Biotinylated Rh(III) complexes in engineered streptavidin for accelerated asymmetric C-H activation

Article abstract of DOI:10.1126/science.1226132

Enzymes provide an exquisitely tailored chiral environment to foster high catalytic activities and selectivities, but their native structures are optimized for very specific biochemical transformations. Designing a protein to accommodate a non-native transition metal complex can broaden the scope of enzymatic transformations while raising the activity and selectivity of small-molecule catalysis. Here, we report the creation of a bifunctional artificial metalloenzyme in which a glutamic acid or aspartic acid residue engineered into streptavidin acts in concert with a docked biotinylated rhodium(III) complex to enable catalytic asymmetric carbon-hydrogen (C-H) activation. The coupling of benzamides and alkenes to access dihydroisoquinolones proceeds with up to nearly a 100-fold rate acceleration compared with the activity of the isolated rhodium complex and enantiomeric ratios as high as 93:7.

Full text of DOI:10.1126/science.1226132

Biotinylated Rh(III) Complexes in Engineered Streptavidin for
Accelerated Asymmetric C−H Activation
Todd K. Hyster et al.
Science 338, 500 (2012);
DOI: 10.1126/science.1226132
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References and Notes
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(even noisy ones) where Takens’ theorem applies
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for membership to a common dynamical system.
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it is not surprising that as a further check, the GC
calculations for all the model and real data exam-
ples considered in this work were largely unsuc-
cessful (table S2 and GC calculations S1 to S5).
Although many empirical measures of species
interactions exist (e.g., inferring interaction proxies
from diet matrices), we suggest that causation in-
ferred from time-series information provides a
“bottom-line” picture of interactions that is more
direct than those possible with proxies. The ability
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Acknowledgments: We thank S. Sandin, L. Kaufman, A. MacCall,
J. Melack, J. Schnute, L. Richards, A. Rosenberg, M. Kumagai,
H. Liu, I. Altman, B. Fissel, S. Glaser, B. Carr, E. Klein, L. Storch,
S. Kealhofer, M. Kogler, and C. Perretti for their comments
and assistance with this work, and P. Sugihara for developing
and producing the initial animations for movies S1 and S2.
Supported by NSF grant DEB-1020372, NSF/NOAA CAMEO Award
NA08OAR4320894, the McQuown Chair in Natural Science,
the Sugihara Family Trust, and Quantitative Advisors (http://
qadvisors.com) (G.S.); National Taiwan University and the National
Science Council of Taiwan (C.H.); NSF graduate research fellowships
(H.Y. and E.D.); and an EPA STAR graduate fellowship (E.D.).
Supplementary Materials
www.sciencemag.org/cgi/content/full/science.1227079/DC1
Materials and Methods
Figs. S1 to S5
Tables S1 to S26
Box S1
GC Calculations S1 to S5
References (35–76)
Movies S1 to S3
6 July 2012; accepted 4 September 2012
Published online 20 September 2012;
10.1126/science.1227079
REPORTS
alytic scaffold remains a major challenge (2–5).
One class of strategies has relied on the incor-
poration of non-natural metal cofactors within
a protein scaffold to afford artificial metalloen-
zymes (6–9). The main focus in the area has been
improving the selectivity of the hybrid catalysts,
rather than reaction rates, which are, by and large,
dictated by the first coordination sphere interac-
tions around the metal (10–12). Among the var-
ious cofactor localization strategies (13, 14), the
biotin-(strept)avidin technology has proven ver-
satile: The geometry of the biotin-binding pocket
is ideally suited to accommodate organometallic
moieties, leaving enough room for substrate bind-
ing and activation (15–19).
Biotinylated Rh(III) Complexes in
Engineered Streptavidin for Accelerated
Asymmetric C–H Activation
Todd K. Hyster,1,2 Livia Knörr,2 Thomas R. Ward,2* Tomislav Rovis1*
Enzymes provide an exquisitely tailored chiral environment to foster high catalytic activities and
selectivities, but their native structures are optimized for very specific biochemical transformations.
Designing a protein to accommodate a non-native transition metal complex can broaden the scope of
enzymatic transformations while raising the activity and selectivity of small-molecule catalysis. Here, we
report the creation of a bifunctional artificial metalloenzyme in which a glutamic acid or aspartic acid
residue engineered into streptavidin acts in concert with a docked biotinylated rhodium(III) complex to
enable catalytic asymmetric carbon-hydrogen (C–H) activation. The coupling of benzamides and alkenes
to access dihydroisoquinolones proceeds with up to nearly a 100-fold rate acceleration compared with the
activity of the isolated rhodium complex and enantiomeric ratios as high as 93:7.
