Efficient conversion of primary azides to aldehydes catalyzed by active site variants of myoglobin

Article abstract of DOI:10.1039/c5sc02857d

The oxidation of primary azides to aldehydes constitutes a convenient but underdeveloped transformation for which no efficient methods are available. Here, we demonstrate that engineered variants of the hemoprotein myoglobin can catalyze this transformation with high efficiency (up to 8500 turnovers) and selectivity across a range of structurally diverse aryl-substituted primary azides. Mutagenesis of the 'distal' histidine residue was particularly effective in enhancing the azide oxidation reactivity of myoglobin, enabling these reactions to proceed in good to excellent yields (37-89%) and to be carried out at a synthetically useful scale. Kinetic isotope effect, isotope labeling, and substrate binding experiments support a mechanism involving heme-catalyzed decomposition of the organic azide followed by alpha hydrogen deprotonation to generate an aldimine which, upon hydrolysis, releases the aldehyde product. This work provides the first example of a biocatalytic azide-to-aldehyde conversion and expands the range of non-native chemical transformations accessible through hemoprotein-mediated catalysis.

Full text of DOI:10.1039/c5sc02857d

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Efficient conversion of primary azides to aldehydes
catalyzed by active site variants of myoglobin†
Cite this: DOI: 10.1039/c5sc02857d
Simone Giovani,‡ Ritesh Singh‡ and Rudi Fasan*
The oxidation of primary azides to aldehydes constitutes a convenient but underdeveloped transformation
for which no efficient methods are available. Here, we demonstrate that engineered variants of the
hemoprotein myoglobin can catalyze this transformation with high efficiency (up to 8500 turnovers) and
selectivity across a range of structurally diverse aryl-substituted primary azides. Mutagenesis of the ‘distal’
histidine residue was particularly effective in enhancing the azide oxidation reactivity of myoglobin,
enabling these reactions to proceed in good to excellent yields (37–89%) and to be carried out at
a synthetically useful scale. Kinetic isotope effect, isotope labeling, and substrate binding experiments
support a mechanism involving heme-catalyzed decomposition of the organic azide followed by alpha
hydrogen deprotonation to generate an aldimine which, upon hydrolysis, releases the aldehyde product.
This work provides the first example of a biocatalytic azide-to-aldehyde conversion and expands the
range of non-native chemical transformations accessible through hemoprotein-mediated catalysis.
Received 3rd August 2015
Accepted 28th September 2015
DOI: 10.1039/c5sc02857d
www.rsc.org/chemicalscience
few methods currently available to promote this trans-
formation require high catalyst loadings (e.g., 10 mol%
Introduction
9
9
a,9b
Organic azides constitute one of the most versatile functional MoO
2
(S
2
CNEt
2
)
2
)
and harsh reaction conditions (reux in
9a
groups in organic chemistry, being stable in the presence of toluene/water mixture), or they lack of selectivity for formation
moisture and air but unveiling peculiar reactivity upon expo- of the aldehyde product.
6
c
1
sure to transition metals, light or heat. Important and well
Our group has recently reported the ability of heme-depen-
established applications of azides in chemical synthesis include dent metalloproteins such as cytochrome P450s and myoglobin
2
3
their use in Staudinger reductions, Schmidt reactions, Curtius to activate azide substrates such as sulfonyl azides and carbo-
3
4
rearrangements, and metal-catalyzed dipolar cycloadditions nazidates in the context of intramolecular C(sp )–H amination
5
1
7h,7i,10
with alkynes to give triazoles. More recently, azides have reactions.
Myoglobin and engineered variants thereof have
provided attractive substrates for the synthesis of nitriles and also proven useful toward promoting carbene transfer reactions
6
7
11
C–H amination reactions via nitrene insertion. A synthetically starting from diazo-containing substrates. The reactivity of
valuable but largely underexplored application of primary alkyl myoglobin on these classes of substrates prompted us to
azides involves their oxidative transformation to aldehydes. The explore its ability to promote the oxidation of primary azides to
latter are classically prepared via the oxidation of primary the corresponding aldehydes. Here, we report that engineered
alcohols with stoichiometric chromium- and manganese-based variants of sperm whale myoglobin can catalyze this trans-
oxidizing agents (e.g., pyridinium chlorochromate, MnO
2
) or via formation with high efficiency and selectivity and across
Swern oxidation, or, alternatively, via partial reduction of esters, a broad range of substrates, thus providing a rst example of
acid chlorides, or nitriles with metal hydrides. In this context, a mild, biocatalytic route to the conversion of primary azides to
catalytic strategies to afford aldehydes from azido groups, aldehydes.
which can be readily introduced into organic molecules via S 2
N
8
displacement of readily available halides, would be attractive.
