Exploration of Structural Frameworks for Reactive and Enantioselective Peptide Catalysts by Library Screenings

Article abstract of DOI:10.1021/acs.joc.6b01591

By screening large-scale N-terminal l-prolyl peptide libraries, we explored efficient catalysts for asymmetric Michael addition of a malonate to an enal. The catalytically active peptides obtained by the screening could be categorized into two groups base

Full text of DOI:10.1021/acs.joc.6b01591

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Note
Exploration of Structural Frameworks for Reactive and
Enantioselective Peptide Catalysts by Library Screenings
Kengo Akagawa, Junichi Satou, and Kazuaki Kudo
J. Org. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.joc.6b01591 • Publication Date (Web): 23 Sep 2016
Downloaded from http://pubs.acs.org on September 25, 2016
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Exploration of Structural Frameworks for Reactive and Enantioselective Peptide Catalysts by Library
Screenings
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Kengo Akagawa, Junichi Satou, and Kazuaki Kudo*
Institute of Industrial Science, University of Tokyo, 4ꢀ6ꢀ1 Komaba, Meguroꢀku, Tokyo 153ꢀ8505, Japan
*kkudo@iis.uꢀtokyo.ac.jp
ABSTRACT: By screening largeꢀscale Nꢀterminal
Lꢀprolyl peptide libraries, efficient catalysts for asymmetric
Michael addition of a malonate to an enal were explored. The catalytically active peptides obtained by the screening
could be categorized into two groups based on the similarity of amino acid sequences. One group of the peptides
selectively gave an (S)ꢀproduct while the other gave a (R)ꢀproduct, despite all the peptides had a common Nꢀterminal
sequence, Proꢀ
D
ꢀPro.
Further optimization by secondꢀgeneration screenings afforded more reactive and
enantioselective catalysts. It was found that the peptides having a histidine residue at the seventh position were good
catalysts, and their reaction efficiencies were correlated with the abilities of entrapping a substrate into resin beads.
Peptides are promising candidates for efficient and selective organocatalysts.1,2 One of the distinct features of
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peptide catalysis is that the structural diversity of peptides can be easily attained by changing amino acid sequences.
While such a tunability of peptides assures the high potency for the development of capable catalysts, identifying an
optimal sequence from numerous combinations of amino acids is difficult and timeꢀconsuming. A powerful solution
to this problem is the screening of a peptide library in a highꢀthroughput manner.3,4 We have recently reported a
novel highꢀthroughput screening method,5 in which resinꢀbound Nꢀterminal prolyl peptides can be assayed for an
amineꢀcatalyzed Michael reaction6 (Scheme 1). The procedure is as follows: 1) stirring a mixture of an enal, a
dyeꢀlabeled nucleophile, and a peptide library on beads, 2) anchoring the dyeꢀlabeled product to the peptide by
addition of a reducing agent, 3) collecting colored beads under the microscope, and 4) analyzing sequences by
electrospray ionization tandem mass spectrometry (ESIꢀMS/MS) after the cleavage of peptides from the beads.
Because active peptides can be colored through the inꢀsitu reductive amination, a premodification of a peptide library
is not necessary. By using this method, we successfully obtained a histidineꢀcontaining peptide which had the
function of enhancing the substrateꢀbinding to the catalyst.5 In our previous study, however, peptide libraries were
constructed based on a reported sequence and only a few residues were varied, while main secondary structures such
as a βꢀturn and a helix were retained. It was expected that new structural frameworks for more efficient catalysts
could be found by screening peptide libraries with fully randomized residues, which would cause dramatic changes in
enantioselectivity due to the diverse threeꢀdimensional structures of screened peptides. In this paper, a pair of
peptides with complementary enantioselectivity was developed based on the consensus sequences obtained by the
screenings, and was further optimized with secondꢀgeneration libraries.
