Toluene Dioxygenase-Catalysed Oxidation of Benzyl Azide to Benzonitrile: Mechanistic Insights for an Unprecedented Enzymatic Transformation

Article abstract of DOI:10.1002/cbic.201500653

Enzymatic dioxygenation of benzyl azide by toluene dioxygenase (TDO) produces significant amounts of the cis-cyclohexadienediol derived from benzonitrile, along with the expected azido diols. We demonstrate that TDO catalyses the oxidation of benzyl azide to benzonitrile, which is further dioxygenated to produce the observed cis-diol. A proposed mechanism for this transformation involves initial benzylic monooxygenation followed by a nitrene-mediated rearrangement to form an oxime, which is further dehydrated to afford the nitrile. To the best of our knowledge, this is the first report of enzymatic oxidation of an alkyl azide to a nitrile. In addition, the described oxime-dehydration activity has not been reported for Rieske dioxygenases.

Full text of DOI:10.1002/cbic.201500653

DOI: 10.1002/cbic.201500653
Communications
Toluene Dioxygenase-Catalysed Oxidation of Benzyl Azide
to Benzonitrile: Mechanistic Insights for an
Unprecedented Enzymatic Transformation
María Agustina Vila+,[a] Mariana Pazos+,[a] CØsar Iglesias,[b] Nicolµs Veiga,[c] Gustavo Seoane,[a]
and Ignacio Carrera*[a]
Enzymatic dioxygenation of benzyl azide by toluene dioxyge-
nase (TDO) produces significant amounts of the cis-cyclohexa-
dienediol derived from benzonitrile, along with the expected
azido diols. We demonstrate that TDO catalyses the oxidation
of benzyl azide to benzonitrile, which is further dioxygenated
to produce the observed cis-diol. A proposed mechanism for
this transformation involves initial benzylic monooxygenation
followed by a nitrene-mediated rearrangement to form an
oxime, which is further dehydrated to afford the nitrile. To the
Scheme 1. Biotransformation of benzyl azide by E. coli JM109 (pDTG601) in
a 5 L bioreactor by the previously described bi-phasic protocol.
best of our knowledge, this is the first report of enzymatic oxi-
dation of an alkyl azide to a nitrile. In addition, the described
oxime-dehydration activity has not been reported for Rieske di-
oxygenases.
benzyl azide by the toluene dioxygenase (TDO) enzymatic
complex from Pseudomonas putida F1 expressed in Escherichia
coli JM109 (pDTG601).[9] In addition to the expected diol 1, the
exocyclic diene 2 was found; its formation was explained by
an spontaneous stereoselective double sigmatropic [3,3] shift
from 1. Significant amounts of cis-cyclohexadienediol, 3, were
obtained as a third product, derived from benzonitrile.
Research on new methodologies for the preparation of nitriles
is of high interest to the organic chemistry community, be-
cause they are considered very useful building blocks for the
preparation of pharmaceuticals and functional material.[1] In
recent years, the oxidative transformation of alkyl azides into
nitriles has attracted much attention; it is an interesting ap-
proach for the synthesis of nitriles because it does not require
elongation of the skeletal carbon chain. Several methods for
this transformation have used stoichiometric oxidant, with or
without a catalyst, for example, BrF3,[2] bis(acetoxy)iodoben-
zene,[3] 2,3-dichloro-5,6-dicyanobenzoquinone,[4] tert-butylhy-
Here we demonstrate that TDO catalyses the oxidation of
benzyl azide to benzonitrile, which is further dioxygenated to
produce the observed cis-diol 3. To the best of our knowledge,
this is the first report of enzyme-catalysed oxidation of a ben-
zylic azide to a nitrile. Although it is well known that bacterial
dioxygenases are able to catalyse oxidations other than the
widely described dihydroxylation of aromatic rings (e.g., mono-
oxygenation of benzylic and allylic CÀH bonds, sulfoxidations,
O- and N-dealkylations and oxidative carbocyclisations),[10,11]
the described transformation cannot easily be explained by
these activities. A plausible mechanism is presented.
