Enzymatic C(sp3)-H amination: P450-catalyzed conversion of carbonazidates into oxazolidinones

Article abstract of DOI:10.1021/cs5018612

Cytochrome P450 enzymes can effectively promote the activation and cyclization of carbonazidate substrates to yield oxazolidinones via an intramolecular nitrene C-H insertion reaction. Investigation of the substrate scope shows that while benzylic/allylic C-H bonds are most readily aminated by these biocatalysts, stronger, secondary C-H bonds are also accessible to functionalization. Leveraging this "non-native" reactivity and assisted by fingerprint-based predictions, improved active-site variants of the bacterial P450 CYP102A1 could be identified to mediate the aminofunctionalization of two terpene natural products with high regio- and stereoselectivity. Mechanistic studies and KIE experiments show that the C-H activation step in these reactions is rate-limiting and proceeds in a stepwise manner, namely, via hydrogen atom abstraction followed by radical recombination. This study expands the reactivity scope of P450-based catalysts in the context of nitrene transfer transformations and provides first-time insights into the mechanism of P450-catalyzed C-H amination reactions.

Full text of DOI:10.1021/cs5018612

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Article
Enzymatic C(sp3)—H amination: P450-catalyzed
conversion of carbonazidates into oxazolidinones
Ritesh Singh, Joshua N. Kolev, Philip A. Sutera, and Rudi Fasan
ACS Catal., Just Accepted Manuscript • DOI: 10.1021/cs5018612 • Publication Date (Web): 29 Jan 2015
Downloaded from http://pubs.acs.org on February 10, 2015
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Enzymatic C(sp3)ꢀH amination: P450ꢀcatalyzed
conversion of carbonazidates into oxazolidinones
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Ritesh Singh, Joshua N. Kolev, Philip A. Sutera and Rudi Fasan*.
Department of Chemistry, University of Rochester, Rochester, New York 14627
KEYWORDS: CꢀH amination • cytochrome P450 • biocatalysis • carbonazidate • oxazolidinones
ABSTRACT. Cytochrome P450 enzymes can effectively promote the activation and cyclization
of carbonazidate substrates to yield oxazolidinones via an intramolecular nitrene C—H insertion
reaction. Investigation of the substrate scope shows that while benzylic/allylic C—H bonds are
most readily aminated by these biocatalysts, stronger, secondary C—H bonds are also accessible
to functionalization. Leveraging this 'nonꢀnative' reactivity and assisted by fingerprintꢀbased
predictions, improved activeꢀsite variants of the bacterial P450 CYP102A1 could be identified to
mediate the aminofunctionalization of two terpene natural products with high regioꢀ and
stereoselectivity. Mechanistic studies and KIE experiments show that the C—H activation step in
these reactions is rateꢀlimiting and proceeds in a stepwise manner, namely via hydrogen atom
abstraction followed by radical recombination. This study expands the reactivity scope of P450ꢀ
based catalysts in the context of nitrene transfer transformations and provides firstꢀtime insights
into the mechanism of P450ꢀcatalyzed C—H amination reactions.
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1. INTRODUCTION
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Expanding the scope of proteinꢀbased catalysts beyond the reaction space encompassed by
natural enzymes constitutes an important, and increasingly soughtꢀafter goal in biocatalysis.1ꢀ11
A
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synthetically most valuable transformation is the direct functionalization of aliphatic C—H
bonds, which occur ubiquitously in manꢀmade and natural compounds. Of particular interest are
methods for the direct conversion of aliphatic C—H bonds into C—N bonds, owing to the
abundance of nitrogen functionalities in biologically active molecules.12ꢀ13 Accordingly,
significant efforts have been devoted over the past several years to the development of synthetic
transition metal catalysts to promote C(sp3)—H amination transformations, especially via nitrene
C—H insertion.14ꢀ18 In stark contrast, natural enzymes capable of supporting C(sp3)—H
amination chemistry via direct C—H bond functionalization are extremely rare (e.g., SAMꢀ
dependent lysine 2,3ꢀaminomutase)19 and none is known to catalyze nitrene C−H insertion
reactions. This 'gap' prompted us and others to develop engineered and artificial biocatalytic
systems for promoting this transformation.7, 20ꢀ22 In independent studies, our laboratory and the
Arnold group demonstrated that engineered cytochrome P450 enzymes containing either a
cysteineꢀ7 or a serineꢀligated heme20 can catalyze the intramolecular C—H amination of
arylsulfonyl azides with high catalytic efficiency and selectivity. More recently, activation of
arylsulfonyl azide substrates for intramolecular C—H amination using alternative hemoproteinꢀ
based catalysts21 as well as for intermolecular sulfimidation23 was reported.