In particular, this transformation can provide a convenient, Results and discussion
alternative route to the installation of aldehydes from a non-
Our studies began with testing the ability of wild-type sperm
oxygenated precursory functional group. Unfortunately, the very
whale myoglobin to promote the conversion of benzyl azide 1 to
benzaldehyde 2a under anaerobic conditions and in the pres-
ence of sodium dithionite (Na S O ) as a reductant (Table 1).
2 2 4
Gratifyingly, we observed formation of the desired product 2a
albeit only in moderate yield (18%, Entry 3, Table 1). The
reaction also produced small amounts of benzylamine 2b (3%)
and N-benzyl-benzylimine (2c, <2%). 2c likely arises from
Department of Chemistry, University of Rochester, 120 Trustee Rd, Rochester, New
York 14627, USA. E-mail: fasan@chem.rochester.edu
Electronic supplementary information (ESI) available: Experimental details,
synthetic procedures, characterization data for reaction products and additional
†
gures. See DOI: 10.1039/c5sc02857d
‡
These authors contributed equally to this work.
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condensation of the benzaldehyde product with benzylamine, (Table 1). These mutations were designed to vary the geometry
whereas a possible route for formation of the latter is discussed of the active site through decreasing (e.g. L29A) or increasing
further below.
(e.g. F43W) the size of the side-chain group. Among the single-
At a catalyst loading of 0.01 mol%, wild-type myoglobin was site Mb variants, both Mb(F43W) and Mb(H64V) display higher
found to support about 1650 catalytic turnovers (TON) for the catalytic activity than the wild type protein (Entries 5 and 7,
conversion of 1 to 2a. Under identical conditions but at higher Table 1). Notably, Mb(H64V) was found to support about
catalyst loading (0.2 mol%), free hemin gave only 95 TON 3500 TON for the conversion of 1 to 2a. A further improvement
(
Entry 1, Table 1). Given the presence of a histidine-coordinated in activity (ꢀ20%) was then achieved by combining the H64V
12
heme in Mb and the impact of axial ligands in affecting the and V68A mutations, thus dening Mb(H64V,V68A) variant as
13
reactivity of metalloporphyrin systems, the same experiment the most promising biocatalyst for further investigation
was repeated in the presence of imidazole (Entry 2, Table 1). The (3740 TON, Table 1). Using this variant, about half of the azide
low catalytic activity observed also in this case further high- substrate (49%) could be converted to benzaldehyde, this cor-
lighted the critical role of the protein scaffold in endowing the responding to a three-fold higher conversion than that obtained
hemoprotein with high azide oxidation reactivity. Additional with the native hemoprotein. Interestingly, further optimization
control experiments showed a nearly complete loss of this experiments showed a marked pH dependence of the relative
activity in the presence of air, or in the presence of carbon amount and distribution of the products 2a, 2b and 2c, with the
monoxide (which forms a stable complex with ferrous Mb), or in plot of TON for benzaldehyde (2a) versus pH yielding a bell-
the absence of the reductant (Na S O ), indicating the direct shaped curve with a maximal value around neutral conditions
2
2 4
involvement of Mb heme center, in its ferrous state, in the (ESI Fig. S1†). At pH 7, the Mb(H64V,V68A)-catalyzed oxidation
catalytic transformation. of benzyl azide to benzaldehyde proceeds with an improved GC
In our previous studies on myoglobin-catalyzed carbene yield of 77% and correspondingly higher turnovers (6340 TON,
11
transfer reactions, we observed a distinctive effect of active site Table 1), while the amount of the byproduct 2b remains as low
mutations on the efficiency and selectivity of these trans- as or lower than that formed at higher pH (4% vs. 4–11%). Time-
formations. As illustrated in Fig. 1a, ve amino acid residues dependent studies showed that the benzyl azide substrate is
(L29, F43, H64, V68, I107) line up the distal pocket of oxidized at a rate of 250 turnovers per min over the rst
myoglobin. To identify improved Mb-based catalysts for the 10 minutes and completely consumed within 1.5 hours (ESI
reaction investigated here, a panel of Mb variants carrying Fig. S2†). Notably, the product conversion with Mb(H64V,V68A)
either a single or a double amino acid substitution at the level of under these optimized conditions matches those reported for
9a
these residues were tested in the reaction with benzyl azide the same reaction in the presence of the Mo-based complex at
ꢁ
high temperature (100 C) and employing 1000-fold higher
catalyst loadings, thus highlighting the efficiency and mildness
Table 1 Catalytic activity of wild-type myoglobin (Mb) and its variants of the biocatalytic process in the context of this transformation.
