Histidine-containing peptide catalysts developed by a facile library screening method

Article abstract of DOI:10.1002/anie.201410268

Although peptide catalysts have a high potential for the use as organocatalysts, the optimization of peptide sequences is laborious and time-consuming. To address this issue, a facile screening method for finding efficient aminocatalysts from a peptide li

Full text of DOI:10.1002/anie.201410268

Angewandte
Chemie
DOI: 10.1002/anie.201410268
Peptide Catalysis
Histidine-Containing Peptide Catalysts Developed by a Facile Library
Screening Method**
Kengo Akagawa, Nobutaka Sakai, and Kazuaki Kudo*
Abstract: Although peptide catalysts have a high potential for
the use as organocatalysts, the optimization of peptide
sequences is laborious and time-consuming. To address this
issue, a facile screening method for finding efficient amino-
catalysts from a peptide library has been developed. In the
screening for the Michael addition of a malonate to an enal,
Figure 1. The resin-supported peptide catalyst.
a dye-labeled product is immobilized on resin-bound peptides
through reductive amination to visualize active catalysts. This
procedure allows for the monitoring of the reactivity of entire
peptides without modifying the resin beads beforehand.
Peptides containing histidine at an appropriate position were
identified by this method. A novel function of the histidyl
residue, which enhances the binding of a substrate to the
catalyst by capturing an iminium intermediate, was indicated.
reactions of enals. The peptide consists of a b-turn motif, d-
Pro–Aib (Aib: 2-aminoisobutyric acid),[7] and a helical section
that stabilizes the entire peptide structure and accelerates the
reactions.[8] Supporting this peptide on the resin allows a facile
preparation of the catalyst and it can be used without
considering the solubility of the hydrophobic peptide. By
fine-tuning the Trp–Trp part, peptide 1 was successfully
applied to stereoselective reactions that were not accessible
using low-molecular-weight catalysts.[9] However, the optimi-
zation of peptide sequences required time and effort, because
alterations of the AAs were conducted in a step-by-step
manner. This situation led us to develop a new method for
screening a peptide library.
Michael reactions catalyzed by cyclic secondary amines,
such as proline, occur through the following steps:[10] 1) the
formation of an iminium ion intermediate between the
catalyst and the carbonyl group of a substrate, 2) the 1,4-
addition of a nucleophile to the iminium intermediate, and
3) the hydrolysis of the resulting adduct to give the product.
In most cases, the second step is rate-determining in the
catalytic cycle. We chose the Michael addition of a malonate
to an a,b-unsaturated aldehyde as a model reaction, as it
typically proceeds through the above-mentioned reaction
mechanism.[11]
P
eptides can be regarded as simplified forms of enzymes and
possess high potential for organocatalysis.[1,2] Enzymes cata-
lyze reactions efficiently and selectively by utilizing multiple
functional groups of amino acids (AAs), which are spatially
arranged in reaction pockets. In the development of peptide
catalysts, it is important to choose appropriate AAs and
allocate them at a suitable position. Extensive examination is
necessary to identify such an ideal peptide sequence from the
numerous possible combinations of AAs, and this is the most
difficult task. Screening a peptide library constructed in
a combinatorial manner is a powerful approach for solving
this problem. The groups of Miller[3] and Wennemers[4]
separately reported screening methods that could be used to
find catalytically active peptides, in which either a fluorescent
reporter molecule or a substrate is co-immobilized on resin
beads along with the peptides. However, applicable reactions
for such screenings are limited, because modifications on the
resin are required to be compatible with solid-phase peptide
synthesis. Another method is to evaluate individual peptides
supported on beads by placing them in a well plate or tubes
with reagents and then analyzing each sample in turn.[5,6] In
this case, the evaluation step is time-consuming.
