Identification and optimization of short helical peptides with novel reactive functionality as catalysts for acyl transfer by reactive tagging

Article abstract of DOI:10.1039/c3ob41421c

Herein we describe the screening and subsequent optimization of peptide catalysts for ester activation. A combinatorial methodology using dye-tagged substrate analogs is described for determining which components of a His-containing helical library display acyl transfer activity. We found that helical peptides display high activity, and amino acids that reinforce this propensity are advantaged. Through this approach two new structural motifs have been discovered that are capable of activating esters in organic solvents. Unlike most acyl transfer catalysts functioning in organic solvents, these catalysts are histidine- rather than N-alkyl histidine-based. Longer peptides with localization of reactive groups on the C-terminal end of the peptide were found to further enhance catalytic activity up to ～2800-fold over background.

Full text of DOI:10.1039/c3ob41421c

Organic &
Biomolecular Chemistry
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Identification and optimization of short helical
peptides with novel reactive functionality as
catalysts for acyl transfer by reactive tagging†
Cite this: Org. Biomol. Chem., 2014,
12, 1488
Silvia Bezer,a Masaomi Matsumoto,a Michael W. Lodewyk,b Stephen J. Lee,c
Dean J. Tantillo,b Michel R. Gagné*a and Marcey L. Waters*a
Herein we describe the screening and subsequent optimization of peptide catalysts for ester activation.
A combinatorial methodology using dye-tagged substrate analogs is described for determining which
components of a His-containing helical library display acyl transfer activity. We found that helical peptides
display high activity, and amino acids that reinforce this propensity are advantaged. Through this approach
two new structural motifs have been discovered that are capable of activating esters in organic solvents.
Unlike most acyl transfer catalysts functioning in organic solvents, these catalysts are histidine- rather than
N-alkyl histidine-based. Longer peptides with localization of reactive groups on the C-terminal end of the
peptide were found to further enhance catalytic activity up to ∼2800-fold over background.
Received 10th July 2013,
Accepted 9th January 2014
DOI: 10.1039/c3ob41421c
www.rsc.org/obc
Introduction
Minimalist peptide catalysts have long been a goal of chemists
seeking solutions to practical problems in organic synthesis
and to bio-organic chemists seeking to understand how inher-
ently complex enzymes can be simplified to identify their key
catalytic features. The tension between this reductionist push
and the pull of massive structural diversity in polypeptides has
necessarily forced the field towards screening techniques that
enable one to competitively assay, in parallel, individually
encoded library members in one-bead one-peptide format.1–5
At the outset of our own studies we wished to test a hybrid
screening protocol for the discovery of biomimetic imidazole-
based acyl transfer catalysts that functioned in organic sol-
vents. This methodology began with a mechanism-based ana-
lysis of the two-step nucleophilic acylation of an alcohol
substrate (Scheme 1). Since k1 is typically rate limiting in the
presence of imidazole,6 we considered the possibility that
nucleophilic catalysts could be discovered by examining
Scheme 1 Transesterification reaction catalyzed by imidazole and
reactive tagging strategy.
His-containing peptide libraries. In a competitive assay for a
limited quantity of a dye-tagged active ester, the absence of a
nucleophile would prevent k2, thus tagging those His-peptides
with the largest k1 for subsequent identification. The de Clercq
group, in particular, has followed a similar course for the
identification of peptides that accelerate serine O-acylation,
though
a mention of reversible histidine N-acylation is
made.7–9
The focus of our efforts was on peptide secondary struc-
tures that maximized our ability to convert peptide hit
sequences into active site structures that reliably position
critical side-chains into 3-dimensional space. Short peptides
that take on helical structure in organic solvent (α-helix and/or
310-helix) were selected as the scaffold because the variable
i + 3/i − 3 and i + 4/i − 4 positions in the linear sequence place
functionality in close proximity to the i-position, to which a
histidine (His) residue was placed (Fig. 1). As will be discussed,
this strategy enables optimization of the helical scaffold and
the discovery of several previously unidentified functional
group assemblies for accelerating acyl transfer reactions.10–16
aDepartment of Chemistry, University of North Carolina at Chapel Hill, Chapel Hill,
North Carolina 27599-3290, USA. E-mail: mgagne@unc.edu, mlwaters@unc.edu
bDepartment of Chemistry, University of California-Davis, Davis, California 95616,
USA
cU.S. Army Research Office, P.O. Box 12211, Research Triangle Park, North Carolina
27709, USA
†Electronic supplementary information (ESI) available: Peptide and peptide-
library synthesis, peptides’ mass-spectrograms, reactive tag synthesis and their
physical data, library hits mass-spectrograms, CD spectra, NOE spectra, cartoon
representations of peptide structures, kinetic details plots, computational work.
