One-bead-one-catalyst approach to aspartic acid-based oxidation catalyst discovery

Article abstract of DOI:10.1021/co200010v

We report an approach to the high-throughput screening of asymmetric oxidation catalysts. The strategy is based on application of the one-bead-one-compound library approach, wherein each of our catalyst candidates is based on a peptide scaffold. For this purpose, we rely on a recently developed catalytic cycle that employs an acid-peracid shuttle. To implement our approach, we developed a compatible linker and demonstrated that the library format is amenable to screening and sequencing of catalysts employing partial Edman degradation and MALDI mass spectrometry analysis. The system was applied to the discovery (and rediscovery) of catalysts for the enantioselective oxidation of a cyclohexene derivative. The system is now poised for application to unprecedented substrate classes for asymmetric oxidation reactions.

Full text of DOI:10.1021/co200010v

RESEARCH ARTICLE
pubs.acs.org/acscombsci
One-Bead-One-Catalyst Approach to Aspartic Acid-Based
Oxidation Catalyst Discovery
Phillip A. Lichtor and Scott J. Miller*
Department of Chemistry, Yale University, P.O. Box 208107, New Haven, Connecticut 06520-8107, United States
S Supporting Information
b
ABSTRACT: We report an approach to the high-throughput
screening of asymmetric oxidation catalysts. The strategy is
based on application of the one-bead-one-compound library
approach, wherein each of our catalyst candidates is based on a
peptide scaffold. For this purpose, we rely on a recently
developed catalytic cycle that employs an acid-peracid shuttle.
To implement our approach, we developed a compatible linker
and demonstrated that the library format is amenable to screen-
ing and sequencing of catalysts employing partial Edman
degradation and MALDI mass spectrometry analysis. The
system was applied to the discovery (and rediscovery) of
catalysts for the enantioselective oxidation of a cyclohexene derivative. The system is now poised for application to unprecedented
substrate classes for asymmetric oxidation reactions.
KEYWORDS: asymmetric catalysis, asymmetric epoxidation, peptides
’ INTRODUCTION
carbodiimide and hydrogen peroxide (Figure 1). Other additives,
including the nucleophilic cocatalyst N,N-dimethylaminopyri-
dine (DMAP), or its corresponding N-oxide, were found to
facilitate catalytic turnover. The acid catalyst is regenerated upon
O-atom transfer from the peracid to the alkene substrate, forming
the corresponding epoxides. Furthermore, peptide catalyst 1 was
found to be effective for enantioselective epoxidation of 2,
yielding 3 in up to 92% ee.
We sought a method to evaluate catalysts quickly using split-
and-pool synthesis and one-bead-one-compound (OBOC) type
libraries9,10 that are compatible to the oxidizing conditions of the
desired transformation. Our lab has previously found such
methods successful in conjunction with a fluorescent sensor to
quickly distinguish active catalysts for enantioselective acyl
transfer reactions.11 The enantioselectivity afforded by the most
active catalysts was then determined in a secondary assay for ee
using either chiral GC or chiral HPLC. In the present study, we
opted for automated analytical HPLC for analysis of reaction
outcomes. While lower throughput than most fluorescence-
based assays,12 the advantages for the present pilot study include
high information content (e.g., simultaneous determination of
rate and selectivity) and applicability to a variety of reactions. The
focus of this study is thus the adaptation of the OBOC method
for the synthesis, screening, and deconvolution (i.e., hit catalyst
sequence determination) of a combinatorial library of aspartic
acid-based asymmetric epoxidation catalyst candidates.
Peptide catalysts are versatile tools for asymmetric synthesis
that mediate a wide variety of reactions.1 Among the very early
demonstrations of this concept was the application of oligopeptides
to the epoxidation of enones, a manifestation of the Juliꢀa-Colonna
epoxidation reaction.2 While the Juliꢀa-Colonna reaction relies on a
nucleophilic epoxidation mechanism, we subsequently developed
an alternative peptide-catalyzed oxidation reaction that relies on
electrophilic peracids.3,4 Given that peptides offer the potential for
massive catalyst diversity, methods for efficient catalyst discovery
remain in demand. Many high-throughput catalyst screening stra-
tegies exist,5 but these methods are often reaction-specific. For
example, libraries of enzymes for oxidation reactions6 have led to the
discovery of remarkable stereoselective enzymatic catalysts using the
tools of molecular biology. One-bead-one-compound catalyst li-
braries based on metal complexes have been evaluated for oxidation
catalysis,7 and the principle of bead tagging has been applied to
facilitate catalyst deconvolution.8 However, tagging of libraries can
itself be labor intensive, cumbersome, and pose stability issues. Our
goal in the present study was the development of a high-throughput
screening protocol for peptide-based oxidation catalysts. A method
free of tagging could in principle employ direct peptide sequencing
of catalyst hits, provided the catalyst screening and deconvolution
protocols are compatible with the oxidizing conditions of the
targeted processes. As we describe below, we found that extant
methodology for screening peptide-based catalysts required mod-
ification for implementation in the oxidation reactions under study.
