Asymmetric Enzymatic Synthesis of Allylic Amines:A
Sigmatropic Rearrangement Strategy
Christopher K. Prier
,
Todd K. Hyster
,
Christopher C. Farwell
,
Audrey Huang
, and
Frances
H. Arnold
Division of Chemistry and Chemical Engineering, California Institute of Technology, 1200 East
California Boulevard, MC 210-41, Pasadena, CA 91125, United States
Abstract
Sigmatropic rearrangements, while rare in biology, offer opportunities for the efficient and
selective synthesis of complex chemical motifs. A “P411” serine-ligated variant of cytochrome
P450
BM3
has been engineered to initiate a sulfimidation/[2,3]-sigmatropic rearrangement sequence
in whole
E. coli
cells, a non-natural function for any enzyme, providing access to enantioenriched,
protected allylic amines. Five mutations in the enzyme substantially enhance its activity toward
this new function, demonstrating the evolvability of the catalyst toward challenging nitrene
transfer reactions. The evolved catalyst additionally performs the highly enantioselective imidation
of non-allylic sulfides.
Keywords
biocatalysis; amination; nitrene transfer; cytochrome P450; directed evolution
Sigmatropic rearrangements, a class of pericyclic reactions in which one
σ
bond is
exchanged for another, are highly valuable reactions in synthetic chemistry due to their
ability to forge challenging chiral centers with high stereospecificity in complex structural
motifs.
1
While useful in chemical synthesis, sigmatropic rearrangements are remarkably rare
in biology.
2
Only a handful of enzymes that exploit such rearrangements have been
identified,
3
a notable example being chorismate mutase, which catalyzes the Claisen
rearrangement of chorismate to prephenate in the biosynthesis of tyrosine and phenylalanine
(Scheme 1).
4
Alternatively, certain biological pathways involve sigmatropic rearrangements
that are spontaneous once a given precursor has been assembled; for instance, Claisen
5
and
Cope rearrangements
6
have been implicated in the prenylation of aromatic amino acid side
chains.
Biocatalysis requires new enzymes for the green and economical synthesis of chemical
products that are often not accessible using natural enzymatic reactions.
7
The design of
enzymes capable of catalyzing or initiating sigmatropic rearrangements would introduce
valuable, complexity-building reactions into biocatalysis, but work in this area has largely
been limited to catalytic antibodies.
8
,
9
Recently, our laboratory
10
and Fasan’s group
11
Correspondence to: Frances H. Arnold.
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demonstrated that heme-containing proteins can perform nitrene transfer, a class of reactions
for which there is no biological precedent. In these reactions, the ferrous state of the heme
cofactor first reacts with a nitrene precursor (such as a sulfonyl azide) to generate an iron
nitrenoid; this reactive intermediate is then intercepted by a nucleophile to yield an aminated
product. Variants of cytochrome P450
BM3
, a soluble P450 from
Bacillus megaterium
, in
which the axial ligand to iron is mutated from cysteine to serine are particularly active
catalysts for nitrene transfer.
10
As these variants display a ferrous-CO Soret peak at 411 nm,
we term them “cytochrome P411s.”
12
Here, we describe a strategy that merges cytochrome
P411-catalyzed amination with a sigmatropic rearrangement for the synthesis of chiral
allylic amines.
In particular, we aimed to exploit the spontaneous sigmatropic rearrangement of allylic
sulfimides; these species undergo a [2,3]-rearrangement in which an N–S bond is exchanged
for an N–C bond, delivering protected allylic amines.
13
This type of sigmatropic
rearrangement is not a feature of any known biological pathway. Previously, we
demonstrated that cytochrome P411s catalyze nitrene transfer to simple sulfides.
10b
We thus
envisioned using a P411 to perform the enantioselective imidation of prochiral allylic sulfide
1
to generate allylic sulfimide
2
(Scheme 1). This species would then spontaneously undergo
the [2,3]-rearrangement to deliver the protected allylic amine
3
, in which a new chiral center
is forged. Since sigmatropic rearrangements often proceed with high stereospecificity, the
stereochemistry set in the P411-catalyzed sulfimidation step would be transferred to the
allylic amine product.
14
This strategy would establish a biocatalytic route to chiral allylic
amines, compounds that are biologically active
15
as well as valuable synthetic
intermediates.
16
,
17
Furthermore, expanding the breadth of enzymatic amination reactions
will facilitate the construction of non-natural
in vivo
pathways for chiral amine production.
In initial experiments, we evaluated the ability of a variant previously identified for the
imidation of sulfides
10b
to promote the desired reaction (this variant, termed P411
BM3
-CIS
T438S, contains 15 mutations relative to wild-type P450
BM3
and will be abbreviated “P”). In
whole-cell bioconversions using tosyl azide (TsN
3
) as the nitrene precursor, variant P only
efficiently imidates aryl–methyl sulfides such as thioanisole (
5
, Figure 1). This variant
provides essentially no product (<0.5% yield) when reacted with phenyl crotyl sulfide (
1
). In
general, P411-catalyzed nitrene transfer reactions suffer from a competing reduction of tosyl
azide to
p
-toluenesulfonamide (
4
), both via reduction of the azide by cellular agents and via
electron transfer to the metal nitrenoid intermediate from the enzyme’s reductase.
