Aldehyde and Ketone Synthesis by P450-Catalyzed Oxidative Deamination of Alkyl Azides

Article abstract of DOI:10.1002/cctc.201600487

Heme-containing proteins have recently attracted increasing attention for their ability to promote synthetically valuable transformations not found in nature. Following the recent discovery that engineered variants of myoglobin can catalyze the direct conversion of organic azides into aldehydes, we investigated the azide oxidative deamination reactivity of a variety of hemoproteins featuring different heme coordination environments. Our studies show that although several heme-containing enzymes possess basal activity in this reaction, an engineered variant of the bacterial cytochrome P450 CYP102A1 constitutes a particularly efficient biocatalyst for promoting this transformation, and it exhibits a broad substrate scope along with high catalytic activity (up to 11 300 turnovers), excellent chemoselectivity, and enhanced reactivity toward secondary alkyl azides to yield ketones. Mechanistic studies and Michaelis–Menten analyses provided insight into the mechanism of the reaction and the impact of active-site mutations on the catalytic properties of the P450. Altogether, these studies demonstrate that engineered P450 variants represent promising biocatalysts for the synthesis of aryl aldehydes and ketones through the oxidative deamination of alkyl azides under mild reaction conditions.

Full text of DOI:10.1002/cctc.201600487

DOI: 10.1002/cctc.201600487
Communications
Aldehyde and Ketone Synthesis by P450-Catalyzed
Oxidative Deamination of Alkyl Azides
Simone Giovani, Hanan Alwaseem, and Rudi Fasan*[a]
Heme-containing proteins have recently attracted increasing
attention for their ability to promote synthetically valuable
transformations not found in nature. Following the recent dis-
covery that engineered variants of myoglobin can catalyze the
direct conversion of organic azides into aldehydes, we investi-
gated the azide oxidative deamination reactivity of a variety of
hemoproteins featuring different heme coordination environ-
ments. Our studies show that although several heme-contain-
ing enzymes possess basal activity in this reaction, an engi-
neered variant of the bacterial cytochrome P450 CYP102A1
constitutes a particularly efficient biocatalyst for promoting
this transformation, and it exhibits a broad substrate scope
along with high catalytic activity (up to 11300 turnovers), ex-
cellent chemoselectivity, and enhanced reactivity toward sec-
ondary alkyl azides to yield ketones. Mechanistic studies and
Michaelis–Menten analyses provided insight into the mecha-
nism of the reaction and the impact of active-site mutations
on the catalytic properties of the P450. Altogether, these stud-
ies demonstrate that engineered P450 variants represent
promising biocatalysts for the synthesis of aryl aldehydes and
ketones through the oxidative deamination of alkyl azides
under mild reaction conditions.
previous work showed that engineered variants of this hemo-
protein can catalyze the oxidative deamination of organic
azides to generate aldehydes.[8] In comparison to classical
methods involving alcohol oxidation with toxic chromium-
based reagents, this transformation provides a convenient ap-
proach to the synthesis of aldehydes starting from a non-oxy-
genated functional group and by using readily accessible alkyl
azides. Furthermore, synthetic catalysts [e.g., MoO2(S2CNEt2)2]
currently available to promote this reaction suffer from poor
catalytic efficiency (20–200 turnovers) and require harsh reac-
tion conditions (reflux in toluene/water mixture).[9] In the inter-
est of comparing and contrasting the reactivities of hemopro-
teins featuring different heme coordination environments in
the context of non-native reactions, we investigated and
report herein the azide oxidative deamination reactivity of
a panel of different heme-containing enzymes, including a cata-
lase, a peroxidase, and wildtype and engineered variants of
a bacterial cytochrome P450. These studies led to the identifi-
cation of an engineered variant of CYP102A1 as a superior bio-
catalyst for the conversion of a broad range of aryl-substituted
alkyl azides into the corresponding aldehydes and ketones. In
addition, insight into the mechanism and catalytic properties
of this enzyme was gained through a combination of kinetic
studies and isotopic labeling experiments.