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Scheme 1. Screening Procedure of a Prolyl Peptide Library for Michael Addition
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Initially, two kinds of libraries consisting of Nꢀterminal proline and the following variable six residues
were constructed by a splitꢀandꢀmix method (Figure 1).7 At the Cꢀterminus, octaethylene glycol and methionine
were introduced for increasing the sensitivity in ESIꢀMS/MS analyses, and for the cleavage of peptides from resin
beads by cyanogen bromide, respectively. The randomized part of library A contained six kinds of amino acids,
which were the components of previously developed catalysts.5 We expected that a histidineꢀcontaining peptide
would be obtained, but upon screening this library with 4ꢀnitrocinnamaldehyde and dyeꢀlabeled malonate 1 (Scheme
2),8 none of the detected sequences had a histidine residue. Thus, in order to access to a wider conformational space,
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D
ꢀleucine was employed in library B instead of histidine of library A.9 The peptide sequences found by the
screenings of both libraries are shown in Figure 2, and a number of observations are noteworthy: (i) all the peptides
have ꢀamino acids appeared in a higher frequency than statistically expected, (iii) all
ꢀPro at the AA2 position, (ii)
peptides found in library B possess ꢀLeu, (iv) the majority of the peptides detected from library A have a consensus
sequence, Proꢀ ꢀProꢀXꢀ ꢀProꢀXꢀXꢀ{Aib or ꢀPro}, (v) the similar sequences with
ꢀLeu at the AA7 position were
also found in the peptides from library B, (vi) other peptides obtained from library B have a different consensus
sequence, Proꢀ ꢀProꢀXꢀXꢀ ꢀLeuꢀ ꢀProꢀX, and (vii) the preserved parts in the consensus sequences from both libraries
unexceptionally consisted of ꢀamino acids or Aib. Consequently, the detected peptides except for samples 9 and 10
could be classified as type I and type II according to the common sequences with ꢀamino acids (Figure 2).
D
D
D
D
D
D
D
D
D
D
D
D
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Figure 1. Peptide libraries with six randomized residues. AA = amino acid.
Scheme 2. Conditions for Initial Screenings
Figure 2. Detected sequences by initial screenings. Consensus residues are boxed by blue and red lines.
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Four type I peptides (samples 1, 4, 7, and 8) and two type II peptides (samples 15 and 19) were selected,
and the corresponding resinꢀsupported catalysts were prepared by a conventional solidꢀphase peptide synthesis. In the
presence of these catalysts, asymmetric Michael addition of dimethyl malonate to 4ꢀnitrocinnamaldehyde was
conducted (Table 1).10 This reaction hardly proceeded with the peptides obtained from colorless beads during the
screening (Table 1, entries 1 to 3). The reaction was significantly enhanced by the type I peptides (Table 1, entries 4
to 7). The ee values were moderate, and the major isomer of the product had the S configuration. From the fact
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that no amino acid with the
suitable at this position. When the seventh Aib of catalyst 2 was replaced with
increased (Table 1, entries 8 to 10), and in the cases with ꢀAla and ꢀTrp, a higher enantioselectivity was also
L
ꢀform was found at the seventh residue in the type I peptides,
Dꢀamino acids seemed
D
ꢀamino acids, the reactivity slightly
D
D
observed (entries 9 and 10). Next, type II peptides were examined. While the reactivity somewhat lowered
compared to type I peptide 2, the ee values were higher (Table 1, entries 11 and 12). The configuration of the major
product was R, which was the opposite enantioselectivity to the case with the type I peptide. This is of interest,
because the Nꢀterminal two residues of peptides 2 and 3, and even the three residues of peptides 2 and 4 are identical
to each other, but the selectivities are completely altered by the subsequent residues.3c The replacement of
the fifth residue in peptide 3 to ꢀLeu decreased the catalytic efficiency along with a dramatic loss in the
DꢀLeu at
L
enantioselectivity (Table 1, entry 13). Similar to the type I peptide, the seventh residue of peptide 3 also affected the
reaction; the use of alanine instead of tyrosine lowered the conversion (Table 1, entry 14). In our previous study, it
was found that a histidyl side chain allocated in proximity to an Nꢀterminal proline enhances the reaction.5
Accordingly, the seventh tyrosine of peptide 3 was replaced to histidine, which resulted in a marked improvement of
the reaction efficiency (Table 1, entry 15).
