.0). Resuspended cells were frozen and stored at -80 °C.
Cell suspensions were thawed at room temperature, lysed by sonication, and clarified by
centrifugation (14,000 rpm, 50 min, 4 °C). The clarified lysate was transferred to a Ni-NTA
column equilibrated with Ni-NTA Lysis Buffer. The protein was washed with Ni-NTA Wash
Buffer (50 mM KPi, 250 mM NaCl, 20 mM histidine, pH 8.0). Proteins were eluted with Ni-NTA
Elution Buffer (50 mM KPi, 250 mM NaCl, 250 mM histidine, pH 7.0). After buffer exchange (50
mM Kpi, pH 7.0), the proteins were stored at +4 °C. Myoglobin concentration was determined by
UV/Vis spectroscopy using an extinction coefficient of ε410 = 157 mM−1 cm−1.
N—H insertion reactions. Under standard reaction conditions, reactions were carried out at a 400
μL scale using 1 or 20 μM myoglobin, 5 mM amine, 5 or 10 mM diazo compound, and 10 mM
sodium dithionite. In a typical procedure, in an anaerobic chamber, a solution containing the
desired myoglobin variant was mixed with a solution of sodium dithionite in argon purged
potassium phosphate buffer (50 mM, pH 8.0). Reactions were initiated by addition of amine (400
mM stock solution in EtOH) followed by the addition of diazo compound (400 mM stock solution
in EtOH), and the reaction mixtures were stirred in the chamber for 12 h at room temperature. For
whole cell experiments, the cell suspensions stored at -80 °C were thawed at room temperature,
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centrifuged, resuspended in reaction buffer (potassium phosphate buffer (50 mM, pH 7.2)
supplemented with micronutrients) and adjusted to the desired OD600. Under standard reaction
conditions, reactions were carried out at a 400 μL scale using E. coli whole cells expressing the
desired myoglobin variant, 5 mM amine and 5 or 10 mM diazo compound. The cells were
transferred to an anaerobic chamber, and reactions were initiated by addition of amine (400 mM
stock solution in EtOH) followed by the addition of diazo compound (400 mM stock solution in
EtOH), and the reaction mixtures were stirred in the chamber for 12 h at room temperature.
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Product inhibition experiment. Reaction was carried out at a 400 μL scale using 5 μM
myoglobin, 2.5 mM benzyl phenylalaninate (5b), 2.5 mM 4-bromoaniline, 10 mM diazo
compound, and 10 mM sodium dithionite. In a typical reaction, a solution containing sodium
dithionite (100 mM stock solution) in potassium phosphate buffer (50 mM, pH 7.2) was degassed
by bubbling argon into the mixture for 3 min in a septum-capped vial. A buffered solution
containing the myoglobin variant was carefully degassed in a similar manner in a separate vial.
The two solutions were then mixed together via cannula. Benzyl phenylalaninate was then added
(2.5 μL from a 0.4 M stock solution in EtOH) and the reaction was stirred at room temperature.
After 15 min, 5 μL of 4-bromoaniline (from a 0.4 M stock solution in EtOH) and 10 μL of EDA
(from a 0.4 M stock solution in DMF) were added with a syringe. The reaction was stirred for 16
hours at room temperature, under positive argon pressure.
Product analysis. The reactions were analyzed by adding 8 µL of internal standard (fluorenone,
50 mM in DMSO) to the reaction mixture, followed by extraction with 400 µL of dichloromethane.
The organic layer was removed via evaporation and the residue was dissolved in 300 μL methanol,
filtered through 0.22 μm syringe filters, and analyzed by SFC and HPLC (see the Supporting
Information Reagents and Analytical Methods section for details on SFC and HPLC analyses).
Calibration curves for quantification of the different N—H insertion products were constructed
using authentic standards prepared as described in the Supporting Information Synthetic
Procedures. All measurements were performed at least in duplicate. For each experiment, negative
control samples containing either no enzyme or no reductant were included.
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Synthetic Procedures and Product characterization. Detailed procedures and characterizations
for the synthesis of aryl diazopropanoates (6a-15a), intermediates and N—H insertion products
(6b-13b, 17a-17m) are provided in the Supporting Information.
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Associated Content
Supporting figures, oligonucleotide sequences, compound characterization data and NMR spectra.
This material is available free of charge via the Internet at http://pubs.acs.org.
Acknowledgements
This work was supported by the U.S. National Institute of Health grant R01 GM098628. N.R.J.
would like to thank RIT COS D-RIG for summer support. The authors are grateful to Kathryn
Goerl for technical support. MS instrumentation at the University of Rochester was supported by
the National Science Foundation grants CHE-0840410 and CHE-0946653.
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Synopsis
A first biocatalytic strategy for the asymmetric synthesis of chiral amines via carbene N—H
insertion is reported.
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