Based on the promising nitrene transfer reactivity exhibited by engineered variants of
CYP102A1 (P450BM3)24 with arylsulfonyl azides7, we envisioned the possibility to exploit P450
catalysts for engaging alternative nitrene donors in intramolecular C—H amination processes. In
this regard, the construction of oxalidinone rings via cyclative nitrene C−H insertion25ꢀ27 is of
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particular relevance as these compounds can provide rapid access, upon hydrolysis, to valuable
(chiral) 1,2ꢀaminoalcohols, key functional motifs in many bioactive molecules28 and chiral
auxiliaries29. To date, this type of transformation have been achieved with rhodium or silverꢀ
based catalysts in the presence of carbamateꢀderived iminoiodinanes25ꢀ26 or Nꢀ
tosyloxycarbamates27 as the substrates. Compared to the latter, organic azides represent
considerably more attractive nitrene precursors, owing to their high atom economy and the
release of environmentally benign N2 gas as the byꢀproduct of the amination reaction.18, 30 Here,
we report that engineered cytochrome P450 enzymes can effectively catalyse the cyclization of
carbonazidate substrates to yield oxazolidinones via an intramolecular C−H amination reaction.
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2. EXPERIMENTAL SECTION
2.1. Chemical synthesis. Synthentic procedures and characterization data for the
acyloxycarbamates 1ꢀ3, carbonazidates 4, 6-14, 23-24, Z-12, deuterated probe compounds D-6,
D1-11, D2-11, and the corresponding oxazolidinone and carbamate products are provided as
Supplementary Information.
2.2. Protein expression and purification. Wildꢀtype CYP102A1 and its engineered variants
were expressed from pCWoriꢀbased plasmids containing the P450 gene under the control of an
IPTGꢀinducible promoter (BamHI / EcoRI cassette) according to procedures described
previously.31 Typically, cultures of recombinant DH5α cells were grown at 37 °C (200 rpm) in
Terrific Broth (TB) medium supplemented with ampicillin (100 mg/L) until OD600 reached 1.0
and then induced with 0.25 mM βꢀDꢀ1ꢀthiogalactopyranoside (IPTG) and 0.3 mM δꢀ
aminolevulinic acid (ALA). After induction, cultures were shaken at 150 rpm and 27 °C and
harvested after 20 hours by centrifugation at 4,000 rpm at 4 °C. Cell lysates were prepared by
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sonication and loaded on Q Sepharose resin. P450s were eluted using 20 mM Tris, 340 mM
NaCl, pH 8.0. After buffer exchange (50 mM potassium phosphate buffer, pH 8.0), the enzymes
were stored at ꢀ80 °C. P450 concentration was determined from COꢀbinding difference spectra
(ε450ꢀ490 = 91,000 Mꢀ1 cmꢀ1). The vector encoding for the thermostable phosphite dehydrogenase
(PTDH) variant Opt13 was kindly provided by the Zhao group.32 PTDH was overexpressed from
pETꢀ15bꢀbased vector in BL21(DE3) cells and purified using Niꢀaffinity chromatography
according to the published procedure.32
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2.3. P450 reactions. The enzymatic reactions were carried out at a 400 ꢀLꢀscale using 5ꢀ20
µM purified P450 and 10 mM substrate in the presence of a PTDHꢀbased cofactor regeneration
system (1 ꢀM PTDH, 100 ꢀM NADP+, 50 mM sodium phosphite). In a typical procedure, a
solution containing the cofactor regeneration system in potassium phosphate buffer (50 mM, pH
8.0) was degassed by bubbling argon into the mixture for 5 min in a sealed vial. A buffered
solution containing the P450 enzyme was carefully degassed in a similar manner in a separate
vial. The two solutions were then mixed together via cannulation. Reactions were initiated by
addition of 8 µL of azide (from a 0.5 M stock solution in methanol) with a syringe, and the
reaction mixture was stirred for 16 h at room temperature under positive argon pressure. Control
reactions with hemin were conducted following an identical procedure with the difference that
the P450 was replaced by 80 µL of a hemin solution (100 µM in DMSO:H2O, 1:1) and dithionate
(10 mM) was used as the reductant. Prior to chromatographic analysis (see Analytical Methods
below), the reaction mixtures were extracted with 400 µL CH2Cl2, followed by evaporation of
the organic layer and dissolution of the residue in 100 µL MeOH. Calibration curves for
quantification of the different oxazolidinones (Figure S4) were generated using authentic
standards produced synthetically (see Synthetic Procedures in SI). All measurements were
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performed in triplicate. For each experiment, negative control samples containing either no
enzyme or no reductant were included. For the COꢀinactivation experiment, carbon monoxide
was bubbled through the reaction mixture containing the P450 enzyme and sodium dithionite
prior to addition of the azide substrate. For the natural product substrates (mentholꢀ and borneolꢀ
derived carbonazidates), the reaction was quenched by adding excess dithionite, followed by
stirring for 24 h before the addition of internal standard and extraction with CH2Cl2. The organic
layer was then analyzed by GCꢀMS.