Table 1. Evaluation of Peptide Sequences for Asymmetric Michael Addition of a Malonate
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entry
1
peptide
conversion (%) ee (%)a
ProꢀTyrꢀHisꢀ(Trp)2ꢀ
D
ꢀProꢀTyr
1
n.d.b
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(from colorless bead)
2
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ProꢀHisꢀLeuꢀTyrꢀLeuꢀTyrꢀLeu
(from colorless bead)
<1
2
n.d.b
Proꢀ(Tyr)3ꢀ(Leu)2ꢀTrp
(from colorless bead)
n.d.b
Proꢀ
(Sample 1 in Library A)
Proꢀ ꢀProꢀTyrꢀ ꢀProꢀLeuꢀTyrꢀAib
(Sample 4 in Library A)
Proꢀ ꢀProꢀTrpꢀ ꢀProꢀTyrꢀTrpꢀAib
(Sample 7 in Library A)
Proꢀ ꢀProꢀTrpꢀ ꢀProꢀTrpꢀTrpꢀ
(Sample 8 in Library A)
D
ꢀProꢀLeuꢀ
D
ꢀProꢀAibꢀTyrꢀAib (2)
55
53
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75 (S)
45 (S)
61 (S)
52 (S)
D
D
D
D
D
D
DꢀPro
8
Proꢀ
Proꢀ
Proꢀ
Proꢀ
D
D
D
D
ꢀProꢀLeuꢀ
ꢀProꢀLeuꢀ
ꢀProꢀLeuꢀ
D
D
D
ꢀProꢀAibꢀTyrꢀ
D
-Pro
-Ala
-Trp
66
74
66
40
71 (S)
85 (S)
82 (S)
96 (R)
9
ꢀProꢀAibꢀTyrꢀ
D
D
10
11
ꢀProꢀAibꢀTyrꢀ
ꢀProꢀAibꢀTyrꢀDꢀLeuꢀDꢀProꢀTyr (3)
(Sample 15 in Library B)
Proꢀ ꢀProꢀLeuꢀTrpꢀ ꢀLeuꢀ
(Sample 19 in Library B)
12
D
D
D
ꢀProꢀTyr (4)
30
82 (R)
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Proꢀ
Proꢀ
Proꢀ
D
D
D
ꢀProꢀAibꢀTyrꢀLeuꢀ
D
ꢀProꢀTyr
25
29
69
12 (R)
95 (R)
95 (R)
ꢀProꢀAibꢀTyrꢀ
D
D
ꢀLeuꢀ
D
D
ꢀProꢀAla
ꢀProꢀAibꢀTyrꢀ
ꢀLeuꢀ
ꢀProꢀHis (5)
9
a The symbol in parentheses indicates the absolute configuration of the major enantiomer. b Not determined.
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Next, for a further enhancement of the catalytic capability, second generation libraries were constructed
based on the initial screenings and evaluations (Figure 3). As for the type I peptide, an octapeptide library with four
randomized residues was designed (library Aꢀ2nd). The variable positions were constituted by ten kinds of amino
acids, and the Dꢀforms of amino acids were employed at the seventh residue. A different approach was employed for
the type II peptide; additional six residues were introduced to the Cꢀterminus of peptide 5 by using the same
components of the initial library (library Bꢀ2nd). Such a stepwise introduction of the randomized block facilitates
the screening of long peptide sequences by suppressing the scale of a library not to exceed a practically affordable
number of beads. These secondꢀgeneration libraries were screened as well, but the reaction time for coloring was
shortened to 10 min from 3 h for the initial screening. Although the detected sequences from library Aꢀ2nd did not
show a noticeable tendency, the amino acids with bulky side chains such as tyrosine and tryptophan were frequently
observed in all of the randomized positions (see, Figure S1 in Supporting Information). The peptides with these
sequences significantly accelerated the Michael reaction (Table 2, entries 1 and 2), compared to peptide 2 (Table 1,
entry 4). Among them, peptide 6 containing
DꢀHis at the seventh residue was most reactive, which was clearly
indicated by the data with 10 mol % catalyst loading (Table 2, entries 1 and 2, conversions in parentheses).11 The
importance of each amino acid of peptide 6 at the AA6 to AA8 positions was confirmed by alanine scanning (Table 2,
entries 3 to 5). When the side chain of
a lower reactivity (Table 2, entry 6). It is assumed that
residue, but the imidazole group is actively engaged in a catalytic cycle. The residues at sixth and eighth positions
D
ꢀHis at the AA7 position was protected by a trityl group, the catalyst showed
D
ꢀHis of peptide 6 simply does not function as a bulky
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are considered to have a role for fixing the histidyl side chain in an appropriate threeꢀdimensional space. By
exchanging the eighth Trp of peptide 6 with more bulky ꢀ3ꢀ(1ꢀpyrenyl)alanine (Pya), a comparable reactivity with an
improved enantioselectivity was observed (Table 2, entry 7). Meanwhile, the screening of library Bꢀ2nd afforded the
sequences highly enriched with ꢀPro and ꢀLeu residues (see, Figure S2 in Supporting Information). The detected
L
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D
D
sequences showed slightly higher conversions and ee values for the Michael reaction (Table 2, entries 8 and 9),
compared to peptide 5 (Table 1, entry 15). The enhancement of the reaction efficiency by the second screening is not
so remarkable, and it seems that such a long peptide chain is unnecessary. However, when the Cꢀterminal residues of
peptide 8 were truncated, the conversion was gradually decreased as the peptide shortened (Table 2, entries 10 to
12).12
Figure 3. Design of secondꢀgeneration libraries.