The asymmetric oxidation process we recently described
depends on the catalytic action of an aspartic acid residue’s
carboxylic acid, which is transformed to the peracid with a
Received: January 17, 2011
Revised:
March 18, 2011
Published: March 22, 2011
r
2011 American Chemical Society
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ACS Combinatorial Science
RESEARCH ARTICLE
Figure 1. A. Catalytic cycle of aspartic acid-mediated epoxidations. B. Peptide-catalyzed reaction.
Figure 2. Schematic diagram of screening approach.
Our workflow for the proposed catalyst discovery protocol is
presented in Figure 2. In the initial stage, the sequence space to
be explored in the OBOC library is defined and the library is
synthesized using the split-and-pool method. The protocol then
calls for sorting the beads into individual reaction vessels (e.g.,
wells of a multiwell plate) and epoxidation reactions are then
performed in the individual wells through the addition of
reagents. Reaction contents are then analyzed by chiral HPLC,
which allows for simultaneous determination of reaction con-
version and enantioselectivity. Hit catalysts are then selected and
subjected to single bead sequencing using partial Edman degra-
dation-mass spectrometry (PED/MS),13 which can either be
performed on a single bead or a batch of beads to determine the
catalyst structure. Hit catalyst sequences may then be resynthe-
sized and evaluated/validated for their performance in epoxida-
tion reactions while in solution or remaining on-bead.
to polystyrene resin. Our previous studies14 had shown that this
substitution of phenylalanine for R-methylbenzylamine in peptide 1
when attached to Merrifield resin affords 3 with reduced but still
significant enantioselectivity (70% ee; Table 1, entry 1). This
reaction was scaled down to a single functionalized polystyrene
macrobead-catalyzed reaction, and the product was obtained with
57% ee (Table 1, entry 2). A slight background reaction can occur
without a bead, perhaps due to oxidation induced by the carbodii-
mide-hydrogen peroxide adduct.15 Further studies suggest that rate
of the background reaction may be attenuated by varying conditions
and substrate choice.
A critical issue proved to be the choice of linker between the
peptide catalyst and the polystyrene bead. Sequencing peptides
using PED/MS requires a linker between the resin and the
peptides to increase the mass of the cleaved peptide fragments,
thus avoiding interference from MALDI matrix peaks. In addi-
tion, a methionine residue incorporated at the beginning of the
linker provides an acid/base-stable moiety that may be cleaved
To examine the viability of our approach, we began our
investigation by testing a variant of peptide 1 attached directly
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RESEARCH ARTICLE
Table 1. Comparison of Linkers and Reaction
Conditions
Table 2. Effect of Methionine on Selectivitya
Boc-Asp-Pro-DVal-Phe-Linker-resin
2
3
f
DIC;H2O2;DMAP and=or HOBt;Solvent
Xxx
entryb
Met
Ala
av yieldc
av ee
1
1
1
1
0
0
:
0
1
4
1
0
13%
22%
22%
12%
6%
37%
37%
37%
32%
33%
50%
2
:
3
:
4
5d
:
:
6
no linker
38%
a Uncorrected HPLC yield is defined as the ratio of the area under the
product peaks to the sum of the total area of the products and starting
material. b Average of three replicates. c Uncorrected, crude HPLC yield.
d No Xxx in peptide.