Cytochrome P450s constitute a superfamily of iron-dependent,
heme-containing oxygenases that play an important role in
drug metabolism, biodegradation, and the biosynthesis of sec-
ondary metabolites.[1] These enzymes have received significant
attention for their ability to hydroxylate aliphatic and aromatic
CꢀH bonds, a challenging reaction to achieve by chemical
means.[2] Furthermore, P450s are known to promote a variety
of other oxidative transformations, including heteroatom deal-
kylation, carbon–carbon bond cleavage, rearrangement reac-
tions, and Baeyer–Villiger reactions.[3] More recently, the scope
of cytochrome P450s was extended to a number of important,
“non-native” reactions useful for the construction of carbon–
carbon,[4] carbon–nitrogen,[5] and nitrogen–sulfur[6] bonds.
Our group recently reported that the heme-containing pro-
tein myoglobin (Mb) constitutes a promising scaffold for pro-
moting a variety of synthetically useful transformations mediat-
ed by metal–carbenoid and –nitrenoid species.[7] In particular,
To investigate the scope of hemoprotein-catalyzed azide-to-
aldehyde oxidation, we initially selected a panel of different
heme-containing enzymes consisting of bovine catalase (Cat),
horseradish peroxidase (HRP), and the bacterial cytochrome
CYP102A1[10] (also known as P450BM3).[11] The heme coordina-
tion environments among these enzymes are significantly dif-
ferent and, additionally, are different to that of the previously
investigated Mb. In Cat and CYP102A1, the amino acid ligands
involved in coordinating the heme iron are tyrosine and cys-
teine residues, respectively,[12] as opposed to histidine in Mb.[13]
The heme group in HRP is also bound through a histidine resi-
due,[14] but a strong H-bonding interaction between this proxi-
mal His and a neighboring aspartic acid residue confers consid-
erable anionic character to the former, a structural feature that
is not present in Mb. Finally, hemoglobin (Hb) was also consid-
ered to evaluate the effect of the tetrameric structure on the
target reactivity as opposed to the monomeric structure of
Mb.
As a model reaction, the conversion of benzyl azide (1) into
benzaldehyde (2a) under anaerobic conditions in phosphate
buffer (KPi, pH 7.0) and in the presence of sodium dithionite
(Na2S2O4) as a reductant (Figure 1a) was used to assess the rel-
ative efficiency of these hemoproteins in promoting azide oxi-
dative deamination. Under these conditions, Cat, HRP, and Hb
showed only low to moderate reactivity, which supported ap-
[a] Dr. S. Giovani, H. Alwaseem, Prof. Dr. R. Fasan
Department of Chemistry
University of Rochester
120 Trustee Road, Rochester, NY 14627 (USA)
E-mail: rfasan@ur.rochester.edu
Supporting Information and the ORCID identification number(s) for the
author(s) of this article can be found under http://dx.doi.org/10.1002/
cctc.201600487.
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coordination environment on the reactivity of the different he-
moproteins toward organic azides, thereby expanding previous
findings from our group.[7e] Indeed, whereas HRP was previous-
ly found to promote the intramolecular CꢀH amination of aryl-
sulfonyl azides with comparable or higher activity than P450s
or Mb,[5a,b,7e] this enzyme showed significantly reduced activity
toward azide-to-aldehyde conversion compared to the latter
hemoproteins. Conversely, the data in Figure 1b,c suggested
that, despite the inherent differences between their active
sites, the thiolate-bound heme in P450 is equally efficient as
the histidinyl-bound heme cofactor in Mb in promoting the ox-
idative deamination of alkyl azide 1 (1400 TONs vs. 1650 TONs
for wildtype Mb[8]).
Given the promising activity of wildtype CYP102A1, we next
extended our investigations to two engineered variants of this
enzyme, namely, CYP102A1(F87A) and FL#62. The former var-
iant was chosen because of the well-documented ability of the
F87A mutation to expand the substrate profile of CYP102A1
toward non-native substrates in the context of monooxygena-
tion reactions.[11,15] In addition to possessing a broad substrate
profile,[16] the FL#62 variant, which carries 6 active-site muta-
tions and 10 additional mutations within its heme domain, was
chosen because of its high reactivity in the context of the CꢀH
amination of azide-based reagents.[5a,b] As shown by the data
summarized in Figure 1, CYP102A1(F87A) showed only slightly
improved TON values for the conversion of benzyl azide (1)
into benzaldehyde (2a) relative to the wildtype P450 (1600 vs.