See DOI: 10.1039/c3ob41421c
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Fig. 1 Spartan generated model for α- and 310-helices based on a poly-
alanine/His 9-mer peptide showing the relative positions of i, i + 3, and
i + 4 residues in the helix.
Fig. 3 Two-step screening procedure for discovering the most reactive
peptide-catalysts.
The screening was achieved by exposing Tentagel resin
beads containing His-peptides to a dichloromethane (DCM) or
trifluoroethanol (TFE) solution of the reactive tag until
approximately 1% of the beads were visibly colored. It is these
beads which should contain the most reactive peptides
(Fig. 3a). The reactive tagging scheme leads to a subset of
beads that are intensely red in the case of Disperse Red 1-tag-
gents RT1–3 and blue for Bodipy-based agent RT4. Control
experiments with (i) acetylated Tentagel resin, (ii) N-terminus
capped polyalanine peptide containing no His on Tentagel,
and (iii) N-terminus capped Trt protected-His/polyalanine
peptide on Tentagel, established that binding/coloration does
not occur without a reactive imidazole. Hit precision was
established via duplicate or triplicate runs and each peptide
hit was identified by MALDI MS analysis of the photolytically
cleaved product in MeOH (see ESI†).8 In the case of ambiguous
MS assignments, the candidates were resynthesized and
screened independently. This screening procedure identifies
members of the library with rapid N-acylation rates (k1 in
Scheme 1).
Information on the competitive rate of library deacylation
was obtained by carrying out a second screening on the
colored beads, those that previously displayed a competitive k1
step (Scheme 1). This was accomplished by pooling the dye-
tagged beads from the first screening (library L3 only, vide
infra) and exposing them to deacylation conditions (added
TFE) while now searching for those with rapid bleaching kine-
tics (Fig. 3b). This 2-round screening procedure is similar to
that reported by De Clercq, who sought catalysts functioning
by a serine acylation mechanism.8
The viability of the described approach is validated by the
identification of new non-biological catalytic moieties that
function in non-aqueous solvents.12,13,15
Results and discussion
Library synthesis and screening procedure
Based on the above criteria, a library of helical imidazole-con-
taining peptides was synthesized on solid support. The library
was prepared on amino functionalized Tentagel resin with a
standard photo-cleavable linker following the protocol for
split-and-mix synthesis (see ESI†).17–19 To guard against select-
ing hits that were influenced by the dye-component or the
leaving group, four dye-labeled active esters were used RT1–4
(Fig. 2). These differ as follows: RT1 is the succinimide ester of
Disperse Red 1; RT2 is the p-nitrophenyl ester of Disperse Red
1;7–9 RT3 is the p-nitrophenyl ester of Disperse Red 1 separated
by a linker; and RT4 is the succinimide ester of Bodipy plus
linker. The expected relative activity of these esters is: RT1 >
RT2 > RT4 > RT3 based on known reactivities of succinimidyl
esters relative to nitrophenyl esters coupled with differences in
inductive effects of the linkers between the ester group and
dye.20
Peptide-catalyst structure optimization
The strong α-helix preference of polyalanine led us to initiate
our study with sequences rich in Ala.21–26 Because Ala-rich
peptides have been shown to increase their helicity in organic
solvents, such as TFE, a 9-residue scaffold was selected corres-
ponding to two full α-helical turns as the basis of the
library.27,28 Library 1, L1, had His at the i-residue and 34 vari-
able substituents at the i + 3 position (Charts 1 and 2). The
Fig. 2 Reactive tags RT1–4 for library screening.
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Chart 3 Amino acid sequence for the identified hits in the library L2.
Chart 1 Amino acid sequence for libraries L1–L3.a a. In L1 and L3,
X and Y are shown in Chart 2. In L2, X corresponds to either Aib or Ala;
Y corresponds to His, Aib, or Ala.
whether the Aib effect was localized to the i + 3 position or if
other positions or stabilization schemes might lead to
improved tagging activity. Library screening identified two
high activity peptides (Chart 3), one containing three Aib resi-
dues, Hit2a, and a second containing two Aib residues, Hit2b;
both contain an N-terminal Aib. As discussed below, both
sequences similarly fold into a hybrid 310/α-helix, which is
common for peptides of similar length and Aib content.30
Hit2a was chosen for further investigation as it allows the posi-
tioning of variable residues in the i + 3 and i + 4 positions
without altering the number of Aib residues in the peptide.