First, we identified the rate-determining step for the
reaction with a resin-supported prolyl catalyst. The reaction
rate between 4-nitrocinnamaldehyde and dimethyl malonate
was measured in the presence of a catalytic amount of resin-
supported proline (Figure 2A). A first-order dependence on
the concentration of the malonate was observed for the initial
production rate of 2 (Figure 2B). This indicates that the step
À
We have previously demonstrated that the resin-sup-
ported peptide 1 (Figure 1) having a prolyl residue at the N-
terminus is an effective organocatalyst for Michael-type
of the C C bond formation is rate-determining in the catalytic
cycle.[12,13]
Next, a strategy for screening the active peptides in
a library was established. We envisaged that a catalytically
active peptide could be visualized by anchoring the Michael
product that was labeled with a dye chromophore. For this
purpose, we attempted to covalently attach the product on the
amine catalyst.[14] It was found that the reaction of a malonate
and an enal with one equivalent of pyrrolidine afforded
enamine 3 as the Michael adduct (Scheme 1). Although the
hydrolysis of enamine 3 occurs much faster than the
nucleophilic attack of the malonate,[15] the enamine formation
between product 2 and the amine catalyst is thermodynami-
[*] Dr. K. Akagawa, N. Sakai, Prof. Dr. K. Kudo
Institute of Industrial Science, University of Tokyo
4-6-1 Komaba, Meguro-ku, Tokyo 153-8505 (Japan)
E-mail: kkudo@iis.u-tokyo.ac.jp
[**] This work was supported by grants from Sanyo Chemical Industries
Ltd, Kaneka Corp., and MEXT KAKENHI (24105506). We thank
Tomokuni Kai for experimental assistance.
Supporting information for this article is available on the WWW
under http://dx.doi.org/10.1002/anie.201410268.
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Scheme 2. Procedure for screening a peptide library.
Figure 2. Michael addition of a malonate with a resin-supported prolyl
catalyst: A) reaction conditions for measuring the conversion by
1H NMR spectroscopy, and B) dependence of the malonate concentra-
tion on the initial reaction rate.
Figure 3. Results of the screening: A) initial peptide library and the
multiply detected sequences, B) microscope image of the peptide
library after the Michael reaction, reduction, and washing (average
particle size=90 mm), and C) second library including amino acids
with functional groups and multiply detected sequences.
position.[7b] AAs possessing side chains with different sizes
and structures were chosen for the fourth and fifth positions.
A standard split and mix method was employed to provide
a one-bead-one-peptide library.[18] With this library contain-
ing 108 different peptides, the above screening was con-
ducted. After the Michael addition, reduction, and washing[19]
it was observed that some beads were colored (Figure 3B).
Mass spectrometry analysis of peptides cleaved from the
positive beads revealed that the Aib–Trp–Trp sequence, the
same sequence as in peptide 1, was the most frequent
sequence (Figure 3A). Interestingly, other multiply detected
peptides had the Trp–Trp sequence at the fourth and fifth
residues. These results are consistent with our previous
observation that the introduction of tryptophans to these
positions was effective for promoting reactions. With the aim
of achieving a marked enhancement of the catalytic activity
by the participation of functional groups of the AAs in the
reaction cycle, a second-generation library was constructed
(Figure 3C).[20] After the screening with this library contain-
ing 100 different peptides, a Phe–His sequence was detected
with a high frequency, and a similar sequence, Tyr–His, was
also observed multiple times (Figure 3C).
Scheme 1. Michael reaction with an equimolar amount of pyrrolidine
followed by reduction.
cally favorable. Addition of NaBH(OAc)3 to this mixture
gave the three-component assembly 4.[16] Based on this
reaction scheme, it was considered that the resin beads were
colored, depending on their catalytic activity, when a dye-
labeled nucleophile was employed. The procedure for screen-
ing a peptide library is shown in Scheme 2: 1) stirring of
a mixture of the a,b-unsaturated aldehyde, dye-labeled
malonate 5, and the peptide library in chloroform, 2) anchor-
ing the product to the resin by addition of the reductant and
washing-off any unreacted reagents, 3) collecting the colored
beads, while observing them under the microscope, and
4) detaching peptides from the beads and analyzing their
sequences using matrix-assisted laser desorption ionization
time-of-flight mass spectrometry (MALDI-TOF-MS) and
electrospray ionization (ESI) MS/MS. The initial peptide
library was constructed by randomizing the third to fifth
residues of peptide 1 (Figure 3A).[17] AAs suitable for the
formation of a turn structure were introduced at the third
The catalytic activity of each peptide identified in the
screening was evaluated. Peptide 1 containing Trp–Trp at the
fourth and fifth positions,[21] peptides 6 and 7 with Phe–His
and Tyr–His sequences, respectively, and peptide 8 with
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Figure 4. Evaluation of peptide catalysts: A) reaction conditions for
measuring the conversion by 1H NMR spectroscopy, and the enantio-
selectivity for each catalyst, and B) time course of the reactions.