a
in the oxidation of benzyl azide to benzaldehyde
To examine the scope of the Mb-mediated transformation in
more detail, various aryl substituted azidomethyl substrates
(3a–19a) were prepared and subjected to Mb(H64V,V68A)-cata-
lyzed oxidation. As summarized in Scheme 1, the majority of
these substrates could be converted to the corresponding
aldehyde in good to excellent yields (44–89%), with the Mb
catalyst supporting from 3740 (3b) to over 8500 turnovers (4b).
These experiments also revealed a signicant impact of both
electronic and steric factors on the efficiency of the reaction.
The synthesis of 3b, 4b, 6b, and 10b in 44–89% yields indicated
that substitutions at the para and meta positions of the phenyl
ring, including double meta substitutions (12b), are generally
well tolerated by the Mb catalyst. Signicantly less so are ortho
substitutions, these resulting in generally lower conversions as
illustrated by the results with 7b (27%) and in particular with 9b
and 13b, for which nearly no product formation was observed.
Notable exceptions to this trend are substrates containing ortho
modications with small uorine substituents (8a, 11a) or fused
ring, such as 1-(azidomethyl)naphthalene 15a, all of which
could be efficiently oxidized (51–66%) to the corresponding
aldehydes (8b, 11b, and 15b, respectively).
b
c
Entry Catalyst
[Catalyst]/mM pH Conv. TON
1
2
3
4
5
6
7
8
9
1
1
1
1
Hemin
0.02
8.0 19%
8.0 22%
8.0 18%
8.0 19%
8.0 23%
8.0 32%
8.0 41%
8.0 15%
8.0 6%
8.0 36%
8.0 42%
8.0 49%
7.0 77%
95
Hemin + imidazole (1 mM) 0.02
WT Mb
Mb(L29A)
Mb(F43W)
Mb(F43V)
Mb(H64V)
Mb(V68A)
Mb(V68F)
Mb(L29A,H64V)
Mb(F43V,V68A)
Mb(H64V,V68A)
Mb(H64V,V68A)
110
1650
1630
2190
2980
3500
1420
580
3110
3380
3740
6340
0.001
0.001
0.001
0.001
0.001
0.001
0.001
0.001
0.001
0.001
0.001
0
1
3
4
a
Reactions (400 mL) were conducted under anaerobic conditions with
0 mM BnN and 10 mM Na for 24 hours at room temperature.
Product yield based on conversion of initial 1a to 2a as determined
by gas chromatography. ¼ nmol aldehyde/nmol catalyst. Errors in
1
3
2
S
2
O
4
In addition to steric factors, the inuence of electronic
effects also became apparent from these studies. Interestingly,
high conversions and catalytic turnovers (4440–8560) were
observed in the presence of benzyl azide substrates with
b
c
reported values are within ꢂ 10%.
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Scheme
1
Substrate scope for Mb(H64V,V68A)-catalyzed azide
oxidation. Reaction conditions: 10 mM azide, 10 mM Na
2 2 4
S O , 1 mM
Mb(H64V,V68A).
reaction occurred in the presence of 18a. Of note, the
Mb-mediated oxidation of cinnamyl azide (17a) gave only
E-citral, while a 2 : 1 mixture of E- and Z-citral was obtained by
12
Fig. 1 (a) Distal pocket of sperm whale myoglobin (pdb 1A6K ). Active
site residues (yellow), heme (wheat), ‘proximal His’ (green), and heme-
14
PCC oxidation of geraniol as reported previously. As indicated
bound water molecule (grey sphere) are highlighted. (b) Electronic by the catalytic turnovers for the corresponding aldehyde
absorption spectra for wild-type Mb and Mb variants Mb(H64V) and
Mb(H64V,V68A) in phosphate buffer at pH 7.0. The Soret bands have
maxima at 408, 398, and 403 nm, respectively. (c) Ligand-induced
decrease of Soret band intensity upon incubation of Mb in phosphate
products (700 (17b) > 400 (19b) > 0 TON (18b)), the order of
reactivity of the substrates above is consistent with the higher
reactivity of electron rich azides observed across the substituted
benzyl azide derivatives. Altogether, these results suggest a clear
buffer at pH 7 with 20 mM benzyl amine (BnNH
BnNH
2
) and para-substituted
2
derivatives (lmax ¼ 408 nm (WT); 409 nm (WT + BnNH
10 nm (WT + 4-(CF )–BnNH ); 409 nm (WT + 4-(NO )–BnNH
12 nm (WT + 4-(OMe)–BnNH ).