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2.4. Analytical Methods. GC/MS analyses were performed on a Shimadzu GCMSꢀQP2010
equipped with a RTXꢀXLB column (30 m x 0.25 mm x 0.28 ꢁm) and a quadrupole mass
analyzer. Separation method: 1 ꢁL injection, inj. temp.: 250 ºC, detector temp.: 220 ºC. Gradient:
column temperature set at 50 ºC for 1 min, then to 280 ºC at 30 ºC/min, then to280 ºC for 4 min.
Total run time was 13.67 min. Enantiomeric excess was determined by chiral gas
chromatography (GC) using a Shimadzu GCꢀ2010 gas chromatograph equipped with a FID
detector and a Agilent CYCLOSILꢀB column (30 m x 0.25 mm x 0.25 ꢁm film). Separation
method for 4ꢀmethylꢀ4ꢀ(pꢀtolyl)oxazolidinꢀ2ꢀone (15): 1 ꢁL injection, injector temp.: 200 ºC,
detector temp: 300 ºC. Gradient: column temperature set at 150 ºC for 0 min, then to 200 ºC at
4.5 ºC/min, then to 230 ºC at 1.5 ºC/min, then to 245 ºC at 15 ºC/min for 8 min. Separation
method for 4ꢀphenyloxazolidinꢀ2ꢀone (20): 1 ꢁL injection, injector temp.: 200 ºC, detector temp:
300 ºC. Gradient: column temperature set at 150 ºC for 0 min, then to 200 ºC at 4.5 ºC/min, then
to 220 ºC at 1 ºC/min, then to 245 ºC at 15 ºC/min for 8 min. HPLC analyses were performed
using a Shimadzu LCꢀ2010AꢀHT equipped with a VisionHTꢀC18 reverseꢀphase column and a
multidiode UV–VIS detector. Injection volume: 10 uL. Flow rate: 1 mL/min. Gradient: 10%
acetonitrile in water (0.1% TFA) for 3 min, then increased to 90% over 24 min.
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2.5. Kinetic isotope effect (KIE) experiments. P450 reactions were carried out at a 400 ꢀLꢀ
scale using 20 µM P450, 10 mM NADPH and stopped after 1 hour. For KIE determination with
6 and D-6, parallel reactions with either of the carbonazidate substrates were carried out at
varying substrate concentration (100 ꢀM to 2 mM). The reactions were analyzed by HPLC and
KIE values were calculated from the resulting plots of initial rate against substrate concentration.
For the intraꢀ and intermolecular KIE analyses with D1-11 and with equimolar mixtures of 11
and D2-11, respectively, the reactions were carried out as described above using 10 mM total
substrate concentration, followed by GCꢀMS analysis. KIE values were determined based on the
ratios between the relative abundances of the characteristic fragments with m/z of 105 (nonꢀ
deuterated) and m/z of 106 (deuterated) (Figure S5). Authentic standards of product 20 and D-20
injected individually and in equimolar mixture were used as references (Figure S5).
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3. RESULTS AND DISCUSSION
In preliminary experiments, a series of oxycarbonylꢀbased nitrene donors were prepared using
2ꢀphenylpropanol derivatives as model substrates (Table 1). These compounds comprised
acetoxyꢀ (1) and aryloxyꢀcarbamate (2, 3) derivatives which have been previously investigated in
the context of rhodiumꢀcatalyzed nitrene transfer reactions.27 Given the ability of P450s to
activate sulfonyl azides,7 the carbonazidate derivative 4 was also synthesized. In addition to
being readily accessible from a synthetic standpoint, carbonazidates are most desirable
precursors for oxazolidinone formation via nitrene C—H insertion because of their high atom
economy, with N2 being produced as byproduct as opposed to AcOH or ArOH with 2 or 3.