1Department of Chemistry, Colorado State University, Fort
Collins, CO 80523, USA. 2Department of Chemistry, Uni-
versity of Basel, Basel CH-4056, Switzerland.
hanks to the advent of genetic engineer- displacing established organometal-catalyzed
ing, enzymes are attracting increasing at- industrial processes (1). However, creating an
*To whom correspondence should be addressed. E-mail:
rovis@lamar.colostate.edu (T.R.); thomas.ward@unibas.ch
T
tention as versatile synthetic tools, even enzyme for an abiotic reaction from a noncat- (T.R.W.)
500
26 OCTOBER 2012 VOL 338 SCIENCE www.sciencemag.org

REPORTS
In recent years, [Cp*RhCl2]2, where Cp* is pen- we were pleased to find that the reaction pro- ity of the catalyst (Table 1, entry 5). Introduction
tamethylcyclopentadienyl, has emerged as a ceeds to completion in a 4:1 mixture of H2O/MeOH of a glutamate residue at position 121 (i.e.,
versatile catalyst for electrophilic aromatic C–H under basic conditions (200 mole % CsOAc), de- K121E) again gives low conversion (Table 1, en-
activation reactions (20). Elegant work by the spite the sparing solubility of the substrates in this try 7). Mutation to an aspartate at position 121
groups of Fagnou and Glorius showed that solvent mixture. Next, we designed a biotinylated (i.e., K121D) improved the conversion to 89%
pivaloyl-protected benzhydroxamic acids may analog [RhCp*biotinCl2]2 (26) for incorporation after 72 hours (Table 1, entry 8). To confirm that
be efficiently coupled with alkenes to access within streptavidin (Sav) (Fig. 1). Two equivalents this increase in activity was indeed caused by
dihydroisoquinolones in good yield at room tem- of [RhCp*biotinCl2]2 were required to displace the presence of a carboxylate residue, we intro-
perature (21, 22). An exogeneous base is required weakly bound 2-(4-hydroxyphenylazo)benzoic duced an asparagine residue (i.e., K121N). As-
for the orthometallation step (23). Computations acid (HABA) in tetrameric streptavidin (27). This paragine is sterically and electronically similar
suggest that the C–H activation process occurs suggests that the dimeric catalyst precursor dis- to aspartic acid but lacks the ability to facil-
via a concerted metalation-deprotonation (CMD) sociates in aqueous solution to RhCp*biotinCl2(H2O) itate the critical C–H activation step. As antici-
mechanism (24). The presence of a base signif- and that the four biotin-binding sites of Sav can pated, K121N gave low conversion after 36 hours
icantly lowers the activation energy of this step. be loaded with the monomeric biotinylated cat- (Table 1, entry 9). The data thus suggest that the
As three coordination sites are required around alyst precursor (28).
reaction is critically dependent on the precise
the Cp*Rh moiety for catalysis (25), it has been When we combined benzhydroxamic acid 1a localization of a carboxylate residue provided
difficult to introduce an asymmetric ligand, and with 1.1 equivalents of methyl acrylate 2a in a 4:1 by Sav. We speculated that if the position of
no enantioselective version of this attractive mixture of H2O:MeOH in the presence of tetra- the carboxylate residue at position 121 could be
benzannulation reaction has been reported thus meric wild-type (WT) Sav and [RhCp*biotinCl2]2, further fine-tuned, increased conversions might
far. In a biomimetic spirit, we hypothesized that only a trace amount of product was observed result. This was realized upon combining a glu-
incorporation of a biotinylated [Cp*RhX2]2 com- after 36 hours at room temperature (Table 1, tamate at position 121 with a lysine at position
bined with an engineered aspartate or glutamate entry 3). To increase conversion, we introduced 118 (i.e., N118K-K121E). In addition to increased
residue might yield an asymmetric catalyst for a basic residue in the proximity of the rhodium activity, this catalyst also gave enhanced levels
the production of enantioenriched dihydroisoqui- moiety. As highlighted in an Auto-Dock (29) mod- of regioselectivity for alkene insertion (15:1)
nolones (Fig. 1).
eling study (Fig. 1D), residues S112A and K121B by comparison to the reaction in the absence
We initially examined the viability of the (of the adjacent Sav monomer B) lie closest to of protein (4:1) (compare Table 1, entry 1 versus
[RhCp*Cl2]2-catalyzed reaction between pivaloyl- the metal center upon incorporation within WT entry 10).