2
); requirement for the (hetero)aromatic ring linked to the azido-
);
4
4
3
2
2
2
methyl group for efficient transformation by the Mb catalyst.
2
Finally, the poor conversion of (1-azidoethyl)benzene (14a) to
acetophenone (14b) showed that oxidation of secondary azides
by Mb(H64V,V68A) is considerably less efficient than that of
primary azides (6340 vs. 95 TON for 2b vs. 14b).
To assess the scalability of these transformations, a large
scale reaction with benzyl azide 1 (30 mg) was carried out in the
presence of 0.05 mol% Mb(H64V,V68A) in phosphate buffer at
room temperature. Aer a simple extraction and purication
step, the desired benzaldehyde product 2a could be obtained in
electron-rich aromatic rings by virtue of electron donating
substituents (4b), heteroatoms (16b), or fused rings (15b vs. 7b),
whereas reduced TTN as compared to benzyl azide (1) were
observed in the presence of substrates with electron poor
aromatic rings (5b). Notably deviating from this trend are the

uorinated substrates, 4-triuoromethyl- (6a), 2-uoro- (8a),
and 2,4-diuoro-benzyl azide (11a), which in spite of the elec-
tron withdrawing effect of the uorine substituents are effec-
tively oxidized by the Mb catalyst (4930–6890 TON, Scheme 1).
Next, we tested substrates such as cinnamyl azide (17a),
8
0% isolated yield (19 mg), thus providing a proof-of-principle
demonstration of the synthetic utility of this biocatalytic
transformation.
Scheme 2 outlines our proposed mechanism for the
Mb-catalyzed azide-to-aldehyde oxidation reaction reported
here. Aer reduction of ferric Mb to the catalytically active
ferrous form (step ‘a’), we envision formation of an initial alkyl
azide–heme complex following binding of the substrate to the
distal pocket of the protein (step ‘b’). This species could then
(2-azidoethyl)benzene (18a), and geranyl azide (19a) to investi-
gate the scope of the Mb-catalyzed reaction across compounds
lacking an aryl substituent in alpha to the azido group.
Although in moderate yields (ꢀ10%), formation of cinna-
maldehyde (17b) and citral (19b) was observed, whereas no
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undergo loss of N
2
to yield a nitrene-heme species, possibly in spectrum of the hemoprotein, in line with previous observa-
19
the form of an imido-iron(IV) intermediate (step ‘c’). These steps tions. Furthermore, incubation of the amine-treated protein
are analogous to those envisioned to precede the nitrene C–H with dithionite and CO yielded a Mb–CO complex spectro-
insertion step in the context of our previously investigated scopically superimposable with that of untreated Mb (ESI
Mb- and P450-catalyzed intramolecular C–H amination reac- Fig. S3†), conrming that the amine complex is reversible and
7h,7i,10
tions with arylsulfonyl azides and carbonazidates.
Unlike that no protein denaturation had occurred. Importantly, the
the latter, however, the presence of an a-hydrogen in the azide amine-induced effect was found to increase in the order p-nitro-
substrates employed here could allow for the tautomerization of benzyl amine > benzyl amine > p-methoxy-benzyl amine
the imido-iron(IV) species to an imine-iron(II) complex (step ‘d’), (Fig. 1c), which is in excellent agreement with the activity trend
whose dissociation releases an aldimine and regenerates observed for the oxidation of the corresponding azides (5a < 1 <
ferrous Mb (step ‘e’). Hydrolysis of the aldimine either in free 4a). In this regard, the large activity enhancement associated
form (step ‘f’), or while still bound to the heme (step ‘g’), then with the H64V mutation is also interesting (Table 1). In deoxy
yields the aldehyde product. An alternative path for the forma- Mb, the ‘distal’ His64 residue contributes to stabilize a water
tion of the imine directly from the alkyl azide–heme complex molecule ligand occupying the sixth coordination site of the
0
20
(
step ‘c ’) is through a 1,2 hydrogen shi analogous to that heme group via hydrogen bonding (Fig. 1a). Substitution of
15
observed for free alkyl nitrenes obtained by azide photolysis.
