Chemoselective, enzymatic C-H bond amination catalyzed by a cytochrome P450 containing an Ir(Me)-PIX cofactor

Article abstract of DOI:10.1021/jacs.6b11410

Cytochrome P450 enzymes have been engineered to catalyze abiological C-H bond amination reactions, but the yields of these reactions have been limited by low chemoselectivity for the amination of C-H bonds over competing reduction of the azide substrate t

Full text of DOI:10.1021/jacs.6b11410

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Communication
Chemoselective, Enzymatic C-H Bond Amination Catalyzed
by a Cytochrome P450 Containing an Ir(Me)-PIX Cofactor
Pawel Dydio, Hanna M. Key, Hiroki Hayashi, Douglas S. Clark, and John F. Hartwig
J. Am. Chem. Soc., Just Accepted Manuscript • Publication Date (Web): 12 Jan 2017
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Chemoselective, Enzymatic C-H Bond Amination Catalyzed
by a Cytochrome P450 Containing an Ir(Me)-PIX Cofactor
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‡
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‡1,2
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1,2
Paweł Dydio, Hanna M. Key, Hiroki Hayashi, Douglas S. Clark, John F. Hartwig*
1
Department of Chemistry, University of California, Berkeley, California 94720, USA.
2
Chemical Sciences Division, Lawrence Berkeley National Laboratory, 1 Cyclotron Road, Berkeley, California 94720,
USA.
3
Department of Chemical and Biomolecular Engineering, University of California, Berkeley, California 94720, USA.
4
Molecular Biophysics and Integrated Bioimaging Division, Lawrence Berkeley National Laboratory, 1 Cyclotron Road,
Berkeley, California 94720, USA.
‡
These authors contributed equally.
Supporting Information Placeholder
and product of overall reduction of the sulfonyl azides to
the sulfonamides. These side products form in comparable
to or greater yields than that of the products of the nitrene
ABSTRACT: Cytochrome P450 enzymes have been engi-
neered to catalyze abiological C-H bond amination reac-
tions, but this abiological process is limited by the low
chemoselectivity for the amination of C-H bonds over
competing reduction of the substrate to a sulfonamide. Here
we report that P450s derived from a thermophilic organism
and containing an iridium porphyrin cofactor (Ir(Me)-PIX)
in place of the heme catalyze C-H bond amination reactions
with high chemoselectivity for intramolecular insertion of
the resulting nitrenes into C-H bonds over reduction of the
azide to the sulfonamide and with broader substrate scope
than that of enzymes containing iron porphyrins. These
insertions into C-H bonds occur in yields up to 98% and
TON of ~300. In one case, the enantiomeric excess reaches
7-10
insertion into C-H bonds in all cases.
Recently, we described an efficient method for the re-
constitution of apo-(PIX)-proteins with various abiological
metal complexes of protoporphyrin and mesoporphyrin
1
1,12
IX.
The reactivity of these artificial PIX proteins is
distinct from that of the analogous Fe-containing heme
1
1,12
proteins, affording abiological reactivity.
We hypothe-
sized that some of the P450 proteins containing non-native
metals would catalyze the reaction of sulfonyl azides with
chemoselectivity for C-H bond amination over azide reduc-
tion that is higher than that of the P450s containing iron in
the active site. Many metal porphyrin complexes are re-
ported to catalyze the amination of C-H bonds with sul-
fonyl azides, although such reactions are often conducted
9
5:5 er, and the reactions can occur with divergent site
selectivity. The chemoselectivity for C-H bond amination
is greater than 20:1 in all cases. Variants of the Ir(Me)-PIX
CYP119 displaying these properties were identified rapidly
by evaluating Ir(Me)-PIX variants in cell lysates, rather
than as purified enzymes, accelerating the catalyst optimi-
zation. This study sets the stage to discover suitable en-
zymes to catalyze challenging C-H amination reactions.
1
3-15
above room temperature.
To enable the use of prospec-
tive artificial metalloenzymes at elevated temperatures, we
focused on the hybrid catalysts generated from the protein
12,16
CYP119,
which is a thermally stable P450 from the
archaeon Sulfolobus solfataricus.