protected benzhydroxamic acid (1a) and methyl Sav. We thus introduced by site-directed muta-
With a mutant exhibiting superior activity in
acrylate (2a) to dihydroisoquinolone (3a) under genesis a basic residue at either of these positions. hand, we were eager to determine whether the
aqueous conditions. Although this reaction is The presence of a glutamate residue at position transformation could be rendered asymmetric
typically performed in MeOH or EtOH (22, 23), 112 (S112E) has a marginal effect on the activ- thanks to the chiral nature of the active site. A
A
B
D
Active Site
Tailoring
Base
Metal
Asn 118A
Metal
Minimally Active
Artificial
Metalloenzyme
Highly Active
Artificial
Metalloenzyme
Lys 121A
Ser 112A
O
O
[RhCp*biotinCl2]2
Streptavidin
with engineered
carboxylate
OPiv
N
NH
+
H
R
R
H
N
Me
S
H
Me
Me
Me
Rh
O
HN
H
NH
Ser 112B
[RhCp*biotinCl2]2
Cl
Cl
O
2
Lys 121B
C
BiotinNH
Sav
1-2
Cp*
H
O
Rh
O
N
OPv
Asn 118B
O
Fig. 1. (A) Synergistic action of a basic residue introduced by site-directed Postulated transition state for the C–H activation step. (D) Auto-Dock model
mutagenesis and a biotinylated RhCp*biotinCl2 moiety acting as catalyst for of biotinylated RhCpbiotin(OAc)2 complex anchored in the proposed active site
an abiotic reaction. (B) Benzannulation reaction catalyzed by the artificial me- of the streptavidin tetramer with key residues highlighted (adjacent complex
talloenzyme for the synthesis of enantioenriched dihydroisoquinolones. (C) in Sav monomer B omitted for clarity).
www.sciencemag.org SCIENCE VOL 338 26 OCTOBER 2012
501

REPORTS
survey of our arsenal of Sav mutants revealed
Table 1. Optimization of the performance of the artificial benzannulase.
that the mutant with superior activity was also
reasonably enantioselective (Table 1, entry 10).
Because position 121 was identified as the best
location for the carboxylate residue, we focused
on position 112 to modify the chiral environ-
ment. A screen of nine mutants at position 112
using an acetate buffer revealed that aromatic
residues gave superior reactivity by comparison
to nonaromatic residues (30 to 50% yield), as
well as enhanced enantioselectivity (Table 1,
entries 12 to 20). Prolonged reaction times were
required to achieve enhanced conversion. The
best mutant under our conditions proved to be
S112Y with 30% yield, 12:1 regioselectivity,
and 88:12 enantiomeric ratio (er) (Table 1, entry
19). We hypothesized that if the superior ac-
tivity imparted by the carboxylate mutation at
position 121 could be combined in a synergistic
way with the improved selectivity provided by
S112Y, a highly active and selective artificial
metalloenzyme might result (30). The use of
S112Y-K121D gave good er but only in fair yield
(Table 1, entry 21). Substituting the aspartic acid
residue with glutamic acid resulted in a su-
perior mutant that gave 80% yield with 20:1
regiomeric ratio and 90:10 enantiomeric ratio
(Table 1, entry 22). The yield and er could be
further improved by exchanging H2O with 3-(N-
morpholino)propanesulfonic acid buffer to yield
the desired product in 95% yield, 19:1 regioiso-
meric ratio (rr), and 91:9 er (Table 1, entry 23).
This transformation proved applicable to a
variety of substrates (Fig. 2). Ethyl vinyl ketone
was highly reactive under the reaction conditions,
resulting in benzannulated product in good yield,
high regioselectivity, but poor enantioselectivity.
When methyl acrylate was replaced with benzyl
acrylate, yield and enantioselectivity were di-
minished. Substitution on the benzamide was better
tolerated under the reaction conditions. Bromo-
substituted and naphthyl benzamides delivered
product in good yield, with a modest erosion of
er in comparison to the parent system. The enan-
tioselectivity increased with para-substituted nitro-
benzamides, albeit at the expense of lower yield.
The remaining mass balance is represented by
unreacted starting material.