Figure 5. Reaction profile for the generation of imidazole adduct 9 and
the exchange with deuterated imidazole.
a His–Phe sequence as a negative control for peptide 6 were
tested in the Michael reaction of dimethyl malonate (Fig-
ure 4A). Compared to peptide 8, a slightly higher reactivity
was observed for peptide 1, and the reaction was markedly
enhanced by peptides 6 and 7 possessing a histidyl residue at
the fifth position (Figure 4B).[22] A high enantiomeric excess
was obtained using the peptide catalysts detected in the
screening, whereas control peptide 8 only achieved a poor
enantioselectivity. Although the goal of the present screening
was not to identify stereoselective catalysts, this method
provided peptides generating a high enantioselectivity in the
investigated reaction. It should be noted that the groups of
Miller and Wennemers also reported the common finding that
reactivity correlates with stereoselectivity in the screening of
peptide libraries.[3,4]
To clarify the reason for the high reactivity of the peptides
with histidine at the fifth position, the following experiments
were conducted.[23] When the enal, pyrrolidine, and imidazole
were mixed, the Michael addition of imidazole took place to
give enamine 9 (Figure 5).[24] The rapid formation of enamine
10 was observed upon addition of deuterated imidazole to this
mixture, and the reaction reached equilibrium at which the
concentrations of enamines 9 and 10 were identical. The facile
exchange of the imidazole group indicates that the addition
reaction of imidazole is reversible, and that the adduct quickly
interconverts with the iminium ion intermediate. It is assumed
that a peptide with a histidyl residue at an appropriate
position captures an enal by the Michael addition of the
imidazole group (Figure 6). Because this state can provide the
reactive iminium intermediate through retro Michael addi-
tion, an acceleration of the reaction is expected due to the
increased amount of the substrate in the resin. To obtain
Figure 6. Proposed model for the reversible addition of the histidyl
side chain.
supporting evidence for this hypothesis, the enal and peptide
catalysts were mixed, and the ratio of the enal taken up by the
resin was estimated by 1H NMR spectroscopy (Figure 7).
Whereas peptide 1 with the Trp–Trp sequence and the control
peptide 8 did not show a clear interaction with the enal, its
uptake into the resin was observed with peptides 6 and 7,
which both had a histidine residue at the fifth position. The
entrapment of the substrate into the resin supposedly leads to
the increase of the amount of the iminium ion intermediate,
which results in the enhancement of the Michael reaction.
Finally, the substrate scope was examined with peptide
catalyst 7 after optimizing the reaction conditions. Regardless
of the nature of the substituents on the phenyl ring, the use of
aromatic enals afforded the products in good yield and
enantioselectivity (Table 1, entries 1–5). Substrates with
naphthyl and thienyl groups gave similar results (entries 6
and 7). As a nucleophile, also dibenzyl malonate could be
employed (Table 1, entries 8 and 9). In the case of aliphatic
aldehydes, the use of S,S’-dibenzyl dithiomalonate was
effective for obtaining products in a good yield, although
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and is potentially applicable to other amine-catalyzed reac-
tions. The development of highly active peptide catalysts is
expected to be accelerated by this screening method.
Received: October 20, 2014
Revised: November 17, 2014
Published online: && &&, &&&&
Keywords: asymmetric synthesis · combinatorial chemistry ·
.
heterogeneous catalysis · peptides · screening
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Figure 7. Evaluation of the substrate-binding capability of a peptide.
Table 1: Peptide-catalyzed Michael addition of malonates to a,b-unsa-
turated aldehydes.