Table 2. Evaluation of Peptide Sequences from Second Screenings
entry peptide
conversion (%)a ee (%)b
1
2
3
4
5
Proꢀ
Proꢀ
Proꢀ
Proꢀ
Proꢀ
D
D
D
D
D
ꢀProꢀLeuꢀ
D
ꢀProꢀAibꢀTrpꢀ
ꢀProꢀAibꢀTrpꢀ
ꢀProꢀAibꢀAlaꢀ
D
ꢀHisꢀTrpc (6)
91 (67)
90 (38)
(38)
82 (S)
81 (S)
84 (S)
81 (S)
76 (S)
ꢀProꢀTrpꢀ
D
D
ꢀTyrꢀTrpc
ꢀProꢀLeuꢀ
ꢀProꢀLeuꢀ
ꢀProꢀLeuꢀ
D
D
D
D
ꢀHisꢀTrp
-AlaꢀTrp
ꢀHisꢀAla
ꢀProꢀAibꢀTrpꢀ
D
(35)
ꢀProꢀAibꢀTrpꢀ
D
(31)
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6
Proꢀ
Proꢀ
Proꢀ
Proꢀ
Proꢀ
Proꢀ
Proꢀ
D
D
D
D
D
D
D
ꢀProꢀLeuꢀ
D
D
ꢀProꢀAibꢀTrpꢀ
D
-His(Trt)ꢀTrp
(23)
91 (65)
74
76 (S)
87 (S)
99 (R)
98 (R)
97 (R)
98 (R)
98 (R)
7
ꢀProꢀLeuꢀ
ꢀProꢀAibꢀTrpꢀ
D
ꢀHisꢀPyad (7)
8
ꢀProꢀAibꢀTyrꢀ
ꢀProꢀAibꢀTyrꢀ
ꢀProꢀAibꢀTyrꢀ
ꢀProꢀAibꢀTyrꢀ
ꢀProꢀAibꢀTyrꢀ
D
D
D
D
D
ꢀLeuꢀ
ꢀLeuꢀ
ꢀLeuꢀ
ꢀLeuꢀ
ꢀLeuꢀ
D
D
D
D
D
ꢀProꢀHisꢀ(
D
ꢀPro)3ꢀ
ꢀLeuꢀ ꢀProꢀAibꢀ
ꢀPro)3ꢀ ꢀLeu
D
ꢀLeuꢀLeuꢀ
D
ꢀLeue (8)
ꢀLeuꢀ
9
ꢀProꢀHisꢀ
ꢀProꢀHisꢀ(
ꢀProꢀHisꢀ(
ꢀProꢀHisꢀ(
D
D
DꢀProꢀ
D
D
ꢀLeue
76
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10
11
D
D
D
D
68
ꢀPro)3
ꢀPro)2
59
12
58
a
b
The value in parentheses indicates the conversion with 10 mol % peptide catalyst. The symbol in parentheses
c
d
indicates the absolute configuration of the major enantiomer. Found by the screening of Library Aꢀ2nd. Pya
indicates
L
ꢀ3ꢀ(1ꢀpyrenyl)alanine. e Found by the screening of Library Bꢀ2nd.