1400 turnovers). In contrast, FL#62 was found to catalyze this
transformation with considerably higher efficiency (Fig-
ure 1b,c), supporting 9700 catalytic turnovers and providing
nearly quantitative product conversion (92%) at a catalyst
loading of merely 0.001 mol%. At lower catalyst loadings
(0.0005 mol%), a TON value as high as 11300 was measured
for this enzyme. Notably, the FL#62 reaction was found to pro-
ceed also with excellent chemoselectivity, which led to the
clean formation of 2a without producing detectable amounts
of byproduct 2b or 2c. Altogether, these properties make
FL#62 not only a considerably more efficient catalytic system
for azide-to-aldehyde conversion than synthetic Mo-based cat-
alysts (50–99% conversions by using 10 mol% at 1008C in
water/toluene mixture)[9a] but also a superior biocatalyst to the
best-engineered Mb variant [Mb(H64V,V68A)] identified in the
course of our previous studies.[8]
Figure 1. a) Catalytic activity of various hemoproteins in the oxidative deam-
ination of benzyl azide (1) to benzaldehyde (2a). b,c) Reactions (400 mL)
were conducted under anaerobic conditions with 10 mm BnN3, 10 mm
Na2S2O4, and 1 mm protein for 24 h at room temperature. TON=nmol alde-
hyde/nmol catalyst. Product yield is based on conversion of 1 into 2a, as
determined by GC. d) Reaction rates as determined over the first 10 min of
the reaction. e) TON versus pH plot for FL#62 reaction.
proximately 5, 63, and 195 turnovers (TONs), respectively (Fig-
ure 1b,c). These TON values are lower or comparable to that
achieved with free hemin under similar reaction conditions (
ꢁ100 TONs).[8] The poor performance of Hb was somewhat un-
expected in light of the high reactivity of the structurally relat-
ed Mb in this transformation (1650 TONs)[8] and it could stem
from unfavorable allosteric effects in the case of the oligomeric
protein. In contrast, wildtype CYP102A1 showed considerably
higher azide oxidation activity, which supported 1400 catalytic
turnovers for the formation of desired product 2a and provid-
ed a product conversion ratio of 18% (Figure 1b,c) The reac-
tion also produced small amounts of benzylamine (2b, 0.5%)
and N-benzylbenzylimine (2c, <3%), as observed in the Mb-
catalyzed reaction.[8] Formation of byproduct 2b was ascribed
to an unproductive pathway resulting from decomposition of
the azide followed by reduction and protonation of the result-
ing nitrene-heme intermediate, whereas 2c originates from
condensation of 2b with the aldehyde product.[8] Importantly,
these initial results clearly evidenced the impact of the heme
Further characterization studies revealed a noticeable effect
of pH on FL#62 azide oxidative deamination activity, and maxi-
mal TON values were achieved around neutral pH (Figure 1e).
A similar pH dependence was observed for the Mb-catalyzed
reaction.[8] From time-course experiments, the FL#62-catalyzed
conversion of 1 into 2b was determined to proceed with
a rate of approximately 14 turnovers per minute over the first
10 min (Figure 1d). This rate is lower than that observed for
the Mb-based catalysts (250 minꢀ1),[8] but the FL#62-catalyzed
reaction proceeded with higher selectivity (i.e., no 2b/2c for-
mation) in addition to the higher TON value. The initial prod-
uct formation rates for FL#62 and the other hemoproteins in-
vestigated also correlated well with the TON values observed
in the corresponding reactions (Figure 1b,d).
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Next, the substrate scope of the FL#62 biocatalyst was inves-
tigated by using a wide panel of different organic azides. As
shown in Table 1, the majority of these compounds were readi-
ly oxidized to the corresponding aldehydes in good to excel-
To further evaluate the synthetic utility of FL#62 in the con-
text of this reaction, a larger scale reaction with benzyl azide
(1; 50 mg, 0.38 mmol) was performed in the presence of
0.05 mol% FL#62 in phosphate buffer (pH 7.0) at room temper-
ature. After simple extraction and purification steps, the de-
sired benzaldehyde product 2a was isolated in 87% yield
(35 mg, 0.33 mmol), which thus supported the scalability of
this biocatalytic transformation.