entryd
linker
solvent
DCMb
DCMb
DCMb
DCMb
Tolueneb
Tolueneb
THF/H2Oc
THF/H2Oc
MeOHc
MeOHc
av ee
1
2
Nonea
None
4
70%
57%
<15%
24%
54%
16%
50%
35%
41%
21%
3
catalyst. Furthermore, overoxidation of the methionine sulfide to
a sulfone interferes with our ability to sequence the peptides since
the fully reduced sulfide is optimal for cleavage from resin.20
Thus, we studied the effect of methionine on selectivity by
making linkers that are cofunctionalized with differing amounts
of methionine and alanine at the position adjacent to the resin
(Table 2). Though reducing the methionine content increases
the yield of 3, the selectivity remains constant when the reaction
is performed in THF/H2O. In DCM, however, there is an
increase in selectivity as the methionine ratio declines, with a
maximum of 37% ee for the 1:4 Met:Ala peptide (see Supporting
Information for complete data set). These data suggest that the
methionine is indeed deleterious under certain conditions. Low-
ering the ratio of Met:Ala to 1:4 produces adequate results to
proceed and was used in subsequent studies.
Despite having lower amounts of methionine, the beads recovered
from the oxidation reactions of Table 2, entry 3 were successfully
sequenced using established PED/MS techniques.21 After analyzing
several beads recovered from other reactions, we found that the
peptide fragments present in the MALDI-TOF often had two
masses for each fragment, one with the expected value and another
less ∼83 amu. This mass discrepancy is consistent with hydrolysis of
the amide of the C-terminal homoserine lactone that results from
cleavage of the methionine with CNBr/TFA. While we are uncertain
of the mechanism of this loss, treatment of peptide/linker construct
on resin with hydrogen peroxide before cleavage results in the
reduced mass. However, treatment with hydrogen peroxide followed
by treatment with a cocktail of NH4I, DMS, and TFA21 mostly gives
the expected masses upon cleavage from resin. With this knowledge
and an expanded set of masses for which to search, we have success-
fully sequenced some peptides without reducing the methionine.
Nonetheless, sequencing of peptides after the oxidation reaction was
challenging at times, possibly because of peptide oxidation or other
modifications of certain peptide sequences during reactions. It is also
possible that the truncation products produced after PED are more
hydrophobic and not as easily ionized as with the traditional linker.17
These challenges are often surmountable, but they generally required
more data analysis to complete a sequence assignment. The risk of
losing a hit bead due to sequencing difficulties is a standard liability in
most HTS assays involving bead-based peptide synthesis. That said,
4
5
5
None
5
6
7
None
5
8
9
None
5
10
a Merrifield resin, many beads, single run. b With DMAP. c With HOBt
and DMAP. d Average of three runs.
using CNBr with aqueous 70% TFA.16,17 Initially, we examined a
tailored peptide linker (4, Table 1, entry 3), containing the βala-
Ala-Leu-Ala-Met sequence attached to the Boc-Asp-Pro-DVal-
Phe sequence. Unfortunately, single bead reactions employing
this catalyst-linker construct provided 3 in <15% ee, which we
judged too low for a peptide that will eventually be our positive
control. The low selectivity may be attributed to an unfavorable
linker conformation, low peptide loading, low solubility, and/or
methionine oxidation (vide infra).
We then attempted to address these issues by using a PEG-linker
(5) based upon amino acid 6,18 which we reasoned would provide
solubility in analogy to PEG-based resins. The peptide with the PEG-
linker provides improved selectivity (24% ee; Table 1, entry 4) relative
to linker 4, although the results are still markedly less impressive in
comparison to single bead reactions performed with beads lacking a
linker altogether. Switching reaction solvents from DCM to toluene
gives lower enantioselectivity, suggesting that screening should be
avoided in toluene as the linker is deleterious to selectivity, possibly
because of low solubility (Table 1, entries 5 and 6). However, use of
THF/H2O as a solvent increases the selectivity of the peptide attached
to linker 5 (35% ee; Table 1, entry 8), though without the linker there
is a slight increase (50% ee; Table 1, entry 7). Interestingly, the reac-
tion proceeds in methanol, a nucleophilic solvent that could compete
with H2O2 to intercept aspartic acid intermediates in the catalytic cycle
or break catalyst-substrate associations by competing for hydrogen
bonds (Table 1, entries 9 and 10).
