Directing group-controlled regioselectivity in an enzymatic C-H bond oxygenation

Article abstract of DOI:10.1021/ja5016052

Highly regioselective remote hydroxylation of a natural product scaffold is demonstrated by exploiting the anchoring mechanism of the biosynthetic P450 monooxygenase PikC_D50N-RhFRED. Previous studies have revealed structural and biochemical evidence for the role of a salt bridge between the desosamine N,N-dimethylamino functionality of the natural substrate YC-17 and carboxylate residues within the active site of the enzyme, and selectivity in subsequent C-H bond functionalization. In the present study, a substrate-engineering approach was conducted that involves replacing desosamine with varied synthetic N,N-dimethylamino anchoring groups. We then determined their ability to mediate enzymatic total turnover numbers approaching or exceeding that of the natural sugar, while enabling ready introduction and removal of these amino anchoring groups from the substrate. The data establish that the size, stereochemistry, and rigidity of the anchoring group influence the regioselectivity of enzymatic hydroxylation. The natural anchoring group desosamine affords a 1:1 mixture of regioisomers, while synthetic anchors shift YC-17 analogue C-10/C-12 hydroxylation from 20:1 to 1:4. The work demonstrates the utility of substrate engineering as an orthogonal approach to protein engineering for modulation of regioselective C-H functionalization in biocatalysis.

Full text of DOI:10.1021/ja5016052

Communication
pubs.acs.org/JACS
Directing Group-Controlled Regioselectivity in an Enzymatic C−H
Bond Oxygenation
Solymar Negretti,†,‡ Alison R. H. Narayan,§,‡ Karoline C. Chiou,⊥ Petrea M. Kells,# Jessica L. Stachowski,⊥
Douglas A. Hansen,† Larissa M. Podust,*,# John Montgomery,*,†,⊥ and David H. Sherman*,†,§,⊥,∥
§
⊥
∥
†Department of Medicinal Chemistry, Life Sciences Institute, Department of Chemistry, and Department of Microbiology &
Immunology, University of Michigan, Ann Arbor, Michigan 48109, United States
#Department of Pathology and Center for Discovery and Innovation in Parasitic Disease, University of California, San Francisco,
California 94158, United States
S
* Supporting Information
this challenge have included the utilization of supramolecular
ABSTRACT: Highly regioselective remote hydroxylation
of a natural product scaffold is demonstrated by exploiting
the anchoring mechanism of the biosynthetic P450
monooxygenase PikCD50N-RhFRED. Previous studies
have revealed structural and biochemical evidence for the
role of a salt bridge between the desosamine N,N-
dimethylamino functionality of the natural substrate YC-
17 and carboxylate residues within the active site of the
enzyme, and selectivity in subsequent C−H bond
functionalization. In the present study, a substrate-
engineering approach was conducted that involves
replacing desosamine with varied synthetic N,N-dimethyl-
amino anchoring groups. We then determined their ability
to mediate enzymatic total turnover numbers approaching
or exceeding that of the natural sugar, while enabling ready
introduction and removal of these amino anchoring groups
from the substrate. The data establish that the size,
stereochemistry, and rigidity of the anchoring group
influence the regioselectivity of enzymatic hydroxylation.
The natural anchoring group desosamine affords a 1:1
mixture of regioisomers, while synthetic anchors shift YC-
17 analogue C-10/C-12 hydroxylation from 20:1 to 1:4.
The work demonstrates the utility of substrate engineering
as an orthogonal approach to protein engineering for
modulation of regioselective C−H functionalization in
biocatalysis.
assemblies or specialized directing groups designed to reach
remote C−H bonds of the substrate;9−19 however, this type of
approach remains relatively unexplored.
In nature, C−H bonds can be oxygenated by enzymes that
have evolved for selectivity and efficiency. Previous work has
shown that the selectivity of wild-type monooxygenases can be
altered via directed evolution strategies.20 Typically, this protein-
engineering approach leads to mutants with altered regioselec-
tivity or variants that are specific for a new substrate. For example,
wild-type P450 BM3 hydroxylates straight chain fatty acids and
has been evolved to variants capable of oxidizing propane21 or
artemisinin.22 Alternatively, understanding the mechanism of
substrate binding for a particular enzyme enables substrate-
engineering strategies, in which the same enzyme can be used as
the catalyst for a wide range of substrates. Using these
approaches, biological catalysts provide an attractive opportunity
for regioselective oxidation of chemically similar bonds that are
remote from directing groups.