Despite these favorable features and the successful application of carbonazidates in the context
of sulfimidation33ꢀ34 and olefin aziridination35ꢀ36 and aminohalogenation33, 37, catalytic
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intramolecular C—H amination reactions involving these compounds have not been reported to
our knowledge. CYP102A1 variant FL#62 was chosen for the initial scouting experiments.
Compared to wildꢀtype CYP102A1, FL#62 contains five active site mutations (V78A, F81S,
A82V, F87A, A184V), which contribute to expand its active site and make it accessible to large
and bulky substrates.31, 38ꢀ39 In addition, FL#62 exhibited the highest C—H amination activity in
the presence of arylsulfonyl azides in our previous studies.7
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P450 reactions with substrates 1-4 were performed in the presence of NADPH, to reduce the
enzyme to its ferrous state, and anaerobically, both of which conditions were previously found to
be critical for supporting P450ꢀdependent nitrene transfer reactivity.7 Gratifyingly, the P450s
were found to yield appreciable amounts of the desired oxazolidinone 5 in the presence of the
acetoxyꢀ and aryloxyꢀcarbamates 1-3 (turnover number (TON): 4ꢀ7, Table 1). Importantly, very
good conversion to the oxazolidinone product could also be achieved from the carbonazidate
substrate 4 (Table 1) with FL#62 as the catalyst. In contrast, little or no cyclic product was
formed in the presence of wildꢀtype CYP102A1 or free hemin. Notably, no reaction was
observed in the presence of 4 under standard amination conditions in the presence of Rh2(OAc)4,
as noted previously27, or Coꢀ(TPP) (TPP = tetraphenylporphyrin), two prominent organometallic
catalysts investigated in the context of C—H amination reactions.16, 40
Toward optimization of the reaction conditions, the importance of the source of reducing
equivalents in the P450ꢀcatalyzed conversion of carbonazidate 4 was investigated. These
experiments showed that cyclization of 4 could be achieved by replacing NADPH with the
inexpensive dithionite (Na2S2O4), albeit only with lower efficiency (5 vs. 20 TON). This
difference could be attributed to the kinetically competitive reduction of the carbonazidate
substrate to the corresponding carbamate by action of the inorganic reductant as compared to the
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enzymatic C—H amination reaction (0.2 turnovers/min). More promisingly, comparable number
of turnovers as observed in the presence of an excess of NADPH (10 mM) could be obtained
using catalytic amounts of NADPH (100 ꢀM) in combination with a phosphite dehydrogenase32
based cofactor regeneration system. Thus, under optimized conditions, over 70 catalytic
turnovers were achieved for the FL#62ꢀcatalyzed transformation of 4 into 5 (Entry 1, Table 2).
These improvements notwithstanding, the yield of the C−H amination product remained
moderate (~4%), with the remainder of the carbonazidate substrate being converted to a
carbamate and alcohol products as discussed in more details later.
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3.2. Investigation of substrate scope. To gain insights into the influence of electronic and
steric effects on the P450ꢀcatalyzed C−H amination reaction, a range of carbonazidates (6-10)
were then prepared and tested for cyclization in the presence of FL#62. As shown in Table 2,
various substitutions at the level of the aromatic ring of the substrate were tolerated by the
enzyme, resulting in turnover numbers ranging from 10 to 100 (Entries 1ꢀ6, Table 2).
Interestingly, an increase in TON was observed upon substitution of the para position with a
methyl group (6; 100 vs. 74 TON), whereas a reduction in C—H amination activity resulted
from substitution of this site with an isosteric but electronwithdrawing trifluoromethyl group (7;
43 TON). This trend in reactivity is consistent with the electrophilic character of the reactive
species envisioned to mediate these reactions (vide infra). Notably, derivatives with a bulky
substituent (tertꢀbutyl) at the para position (8) as well as double substitutions on the aromatic
ring (10) were also processed by the enzyme.