Thus, two types of catalysts, peptides 7 and 8, with opposite enantioselectivities were successfully
developed through the screenings and optimization. These peptides possess Dꢀ or Lꢀhistidine at the seventh residue,
and this amino acid is considered to be important for promoting the reaction. In our previous study, it was suggested
that a histidineꢀcontaining prolyl peptide had the ability to bind an α,βꢀunsaturated aldehyde through the formation of
an iminium intermediate at the Nꢀterminal proline followed by the Michael addition of the imidazole group.5,13 In
addition to the acceleration of the reaction by entrapping the substrate into resin beads, high enantioselectivity can be
expected because one face of the substrate is covered by the histidyl residue. To examine such abilities of the
present catalysts, the substrate aldehyde and the resinꢀsupported peptides were mixed, and the amount of the aldehyde
1
remaining in solution phase was monitored by H NMR (Figure 4). The entrapment of the aldehyde into the resin
beads was observed, and a more reactive peptide in the Michael addition showed a faster uptake of the substrate.
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Figure 4. Evaluation of substrateꢀbinding capability of peptides.
Finally, other substrates were tested for the asymmetric Michael addition of a malonate (Table 3).
Peptide 8 was chosen for the examination due to the highest enantioselectivity. In the presence of 2 mol % of the
catalyst, the reaction proceeded sufficiently and afforded the product with excellent enantioselectivity (Table 3, entries
1 to 6).
Table 3. Asymmetric Michael Addition of a Malonate with Peptide 8
entry
R
9
a
b
c
d
e
f
yield (%)
ee (%)
99
1
4ꢀNO2C6H4
4ꢀClC6H4
4ꢀBrC6H4
4ꢀMeOC6H4
C6H5
84
83
80
81
81
81
2
99
3
98
4a
5
96
98
6
2ꢀnaphthyl
97
a The concentration of the aldehyde was 0.6 M.
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In conclusion, efficient and enantioselective peptide catalysts for Michael addition of a malonate were
developed by the screening of fully randomized libraries followed by the optimization with secondꢀgeneration
libraries. Two types of peptides obtained in the screenings showed an opposite enantioselectivity to each other,
although they possessed common Nꢀterminal residues. A rational design for such sequences and optimization of
them in a conventional way are considered to be difficult. This study demonstrates the potency of the present
screening method, and further applications to other reactions are expected.
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EXPERIMENTAL SECTION
Procedure for constructing peptide libraries: For resin beads, TentaGel MB NH2 (Rapp Polymere GmbH, product
number: MB 250002, 0.23 mmol/g amine loading) was used. The coupling reaction of an amino acid was performed
in an empty column with a filter at the bottom. Before the reaction, the resin was swollen with dichloromethane
(DCM) for 20 min, and washed with N,Nꢀdimethylformamide (DMF). A typical procedure for the elongation of an
amino acid was as follows.
A DMF solution of an Fmocꢀprotected amino acid (3.0 equiv.),
Oꢀbenzotriazolꢀ1ꢀylꢀN,N,N’,N’ꢀtetramethyluronium hexafluorophosphate (HBTU, 3.0 equiv.), 1ꢀhydroxybenzotriazole
(HOBt, 3.0 equiv.), and diisopropylethylamine (DIEA, 6.0 equiv.) was added to the resin. The resulting mixture was
stirred in every 10 min with a vortex mixer. After 1 h, the resin was washed with DMF. To remove the Fmoc group,
the resin was washed once with a DMF solution of piperidine (20 vol %), and this DMF/piperidine solution was added
afresh to the resin. The resulting mixture was stirred in every 5 min with a vortex mixer. After 20 min, the resin was
washed with DMF, and used for a next coupling.
Preparation of library A:
After the coupling of methionine and the removal of its Fmoc group,
FmocꢀNHꢀ(CH2ꢀCH2ꢀO)8ꢀ(CH2)2ꢀCO2H (FmocꢀOEGꢀOH, ChemPep, Inc., product number: 280112) was introduced.
The coupling of this amino acid was performed with each 2 equiv. of FmocꢀOEGꢀOH, HBTU, and HOBt, and 4 equiv.
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of DIEA for 4 h. After the removal of the Fmoc group, the resin beads were divided into six groups, and they were
transferred into different columns. For these groups, Fmocꢀprotected Aib, His(Trt), Leu, ꢀPro, Trp, and Tyr(tBu)
D
6
7
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9
were attached, respectively. After the coupling reactions, all beads were combined together, and the terminal Fmoc
protection was removed. The series of the above manipulations, splitting the beads, coupling amino acids, mixing
the beads, and removing the Fmoc group, was repeated, until six randomized residues were introduced. Finally,
FmocꢀProꢀOH was attached to the combined mixture of the beads. After the removal of the terminal Fmoc group,
the resin was washed with DCM and dried under reduced pressure. To remove the protecting groups of side chains,
the resin was soaked in trifluoroacetic acid (TFA) containing water (3 vol %), and the resulting mixture was stirred in
every 10 min with a vortex mixer. After 1 h, the resin was successively washed with DCM, DMF,
triethylamine/DCM (1:1), DMF, and DCM, and was dried under reduced pressure.