His64 with valine removes this interaction, leading to a ve
This type of rearrangement has been predicted to be energeti- coordinate heme center as determined by X-ray crystallog-
21
cally barrierless for singlet nitrene species, but it is associated raphy. Spectroscopically, this feature is evidenced by the blue
16
with an energy barrier for triplet nitrenes. Since the putative shi of the Soret band from 408 nm for ferric wild-type Mb to
imido-iron(IV) intermediate in Scheme 2 is likely to have a non- 398–402 nm for Mb(H64V) and Mb(H64V,V68A), as shown in
singlet electronic state (i.e., S ¼ 2) according to Density Func- Fig. 1b. Thus, the H64V mutation could promote catalysis by
17
18
tional Theory (DFT) calculations on related species, a path favouring binding of the alkyl azide substrate to the axial
proceeding through this intermediate appears to be more coordination site of the heme group. Furthermore, removal of
plausible. In addition, a barrierless nitrene-to-imine conversion the heme-bound water molecule as a result of the H64V muta-
would not be consistent with the kinetic isotope effect (KIE) tion makes the distal pocket of the hemoprotein largely
21
observed upon deuteration of the a-position (vide infra).
hydrophobic. This phenomenon can account for the efficient
Several experimental data were found to be supportive of the oxidation of the uorine-containing azide substrates (6a, 8a,
mechanism presented in Scheme 2. Reasonably, formation of 11a), whose increased lipophilicity (compared to benzyl azide)
the initial azide–heme complex (step ‘b’) is favoured for elec- is thus expected to favour binding to the hydrophobic active site
tron-rich alkyl azides, which is consistent with the generally of the H64V-containing Mb variants. This conclusion is
higher TON supported by Mb(H64V,V68A) for the oxidation of consistent with the larger heme spin shi for binding of
azide substrates containing electron-rich aryl substituents (e.g., 4-(triuoromethyl)benzyl amine to Mb as compared to the
4
a, 15a, 16a). To further examine this aspect, binding studies benzyl amine (Fig. 1c).
were carried out with Mb and different para-substituted benzyl a-Hydrogen deprotonation constitutes a rate-limiting step in
amines as unreactive mimics of the corresponding azides the enolization of ketones and aldehydes, as demonstrated by
2
2
(
Fig. 1c). Binding of the amine ligands to the heme cofactor in kinetic isotopic effect (KIE) experiments (k /k ¼ 4–6). By
H
D
Mb resulted in a red shi (Dlmax ¼ 2–3 nm) and decrease in analogy, the mechanism outlined in Scheme 2 would predict
intensity of the Soret band (410 nm) in the electronic absorption a decrease in rate upon H / D substitution of the a-hydrogen
Scheme 2 Proposed mechanism and catalytic steps for the myoglobin-catalyzed oxidation of alkyl azides to aldehydes (black). The pathway
leading to the minor alkyl amine byproduct is also shown (brown).
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1
8
in the azide substrate. To examine this aspect, a competition benzyl azide in the presence of O-labeled water (Fig. 2b).
1
8
experiment was performed using a mixture of benzyl azide 1 GC-MS analysis showed a degree of O incorporation into the
and its deuterated analog d2-1 in the presence of benzaldehyde product (44%) that closely matched the ratio of
1
8
Mb(H64V,V68A) as the catalyst (Fig. 2a). LC-MS analysis of this H2 O in the reaction mixture (50%) (ESI Fig. S5†), thus
ꢁ
reaction yielded a KIE value (k
H
/k
D
) of 1.7 ꢂ 0.3 at 22 C (ESI providing direct evidence for the occurrence of the hydrolysis
Fig. S4†), thus demonstrating that deprotonation of the aC–H step as part of the mechanism of the reaction. Combined
bond is part of the rate-determining step of the reaction. Inci- together, the KIE, isotopic labelling, and Mb binding experi-
dentally, this KIE value is comparable to that measured for the ments support the reaction pathway described in Scheme 2.
imine-to-aldimine tautomerization step implicated in the L-Ala Interestingly, this reaction sequence is fundamentally distinct
4
D-Ala isomerization reaction catalyzed by the PLP-dependent from that proposed for azide oxidation mediated by a molyb-
23
alanine racemase (k /kD ¼ 1.3–1.9). The mechanistic insights denum-based catalyst, which requires oxygen and purportedly
H
9a
gathered from the KIE studies suggested that increasing the involves an oxoaziridine and oxime intermediates.