Table 1. Substrate scope for the FL#62-catalyzed oxidative deamination
of alkyl azides.[a]
With respect to the mechanism, we previously proposed
that this reaction involves heme-catalyzed decomposition of
the heme-bound alkyl azide to give an imido–iron(IV) inter-
mediate. The latter could then undergo tautomerization to an
imine–iron(II) complex, followed by hydrolysis of the imine (in
either free or heme-bound form) to yield the aldehyde product
(Figure 2). Experimental evidence in support of the imine tau-
Product R1
R2
Conversion [%] Selectivity [%] TON
3b
H
4-MeC6H4
4-MeOC6H4
4-O2NC6H4
4-F3CC6H4
2-MeC6H4
2-FC6H4
2-O2NC6H4
3-O2NC6H4
2,4-F2C6H3
3,5-Me2C6H3 36
2,6-Cl2C6H3
Ph
Ph
58
99
25
69
22
96
1
94
99
84
93
79
98
18
94
90
88
72
51
98
91
94
98
85
6000
9440
2670
6820
2000
9660
60
4b
H
5b
H
6b
H
7b
H
8b
H
9b
H
10b
11 b
12b
13b
14b
15b
16b
17b
18b
19b
H
H
H
H
67
51
6600
5850
3540
720
7
5
Me
CF3
CO2Me Ph
H
H
H
490
76
49
70
97
19
7470
4650
7480
9500
1970
1-naphthyl
2-thienyl
PhCH=CH
Figure 2. Proposed mechanism and catalytic steps for the P450-catalyzed ox-
idation of alkyl azides to aldehydes.
[a] Reaction conditions: azide (10 mm), FL#62 (1 mm), Na2S2O4 (10 mm).
tomerization step included the observation of a significant ki-
netic isotope effect (KIE) upon H!D substitution at the level
of the a carbon atom in the azide substrate.[8] Interestingly,
a related imine intermediate was recently invoked in the azide-
to-nitrile conversion catalyzed by non-heme iron-dependent
dioxygenases.[17] Furthermore, 18O-labeling experiments indicat-
ed that the oxygen atom in the aldehyde product derives from
a hydrolytic process.[8] In light of the higher catalytic activity
(i.e., TON) and slower rate of the P450-catalyzed reaction
versus the Mb-catalyzed one, we thus wondered whether
these biocatalytic transformations shared an analogous mecha-
nism or not. To this end, we measured the KIE for the FL#62-
catalyzed oxidative deamination of benzyl azide (1) from com-
petition experiments in the presence of an equimolar concen-
tration of the deuterated analogue, [D2]-1. GC–MS analysis of
this reaction (Figure S3a, Supporting Information) yielded a KIE
value (kH/kD) of 1.9ꢂ0.2 at 228C, which is comparable to that
observed in the presence of Mb(H64V,V68A) as the catalyst (kH/
kD =1.7ꢂ0.3).[8] In addition, the FL#62-catalyzed conversion of
benzyl azide (1) in the presence of 18O-labeled water resulted
in the formation of 18O-labeled aldehyde product 2a(18O) (Fig-
ure S3b), as observed for the Mb-catalyzed reaction. Altogeth-
er, these results hint at important mechanistic similarities be-
tween the two systems, although differences between them
are also apparent. Indeed, the low formation of the amine by-
product in the FL#62 reactions across all the tested azide sub-
lent yields (49–99%), as determined by GC. In these reactions,
P450 was determined to support from 4650 to 9660 TONs. In
addition, the reaction proceeded with high selectivity (>90–
95%), in particular for the alkyl azides most efficiently pro-
cessed by the enzyme. The viable substrates included variously
mono- and disubstituted benzyl azide derivatives (i.e., 3a, 4a,
6a, 8a, 10a, and 11 a), (hetero)aryl-substituted primary alkyl
azides (i.e., 17a and 18a), and secondary alkyl azides (i.e., 15a
and 16a), which supported the broad substrate scope of the
FL#62 catalyst. Modest (20–30%) to low (<20%) product con-
versions were observed instead with benzyl azide derivatives
that carried one or two substituents at the ortho position(s)
(i.e., 7b, 9b, and 13b). These results suggested a negative
effect on the enzymatic transformation of increased steric hin-
drance in proximity to the azido group. Whereas the conver-
sion yields of these P450-catalyzed reactions were consistently
higher than those achieved with engineered myoglobins,[8]
particularly striking is the considerably higher efficiency of the
P450 catalyst in the synthesis of ketones 15b and 16b from
secondary alkyl azides 15a and 16a, respectively. Indeed,
whereas this reaction proceeded inefficiently in the presence
of the Mb catalyst (280–400 TONs, <2% conversion),[8] it was
effectively catalyzed by the engineered P450 (4650–
7470 TONs), which resulted in much improved product conver-
sions (49–76%).