O
O
[RhCp*biotinCl2]2 (1 mol %)
O
CO2Me
Sav Mutant (0.66 mol %)
OPiv
NH
CO2Me
NH
N
H
Solvent/MeOH (4:1)
1a
2a
3a
3a'
23 °C, 72h
CO2Me
major
minor
regioisomeric
ratio (rr)
enantiomeric
ratio (er)
entry
Sav Mutant
Solvent
yield (%)*
-
-
80
< 5%
< 5%
46
10
< 5%
7
1
2
4:1
-
51.5:48.5
-
Acetate Buffer
H2O
WT†
WT†
H2O
3
-
-
4
9:1
15:1
-
75:25
78:22
-
Acetate Buffer
H2O
S112E†
5
S112D†
H2O
6
K121E†
H2O
7
15:1
15:1
-
78:22
78:22
-
89
< 5%
99
8
8
K121D
H2O
K121N†
H2O
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
N118K-K121E
N118K-K121E†,
S112A
15:1
6:1
6:1
10:1
6:1
6:1
6:1
6:1
5:1
12:1
12:1
20:1
20:1
19:1
82:18
52:48
75:25
71:29
86:14
76:24
81:19
79:21
81:19
88:12
86:14
90:10
90:10
91:9
H2O
‡
H2O
12
6
Acetate Buffer
Acetate Buffer
Acetate Buffer
Acetate Buffer
Acetate Buffer
Acetate Buffer
Acetate Buffer
Acetate Buffer
Acetate Buffer
H2O
S112C
S112F
50
6
S112K
S112M
1
35
4
S112T
S112V
30
32
30
80
95
S112Y
S112W
S112Y-K121D
S112Y-K121E
S112Y-K121E
H2O
MOPS Buffer
*Yield determined by gas chromatography integration.
†Reaction conducted for 36 hours.
‡Sav preloaded with excess biotin for 10 min and then treated with [RhCp*biotinCl ] .
2 2
[RhCp*biotinCl2]2 (1 mol %)
O
O
S112Y-K121E (0.66 mol %)
R
yield (%)
r.r.
OPiv
N
NH
H
MOPS Buffer/MeOH (4:1)
23 °C, 72h
e.r.
R
R'
R'
1a-d
2a-c
3b-f
The data presented above argue for the syn-
ergistic action of both the carboxylate side
chain and the chiral cavity inside the metallo-
enzyme for optimal reactivity and selectivity.
We sought further support for the role of the
critical basic amino acid residue by conduct-
ing mechanistic studies. Using the monodeu-
terated benzamide d1-1a, we observed a kinetic
isotope effect (kH/kD) (KIE) value of 3.8 for the
reaction with [RhCp*biotinCl2]2 under buffered
conditions [1:4 MeOH:acetate buffer (pH = 5.9,
0.69 M) for the internal competition KIE study]
(figs. S1 to S3). In the presence of WT Sav un-
der identical conditions, a KIE value of 2.8 was
obtained. With the most active mutant, N118K-
K121E, a KIE value of 4.8 was found under
O
O
O
NH
NH
NH
OMe
Et
OBn
Br
O
95% yield
O
64% yield
14:1
61% yield
15:1
73:27
O
3c
10:1
3d
3b
56:44
88:12
O
O
NH
NH
OMe
OMe
O2N
30% yield
32:1
80% yield
22:1
O
O
3e
3f
93:7
89:11
acetate-free conditions (4:1 H2O:MeOH). These Fig. 2. Substrate scope.
502
26 OCTOBER 2012 VOL 338 SCIENCE www.sciencemag.org

REPORTS
O
[RhCp*biotinCl2]2 (1 mol %)
O
CO2Me
Sav Mutant (0.66 mol %)
OPiv
NH
CO2Me
N
H
H2O/MeOH (4:1)
23 °C, 24h
1a
2a
3a
wild-type
N118K-K121E
S112Y-K121E
A
B
C
100
90
80
70
60
50
100
100
90
80
70
60
50
90
80
70
60
50
bound - 3x more reactive
bound - 92x more reactive
bound - 34x more reactive
0
2
4
6
8
10
12
0
2
4
6
8
10
12
0
2
4
6
8
10
12
equivalents of metal monomer
Fig. 3. Determination of the relative catalytic rates of free and Sav-bound catalysts using (A) WT Sav, (B) N118K-K121E, and (C) S112Y-K121E. Blue crosses,
experimentally determined er; orange line, predicted er in the case of no protein rate acceleration [i.e., krel = (kbound)/(kfree) = 1]; green line, fitted er allowing
the determination of krel.