We were motivated to assess the utility of biochemically
characterized bacterial P450 monooxygenase PikC from the
pikromycin biosynthetic pathway as an oxidizing agent based on
its known function and mechanism of substrate recognition.23 In
nature, PikC is involved in the hydroxylation of the 12-
membered-ring macrolide YC-17 (1) and the 14-membered-
ring macrolide narbomycin (4) (Figure 1). Analysis of X-ray
cocrystal structures of PikC with its natural substrates revealed
largely nonspecific hydrophobic interactions between the
macrolactone substrates and active-site residues. This mode of
binding was leveraged in a previous study that demonstrated the
ability of PikC to hydroxylate a range of substrates with an
appended desosamine sugar (see 6 → 7, Figure 1c).24
he regioselective oxidation of unactivated C−H bonds in
T
complex molecules remains a substantial synthetic
challenge.1−6 In the absence of a covalently installed directing
group, the intrinsic influences of the substrate play the major role
in governing regioselectivity of oxygenation. In contrast, directed
methods are highly effective at overcoming the inherent
reactivity of activated C−H bonds by relying on a specific
binding event between the directing group and catalyst that often
leads to functionalization of a C−H bond proximal to the
directing group. Directed processes of this type typically involve a
5- or 6-membered transition state for the interaction of the metal
with the reactive C−H bond.5,7,8 Functionalization of a region
within a molecule that is remote to a directing group, while
overriding the inherent reactivity of specific bonds, is not
possible with these well-studied methods. Strategies to address
While desosamine proved to be an effective anchoring group
for unnatural substrates, several practical challenges limit its
utility.25 We anticipated that a simplified anchoring group
capable of active-site salt bridge formation could lead to effective
PikC-catalyzed C−H bond functionalization. Herein, we report
in vitro implementation of a substrate-engineering approach that
replaces desosamine with a range of more accessible anchoring
Received: February 14, 2014
Published: March 14, 2014
© 2014 American Chemical Society
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Scheme 1. Hydroxylation of YC-17 Analogues Possessing
a
Synthetic Anchoring Groups
Figure 1. Reactions of PikC.
moieties and enables tuning the regioselectivity of C−H bond
oxygenation with PikCD50N-RhFRED.
To gain insight on the factors required for an effective
anchoring group, several YC-17 (1) analogues were synthesized
in which the natural anchoring group, desosamine, was replaced
by various tertiary-amine-containing elements (Scheme 1). For
ease of synthesis and removal, synthetic anchoring groups were
appended to the C-3 hydroxyl group of the YC-17 aglycone
through an ester linkage. These YC-17 analogues with linear
anchors (8a, 8b, and 10) were reacted with PikCD50N-RhFRED
in the presence of an NADPH regeneration system to afford
monohydroxylated products. The three-carbon anchoring group
(see 8b) led to greater than 99% conversion of the starting
material to monohydroxylated products, while anchors one
carbon shorter (8a) or longer (10) resulted in slightly
diminished conversions of 80% and 91%, respectively. The
total turnover numbers (TTNs, no. of moles of product/no. of
moles of enzyme) for 8a, 8b, and 10 were 260, 544, and 452,
lower than the TTN of 896 for the natural substrate (1) of PikC.
The binding affinities for 8a, 8b, and 10 also suggest that 8b is a
superior substrate compared to 8a or 10, with Kd = 81 μM,
approximately 4 times greater than the Kd for natural substrate
YC-17 (1). To gauge the importance of the amine moiety within
the synthetic anchoring groups, the dimethylamino group in 8a
and 8b was replaced with an isopropyl group.26 Consistent with
PikC E94 mutant studies previously reported,27 these substrates
were unable to participate in salt bridge formation and were not
hydroxylated.
In addition to achieving reactivity of PikC with YC-17
analogues, we envisioned that synthetic anchoring groups would
be able to control the regioselectivity of C−H bond oxygenation
in a macrolactone system. PikC hydroxylates its natural substrate,
YC-17 (1), at C-10 or C-12 to produce a 1:1 mixture of
methymycin (2) and neomethymycin (3). We sought to identify
unnatural anchors that could favor the production of
methymycin (2) or neomethymycin (3) analogues. The 3-
(dimethylamino)propanoate derivative 8b displayed an altered
regioselectivity compared to the enzyme’s natural substrate, YC-
17 (1), with C-12 oxygenation favored over hydroxylation of the
allylic C-10 position (1:3).28 Shortening the linker by one
methylene unit, N,N-dimethylamino ethanoate derivative 8a
afforded a 1:1.6 ratio of C-10 and C-12 hydroxylated products.