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strates suggests that the unproductive pathway leading to this
reduction is more disfavored in the case of the P450 system. In
addition, the significantly higher (10- to 15-fold) conversions
and TON values measured for the FL#62-catalyzed formation of
15b and 16b versus 14b (Table 1) suggest that the P450 cata-
lyst is significantly more sensitive to an increase in the acidity
of the a protons than the Mb catalyst (three- to fourfold
higher TON for 15b and 16b, respectively, relative to the TON
for 14b).[8]
hormone metabolism (e.g., P4501B1/retinol, kcat/KM ꢁ7ꢁ
103 mꢀ1 ꢀ1).[19] For all the P450s, incubation with benzyl azide
s
resulted in a small (<5–10%) but detectable heme spin shift
(Figure S4), which is indicative of the displacement of the axial
water ligand on the heme by the bound substrate. Titration ex-
periments revealed that FL#62 has a higher binding affinity for
benzyl azide than wildtype CYP102A1 and CYP102A1(F87A)
(dissociation constant, KD, of 68 mm vs. 93 and 84 mm, respec-
tively; Table 2; Figure S4).
Kinetic
experiments
with
wildtype
CYP102A1,
Another interesting result emerging from our substrate
scope investigations with FL#62 is the noticeable difference
between the TON values associated with the synthesis of elec-
tron-deficient benzyl aldehydes (e.g., 5b; 2670 TONs) and
those associated with electron-rich ones (e.g., 4b; 9440). To
elucidate the basis of this trend, we extended the substrate
binding experiments and Michaelis–Menten analyses to the
FL#62-catalyzed oxidative deamination of p-nitrobenzyl azide
(5a) and p-methoxybenzyl azide (4a). Whereas p-nitrobenzyl
azide had weaker affinity for FL#62 than benzyl azide and p-
methoxybenzyl azide (Table 2), no significant differences were
found among the kinetic parameters (KM, kcat) and catalytic effi-
ciency (kcat/KM) of the enzyme for the transformation of these
substrates, which suggested that other factors must be at the
basis of the less efficient conversion of 5a compared to 4a
and 1.
CYP102A1(F87A), and FL#62 were performed to gain further in-
sight into the catalytic properties of these enzymes. In the re-
action with model substrate 1, all of these P450s were found
to follow Michaelis–Menten kinetics (Figure 3). Similar kinetic
parameters (Michaelis constant, KM; turnover number, kcat) were
To further examine this aspect, we performed product inhibi-
tion experiments in which reaction mixtures containing FL#62
and 1, 4a, or 5a as the substrate were spiked with increasing
concentrations (0–10 mm) of the corresponding aldehyde
products. Interestingly, the addition of para-nitrobenzaldehyde
(5b) significantly reduced the conversion of 5a (Figure 4a),
which evidenced the occurrence of product inhibition. In con-
trast, no product inhibition was observed within the concen-
tration range tested for both benzyl azide (1) (Figure S5) and
para-methoxybenzyl azide (4b) (Figure 4b), thus explaining
the greater yields observed in the presence of these substrates
(Table 1).
Figure 3. Overlay plot of initial velocity (V0) versus substrate concentration
for the reactions of CYP102A1 and its variants with selected alkyl azide sub-
strates. The corresponding kinetic parameters (kcat, KM, kcat/KM) are reported
in Table 2.
determined for the parent enzyme and its single mutant var-
iant, CYP102A1(F87A), which is consistent with their similar
performance in terms of TON and product conversion (Table 1).
Conversely, the superior catalytic performance of FL#62 is re-
flected by the eight fold higher catalytic efficiency (kcat/KM =
4.6ꢁ102 msꢀ1) compared to that of the parent enzyme and
F87A variant, which derives as a result of a twofold lower KM
value and a fourfold faster turnover number, kcat (Table 2).