With compounds 11-14, the influence of the relative strength of the target C—H bond on the
efficiency of the reaction was investigated. Interestingly, FL#62ꢀmediated amination of the
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secondary, benzylic C—H bond in 11 as well as in the more conformationally rigid 13 was found
to occur with significantly lower efficiency (10ꢀfold) as compared to the tertiary, benzylic C—H
site in 4 (5ꢀ6 vs. 74 turnovers). These results suggest a clear dependence of the P450ꢀdependent
amination activity on the C—H bond strength, a conclusion further supported by the
comparatively higher activity of the enzyme on 12 (15 TON), and lack of activity on 14, in
which the C—H bonds in β to the carbonazidate group have estimated bond dissociation energies
(BDE) of 86 and 98 kcal/mol, respectively.41 This bias notwithstanding, the catalytic turnovers
achieved by these P450 catalysts with carbonazidates compare very favorably with those
reported for rhodium or silver catalysts in the context of oxalidinone synthesis from carbamateꢀ
iminoiodinane or Nꢀtosyloxyꢀcarbamate precursors (5ꢀ25 turnovers).25ꢀ27 Furthermore, the
absence of aziridine formation in the reaction with 21 supports the chemoselectivity of the P450
catalyst toward nitrene C—H insertion over the potentially competing aziridination reaction.27, 42
Chiral analysis of the oxazolidinone products generated from 4, 8, and 11 in the presence of
FL#62 showed only low levels of enantioinduction (2ꢀ5% e.e.). These results were rather
surprising as moderate to high degree of stereoꢀ or enantioselectivity (40ꢀ91% e.e.) were
observed in the cyclization of arylsulfonyl azides by means of FL#62 and related CYP102A1
variants.7 Screening of a panel of 15 FL#62ꢀderived active site variants against carbonazidates 4,
8, and 11 gave similar results, suggesting that the limited chiral induction observed with these
substrates is unlikely to be dependent upon the active site configuration of the enzyme but it may
rather be linked to the mechanism of the C—H activation step operating in these processes as
discussed further below.
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3.3. Natural product substrates. To further investigate the scope and potential utility of the
P450 catalysts in the context of oxalidinone synthesis, carbonazidate derivatives of two natural
product terpenes, menthol (23) and borneol (24), were subjected to FL#62 catalysis (Figure 1).
Notably, both compounds could be converted to a single fiveꢀmembered cyclic product, namely
the transꢀoxazolidinone 25 and the cisꢀoxazolidinone 26, respectively, with high regioꢀ and
stereoselectivity. The high degree of siteꢀselectivity obtained in the reactions with these
substrates is contrast with the unselective nitrene C—H insertion events observed upon pyrolysis
or photolysis of related compounds.43ꢀ44
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Despite the successful cyclization of 23 and 24, we noted that the catalytic efficiency of FL#62
on these substrates was only modest (TON ~ 4). Recently, we reported a fingerprintꢀbased
method for predicting the substrate reactivity of engineered P450s on target molecules that are
structurally related to the probe compounds used for their fingerprinting (referred to as
"fingerprint single component analysis" or SCA).38ꢀ39 Substrates 23 and 24 share significant
scaffold similarity with two of said fingerprint probes, namely the cyclohexaneꢀ and the bornaneꢀ
based probes (Figure S1). Using SCA, four candidate P450 catalysts with potentially improved
activity for the cyclization of these substrates were identified from our previously assembled
collection of FL#62ꢀbased variants, which were derived from this P450 via simultaneous
mutagenesis of 2 to 6 active site positions.31, 39 Gratifyingly, these P450s were found capable of
processing 23 and 24 supporting 3ꢀ to 5ꢀfold higher catalytic turnovers than FL#62 (Figure 2).
Altogether, these studies demonstrate the possibility to apply these P450 catalysts for C—H
amination transformations in the context of structurally and stereochemically complex
molecules, also showing how the activity on a particular substrate can be improved by active site
mutagenesis. Furthermore, these results support the functionality of our fingerprintꢀbased
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methods for P450 substrate reactivity prediction also in the context of P450ꢀmediated C—H
aminations.
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3.4. Mechanistic studies. Further experiments were conducted to shed light into the
mechanism of these reactions. The requirement for a reductant (i.e. dithionite or NADPH) and
complete loss of carbonazidate C—H amination activity upon complexation of the reduced P450
with CO provided clear evidence in support of the involvement of the ferrous heme in catalysis.
Accordingly and building upon our investigations with arylsulfonyl azides7 and previous studies
with metalloporphyrin systems45ꢀ48, we envision the catalytic cycle to initiate with the binding of
the carbonazidate substrate to the ferrous heme to form an azidoꢀiron(II) complex, which would
then undergo nitrogen loss to give a putative imidoꢀiron(IV) intermediate (Figure 2).