10
However, we reasoned that the enzyme could be engineered to accommodate allylic sulfide
substrates, orient them in a manner suitable for productive bond formation with the iron
nitrenoid, and in such a way outcompete undesired azide reduction pathways.
To evolve catalysts for the desired reaction we performed a substrate walk, in which P411
variants were first evaluated for their ability to imidate phenyl ethyl sulfide (
6
) and the most
active variants subsequently assayed for activity toward the larger sulfides
7
and
1
(Figure
1). We first found that introducing the active site mutation I263F into variant P is activating
toward sulfimidation; this mutation was identified during studies on P411-catalyzed
regioselective C–H amination.
10c
Importantly, P-I263F displays a sufficient level of activity
on phenyl ethyl sulfide (
6
, 12% yield) for screening of enzyme variants. Taking P-I263F as
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the parent, we evaluated site-saturation libraries at residues V87, L181, A328, and S438 (all
located near the active site on the distal face of the heme) for the ability to imidate sulfide
6
,
from which we identified the beneficial mutations V87A and A328V (Figure 1).
Gratifyingly, in addition to displaying improved activity on sulfide
6
, the variants
incorporating these mutations also display appreciable activity for transformation of the
desired substrate
1
. Combining these mutations yielded an enzyme (P-I263F V87A A328V,
or P-3) having further improved activity toward sulfide
1
, delivering the rearrangement
product in 7% yield with 220 turnovers, while also validating our reaction design and
directed evolution strategy.
The observed product of these reactions, however, is not the phenylthiosulfonamide
8
(the
initial product of the rearrangement) but rather the sulfonamide
9
, in which the S–N bond of
8
has undergone reductive cleavage, presumably by cellular reductants such as NAD(P)H or
glutathione. Furthermore, for the experiments described above, we employed sulfide
1
as a
3:1
E:Z
mixture of alkene isomers.
18
Considering that the P411 variants likely display
differing activities toward the two alkene isomers, we prepared a batch of sulfide
1
enriched
in the
Z
olefin (
Z
-
1
, >15:1
Z:E
). Employing
Z
-
1
in the amination reaction with P-3 improves
the yield two-fold to 14% (490 turnovers), demonstrating that the enzyme prefers the
Z
-
olefin over the
E
-olefin (Figure 2).
Having identified a catalyst active on the desired substrate for the rearrangement, we then
performed a second round of evolution using P-3 as the parent and screening for activity on
sulfide
1
(see SI for details). Upon evaluating site-saturation libraries at positions A268,
L437, and S438, the mutation A268G was found to provide a 3-fold improvement in yield
(Figure 2). The wild-type threonine residue at this position plays a critical role in the natural
monooxygenation reaction,
19
and the mutation T268A was previously shown to be highly
activating toward nitrene as well as carbene transfer by P450
BM3
variants.
10
,
20
We then
performed a further round of evolution by saturating active-site residues A78, A82, T260,
and P329 in the variant P-I263F V87A A328V A268G (P-4). Mutagenesis at A82 yielded
two variants that improve the reaction yield more than 1.5-fold: the mutations A82L and
A82I both improve protein expression in
E. coli
compared to P-4, with the variant having the
A82I mutation showing higher specific activity (Figure 2). This variant, P-5, promotes the
sulfimidation/[2,3]-rearrangement of sulfide
Z
-
1
in 77% yield with greater than 2,000
turnovers.
21
The product
10
is obtained in moderate enantiomeric excess (68% ee, Table 1,
entry 1). Notably, the mutations identified in this study both introduce (A328V, A82L/I) and
remove (V87A, A268G) steric bulk in the active site. It thus seems likely that the new
mutations not only enlarge the active site to accommodate substrates, but also constrict
substrates and intermediates in a manner that facilitates bond formation.
We then evaluated the ability of the best evolved variant (P-5) to perform the sulfimidation/
rearrangement of other allylic sulfides. While the enzyme shows a strong preference for
amination of the substrate for which it was evolved (
Z
-
1
), it also catalyzes the amination of a
range of other sulfides to form both chiral (entries 1–3, Table 1) and achiral (entries 4 and 5)
tosyl-protected allylic amines. Interestingly, given that the parent variant (P) is a poor
catalyst for the amination of sulfides as small as phenyl ethyl sulfide (
6
), the evolved variant
P-5 is capable of nitrene transfer to substrates having alkyl chains as long as six carbons
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(Table 1, entry 3). The reaction may be carried out on semi-preparative scale with reduced
catalyst loadings (whole cells at OD
600
= 10, ~0.6 μM enzyme) to afford amine
10
in 71%
isolated yield and 6,100 turnovers (see SI for details). Finally, the nitrene transfer activity of
the variant P and other cytochrome P411s is generally inhibited in the presence of oxygen,
leading us to employ anaerobic conditions for the above biotransformations. In contrast, the
evolved variant P-5 performs nitrene transfer under aerobic conditions with no loss of
activity, delivering allylic amine
10
in 79% yield.