droperoxide,[5] Pd(OAc)2,[6] Ru(OH)x/Al2O3 and electrochemical
anodic oxidation.[8]
[7]
Recently, we reported the structures of novel interesting
azido diols (Scheme 1) obtained from the biotransformation of
The formation of nitrile 3 involves two processes: oxidation
of the primary azide functionality and dioxygenation of the
aromatic ring by TDO. This chained oxidation can occur from
either the benzylic azide in the starting material or from the
allyl azide in the cis-cyclohexadienediol 2, and can be chemi-
cally or enzymatically mediated (by the TDO complex or anoth-
er enzyme in the biocatalyst). In order to answer these ques-
tions we carried out the biotransformation of 1 in resting cells
of E. coli JM109 (pDTG601) (Figure 1A); only diol 2 was detect-
ed as a product, formed by spontaneous two [3,3] sigmatropic
shifts, as previously described.[9] Because 3 cannot be pro-
duced from 1, benzonitrile (a known substrate for TDO)[12]
should be formed as a first intermediate prior to dioxygena-
tion. We carried out a control experiment with E. coli JM109
(lacking the genes for TDO) to assess other enzymes in nitrile
generation (Figure 1A). Benzonitrile was not detected, and
[a] M. A. Vila,+ M. Pazos,+ Dr. G. Seoane, Dr. I. Carrera
Departamento de Química Orgµnica
Facultad de Química, Universidad de la Repfflblica
Av. General Flores 2124, C.P. 11800 Montevideo (Uruguay)
E-mail: icarrera@fq.edu.uy
Homepage: http://www.secobi.fq.edu.uy/
[b] C. Iglesias
Cµtedra de Microbiología, Departamento de Biociencias
Facultad de Química, Universidad de la Repfflblica
Av. General Flores 2124, C.P. 11800 Montevideo (Uruguay)
[c] Dr. N. Veiga
Cµtedra de Química Inorgµnica, Departamento Estrella Campos
Facultad de Química, Universidad de la Repfflblica.
Av. General Flores 2124, C.P. 11800 Montevideo (Uruguay)
[+] These authors contributed equally to this work.
Supporting information for this article is available on the WWW under
http://dx.doi.org/10.1002/cbic.201500653.
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ucts (overall yield, 40%) along with unreacted starting material.
To our surprise, no nitrile functionality was found in any prod-
uct. The expected diol 4 accounted for 80% of the products,
but the formation of triols 5 and 6 deserves some comment.
The formation of 5 could be explained by the known benzylic
monooxygenation activity of TDO followed by ring dioxygena-
tion.[10e] Formation of 6 is intriguing, given that it was not
a result of azide displacement by water, as phenethylic alcohol
was not detected in the E. coli JM109 control; this is explained
below.
It was evident that nitrile formation by the TDO complex re-
quires the azide functionality to be placed at the benzylic posi-
tion. Given the formation of 5, for the case of benzyl azide we
hypothesized that benzylic monooxygenation could occur as
the first step in the production of benzonitrile. Benzylic CÀH
oxygenation by TDO has been extensively studied for alkyl
arenes.[10e,14] Usually benzene rings that contain a benzylic CÀH
bond can be monooxygenated by TDO to give alcohols; these
are better substrates than their alkyl precursors, and thus un-
dergo rapid cis-dihydroxylation to yield enantiopure triols. This
monooxygenation activity is highly sensitive to the arene sub-
stitution pattern; for example 1,4-disubstituted arenes are not
good substrates for benzylic monooxygenation by TDO.[10e]
In order to obtain evidence that initial monooxygenation at
the benzylic position mediates nitrile formation by TDO, E. coli
JM109 (pDTG601) was supplied with p-chlorobenzyl azide as
a substrate (Figure 2A). Nitrile formation was completely abol-
ished: diol 7 was the only product found in the reaction, along
with unreacted starting material. Also, no azide-rearranged
product was found.
Figure 1. A) Diol 1 is not a substrate for TDO. E. coli JM109 does not oxidize
benzyl azide to benzonitrile. B) Proposed sequence for the generation of
diol 3. C) HPLC product formation profiles for benzyl azide biotransformation
in a 5 L bioreactor by using a biphasic protocol. Formations of diols 1+2
are expressed together to simplify data analysis.
only unreacted benzyl azide was recovered. This strongly sug-
gested the participation of the TDO complex in the oxidation.