Reasonably, the subsequent C—H activation event could then proceed through (a) a stepꢀwise
hydrogen atom abstraction (HAA)/radical rebound mechanism, analogous to that mediated by
oxoꢀiron(IV)porphyrin π cation radical (Compound I) in P450ꢀcatalyzed C—H oxidations49ꢀ50, or
(b) a concerted nitrene C—H insertion, analogous to that operating in rhodiumꢀcatalyzed C—H
aminations51. To probe this step, the cis isomer of carbonazidate 12 ((Z)-12, Figure 3) was
subjected to FL#62ꢀcatalyzed amination conditions, with scrambling of the double bond
geometry upon cyclization being revealing of a radical mechanism. Interestingly, only formation
of the rearranged trans oxazolidinone (E)-21 was observed (TON: 16), a product identical to that
generated starting from (E)-12 (Figure 3). In contrast, rhodiumꢀcatalyzed cyclization of
carbamate (Z)-27 activated in situ with PhI(OAc)2 yielded the cis oxazolidinone product (Z)-21.
Thus, these results unambiguously revealed that a stepwise mechanism, involving HAA followed
by radical recombination, is operative in the P450 amination reaction (Figure 2). Furthermore,
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the complete Z→E rearrangement of the olefinic bond in (Z)-12 is suggestive of the formation of
a relatively longꢀlived carbonꢀcentered radical, which could account for the observed lack of
stereo/enantioselectivity upon amination of the benzylic C—H sites in 4 and 11 by FL#62 and its
variants. On the other hand, it is possible that the high stereoselectivity observed in the case of
the menthol and borneol derivatives (Figure 1) could derive from a faster rebound step for the
less stabilized secondary alkyl radical intermediate formed during the intramolecular C−H
amination of these substrates.
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3.5. Kinetic isotope effect (KIE) experiments. According to the mechanistic model of Figure
2, cleavage of the C—H bond was expected to be rate limiting during oxazolidinone formation.
To examine this aspect, the effect of H/D substitution on the C—H amination rate of substrate 6
was determined (Figure 4a). The positive primary kinetic isotope effect observed in these
experiments (kH/kD: 2.7 ± 0.3 at 20°C) is consistent with the involvement of C—H bond cleavage
in the rateꢀdetermining step of the reaction. At the same time, this value is considerably smaller
than and comparable to, respectively, that observed for C—H amination reactions with Ruꢀ
porphyrins (kH/kD: 6ꢀ12)46, 52 and rhodium catalysts (kH/kD: 1.8ꢀ4.5)51, 53, which were established
to operate via a stepwise and concerted C—H activation pathway, respectively. In light of the
large KIE difference compared to the Ruꢀporphyrin system, we wondered whether other steps
prior to C—H bond cleavage, e.g., activation of the azide via N2 extrusion (Figure 2), may be
also kinetically relevant in the P450 reaction, thus resulting in partial ‘masking’ of the actual
KIE associated with the C—H activation step. A powerful approach to examine this aspect is
54ꢀ55
through comparison of KIEintra versus KIEinter
.
Accordingly, the monoꢀ (D1-11) and gemꢀ
dideuterated (D2-11) analogs of compound 12a (Figure 3b) were synthesized (see SI for details)
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and utilized in intraꢀ and intermolecular competition experiments, respectively. Expectedly, a
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KIEintra > KIEinter would reveal that azide activation is also rateꢀlimiting, whereas KIEintra
≈
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KIEinter would indicate that C—H bond cleavage is the only rateꢀdetermining step in the reaction.
Analysis of the FL#62ꢀcatalyzed cyclization of D1ꢀ11 yielded a KIEintra = 4.7 ± 0.3, whereas the
competition experiment with a 1:1 mixture of 11 and D2-11 yielded a KIEinter value of 5.3 ± 0.4
(Figure 3b), as determined by mass spectrometry (Figure S5). The close similarity between
these values (within experimental error) thus supports a scenario in which C—H bond cleavage
is likely to be the predominant rateꢀdetermining step in these P450ꢀcatalyzed C−H amination
reactions. At the same time, it is possible that in the presence of weaker C−H bonds (e.g., tertiary
vs. secondary C−H bond in 6 vs. 11) other steps may become kinetically important, thereby
affecting the extent of the KIE associated with the H/D substitution at the level of the aminated
C−H bond.
