Asymmetric Enzymatic Synthesis of Allylic Amines: A Sigmatropic Rearrangement Strategy

Article abstract of DOI:10.1002/anie.201601056

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.

Full text of DOI:10.1002/anie.201601056

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International Edition: DOI: 10.1002/anie.201601056
German Edition: DOI: 10.1002/ange.201601056
Directed Evolution
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*
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 P450BM3 has been engineered to initiate a sulfimi-
dation/[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 enantiose-
lective imidation of non-allylic sulfides.
S
igmatropic rearrangements, a class of pericyclic reactions
in which one s 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 rear-
rangement of chorismate to prephenate in the biosynthesis of
tyrosine and phenylalanine (Scheme 1).[4] Alternatively, cer-
tain 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.
Scheme 1. Design of an enzymatic sulfimidation/sigmatropic rear-
rangement sequence.
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 inter-
cepted by a nucleophile to yield an aminated product.
Variants of cytochrome P450BM3, 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.
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-build-
ing reactions into biocatalysis, but work in this area has
largely been limited to catalytic antibodies.[8,9] Recently, our
laboratory[10] and Fasanꢀs group[11] demonstrated that heme-
containing proteins can perform nitrene transfer, a class of
reactions for which there is no biological precedent. In these
In particular, we aimed to exploit the spontaneous
sigmatropic rearrangement of allylic sulfimides; these species
[*] Dr. C. K. Prier, Dr. T. K. Hyster, Dr. C. C. Farwell, A. Huang,
Prof. Dr. F. H. Arnold
À
undergo a [2,3]-rearrangement in which an N S bond is
À
exchanged for an N C bond, delivering protected allylic
Division of Chemistry and Chemical Engineering
California Institute of Technology
1200 East California Boulevard, MC 210-41, Pasadena, CA 91125
(USA)
amines.[13] This type of sigmatropic rearrangement is not
a feature of any known biological pathway. Previously, we
demonstrated that cytochrome P411s catalyze nitrene trans-
fer 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
E-mail: frances@cheme.caltech.edu
Supporting information for this article can be found under:
http://dx.doi.org/10.1002/anie.201601056.
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species would then spontaneously undergo the [2,3]-rear-
rangement to deliver the protected allylic amine 3, in which
a new chiral center is forged. Since sigmatropic rearrange-
ments often proceed with high stereospecificity, the stereo-
chemistry 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.
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 enzymeꢀs reductase.[10] How-
ever, 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
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 P411BM3
-
CIS T438S, contains 15 mutations relative to wild-type
P450BM3 and will be abbreviated “P”). In whole-cell biocon-
versions using tosyl azide (TsN3) as the nitrene precursor,
variant P only efficiently imidates aryl-methyl sulfides such as
thioanisole (5, Figure 1). This variant provides essentially no
À
during studies on P411-catalyzed regioselective C H amina-
tion.[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 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
À
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 Supporting Information (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 P450BM3 variants.[10,20] We then
performed a further round of evolution by saturating active-
Figure 1. Evolution of a P411 catalyst for nitrene transfer to progres-
sively larger aryl-alkyl sulfides. Experiments were performed using
whole cells overexpressing the P411 variant, resuspended to
OD600 =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=P411BM3-CIS T438S.
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tiomeric 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 (Table 1, entry 3). The
reaction may be carried out on semi-preparative scale with
reduced catalyst loadings (whole cells at OD600 = 10, ꢀ 0.6 mm
enzyme) to afford amine 10 in 71% isolated yield and 6100
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 biotransforma-
tions. 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 pro-
ceeds with imperfect stereofidelity, via competing endo and
exo rearrangement transition states.[14] Notably, however,
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]
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 expres-
sion 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]-rearrange-
ment of sulfide Z-1 in 77% yield with greater than 2,000
turnovers.[21] The product 10 is obtained in moderate enan-
Table 1: Substrate scope of the P411-catalyzed sulfimidation/[2,3]-rear-
rangement.[a]
Table 2: Enantioselective imidation of sulfides catalyzed by an evolved
P411 variant.[a]
[a] Reaction conditions as given in Figure 1, with a DTT work-up
employed for reactions giving >30% yield.[21]
[a] Reaction conditions as given in Figure 1.
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Soc. 2005, 127, 15002; c) S. Li, A. N. Lowell, F. Yu, A. Raveh,
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these results also represent the first highly enantioselective
enzymatic imidation of sulfides.
We have thus demonstrated that cytochrome P411BM3
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 enan-
tioenriched 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
P411BM3 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.
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Our research is supported by the National Science Founda-
tion, 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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Keywords: amination · biocatalysis · cytochrome P450 ·
directed evolution · nitrene transfer
How to cite: Angew. Chem. Int. Ed. 2016, 55, 4711–4715
Angew. Chem. 2016, 128, 4789–4793
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Farwell, P. S. Coelho, F. H. Arnold, Angew. Chem. Int. Ed. 2014,
convert the product mixture entirely to the sulfonamide 9
53, 6810; Angew. Chem. 2014, 126, 6928.
prior to analysis (see SI for details).
[21] At the higher conversions (> 30% yield) obtained with the
evolved variants, the cellꢀs endogenous reductants are no longer
sufficient to completely reduce intermediate 8, and therefore
a work-up with dithiothreitol (DTT) was implemented to
Received: February 1, 2016
Published online: March 11, 2016
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