acidity of the a-hydrogen would favour Mb-catalyzed azide As noted earlier, the reaction with benzyl azide also results
oxidation. To test this hypothesis, the oxidation activity of in the formation of 2b and 2c (Table 1) as minor byproducts
Mb(H64V,V68A) on (1-azidoethyl)benzene (14a) was compared (<5%). Small amounts (0.1–10%) of related amine and imine
with that observed in the presence of two related substrates, 20a species were observed also in the presence of the most reactive
and 21a (Scheme 3), in which the benzylic C–H site is rendered substrates described in Scheme 1. Whereas 2c can simply arise
more acidic because of the adjacent triuoromethyl and ester from the condensation of 2a with 2b, the benzylamine
group, respectively. In line with our expectations, three to four- byproduct is akin to the sulfonamide and carbamate products
fold higher TON were measured for the formation of 20b and observed during P450-catalyzed C–H amination with sulfonyl
7
h,7i,10
2
1b as compared to 14b (Scheme 3), thus providing further azides and carbonazidates.
support to the importance of the a-H deprotonation step in the we envision this species originates from overreduction and
Mb-catalyzed alkyl azide oxidation reaction. protonation of the putative imido-iron intermediate (step ‘h’,
In analogy to those reactions,
The step involving the release of the aldehyde product from Scheme 2). It is interesting to note, however, how this side
the aldimine intermediate (step ‘f’ or ‘g’ in Scheme 2) was then reaction is largely disfavored in the context of azide oxidation
probed by repeating the reaction with Mb(H64V,V68A) and as compared to these C–H amination processes. This differ-
ence can be attributed at least in part to the ease by which the
alkyl azide can undergo a-proton elimination as compared to
nitrene C–H insertion, as suggested by our KIE experiments
(
H D
i.e. k /k of 1.7 for azide oxidation as compared to 2.5–5 for
7
i,10
C–H amination ).
Conclusions
In conclusion, we report the rst example of a mild, bio-
catalytic method for the oxidative conversion of primary
azides to aldehydes. Using an engineered active site variant of
myoglobin as the catalyst, this valuable yet underdeveloped
transformation could be afforded with high efficiency and
selectivity across a broad range of aryl-substituted primary
azide substrates. Importantly, the catalytic turnovers sup-
ported by the Mb catalyst at room temperature and in buffer
exceed by orders of magnitudes those previously obtained
with synthetic catalysts in organic solvents and at high
temperature. Kinetic isotope effect, isotope labeling, and
substrate binding experiments support a mechanism that
involves heme-catalyzed decomposition of the alkyl azide
followed by a-hydrogen deprotonation to generate an aldi-
mine which, upon hydrolysis, gives the aldehyde product. As
suggested by the data presented in Scheme 3, these mecha-
nistic insights can prove valuable toward expanding the scope
of these Mb-based catalysts and guiding their further opti-
mization in the context of this and related reactions. Finally,
the Mb-dependent azide oxidase activity reported here
expands the range of synthetically valuable transformations
accessible through engineered and articial metalloprotein
Fig. 2 (a) Measurement of kinetic isotope effect (KIE) for H/D substi-
tution of aC–H bond on Mb(H64V,V68A)-catalyzed oxidation of
benzyl azide. See also ESI Fig. S4.† (b) Isotope labelling experiment
18
demonstrating O incorporation in the aldehyde product in the
18
presence of H
2
O-containing buffer. See also ESI Fig. S5.†
Scheme 3 Mb(H64V,V68A)-catalyzed oxidation of secondary alkyl
azides.
7
h,7j,11,24
catalysts.
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1
2 J. Vojtechovsky, K. Chu, J. Berendzen, R. M. Sweet and
I. Schlichting, Biophys. J., 1999, 77, 2153.
Acknowledgements
This work was supported by the U.S. National Institute of Health 13 (a) T. Higuchi, K. Shimada, N. Maruyama and M. Hirobe, J.
grant GM098628. MS instrumentation was supported by the
U.S. National Science Foundation grant CHE-0946653.
Am. Chem. Soc., 1993, 115, 7551; (b) Y. Urano, T. Higuchi,
M. Hirobe and T. Nagano, J. Am. Chem. Soc., 1997, 119,
12008; (c) A. Franke, N. Hessenauer-Ilicheva, D. Meyer,
G. Stochel, W. D. Woggon and R. van Eldik, J. Am. Chem.
Soc., 2006, 128, 13611; (d) T. Yamane, K. Makino,
N. Umezawa, N. Kato and T. Higuchi, Angew. Chem., Int.
Ed., 2008, 47, 6438.
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