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Preparation of library B: The same procedure for the preparation of library A was conducted using Aib,
Leuꢀd10, DꢀPro, Trp, and Tyr(tBu) as the constituent amino acids for the randomizing part.
DꢀLeu,
Preparation of library A-2nd: After the introduction of methionine and OEG and the removal of the Fmoc group
according to the above procedure, the resin beads were divided into ten groups, and they were coupled with
Fmocꢀprotected Aib, Ala, Glu(OtBu), Gly, His(Trt), Leu, Pro, Thr(tBu), Trp, and Tyr(tBu), respectively. All beads
were combined together, and the terminal Fmoc protection was removed. For the randomized positions of the library,
amino acids were introduced in this way. For the seventh residue from the Nꢀterminus, Aib, Gly, and the
Ala, Glu(OtBu), His(Trt), Leu, Pro, Thr(tBu), Trp, and Tyr(tBu) were used. The common sequences, Proꢀ
first and second residues and ꢀProꢀAib at the fourth and fifth residues, were introduced by the coupling reaction to
D
ꢀforms of
DꢀPro at the
D
the combined mixture of the beads. After the introduction of all residues, the following procedure was the same as
above.
Preparation of library B-2nd: Instead of Nꢀterminal proline of library B, the common six residues were introduced
to the randomized part using Fmocꢀprotected His(Trt), DꢀPro, DꢀLeu, Tyr(tBu), Aib, DꢀPro and Pro, successively.
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Procedure for screening peptide libraries: To a roundꢀbottom flask that contained a peptide library on resin beads
(0.3 g, approximately 0.05 to 0.06 mmol of the terminal prolyl group), a solution of 4ꢀnitrocinnamaldehyde and
dyeꢀlabeled malonate 1 (10 mM for each reagent) in THF (5 mL) was added. After this mixture was stirred for 3 h in
the initial screening, or for 10 min in the second screening, sodium triacetoxyborohydride (0.25 mmol) was added.
After stirring the resulting mixture for additional 15 min, the resin beads were transferred to an empty column with a
filter at the bottom, and washed with DMF and DCM, successively. Then, the resin beads were put on a glass plate,
and highly colored beads were picked up with a needle under a microscopic view. Each selected bead was put into
an Eppendorf tube, separately. To detach peptides from the beads, an aqueous solution (8 ꢀL) of formic acid (70%)
and cyanogen bromide (50 mg/mL) was added to the tube, and this was left in dark at 25 °C for 24 h. The solvent
was removed under a stream of nitrogen gas, and completely dried under reduced pressure. The residue was dissolved
in methanol (100 ꢀL), and the solution was passed through a membrane filter (pore size: 0.45 ꢀm). This sample was
analyzed by an ESIꢀMS/MS measurement in a positiveꢀion mode.
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Preparation of resin-supported peptide catalysts: A similar procedure with the preparation of peptide libraries
was employed. For resin beads, TentaGel S NH2 (AnaSpec, Inc., product number: 22798, 0.25 mmol/g amine
loading) was used. Instead of HBTU and HOBt, Oꢀ(7ꢀazabenzotriazolꢀ1ꢀyl)ꢀN,N,N’,N’ꢀtetramethyluronium
hexafluorophosphate (HATU) and 1ꢀhydroxyꢀ7ꢀazabenzotriazole (HOAt) were used. The reaction time for the
coupling was 30 min with stirring in every 5 min, and the completion of the reaction was checked by the Kaiser test.
The coupling reaction to an Nꢀterminal Aib, Lꢀ or DꢀPro residue was performed twice without the Kaiser test.