16. C.-C. Lin, C.-W. Lin, A. S. C. Chan, Tetrahedron
results suggest a primary kinetic isotope effect eight equivalents, mbound = mfree = 4); %(R)bound
in all cases. Subtle differences in the geometry of and %(R)free are the %(R) for the Sav-bound and
Asymmetry 10, 1887 (1999).
17. M. T. Reetz, J. J. P. Peyralans, A. Maichele, Y. Fu,
the CMD mechanism (Fig. 1C) is a likely source the free catalyst (i.e., %(R)free = 0.515, Table 1,
M. Maywald, Chem. Commun. 41, 4318 (2006).
of the slightly different KIE values.
entry 1). Using Eq. 1 and performing a least-square
18. M. Skander et al., J. Am. Chem. Soc. 126, 14411
(2004).
To provide further support for the critical minimization on the calculated and experimen-
role of the carboxylate residue within the en- tally determined %(R) (green line and blue crosses
zyme’s active site, we conducted competition respectively, in Fig. 3) (also see table S1), we
experiments between protein-bound and free can determine the relative rates krel = (kbound)/
[RhCp*biotinCl2]2 catalyst precursors. The very (kfree). For WT Sav, we compute a 3-fold rate
limited solubility of the starting material precludes acceleration compared with the protein-free cat-
the use of classical Michaelis-Menten kinetic ex- alyst. This phenomenon of protein acceleration
periments. Because the free biotinylated catalyst is enhanced in the presence of carboxylate-bearing
leads to nearly racemic product (51.5:48.5 er) Sav isoforms: In pure water and for N118K-K121E
(Table 1, entry 1), whereas the Sav-bound cat- and S112Y-K121E, we compute rate accelerations
alyst leads to enantioenriched product, a com- of 92 and 34, respectively. The substantially in-
parison of the er as a function of the number of creased rate is diagnostic of the key role of the
equivalents of RhCp*biotinCl2(H2O) versus tetra- active-site carboxylate in the turnover-limiting step,
meric Sav provides an estimate of the relative which we suggest is the C–H activation event.
rates of the protein-bound (kbound) and the free This confirms the hypothesis that the engineered
biotinylated catalyst (kfree) (31). These experi- carboxylate residue within the active site is key
ments were performed with WT Sav, N118K- to generating a highly active and selective arti-
K121E, and S112Y-K121E as host protein (Fig. ficial benzannulase.
19. T. R. Ward, Acc. Chem. Res. 44, 47 (2011).
20. T. Satoh, M. Miura, Chem. Eur. J. 16, 11212 (2010).
21. N. Guimond, S. I. Gorelsky, K. Fagnou, J. Am. Chem. Soc.
133, 6449 (2011).
22. S. Rakshit, C. Grohmann, T. Besset, F. Glorius, J. Am.
Chem. Soc. 133, 2350 (2011).
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to biotinylated rhodium monomer, delivering product
in 23% yield with 9:1 regioselectivity after 36 hours.
Identical product distribution was obtained upon using
3, A, B, and C, respectively). Because the er
[Cp*biotinRh(H2O)3]2+
.
with up to four equivalents RhCp*biotinCl2(H2O)
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References and Notes
remains essentially constant, we conclude that
the four Sav-bound catalysts operate indepen-
dently (i.e., no cooperative effect) and induce the
same level of enantioselectivity. Past four equiv-
alents and if the relative rates kbound and kfree
are identical, the er is expected to sharply and
asymptotically decrease (orange lines in Fig. 3).
The rate acceleration provided by the protein-
environment can be estimated by Eq. 1
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Acknowledgments: We thank the National Institute of General
Medical Sciences (GM80442), the Swiss National Science
Foundation (grant 200020-126366), the National Center of
Competence in Research Nanoscale Sciences, the Marie Curie
Training Network (FP7-ITN-238434), Amgen, and Roche for
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stimulating discussions.
%ðRÞcalculated ¼
k
bound•mbound•%ðRÞ
þ kfree•mfree•%ðRÞfree
bound•m boundþ kfree•m
Supplementary Materials
www.sciencemag.org/cgi/content/full/338/6106/500/DC1
Materials and Methods
Figs. S1 to S3
Table S1
k
bound
free
ð1Þ
where kbound and kfree are the rate constants for
the Sav-bound and the free catalyst, respective-
ly. The parameters mbound and mfree are the
number of Sav-bound and free catalysts (i.e., at
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15 June 2012; accepted 21 September 2012
10.1126/science.1226132
www.sciencemag.org SCIENCE VOL 338 26 OCTOBER 2012
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