Since peracids can oxidize sulfides to sulfones,19 we hypothe-
sized that the peptide may engage in nonproductive oxidation of
the methionine sulfide, eroding selectivity by sequestering active
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ACS Combinatorial Science
RESEARCH ARTICLE
reactions to increase selectivity, facilitate purification and allow
greater control of the catalyst loading. Under the homogeneous
conditions optimized for peptide 1, peptide 9 provides 3with 64% ee
(81% yield), and peptide 10 provides 3 with 65% ee (74% yield).
These results are in line with expectations from our earlier study and
provide validation for our approach to find selective catalysts.
With the validation studies of the process presented herein, we
are now poised to search for catalysts capable of selective oxida-
tion of more complex molecules in at least a medium throughput,
and perhaps a high-throughput mode. The use of general chroma-
tography technologies to analyze complex product mixtures will
continue to prove useful in these studies. Of course, advances in the
rapid analysis of reactions remains an ongoing challenge for this
field. Nonetheless, the present results provide the path forward for
the synthesis, testing, and sequence determination of OBOC
peptide-based oxidation catalyst libraries that function under con-
ditions of electrophilic oxidation catalysis.
Figure 3. Reaction yield and selectivity with catalysts from split-and-
pool library.
’ EXPERIMENTAL PROCEDURES
Peptide Synthesis for on-Bead Screening. Polystyrene
macrobeads (500ꢀ560 μm, Rapp Polymere) were swelled in
DMF for 20 min and then coupled twice for 3 h each to amino
acid coupling partner using Fmoc-protected amino acid mono-
mer (4 equiv), O-benzotriazole-N,N,N0,N0-tetramethyluronium
hexafluorophosphate (HBTU 4 equiv), 1-hydroxybenzotriazole
i
hydrate (HOBt H2O 4 equiv), and Pr2EtN (8 equiv). After
3
coupling, the resin was washed several times with DMF and
DCM. Deprotections commenced with two 20 min treatments of
20% piperidine/DMF and were followed by exhaustive washing
with DMF and DCM. The final coupling was performed with
Boc-Asp(OFm)-OH. The Asp-side chain was deprotected by
treatment with 20% piperidine/DMF four times for 10 min each.
Finally, beads were washed exhaustively with DMF, DCM, and
MeOH; then dried under N2.
Figure 4. Validation of peptides from screen.
Screening Protocol. Polystyrene macrobeads were individu-
ally transferred to 200 μL PCR tubes and inspected for uni-
formity of size. Three solutions were sequentially added to each
tube, each addition followed by centrifugation: H2O (1.84 μL);
2.65 μL of a THF solution containing 2 (1 μmol, 1 equiv), DIC
as long as “winning” beads may be found, the impact of beads not
amenable to sequencing is minimized. In this light, we were con-
fident that we had sufficiently robust methods in place to proceed.
To test the protocol from start to finish, a split-and-pool library
with three variable positions was constructed and screened for selec-
tive oxidation of 2. The library was designed as shown in Figure 3.
The i-position was fixed as Asp. Then iþ 1, iþ 2, and iþ 3 positions
were varied with eight residues, creating a library of 512 theoretical
peptide sequences for screening. To ensure that a successful catalyst
could be identified from the library, one positive control bead
(Table 2, entry 3) was added to 49 other beads from the library in
a double-blind fashion.
As revealed in Figure 3, two beads from the screen outperformed
the rest. These were then identified by PED. Significantly, one of the
beads was identified as the positive control (Boc-Asp-Pro-DVal-
Phe); the other proved similar to the control, but with Tyr(OtBu) in
the iþ3 position (Boc-Asp-Pro-DVal-Tyr(OtBu)). Notably, in the
OBOC assay, the positive control and its Tyr-variant afforded 3 with
37% and 40% ee, respectively.
(2 μmol, 2 equiv), HOBt H2O (0.1 μmol, 0.1 equiv), and
3
DMAP (0.1 μmol, 0.1 equiv); and 0.5 μL of a 30% aq. H2O2
solution (5 equiv), bringing the total reaction concentration of 2
to ∼0.2 M. After the last addition, the tubes were closed, centri-
fuged, and allowed to stand for 12 h. Reactions were quenched
with ∼3 drops of sat. aq. Na2SO3, centrifuged, and then 175 μL
HPLC grade hexanes was added. The biphasic mixture was
vortexed, centrifuged, and the organic layer was carefully re-
moved to an HPLC vial with insert. The samples were concen-
trated to dryness under a stream of N2, dissolved in 125 μL
HPLC hexanes, and resolved by HPLC using a Chiralpak AD-H
column, eluting with 1.0% isopropanol in hexanes.