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3.5. Competing pathways. While the studies above provided insights into the productive
pathway leading to oxazolidinone formation, evidence for the occurrence of competing,
unproductive pathways was also gathered. Indeed, in addition to the oxazolidinone product, a
considerable amount of the acyclic carbamate and of the corresponding alcohol was observed in
the P450 reactions with all the substrates tested. Whereas the carbonazidate is completely
consumed in these reactions, the carbamate and alcohol products were found to account for the
majority of the consumed substrate (~ 90%). Nearly complete (>95%) suppression of either
byproduct in the presence of COꢀsaturated buffer proved that they are both generated at the level
of the heme center in the enzyme and not from reaction with the flavins in the reductase domain
or components of the cofactor regeneration system. As proposed in the context of our previous
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studies with arylsulfonyl azides, where a similar reduction byproduct (i.e., sulfonamide) was
observed7, we suspect the acyclic carbamate to originate from overreduction and protonation of
the imidoꢀiron intermediate (blue path, Figure 2), a process potentially favored by the native two
singleꢀelectron/proton transfer mechanism operating in P450s as required for the reductive
activation of O2 to Compound I50. Interestingly, a similar 'uncoupling' phenomenon was
observed during P450ꢀcatalyzed nitrene transfer to sulfides recently investigated by Arnold and
coworkers.23 Possible pathways leading to the alcohol byproduct could involve hydrolysis of the
heme bound carbonylꢀnitrenoid species or radical decarboxylation during catalysis. While these
uncoupling mechanisms currently limit the synthetic utility of these P450ꢀcatalyzed
transformations, we anticipate that targeting structural elements mediating the native
electron/proton transfer pathway in these enzymes could provide a future avenue to further
increase their efficiency as C—H amination catalysts.
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4. CONCLUSIONS
In conclusion, we have reported the successful synthesis of oxazolidinones via the P450ꢀ
catalyzed cyclization of carbonazidates. In addition to expanding the reaction scope of
cytochrome P450 enzymes, this report describes the first example in which these atomꢀ
economical and readily accessible nitrene donors are successfully employed in catalytic
intramolecular C—H amination reactions. Our structureꢀreactivity analyses indicate that whereas
benzylic/allylic C—H bonds with a BDE < 90 kcal/mol are most readily aminated by these
biocatalysts, activation of stronger C—H bonds (i.e., nonꢀbenzylic/nonꢀallylic 2° C—H) is also
feasible. Our mechanistic and KIE analyses indicate that the C—H activation step is rate limiting
and proceeds in a stepwise manner (i.e. via HAA/radical rebound), reminiscent of that involved
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in P450 hydroxylations. Overall, these studies provide firstꢀtime insights into the mechanism of
P450ꢀmediated C—H amination as well as a basis for further optimization of these catalysts. The
experiments with the terpene substrates demonstrate the feasibility of applying these P450ꢀbased
amination catalysts for the transformation of complex natural products, with active site
mutagenesis providing a way to improve the catalytic activity toward a particular target
substrate. Since carbonazidates can be readily synthesized from alcohols, the reactivity reported
here could open exciting opportunities for the application of these enzymes in synthesis, which
may include, for example, coupling selective P450 hydroxylation31, 39, 56 with P450ꢀcatalyzed
carbonazidate cyclization to install 1,2ꢀaminoalcohol functionalities at remote C(sp3)—H
positions in organic molecules.
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Table 1. C—H amination reactivity of wildꢀtype CYP102A1 and variant FL#62 on different
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6
carbonylꢀbased nitrene donors.[a]
7
8
9
O
P450 (0.2 mol%)
HN
10
11
12
13
14
15
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18
19
20
21
22
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27
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O
Y
O
KPi (pH 8.0)
25oC, 16 hrs
O
1-4
5
Entry
Y
Catalyst[a]
Turnovers[b]
1
2
3
Hemin
<0.5
5
4
O
H
CYP102A1
FL#62
N
O
1
NO2
4
5
6
Hemin
CYP102A1
FL#62
0
4
7
H
N
O
2
7
8
9
Hemin
CYP102A1
FL#62
0
0
0
O2N
H
N
O
NO2
3
10
11
12
13
14
Hemin
CYP102A1
FL#62
Rh2(OAc)4
Co(TPP)
0
2
20
0
0
—N3 (4)
[a] Reaction conditions: 20 ꢁM P450, 10 mM substrate, 10 mM NADPH. Hemin reactions: 200
ꢁM hemin, 10 mM substrate, 10 mM Na2S2O4. Rh2(OAc)4/Co(TPP) reactions: 5 mol% catalyst,
200 mM 4 in dry toluene. [b] As determined by HPLC (SD within 15%).