By contrast, extending the linker by one methylene unit,
oxygenation of N,N-dimethylamino butanoate analogue 10
a
Reaction conditions: 5 μM PikCD50N-RhFRED, 1 mM substrate, 1
mM NADP+, 1 U/mL glucose-6-phosphate dehydrogenase, and 5 mM
glucose-6-phosphate for NADPH regeneration in 50 μL of reaction
buffer (50 mM NaH2PO4, pH 7.3, 1 mM EDTA, 0.2 mM
dithioerythritol, and 10% glycerol). Percent conversion was assessed
by loss of starting material (measured by LCMS, quantified by
comparison to standard curve). Isolated yield from preparative-scale
reactions shown in parentheses. Product ratios are given as C10:C12
hydroxylation, and the major product is illustrated.
favored hydroxylation at the allylic C-10 position (1.8:1).
These results demonstrated that anchor length had a modest
effect on the regioselectivity of the oxidation of distal C−H
bonds.
Based on results of the linear anchoring groups, we considered
that more structurally rigid forms might lead to superior substrate
binding and selectivity. Thus, N-methylproline anchoring groups
(see 12a and 12b) were assessed for their reactivity with
PikCD50N-RhFRED. While we envisioned one proline enan-
tiomer would correspond to a matched case with the catalyst and
the opposite enantiomer would be mismatched, both D- and L-
proline derivatives performed equally well as anchoring groups.
Compared to the two-carbon linear anchor (8a) that places the
dimethylamino group the same number of bonds away from the
macrolactone core, proline compounds 12a and 12b possessed
superior TTNs of 456 and 485 and lower Kd values of 47 and 28
μM, respectively. Regioselectivity of 10-dml C−H bond
oxygenation was also influenced by more-rigid cyclic anchoring
groups. 10-dml derivatives containing both enantiomers of N-
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Communication
methylproline (12a and 12b) resulted in preferential C-10
hydroxylation by PikC in contrast to substrate 8a.
Table 1. TTNs and Kd Values of Unnatural Substrates with
PikCD50N and PikCD50NH238A Mutants
A second class of cyclic anchors that employed benzylic amines
as anchoring groups was also investigated (14, 16a, and 16b).
Using 0.5 mol % enzyme, 14, 16a, and 16b were converted to
monohydroxylated products, and TTNs for these aryl substrates
were measured at 0.1 mol % enzyme. The ortho derivative (14)
displayed a low TTN of 152, while the para and meta derivatives
(16a and 16b) displayed greater TTNs than the linear anchors
(602 and 580, respectively).
a
TTN
D50NH238A
Kd (μM)
substrate
D50N
D50N
D50NH238A
1
896 0.71
260 11
544 23
452 5.0
456 5.0
485 21
152 11
602 3.5
580 11
842 16
572 18
870 38
853 38
542 30
538 20
491 26
816 33
922 35
19
nd
8a
118 21
81 15
123 14
47 3.0
28 5.4
nd
11 0.56
161 25
168 19
19 1.3
21 0.70
nd
8b
10
12a
12b
14
A significant improvement in regioselectivity was observed
with benzylic amines. Ortho derivative 14, which places the
anchoring amine group the same number of carbon atoms away
from the macrolactone core as 10, afforded a reversal of
regioselectivity compared to 10 to favor C-12 hydroxylation.
Increasing the distance between the ester carbonyl and the
terminal N,N-dimethylamino group with para and meta
derivatives 16a and 16b led to a highly regioselective allylic
hydroxylation at C-10 with up to 20:1 selectivity for meta
derivative 16b. We hypothesize that the more rigid anchors
restrict the conformational freedom of the substrate in the active
site, thus enhancing the selectivity of the enzymatic reaction.
To establish the substrate binding orientation within the active
site, X-ray structures of catalytically competent compounds 8a
and 8b bound within the active site of PikCD50N were obtained.
The salt-bridge contact between the N,N-dimethylamino group
of the synthetic anchor and E94 was preserved in these
complexes as in natural substrate binding (Figure 2). Addition-
ally, a new hydrogen-bonding interaction between H238 and the
ester carbonyl group of the anchor emerged in the active site for
both 8a and 8b.
16a
16b
nd
nd
nd
nd
a
The measurement of Kd values for the benzylic amine substrates was
inhibited by the background absorbance of these compounds; thus,
dissociation constants were not obtained for 14, 16a, and 16b.