Here we report that variants of CYP119 containing an
Ir(Me)-PIX cofactor catalyze the insertion of nitrenes into
C-H bonds with greater than 25 : 1 chemoselectivity for
insertion over reduction of the sulfonyl azide to the sulfon-
amide. These insertions into C-H bonds occur in yields up
to 98% and TON of ~300. In one case, the enantiomeric
excess reaches 95:5 er, and the reactions can occur with
divergent site selectivity. The high activity of Ir(Me)-PIX
CYP119 enzymes enables the formation of benzo-fused
sulfamates via C-H amination, which have not been formed
by Fe-PIX enzymes.
Cytochrome P450 enzymes (P450s) are native metallo-
proteins containing Fe-protoporphyrin IX (Fe-PIX) cofac-
tors that catalyze reactions including C-H bonds oxidations
1
,2
of a wide range of substrates with exquisite selectivities.
In contrast, natural enzymes that catalyze C-H bond amina-
tion reactions are rare, and they react with narrower scope
3
and lower selectivity.
Recent protein engineering and directed evolution of Fe-
PIX enzymes has created variants that catalyze the intramo-
lecular amination of C-H bonds with good enantioselectivi-
We commenced studies to create an artificial enzyme for
chemoselective C-H amination by assessing the reactivity
of a set of free metalloporphyrins IX (M-PIX) for the mod-
el reactions to convert sulfonyl azides 1 and 2 into sultams
4-6
ty, but the yields of the amination products are limited by
poor chemoselectivity. These reactions form mixtures of
products of insertion of the nitrene unit into the C-H bonds
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and 4 under aqueous conditions at room temperature (Fig
depicts the chemoselectivity for the formation of the C-H inser-
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tion products 3 and 4 over the reduction products 5 and 6 in the
presence of each metal complex under aqueous conditions at room
temperature. The bars reflect the molar ratio of the two products
1
). These model azides would undergo aminations of C-H
bonds catalyzed by metal-porphyrin complex by forming a
metal-nitrene complex and insertion of the nitrene unit into
either tertiary or secondary C-H bonds, respectively. The
metalloporphyrin PIX complexes containing Fe, Cu, and
Mn either did not react with the sulfonyl azides or reacted
with chemoselectivity that strongly favored formation of
sulfonamide products 5 and 6 over the sultam products 3
and 4. Reactions of sulfonyl azide 1 in the presence of the
metal-PIX complexes containing Co, Ru, or Rh preferen-
tially formed sultam 3 over the sulfonamide product 5 with
modest chemoselectivity. However, the reactions of sul-
fonyl azide 2, containing stronger, secondary C-H bonds,
catalyzed by the same complexes formed predominantly
sulfonamide 6 over sultam 4.
(
sultam and sulfonamide) formed, and a comparison of the out-
comes from insertions into a secondary C-H bond (substrate 1,
dark gray bars) and into a tertiary C-H bond (substrate 2, light
gray bars). *Reactions catalyzed by Cu and Mn porphyrins pro-
duced trace sulfonamide product and no observable sultam.
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Although porphyrins containing iridium have not been
reported to catalyze the insertion of nitrenes into C-H
1
7
bonds, we found that Ir(Me)-PIX is the most active and
the most chemoselective catalyst among the series of M-
PIX complexes we tested for the formation of products
from C-H amination under aqueous conditions at room
temperature. The selectivity of Ir(Me)-PIX for formation of
the sultam over the sulfonamide was > 10:1 for both sub-
strates (Fig 1). In the presence of Ir(Me)-PIX, substrate 1
reacted to form sultam 3 in 92% yield, with 14:1 selectivity
for formation of 3 over formation of sulfonamide 5. Under
otherwise identical conditions, substrate 2 containing less
secondary C-H bonds, which are less reactive than the
tertiary C-H bonds of 1, underwent the amination to form 4
in 72% yield, along with 6% of side-product 6. The reac-
tion of substrate 2 conducted at 37 °C occurred to form
sultam 4 in 89% yield and sulfonamide 6 in 8% yield.
Figure 2. Structure of WT Fe-CYP119 (prepared in Chimera
from PDB 1IO7). Left: Structure of Fe-CYP119. Right: Residues
targeted in the evolution of the protein scaffold to increase activi-
ty and selectivity in C-H insertion reactions.