In the proposed formation of 3 (Figure 1B), TDO catalyses the
initial oxidation to form benzonitrile, which is subsequently di-
hydroxylated by the same enzymatic complex. We next studied
product formation profiles in a biotransformation carried out
in a 5 L bioreactor (Figure 1C).[13] The plots show an initial lag
phase for the formation of cyanodiol 3, and then both curves
(which represent the formation of products 1+2 and forma-
tion of cyanodiol 3) display comparable initial slopes. These
profiles are consistent with the proposed initial formation of
benzonitrile from benzyl azide and subsequent dioxygenation
to 3.
In order to deepen our understanding of the benzyl azide
oxidation to benzonitrile by the TDO complex we studied the
biotransformation of phenylethylazide (Scheme 2). Three cis-cy-
clohexadienediols (4, 5 and 6) were isolated as the only prod-
Figure 2. A) Nitrile formation activity was abolished when para substitution
was made in the aromatic ring. B) Model of p-chlorobenzyl azide and benzyl
azide (yellow) docked in the active site of TDO (PDB ID: 3EN1): C (grey), N
(blue), O (red), H (white), Cl (green).
Scheme 2. Biotransformation of phenylethylazide by E. coli JM109
(pDTG601) in resting cells.
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Based on the X-ray structure of TDO,[15] we performed mo-
lecular docking to rationalise this result. p-Chlorobenzyl azide
and benzyl azide were docked into the active site of TDO (Fig-
ure 2B): residues Gln215 and Phe216 form a “wall” at the
active site, thus preventing lateral movement of the substrates.
As a consequence, whereas the benzylic position of the mono-
substituted substrate is 4.54 Š from the dioxygen molecule
bound to the FeII ion, for the 1,4-disubstituted substrate this
distance is greater (5.37 Š, which prevents benzylic monooxy-
genation from taking place). The results are in full agreement
with the prediction of Boyd et al. based on the similarities of
the active sites of TDO and NDO.[10f]
transition metals by the carbon-attached nitrogen (N1) or by
coordination with the terminal nitrogen (N3).[16] N1 coordina-
tion results in a shortening of the terminal N2ÀN3 bond and
a weakening (elongation) of the bond between N1 and N2,
thereby facilitating loss of dinitrogen (N2).[16] For 9 the calculat-
ed Wiberg bond indexes (bond orders) are 1.45 (N1ÀN2 bond)
and 2.34 (N2ÀN3), thus nitrogen loss could be favoured there-
by resulting in a iron(IV)–nitrene intermediate 9a (Scheme 3B).
The generation of an imido–iron(IV) complex in iron-containing
monooxygenases (equivalent to 9a) has been recently de-
scribed for acyl azides in the P450-catalysed conversion of car-
bonazidates to oxazolidinones.[17]
Thus, both the formation of 5 from phenylethylazide and
the lack of nitrile formation in p-chlorobenzyl azide suggest
that initial monooxygenation at the benzylic position to yield
the a-hydroxyl azide 8 mediates nitrile formation. Scheme 3
shows a possible route for the overall oxidation process from
8. According to computational results at the UB3LYP/LANL2DZ
level of theory, coordination of 8 to FeII in the TDO active site
would be highly favoured. By using the metal and amino acid
coordinates of the TDO crystal structure, we obtained the
most stable geometry for the resulting coordination complex 9
(Scheme 3A). The structural constraints imposed by the pro-
tein architecture lead to the formation of a high-spin adduct,
for which the resulting four-membered chelate ring is stable
enough to support bidentate coordination through the hy-
droxylic oxygen and nitrogen N1 of the azide. Azides can bind
Given the high reactivity of iron(IV)–nitrene species, rear-
rangement of the nitrene to provide a more stable FeII-coordi-
nated compound is proposed (Scheme 3B). Migration of the
phenyl group (pathway B1) would afford 10, which would
render the corresponding formylaniline or its dioxygenated de-
rivative (not found in the reaction medium). In the case of OH
or H group migration, the corresponding benzaldoxime 11 or
benzamide’s iminol 12 would be obtained (pathways B2 and
B3, respectively). Both products could be dehydrated to give
benzonitrile, thus implying either oxime or nitrile dehydratase
activity by TDO (or other enzyme present in the microorgan-
ism), as spontaneous dehydration would not be expected in
the reaction medium. In order determine the correct pathway
we used benzaldoxime (E and Z isomers) and benzamide as
substrates with E. coli JM109 (pDTG601). Benzamide gave no
Scheme 3. A) Monooxygenation of benzyl azide by TDO gives hydroxyazide 8, which could coordinate to FeII in the active site of the enzyme. This bidentate
coordination is supported by computational calculations at the UB3LYP/LANL2DZ level of theory. B) The proposed coordination could promote nitrogen loss
to form an iron(IV)–nitrene complex, which could rearrange via three pathways (B1, B2 and B3).