We rationalized that two possibilities exist for the moderate enantioselectivities obtained in
the amination of
1
: either the sulfimidation event is only moderately selective and the
rearrangement proceeds with high stereospecificity, or the sulfimidation is more highly
selective and erosion of enantiopurity occurs in the rearrangement step. In examining the
imidation of non-allylic sulfides by the variant P-5, we found that these substrates react with
very high levels of enantioinduction. For example, phenyl
n
-butyl sulfide, which differs from
sulfide
1
only by saturation of the alkene, is converted to its corresponding sulfimide in 98%
ee (Table 2, entry 3). It is therefore likely that in the sulfimidation/rearrangement reactions,
the imidation event also proceeds with high enantioselectivity, while the rearrangement
proceeds with imperfect stereofidelity, via competing
endo
and
exo
rearrangement transition
states.
14
Notably, however, these results also represent the first highly enantioselective
enzymatic imidation of sulfides.
We have thus demonstrated that cytochrome P411
BM3
variants can promote a sulfimidation/
[2,3]-rearrangement sequence in whole cells, introducing a new sigmatropic rearrangement
into the synthetic repertoire of biocatalysis. While the P411 does not catalyze the
rearrangement itself, the enzyme enables the reaction by assembling the required allylic
sulfimide intermediate; by performing this step with high enantioselectivity, the P411
ultimately delivers enantioenriched allylic amine products. It may be possible to improve the
enantiospecificity of the rearrangement by evolving the enzyme to preferentially bind one of
the competing rearrangement transition states.
8h
The activity of the catalyst was
substantially enhanced by directed evolution: for the transformation of sulfide
Z
-
1
, the five
mutations identified in this study improve the conversion more than 200-fold over the
activity of the parent P as well as confer the ability to function under aerobic conditions.
Starting from a variant (P) that performs azide reduction almost exclusively (>99%) in
preference to nitrene transfer to sulfide
1
, directed evolution delivered a variant (P-5) that
efficiently promotes the desired nitrene transfer process (up to 77% yield for amination of
Z
-
1
). As both nitrene transfer and nitrene reduction are reaction pathways that are absent in
nature, we have demonstrated that only a few mutations in P411
BM3
can partition reactivity
between two competing non-natural pathways. This study thus highlights the ability of
enzymes to adapt, through directed evolution, to facilitate valuable reaction pathways for
which no natural enzymes have evolved.
Supplementary Material
Refer to Web version on PubMed Central for supplementary material.
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Acknowledgments
Our research is supported by the National Science Foundation, Division of Molecular and Cellular Biosciences
(grant MCB-1513007). C.K.P. thanks the Resnick Sustainability Institute for a postdoctoral fellowship. T.H.K. and
C.C.F. were supported by a Ruth L. Kirschstein National Research Service Award (F32GM108143) and an NSF
Graduate Research Fellowship, respectively. We thank Dr. Scott Virgil and the Center for Catalysis and Chemical
Synthesis at Caltech for assistance with chiral chromatography.
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21.
At the higher conversions (>30% yield) obtained with the evolved variants, the cell’s endogenous
reductants are no longer sufficient to completely reduce intermediate
8
, and therefore a work-up
with dithiothreitol (DTT) was implemented to convert the product mixture entirely to the
sulfonamide
9
prior to analysis (see SI for details).
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Figure 1.
Evolution of a P411 catalyst for nitrene transfer to progressively larger aryl-alkyl sulfides.
Experiments were performed using whole cells overexpressing the P411 variant,
resuspended to OD
600
= 30, with 5 mM sulfide and 5 mM tosyl azide. Results are the
average of experiments performed with duplicate cell cultures, each used to perform
duplicate chemical reactions. P = P411
BM3
-CIS T438S.
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Figure 2.
Evolution of a catalyst for sulfimidation/[2,3]-rearrangement of
Z
-
1
. Reaction conditions as
given in Figure 1, with a DTT work-up employed for reactions giving >30% yield.
21
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Scheme 1.
Design of an enzymatic sulfimidation/sigmatropic rearrangement sequence
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Table 1
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Substrate scope of the P411-catalyzed sulfimidation/[2,3]-rearrangement.
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a
Reaction conditions as given in Figure 1, with a DTT work-up employed for reactions giving >30% yield.
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Table 2
Enantioselective imidation of sulfides catalyzed by an evolved P411 variant.
a
a
Reaction conditions as given in Figure 1.
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