Procedure for the evaluation of peptide catalysts (Tables 1 and 2): To a mixture of a resinꢀsupported peptide
(0.006 or 0.003 mmol of the terminal prolyl group), 4ꢀnitrocinnamaldehyde (0.03 mmol), and THF (300 ꢀL), dimethyl
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malonate (0.09 mmol) was added. After stirring the mixture at 25 °C for 12 h, the peptide catalyst was filtered off
4
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1
and washed with chloroform. The conversion was determined by H NMR measurement of the crude mixture in
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CDCl3. The ratio of the starting aldehyde and the product was used for the calculation. Then, the crude mixture
was dissolved in tꢀbutyl alcohol (200 ꢀL), 1 M aqueous solution of sodium dihydrogen phosphate (200 ꢀL), and 1 M
aqueous solution of potassium permanganate (200 ꢀL). After stirring the mixture for 30 min, 1 M aqueous solution
of hydrochloric acid was added. The mixture was extracted with ethyl acetate, and the organic layer was washed
with water three times. After the removal of the solvent under reduced pressure, the residue was dissolved in
methanol (500 ꢀL) and toluene (200 ꢀL). To this solution, trimethylsilyldiazomethane (0.6 M solution in hexane,
200 ꢀL) was added. After stirring the mixture for 10 min, the solvent was removed under reduced pressure. The
residue was purified by preparative TLC (ethyl acetate/hexanes 1:1) to afford the corresponding methyl ester of the
product.10b Enantiomeric excess of this compound was determined by HPLC analysis with Chiralpak IA
(hexane/2ꢀpropanol 7:3, 0.8 mL minꢀ1).5
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Procedure for the peptide-catalyzed Michael addition of a malonate (Table 3): To a mixture of resinꢀsupported
peptide 8 (8.7 mg, 0.0016 mmol of the terminal prolyl group), an α,βꢀunsaturated aldehyde (0.08 mmol), and THF
(200 ꢀL, or 133 ꢀL for 4ꢀmethoxycinnamaldehyde), dimethyl malonate (0.24 mmol) was added. After stirring the
mixture at 20 °C for 60 h, the peptide catalyst was filtered off and washed with chloroform. The solvent was
removed under reduced pressure, and the residue was purified by flash column chromatography on silica gel
(ether/hexanes 3:7 to 9:1) to afford the Michael product. The spectral data of all products (9a,10d 9b,10c 9cꢀe,10b 9f5)
were reported. To determine the enantioselectivity, Michael products were converted to the corresponding methyl
esters according to the previous report.5,10b
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ASSOCIATED CONTENT
Supporting Information
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Supporting Information is available free of charge on the ACS Publications website.
Results of secondꢀgeneration screenings, ESIꢀMS/MS spectra of peptides, NMR spectra of Michael products, and
HPLC traces. (PDF)
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AUTHOR INFORMATION
Corresponding Author
*Eꢀmail for kkudo@iis.uꢀtokyo.ac.jp
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
We thank Ms. Miyuki Okuyama for experimental assistance. This work was financially supported by JSPS
KAKENHI (No. 15K17851 to K.A. and No. 15K05495 to K.K.), and MEXT KAKENHI (No. 26105710 to K.K.).
REFERENCES
(1) For reviews, see: (a) Davie, E. A. C.; Mennen, S. M.; Xu, Y.; Miller, S. J. Chem. Rev. 2007, 107, 5759. (b)
Wennemers, H. Chem. Commun. 2011, 47, 12036.
(2) For recent selected examples, see: (a) Wiesner, M.; Upert, G.; Angelici, G.; Wennemers, H. J. Am. Chem. Soc.
2010, 132, 6. (b) Hrdina, R.; Müller, C. E.; Wende, R. C.; Wanka L.; Schreiner, P. R. Chem. Commun. 2012, 48, 2498.
(c) Kastl, R.; Wennemers, H. Angew. Chem. Int. Ed. 2013, 52, 7228. (d) Pathak, T. P.; Miller, S. J. J. Am. Chem. Soc.
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2013, 135, 8415. (e) Zheng, L.; Marcozzi, A.; Gerasimov, J. Y.; Herrmann, A. Angew. Chem. Int. Ed. 2014, 53, 7599.
(f) Yoganathan, S.; Miller, S. J. J. Med. Chem. 2015, 58, 2367. (g) Wende, R. C.; Seitz, A.; Niedek, D.; Schuler, S. M.
M.; Hofmann, C.; Becker, J.; Schreiner, P. R. Angew. Chem. Int. Ed. 2016, 55, 1433.