Whereas the catalytic efficiency of FL#62 in this non-native re-
action is significantly lower than that of CYP102A1 for the hy-
In conclusion, our results demonstrate that cytochrome
P450s are promising catalysts for the synthesis of aromatic al-
dehydes through the oxidative deamination of alkyl azides. In
particular, we established that the engineered CYP102A1 var-
iant FL#62 represents a superior biocatalyst for this transforma-
tion relative to other heme-containing enzymes and the previ-
ously reported Mb catalysts. In addition to excellent chemose-
droxylation of fatty acids (kcat/KM ꢁ105 mꢀ1
s )
ꢀ1 [18], it falls in the
same range as those exhibited by P450s involved in drug and
Table 2. Substrate binding affinity and Michaelis–Menten parameters corresponding to CYP102A1, CYP102A1(F87A), FL#62, and selected alkyl azide sub-
strates.[a]
Enzyme
Substrate
KD
[mm]
KM
[m]
kcat
[minꢀ1
kcat/KM
[mꢀ1 sꢀ1
]
]
CYP102A1
CYP102A1(F87A)
FL#62
FL#62
FL#62
BnN3 (1)
BnN3 (1)
BnN3 (1)
4-MeOC6H4CH2N3 (4a)
4-O2NC6H4CH2N3 (5a)
93 (ꢂ9)
1.8ꢁ10ꢀ2 (ꢂ0.7)
1.9ꢁ10ꢀ2 (ꢂ0.5)
9.2ꢁ10ꢀ3 (ꢂ1.9)
8.3ꢁ10ꢀ3 (ꢂ1.0)
1.1ꢁ10ꢀ2 (ꢂ0.2)
66 (ꢂ13)
75 (ꢂ11)
255 (ꢂ20)
209 (ꢂ9)
329 (ꢂ29)
61 (ꢂ27)
66 (ꢂ20)
84 (ꢂ13)
68 (ꢂ10)
4.6ꢁ102 (ꢂ1.0)
4.2ꢁ102 (ꢂ0.5)
5.0ꢁ102 (ꢂ1.0)
107 (ꢂ19)
259 (ꢂ36)
[a] Equilibrium dissociation constants (KD) were calculated from substrate-induced heme spin shift experiments (Figure S4). The kinetic parameters (kcat, KM,
kcat/KM) were calculated by nonlinear fitting of the V0 vs. concentration curves (Figure 3) to the Michaelis–Menten equation.
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lectivity and high catalytic activity (up to 11300 TONs) across
a broad range of alkyl azides, the enhanced reactivity of this
P450 enabled extension of this transformation to the synthesis
of ketones from secondary azides. These studies contributed
valuable insight into the differential reactivities of the hemo-
proteins and engineered variants thereof in the context of
non-native reactions, which is expected to aid the future devel-
opment of biocatalysts for a growing number of synthetically
useful transformations not found in nature.
Acknowledgements
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This work was supported in part by the U.S. National Institute of
Health (NIH) grant GM098628 and in part by the U.S. National
Science Foundation (NSF) grant CHE-1609550. MS instrumenta-
tion was supported by the U.S. National Science Foundation
(NSF) grant CHE-0946653.
Keywords: aldehydes · azides · cytochrome P450 · oxidative
deamination · protein engineering
Received: April 25, 2016
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Published online on && &&, 0000
ChemCatChem 2016, 8, 1 – 6
www.chemcatchem.org
5
ꢀ 2016 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
&
These are not the final page numbers! ÞÞ

COMMUNICATIONS
S. Giovani, H. Alwaseem, R. Fasan*
&& – &&
Aldehyde and Ketone Synthesis by
P450-Catalyzed Oxidative
Deamination of Alkyl Azides
Move azide: An engineered variant of
CYP102A1 efficiently catalyzes the oxi-
dative deamination of primary alkyl
azides to yield aldehydes and exhibits
a broad substrate scope along with
high catalytic activity and excellent che-
moselectivity. The enhanced reactivity
of this enzyme also enables the conver-
sion of selected secondary alkyl azides
into ketones.
&
ChemCatChem 2016, 8, 1 – 6
www.chemcatchem.org
6
ꢀ 2016 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
ÝÝ These are not the final page numbers!