On the basis of these results, we used Ir-PIX containing
P450s formed from CYP119, which was identified previ-
12
ously to be thermally stable, as catalysts for C-H amina-
tion reactions (Fig 2). To assess initially the ability of
Ir(Me)-PIX CYP119 variants to catalyze the insertion of
nitrenes into C-H bonds, we evaluated reactions of sul-
fonylazide 2 to form sultam 4 in the presence of the wild-
type Ir(Me)-PIX enzyme, the variant containing the single
mutation C317G to the axial ligand, and the variant
‘
CYP119-Max-L155G’ (Fig 3). CYP119-Max-L155G,
which contains 5 mutations (C317G, T213G, L69V,
V254L, L155G), was identified previously to be highly
Metal-porphyrin
(
0.17%)
O
S
O
O
O
S
O
12
1
00 mM NaPi
pH = 6.0, rt, 16 h
O
active for the insertion of carbenes into C-H bonds. Alt-
S
N3
NH2
+
+
hough the reactivity of the WT and C317G Ir(Me)-PIX
enzymes was low, the reaction catalyzed by the variant
CYP119-Max-L155G formed sultam 4 in high yield (98%
yield, 294 TON) with excellent chemoselectivity (<1%
yield of sulfonamide 6). This result demonstrates that
changing the metal site of an enzyme P450 from native iron
to the abiological iridium-methyl unit creates an active and
distinctly chemoselective catalyst for C-H amination.
NH
H
H
1
2
3
4
5
6
Metal-porphyrin
(
0.17%)
00 mM NaPi
pH = 6.0, rt, 16 h
O
S
O
O
S
O
O
1
O
S
N3
NH2
H
NH
H
O
S
O
O
O O
Ir(Me)CYP119 (0.33%)
CYP119MAX- L155G
O
S
S
N3
NH2
+
NH
H
H
100 mM NaPi, pH = 6.0
3
7 oC, 66 h
9
8% yield
294 TON
3 : 37 er
<1% yield
< 2 TON
6
Figure 3. C-H amination reaction catalyzed by a variant of
Ir(Me)-PIX CYP119 containing the following mutations: C317G,
L69V, T213G, V254L, L155G. Selectivity and yield determined
by SFC using an internal standard.
Figure 1. Reactions of sulfonyl azides 1 and 2 in the presence of a
set of [M]-PIX-IX complexes, the putative catalysts for C-H
amination reactions, forming the C-H insertion sultam products 3
and 4 and the sulfonamide reduction products 5 and 6. The figure
The variant CYP119-Max-L155G formed sultam 4 in
high yield (98% yield), but with a modest 63:37 enantio-
meric ratio (er). To improve the enantioselectivity of this
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The reaction of 2-propyl benzenesulfonyl azide (9) can
reaction, we created a library of plasmids encoding variants
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of CYP119 containing mutations at a subset of eight differ-
ent positions within the active site of the enzyme (Fig 2).
occur by insertion of the nitrene into the C-H bonds located
at the benzylic or homobenzylic positions to form either a
five- or a six-membered ring (10 and 11, respectively; Fig
11,12
In prior studies,
each variant of the Ir(Me)-PIX en-
4
). The free Ir(Me)-PIX cofactor catalyzes the reaction of 9
zyme was purified prior to evaluating its reactivity. To
accelerate the evaluation of mutant enzymes, we developed
an approach to conduct directed evolution of artificial heme
proteins that does not require the purification or concentra-
tion of the enzyme variants. By this approach, the apo form
of the CYP119 variants were overexpressed in E. coli.
After lysis of the cells, removal of the cell debris, and dial-
ysis into tris buffer, the Ir(Me)PIX cofactor was added to
the cell lysate. The resulting solutions containing unpuri-
fied, reconstituted Ir(Me)-CYP119 mutants were used in
catalytic experiments. By this protocol, we evaluated as
catalysts 142 different Ir(Me)-PIX CYP119 systems that
contained 2 to 4 mutations at the positions shown in Fig 2.