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Scheme 4. A) Benzamide is not a substrate for TDO. B) Biotransformation of
Z and E benzaldoximes by E. coli JM109 (pDTG601).
Scheme 5. Proposed mechanism for the formation of 6.
reaction (all starting material recovered; Scheme 4A). However,
Z and E benzaldoximes afforded nitrile 3 along with the ex-
pected oxime diol 13 and triol 14, although in different pro-
portions (Scheme 4B). Nitrile formation was not detected in
a control experiment (same substrates with E. coli JM109), thus
suggesting oxime dehydratase activity of the TDO complex.
However, because of the known E/Z isomerisation of benzal-
doximes in protein preparations,[18] we cannot establish wheth-
er both isomers are substrates for TDO-mediated dehydration.
The formation of 14 can be explained by reduction of the
oxime moiety to the primary alcohol by an alcohol dehydro-
genase in E. coli, according to previous reports.[18] In view of
these results, we propose that pathway B2 of Scheme 3B de-
scribes the formation of benzonitrile.
low overall yield for this transformation could be explained by
the fact that the initial coordination to Fe would not be as
strong as in complex 9, as there is no OH group to produce
a bidentate ligand.
In summary, we present the first example of enzyme-cata-
lysed conversion of an alkyl azide into a nitrile. A proposed
mechanism for this transformation by the TDO complex in-
volves initial benzylic monooxygenation, followed by nitrene-
mediated rearrangement to form an oxime, which is further
dehydrated to afford the corresponding nitrile. This oxime-de-
hydration activity has not previously been reported for Rieske
dioxygenases. The suggested iron-catalysed nitrene generation
is being studied in our group as it could be of great impor-
tance and might contribute to the development of novel enzy-
matic CÀH amination strategies of organic compounds,[22]
a field that has been recently developed for P450 enzymes[17,23]
but not yet for Rieske dioxygenases.
The enzymatic transformation of oximes to nitriles has been
described for FeII-containing enzymes such as aldoxime dehy-
dratases,[19] and cytochrome P450 monooxygenases,[20] but to
the best of our knowledge it has not been previously de-
scribed for TDO or other Rieske dioxygenases. Further studies
regarding this point will be reported in due course by our
group.
Acknowledgements
Another possibility for the transformation of 8 into 3 is that
hydroxyazide 9 could be transformed to benzonitrile by a
Schmidt reaction[21] in the TDO active site, promoted by a pro-
ton transfer. However, the evidence of nitrile formation from
benzaldoximes as well as the formation of 6 in the biotransfor-
mation of phenylethylazide makes this mechanism less proba-
ble and favours our proposed route. A possible explanation for
the formation of 6 is coordination of the starting material with
FeII to promote N2 loss and thus formation of the correspond-
ing metal–nitrene complex (Scheme 5). Rearrangement of the
iron(IV)–nitrene complex would produce the corresponding
imine, which is unstable in water and produces the aldehyde
(Scheme 5B). As stated previously, reductases in E. coli could
reduce this aldehyde to the phenethylic alcohol, which is
a known substrate of TDO, thus finally giving triol 6.[10e] The
This study was financed by the Agencia Nacional de Investiga-
ción e Innovación (Uruguay, Project FCE 6045). The authors
would like to thank Prof. Sonia Rodríguez (Cµtedra de Microbiolo-
gía, Facultad de Química–UdelaR) for her continuous support
and mentoring in the microbiological processes and Prof. Alejan-
dra Rodríguez and Rafael Gonzµlez (Polo Tecnológico de Pando,
Facultad de Química, UdelaR) for the HRMS analysis.
Keywords: azides · biocatalysis · dioxygenases · oxidation ·
Rieske
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Manuscript received: December 3, 2015
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