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Table 2. Scope of P450ꢀcatalyzed C—H amination with carbonazidate substrates.[a]
3
4
5
6
7
8
9
O
R1
HN
FL#62 0.05 mol%
R1
R2
5;15-21
O
N3
O
R2
10
11
12
13
14
15
16
17
18
19
20
21
22
23
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25
26
27
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30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
KPi (pH 8.0)
25oC, 16 hrs
O
4;6-12
Entry
Substrate
4
Product
5
TON
1
74 ± 8
H
N
O
O
2
3
4
R1 = Me; R2 = p-Tol
(6)
100 ± 4
43 ± 2
7 ± 1
O
15
H
N
R1 = Me; R2 = 4-CF3-Ph
(7)
O
16
F3C
tBu
H
O
N
R1 = Me; R2 = 4-tBu-Ph
(8)
O
17
H
O
O
N
5
R1 = H; R2 = m-Tol
(9)
23 ± 2
O
O
18
H
N
6
7
R1 = H; R2 = 3,5-dimethyl-
10 ± 1
5 ± 1
phenyl
(10)
19
O
R1 = H; R2 = Ph
HN
(11)
O
20
O
HN
9
15 ± 2
R1 = H; R2 = CH2-CH=CH-Ph
O
(12)
21
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H
O
O
N
8
6 ± 1
N3
O
O
7
8
9
13
22
O
O
N3
10
11
12
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0
10
no oxazolidinone
product
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[a] Reactions conditions: 5 ꢀM P450, 10 mM carbonazidate, cofactor regeneration system (100
ꢀM NADP+, 1 ꢀM phosphite dehydrogenase, 50 mM Na2PO3), 16 hours. Turnover numbers
(TON) were measured by HPLC from triplicate experiments.
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Figure 1. P450ꢀcatalyzed C—H amination in terpene natural products. The graphs illustrates the
relative activity, based on TON, for the FL#62ꢀderived activeꢀsite variants selected by fingerprint
analysis (SCA). The amino acid mutations in the P450 variants can be found in Table S1. "s.r.c."
= standard reaction conditions as indicated in Table 2.
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Figure 2. Proposed mechanism for P450ꢀcatalyzed conversion of carbonazidates to
oxazolidinones. The competing pathway leading to the carbamate byproduct is indicated in blue.
Y = —OC(O)—.
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FeIII
O
ROC(O)NH2
R
R
Cys
1 e-
O
Y
R
N3
N
H
R
Y
H
FeII
Cys
N
R
Y
R
R
R
R
Y
N
NH2
R
NH
N
H
FeIII
Cys
H
FeIII
Cys
FeII
Cys
R
Y
N
R
H
FeIV
Cys
HAA
N2
1 e-, 2 H+
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Figure 3. Rearrangement studies. a) FL#62ꢀcatalyzed cyclization of (E)-12 (or its isomer (Z)-12)
leads to formation of trans oxazolidinone (E)-21, as determined by HPLC (Figure S3 in SI) and
GCꢀMS analysis. The Z→E rearrangement in case of (Z)-12 is indicative of the formation radical
intermediate via HAA. (b) Rhodiumꢀcatalyzed cyclization of carbamate Z-27 is not accompanied
by scrambling of the double bond configuration, in agreement with the concerted nitrene C−H
insertion mechanism proposed for these catalysts51.
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Figure 4. Kinetic isotope effect (KIE) experiments. (a) KIE value for FL#62ꢀcatalyzed
amination of 6 and its deuterated analog, D-6. The graph reports a plot of initial rate versus
substrate concentration for the two reactions. (b) Comparison of KIEintra and KIEinter values as
determined from intramolecular (D1-11) and intermolecular (1:1 mixture of 11 and D2-11)
competition experiments, followed by GCꢀMS analysis (Figure S5).
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ASSOCIATED CONTENT
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Supporting Information. Synthetic procedures, compound characterization data and NMR
spectra, additional figures and tables. This material is available free of charge via the Internet at
http://pubs.acs.org.
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AUTHOR INFORMATION
Corresponding Author
*Eꢀmail: fasan@chem.rochester.edu.
Author Contributions
The manuscript was written through contributions of all authors. All authors have given approval
to the final version of the manuscript.
Funding Sources
This work was supported by the U.S. National Institute of Health (R01 grant GM098628). MS
instrumentation was supported by the U.S. NSF grant CHEꢀ0946653.
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