Bead Recovery. High-performing beads were recovered from
the leftover aqueous layers into BioRad chromatography tubes.
These beads were washed several times with H2O, MeOH, and
DCM; and then dried. The beads were moved to a glass vessel
and treated with anhydrous TFA once for 20 min, and then again
for 10 min. The resin was washed with DCM, and then the PED
protocol from Thakkar et al. was followed using 10 μL of PITC
with 160 μL of 31 mM Fmoc-OSu in pyridine. After three cycles
of PED, the beads were washed with H2O, MeOH, DCM, then
DMF. They were treated with 20% piperidine in DMF during
The two peptides were then resynthesized as bead-bound peptide
catalysts 7 and 8, respectively, on polystyrene resin and validated as
successful catalysts (Figure 4).22 When reexamined in triplicate in an
epoxidation reaction employing single beads, each gave a satisfactory
recapitulation of the results from the OBOC assay, with 7 affording
3 with 46% ee, and 8 providing 3 with 50% ee. Each was then
synthesized as a C-terminal methyl ester for study in homogeneous
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ACS Combinatorial Science
RESEARCH ARTICLE
two 20 min cycles and then rinsed with DMF, DCM, and MeOH.
Optional Reduction: The beads were treated with about 50 μL of a
1 mL solution of TFA containing 25 mg NH4I (does not dissolve
well) and >20 μL DMS. After 20 min, the bead was removed, and
washed exhaustively with H2O, DCM, and MeOH.
Peptide Cleavage and Analysis. After drying in a PCR tube,
these beads were treated with a few drops of 20 mg/mL CNBr in
70% TFA (aq.) overnight in the dark for at least 12 h. After drying
the beads under vacuum, the resulting white solid was dissolved
in 30% H2O/MeCN. 0.5 μL of the peptide solution was mixed
with a 0.5 μL 30% H2O/MeCN with either 4.5 mg/mL R-cyano-
4-hydroxycinnamic acid or 20 mg/mL sinapinic acid, and dried
on a plate for MALDI-TOF analysis.
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’ ASSOCIATED CONTENT
S
Supporting Information. Synthetic protocols and char-
b
acterization of 6, 9, and 10; experimental details of peptide and
library validation; and additional oxidation studies of library.This
material is available free of charge via the Internet at http://pubs.
acs.org.
(6) For a few examples of enantioselective oxidations mediated by
enzymes, see: (a) Landwehr, M.; Hochrein, L.; Otey, C. R.; Kasravan, A.;
B€ackvall, J.-E.; Arnold, F. H. Enantioselective R-Hydroxylation of
2-Arylacetic Acid Derivatives and Buspirone Catalyzed by Engineered
Cytochrome P450 BM-3. J. Am. Chem. Soc. 2006, 128, 6058–6059. (b)
Peters, M. W.; Meinhold, P.; Glieder, A.; Arnold, F. J. Regio- and
Enantioselective Alkane Hydroxylation with Engineered Cytochromes
P450 BM-3. J. Am. Chem. Soc. 2003, 125, 13442–13450. (c) Kayser,
M. K. “Designer Reagents” Recombinant Microorganisms: New and
Powerful Tools for Organic Synthesis. Tetrahedron 2009, 65, 947–974.
(d) Reetz, M. T.; Wu, S. Laboratory Evolution of Robust and Enantio-
selective BaeyerꢀVilliger Monooxygenases for Asymmetric Catalysis. J.
Am. Chem. Soc. 2010, 131, 15424–15432. (e) For a more general
discussion of stereoselectivity in enzyme engineering, see: Reetz,
M. T. Laboratory Evolution of Stereoselective Enzymes: A Prolific
Source of Catalysts for Asymmetric Reactions. Angew. Chem., Int. Ed.
2011, 50, 138–174.
’ AUTHOR INFORMATION
Corresponding Author
*E-mail: scott.miller@yale.edu.
Funding Sources
This work is supported by National Institutes of Health (R01-
GM096403) to S.J.M. P.A.L. was partially supported by NIH
CBI-TG-GM-067543.
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(18) See Supporting Information for synthesis.
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(22) See Supporting Information for performance data.
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