As a result, the regioselectivity of the PikC C−H bond
oxygenation can be controlled by the structure of the anchoring
group. In contrast to typical synthetic C−H bond activation
strategies that leverage proximal groups or differing C−H bond
strengths, our substrate-engineering approach utilizes removable
anchoring groups to achieve regioselective oxidation of remote
sites within a molecule. The size and rigidity of the anchoring
group correspond to significant changes in regioselectivity of the
C−H bond hydroxylation. This focused study demonstrates the
potential of this strategy as an effective means of expanding the
substrate scope of the PikC monooxygenase. By this approach,
regions of substrates were oxidized distal to the alkyl amino
functionality, rendering the regioselectivity patterns orthogonal
to those achieved by either directed or nondirected small-
molecule oxidation catalysts.1−11 Based on X-ray structure
analysis, substrate orientation within the PikC active site was
controlled by the electrostatic interaction of the N,N-
dimethylamino moiety with the E94 carboxylate group.
Substrate engineering has provided new insights into the
structural parameters that govern productive substrate binding
and conversion of potential P450 substrates. It also provides new
information toward developing a general method for regio- and
stereoselective oxidation of highly complex molecules, including
secondary metabolites. There is a long-standing demand for
enzymes with improved activity and substrate tolerance that can
be applied toward chemoenzymatic synthesis of novel
therapeutics, materials, fuels, and commodity chemicals.
Bacterial biosynthetic P450s offer a unique source of biocatalysts
for the selective oxidation of C−H bonds for a broad range of
substrate classes. Efforts to improve the substrate scope,
regioselectivity, and yields through substrate and protein
engineering are underway.
Figure 2. Active site of PikCD50N with 8a (A) and 8b (B) bound.
Substrates are in yellow, heme in gray, and amino acid residues in pink
sticks; fragments of protein structure are shown as green ribbon.
Distances are in angstroms.
To test impact of the H-bond between H238 and the
anchoring group carbonyl on substrate binding, the double
mutant PikCD50N/H238A-RhFRED was generated. With the
D50N/H238A mutant, TTN for the natural substrate YC-17
(1) was within experimental error compared to that of the D50N
mutant (896 vs 842), whereas substrates containing unnatural
anchoring groups were hydroxylated by the double mutant with
higher TTNs (Table 1). While the TTNs were uniformly
affected by the new mutation, the dissociation constants for 8a,
12a, and 12b decreased, indicating improved binding. In
contrast, the Kd values for 8b and 10 increased, suggesting lack
of benefit from the H-bond for 8b and 10 analogues.
ASSOCIATED CONTENT
* Supporting Information
Experimental details and characterization data. This material is
available free of charge via the Internet at http://pubs.acs.org.
■
S
AUTHOR INFORMATION
■
Corresponding Authors
larissa.podust@ucsf.edu
jmontg@umich.edu
davidhs@umich.edu
In the current study, the inherent substrate flexibility of PikC
was exploited to expand substrate scope using readily available
and easily manipulated desosamine mimics as anchoring groups.
Author Contributions
‡S.N. and A.R.H.N. contributed equally.
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(27) Sherman, D. H.; Li, S.; Yermalitskaya, L. V.; Kim, Y.; Smith, J. A.;
Waterman, M. R.; Podust, L. M. J. Biol. Chem. 2006, 281, 26289.
(28) Borisova, S. A.; Zhao, L.; Sherman, D. H.; Liu, H.-w. Org. Lett.
1999, 1, 133.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
This work was supported by NIH grant GM078553 and the H.
W. Vahlteich Professorship (D.H.S.). S.N. acknowledges training
grant support from the University of Michigan Chemistry-
Biology Interface (CBI) training program (NIH grant
5T32GM008597-14). A.R.H.N. acknowledges the Life Sciences
Research Foundation for a postdoctoral fellowship. K.C.C.
acknowledges training grant support from the Pharmacological
Sciences Training Program (NIH grant GM007767). The
Advanced Light Source is supported by the Director, Office of
Science, Office of Basic Energy Sciences, of the U.S. Department
of Energy under Contract No. DE-AC02-05CH11231. The
authors thank Dr. Shengying Li for helpful discussions and the
staff members of beamline 8.3.1, James Holton, George Meigs,
and Jane Tanamachi, at the Advanced Light Source at Lawrence
Berkeley National Laboratory for assistance with data collection.
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