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(3) (a) Copeland, G. T.; Miller, S. J. J. Am. Chem. Soc. 2001, 123, 6496. (b) Krattiger, P.; McCarthy, C.; Pfaltz, A.;
Wennemers, H. Angew. Chem. Int. Ed. 2003, 42, 1722. (c) Krattiger, P.; Kovasy, R.; Revell, J. D.; Ivan, S.; Wennemers,
H. Org. Lett. 2005, 7, 1101. (d) Matsumoto, M.; Lee, S. J.; Waters, M. L.; Gagné, M. R. J. Am. Chem. Soc. 2014, 136,
15817. (e) Matsumoto, M.; Lee, S. J.; Gagné, M. R.; Waters, M. L. Org. Biomol. Chem. 2014, 12, 8711.
(4) For other examples of library screenings, see: (a) Francis, M. B.; Jamison, T. F.; Jacobsen, E. N. Curr. Opin. Chem.
Biol. 1998, 2, 422. (b) Berkessel, A. Curr. Opin. Chem. Biol. 2003, 7, 409. (c) Revell, J. D.; Wennemers, H. Curr.
Opin. Chem. Biol. 2007, 11, 269. (d) Maillard, N.; Biswas, R.; Darbre, T.; Reymond, J.ꢀL. ACS Comb. Sci. 2011, 13,
310. (e) Lichtor, P. A.; Miller, S. J. Nature Chem. 2012, 4, 990. (f) Sambasivan, R.; Ball, Z. T. Angew. Chem. Int. Ed.
2012, 51, 8568. (g) Giuliano, M. W.; Lin, C.ꢀY.; Romney, D. K.; Miller, S. J.; Anslyn, E. V. Adv. Synth. Catal. 2015,
357, 2301. (h) Gilbertson, S. R.; Collibee, S. E.; Agarkov, A. J. Am. Chem. Soc. 2000, 122, 6522. (i) Tanaka, F.; Fuller,
R.; Barbas, C. F., III, Biochemistry 2005, 44, 7583.
(5) Akagawa, K.; Sakai, N.; Kudo, K. Angew. Chem. Int. Ed. 2015, 54, 1822.
(6) For reviews for aminocatalysis, see: (a) Erkkilä, A.; Majander, I.; Pihko, P. M. Chem. Rev. 2007, 107, 5416. (b)
Mukherjee, S.; Yang, J. W.; Hoffmann, S.; List, B. Chem. Rev. 2007, 107, 5471.
(7) (a) Furka, Á.; Sebestyén, F.; Asgedom, M.; Dibó, G. Int. J. Pept. Protein Res. 1991, 37, 487. (b) Lam, K. S.;
Salmon, S. E.; Hersh, E. M.; Hruby, V. J.; Kazmierski, W. M.; Knapp, R. J. Nature 1991, 354, 82. (c) Lam, K. S.; Lebl,
M.; Krchňák, V. Chem. Rev. 1997, 97, 411.
(8) The number of the beads classified as the most highly colored group was less than one hundred.
(9) To discriminate Lꢀ or Dꢀleucine by mass spectrometry, deuterated leucine for the Lꢀform was used.
(10) For examples of asymmetric Michael reactions of malonates to unsaturated aldehydes by aminocatalysts, see: (a)
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Yamaguchi, M.; Shiraishi, T.; Hirama, M. Angew. Chem. Int. Ed. 1993, 32, 1176. (b) Brandau, S.; Landa, A.; Franzén,
J.; Marigo, M.; Jørgensen, K. A. Angew. Chem. Int. Ed. 2006, 45, 4305. (c) Maltsev, O. V.; Kucherenko, A. S.; Zlotin,
S. G. Eur. J. Org. Chem. 2009, 5134. (d) Fleischer, I.; Pfaltz, A. Chem. Eur. J. 2010, 16, 95. (e) Ghosh, S. K.;
Dhungana, K.; Headley, A. D.; Ni, B. Org. Biomol. Chem. 2012, 10, 8322.
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(11) A possible reason why the peptide with DꢀHis at the seventh residue was not detected other than peptide 6 is that
the ratio of highly active sequences in library Aꢀ2nd was much higher than that in the initial library. Changing
screening conditions such as the concentration and the amount of the substrate might improve the screening
sensitivity.
(12) Some other results with the sequences obtained from the secondꢀgeneration libraries are shown in the Supporting
Information.
(13) For an example of a related intermediate, see: Maltsev, O. V.; Chizhov, A. O.; Zlotin, S. G. Chem. Eur. J. 2011, 17,
6109.
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