Many of the mutants evaluated were inactive; however,
several variants did form product 4 in an enantioselective
fashion (Table S5). Specifically, the mutant C317G,
T213G, V254L, F310L formed product 4 with 84:16 er,
while the mutant C317G, T213A, A152L formed the oppo-
site enantiomer of the product with 26:74 er.
to form a mixture of the five-membered product 10 and the
six-membered product 11 with a slight preference for for-
mation of 10 (60:40, 10 : 11). On the other hand, the vari-
ant C317G, T213G, V254L, F310L of Ir(Me)-PIX CYP119
catalyzes the same reaction of 9 with opposite site selectivi-
ty, forming preferentially sultam 11 over sultam 10 (20:80,
10 : 11), and with 84:16 er of product 11, while producing
less than 1% of the free sulfonamide byproduct. These
results show that directed evolution can create Ir(Me)-PIX
CYP119 catalysts for enantio-, chemo-, and site-selective
C-H amination. Moreover, this study shows that evaluating
variants of artificial metalloenzymes based on PIX-proteins
containing abiological metals can be accomplished rapidly
using cell lysates, without the need for protein purification
or concentration.
0
1
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0
To demonstrate further the potential of the Ir(Me)-PIX
artificial metalloenzymes to catalyze C-H amination, we
evaluated variants of Ir(Me)-PIX CYP119 as catalysts for
the reaction of a sulfamate motif (Fig 5). The aryl sulfa-
mates formed by the amination reaction can undergo sub-
sequent nickel-catalyzed cross-coupling reactions to form
The mutants that formed 4 with the highest enantiomeric
ratios by this protocol were subsequently evaluated as puri-
fied enzymes for the same reaction of 2 to form 4 and in the
reactions of similar sulfonyl azides to form products 7, 8,
and 11 (Fig 4). These experiments revealed that the mu-
tants which were most selective in the cell lysates were
equally selective when used as purified enzymes. In partic-
ular, the mutant C317G, T213G, V254L, F310G catalyzed
the formation of 4 with 84:16 er, 201 TON and 67% yield,
in a reaction conducted at 37 ºC with purified enzyme. The
same mutant also catalyzed the formation of sultams 7, 8,
and 11 in a highly enantioselective fashion, with up to 95:5
er, 192 TON and 64% yield. All reactions occurred with >
1
8,19
various chiral benzyl amines.
We found that the C-H
amination of 12 does not occur in the presence of the vari-
ants of Fe-P450-BM3, which were reported previously for
8
the C-H amination reactions. Moreover, the reaction of 12
in the presence of the free Ir(Me)-PIX cofactor formed only
7% of product 13 from nitrene insertion into the benzylic
C-H bond. However, an evaluation of 20 mutants that were
active or selective for the reaction of substrate 2 revealed
mutants that create more active catalysts. The variant
C317G, T213G, V254L formed cyclized product 13 in 84%
yield, 255 TON, 90:10 er, and >25:1 chemoselectivity for
C-H bond amination over reduction to the sulfonamide.
The reaction in the presence of the mutant C317G, T213G,
L69V occurred to form 13 in even higher enantioselectivity
(95: 5 er), although the yield of the reaction was lower
(10%). For comparison, the reaction of 14 catalyzed by
chiral rhodium catalysts typically used for enantioselective
C-H amination was reported to form product 13 in low
2
0:1 chemoselectivity for C-H amination over reduction to
the sulfonamide.
O
O
O
Et
O
O
O
O
Et
S
O
S
S
S
NH
NH
NH
NH
Et
n
R
4
7
8
10 (n = 0, R = Et)
2
0
11 (n = 1, R = Me)
yields and with low ee (up to 66: 34 er). By incorporating
the abiological Ir(Me)-PIX cofactor into CYP119, we have
created a catalyst that forms selectively a valuable class of
molecules that has not been created previously with any
natural enzymes and transition metal catalysts.