Entry
R1
R2
2
Yield [%][a]
ee [%]
1
2
3
4
5
6
7
4-NO2C6H4
4-ClC6H4
4-BrC6H4
4-MeOC6H4
C6H5
2-naphthyl
2-thienyl
4-NO2C6H4
4-MeOC6H4
Et
OMe
OMe
OMe
OMe
OMe
OMe
OMe
OBn
OBn
SBn
a
b
c
d
e
f
g
h
i
89
84
88
82
84
80
83
70
98
94
96
92
94
94
94
98
96
78
78
8[b,c]
9[b,c]
10[b,d]
11[b,d,f]
76
j
k
80[e]
77[e]
c-Hex
SBn
[a] Yields of isolated products. [b] The reaction was performed with
400 mm of an enal at 08C. [c] The ratio of THF/EtOH used was 1:2.
[d] The amount of a malonate was 1.1 equiv. [e] The product was isolated
as the corresponding carboxylic acid after oxidizing the formyl group.
[f] The amount of catalyst 7 was 10 mol%.
the enantioselectivity was somewhat lower (Table 1,
entries 10 and 11). However, because only a poor selectivity
was attained with a representative low-molecular-weight
catalyst for these substrates,[25] the superior applicability of
the peptide catalyst was demonstrated.
In conclusion, a new peptide catalyst was developed by
a library screening method. It was shown that histidine at the
fifth position plays a critical role in the enhancement of the
reaction by trapping a substrate through the reversible
Michael addition of an imidazole group. In the screening,
a dye-labeled product was immobilized by reducing the
enamine form of the Michael adduct, which allowed us to
monitor the catalytic activity of an entire peptide mixture in
a single event. Thus, the screening could be performed easily,
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[12] For a detailed discussion, see the Supporting Information.
[13] For an example in which a reaction rate does not depend on the
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[19] Because the Michael product was not detected in the filtrate
solution, it was confirmed that the hydrolysis of the Michael
adduct from the peptide did not occur.
[20] In this library, methionine was inserted between the helical part
and the resin to detach peptides through treatment with
À
cyanogen bromide. For the resin beads, TentaGel S NH2
(AnaSpec, Inc., product number: 22798, 0.24 mmolgÀ1 amine
loading) was used. In the following experiments for the
evaluation of catalysts and the reaction scope, this resin was
used.
[21] The peptides with Gly–Trp–Trp and Ala–Trp-Trp for the third to
fifth residues showed a reactivity comparable with that of
peptide 1.
[14] For examples of monitoring catalytic activity by covalent tagging
on peptides, see: a) J. Kofoed, J.-L. Reymond, J. Comb. Chem.
2007, 9, 1046 – 1052; b) M. Matsumoto, S. J. Lee, M. L. Waters,
M. R. Gagnꢂ, J. Am. Chem. Soc. 2014, 136, 15817 – 15820.
[15] See Figure S2 in the Supporting Information.
[22] To check that the coloring of beads in the screening conditions
corresponds to the catalytic activity,
a coloring test was
conducted with catalysts 7 and 8. See Figure S5 in the Supporting
Information.
[16] R. O. Hutchins, W.-Y. Su, R. Sivakumar, F. Cistone, Y. P. Stercho,
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enable the peptide cleavage by trifluoroacetic acid.
[23] For an example of the reaction of proline and an imidazole-
containing peptide, see: M. M. Vasbinder, J. E. Imbriglio, S. J.
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4504 – 4506.
ˇ
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[25] For the results with a low-molecular-weight catalyst, see page S7
in the Supporting Information.
448; b) ꢆ. Furka, F. Sebestyꢂn, M. Asgedom, G. Dibꢇ, Int. J.
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Communications
Peptide Catalysis
K. Akagawa, N. Sakai,
K. Kudo*
&&&& — &&&&
Histidine-Containing Peptide Catalysts
Developed by a Facile Library Screening
Method
Catalytically active peptides were identi-
fied from a peptide library by a facile
screening method. Reactive amino cata-
lysts can be visualized by anchoring a dye-
labeled product. Histidine-containing
peptides were found to be efficient cata-
lysts for the enantioselective Michael
addition of malonates and enals.
6
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