8
4: 16 er
95: 5 er
192 TON
84: 16 er
129 TON
1 : 4 (10 : 11)
84: 16 er (11)
201 TON
1
26 TON
Chemoselectivity Chemoselectivity Chemoselectivity
25 : 1 20 : 1 22 : 1
Chemoselectivity
> 25 : 1
>
Figure 4. Outcome of C-H insertion reactions forming sultams 4,
7, 8, and 11. Conditions: 0.33 mol% Ir(Me)-PIX CYP119 (muta-
tions C317G, T213G, V254L, F310G), 10 mM substrate, 0.5 mL
solvent (100 mM Na-Pi, 100 mM NaCl, pH = 6.0 containing 2
o
vol% DMF), 37 C and 66 h. Chemoselectivity refers to molar
ratio of sultam to sulfonamide products. Yield and TON refer to
the formation of the sultam product.
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O
O
Author Contributions
O
O
O
S
X
O
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
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4
4
4
4
4
4
4
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4
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5
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5
5
5
5
5
6
Catalyst
‡
S
These authors contributed equally.
NH
1
1
2: X = N3
4: X = NH2
13
Notes
J.F.H., H.M.K., P.F.D., and D.S.C. are inventors on PCT Applica-
tion No. PCT/US2016/057032, filed October 14, 2016, by the
Lawrence Berkeley National Laboratory, that covers preparation
and application of the artificial metalloenzymes containing iridi-
um-porphyrins in this paper.
201
220
Fe-P411-CIS-T438S:
No Reaction
12
Ir(Me)-CYP119
C317G, T213G, V254L
90: 10 er, 84% yield,
255 TON,
25 : 1 (13 : 14)
1
2
Ir(Me)-CYP119
C317G, T213G, L69V
95: 5 er, 10% yield,
30 TON
5 : 1 (13 : 14)
ACKNOWLEDGMENT
2
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
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0
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8
9
0
1
2
3
4
5
6
7
8
9
0
14
[Rh {(S)-nttl} ]:
66: 34 er, 38% yield,
1 TON20
The authors gratefully acknowledge Cynthia Mantassas for her
work on related studies that informed aspects of this publication.
This work was supported by the Director, Office of Science, of
the US Department of Energy under contract no. DE-AC02-
2
4
1
Figure 5. C-H amination of aryloxysulfonyl azides 12 to form
aryl sulfamate 13 in the presence of Ir(Me)- and Fe-enzymes and
a Rh-catalyst. The conditions with Ir(Me)CYP119 mutants are the
same as those in Fig 4. The conditions with Fe-P411-CIS mutant:
0.2% Fe-P411-CIS-T438S, 2 mM substrate, 2 mM Na S O , 1 mL
2 2 4
solvent (100 mM KPi, pH 8.0 containing 2.5 vol% DMSO). The
results for the [Rh] catalyzed reaction are those in ref 20.
0
5CH11231, by the NSF (graduate research fellowship to
H.M.K.), the NWO Netherlands Organization for Scientific Re-
search (Rubicon postdoctoral fellowship no. 680-50-1306 to
P.D.), and the Naito Foundation (postdoctoral fellowship to H.H.).
We thank the QB3 MacroLab facility at UC Berkeley (competent
cells) and the UC Berkeley DNA Sequencing Facility (plasmid
sequencing).
In summary, we have shown that Ir(Me)-PIX CYP119
enzymes catalyze C-H amination reactions with high
chemoselectivity for insertion of nitrenes over reduction to
the sulfonamide. Although Ir-containing porphyrins have
not been reported to catalyze C-H amination reactions,
Ir(Me)-PIX enzymes furnish sultams from sulfonyl azides
in high yields, high enantioselectivity and good turnover
numbers, while giving only traces of the sulfonamide by-
products typically observed in substantial amounts from the
reactions catalyzed by Fe-PIX enzymes. Variants display-
ing these favorable selectivities were identified rapidly by
screening mutants in cell lysates, instead of screening iso-
lated purified enzymes. Moreover, Ir(Me)-PIX CYP119
enzymes catalyze chemoselective C-H insertion reactions
of aryloxysulfonyl azides that do not form any C-H amina-
tion products in the presence of natural enzymes and form
the product with low yield and enantioselectivity with rho-
dium catalysts. Together, these results exemplify the merits
of incorporating unnatural metals into PIX enzymes in
order to achieve reaction outcomes previously not achieved
using natural enzymes.
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Corresponding Author
John F. Hartwig (jhartwig@berkeley.edu)
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