Scaffold-optimized library generation and screening
A 425-member third-generation library L3 was constructed on
the Hit2a scaffold (His-scaffold 2T, where 2T indicates two
turns) by independently varying the i + 3 (with X) and the i + 4
(with Y) positions (Charts 1 and 2). Screening of L3 in DCM
with reactive tags RT2–RT4 selected a single peptide (∼0.2% of
L3) that was independent of the reactive tag type. Other pep-
tides were identified with the various reactive tagging agents,
but only Hit3a was observed in each screen. Its MW (Fig. S2b–
d†) was consistent with X = citrulline (Cit, [24] in Chart 2) and
Y = 3-pyridylalanine (3Py, [12] in Chart 2), giving His-3PyCit 2T
(Fig. 4). Repeating the screen in TFE with RT3 again selected
for His-3PyCit 2T, indicating that the hit was robust and
superior in multiple solvents (Table S1 and Fig. S2e†). His-
3PyCit 2T therefore has the highest k1 rate in L3. More impor-
tantly its high rate holds for different leaving groups, and is
independent of dye structure and solvent (DCM and TFE). Its
arrangement of functional groups represents a unique, non-
biological structural motif for accelerating acyl activation reac-
tions in organic solvents.
Chart 2 Amino acids at variable positions in libraries L1 and L3.
i/i + 3 relationship strategically places these two positions in
close proximity regardless of whether the individual peptide
adopted an α- (3.6 residues per turn) or 310-helix (3 residues
per turn).
The variable position explored how H-bond donors and
acceptors, aromatic groups and residues imposing confor-
mational biases influenced the rate of tagging (Chart 2). Poten-
tial nucleophilic residues that could interfere with His
acylation were excluded. As expected, the rate of library tagging
depended on the activity of each reactive tag with RT1–RT3
taking 25 s, 5 min, and 3 h, respectively to tag ∼1% of the
beads. MS analysis of the photo-cleaved colored beads (in
MeOH) indicated that a single unique peptide (Hit1) was
identified from this library (2.8% of total), whose molecular
Library L3 was also subjected to a two-step screening proto-
col with reactive tag RT2 to look for hits that additionally have
competitive deacylation properties. In this mode, the tagging
time was extended so that ∼10% of the most reactive beads
became colored in DCM. These red beads were removed from
the library and subjected to a deacylation screen by the
weight identified it as Ac-AAH̲AAB̲AAA-NH2 (A7HB, X = B,
aminoisobutyric acid or Aib, [28] in Chart 2) (Fig. S2a†). Impor-
tantly, although Aib does not contain potentially reactive func-
tional groups, it is a strong promoter and stabilizer of helicity
in short peptides.29,30
These results led to the design of a second-generation
library, L2 (Chart 1), whose goal was determining an optimum
backbone sequence for high inherent activity. Since the
number and positioning of the Aibs within a poly-Ala sequence
influences both the absolute helicity and helical confor-
mation,30 L2, an 8 membered library, sought to determine Fig. 4 Cartoon representation of the hits from library L3.
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addition of TFE and looking for bleaching of the beads. In prin- entry 7); (d) removal of both the 3-Py and Cit groups (His-
ciple, beads displaying a fast k1 (acylation) and a competent scaffold 2T, entry 1). Each of these variants in addition to
deacylation (k3) should have good catalytic properties. This two N-methylated His (MeHis-3Py-Cit 2T, entry 6) were slower, indi-
step treatment of L3 led to a single peptide corresponding to cating a synergistic arrangement of the functional groups.
X = 2-naphthylalanine (2Nal residue [16] in Chart 2), Y =
Since helical propensity should increase with increasing
glycine (G, residue [30] in Chart 2), (His-2Nal-Gly 2T, Fig. 4). peptide length several variants were examined including a
The peptide de-tagging rates were slower than the tagging, 4-turn His-3Py-Cit 4T (entry 9) and a 4-turn His-2Nal-Gly 4T
requiring 2 h to identify the first bleached beads. Like the (entry 14); each showed significant increases in their initial krel
single-step screening, the 2Nal/Gly combination is a unique rates of 2800- and 2200-fold, respectively (Table 1). These
structural motif for accelerating the acyl transfer reaction.
4-turn analogs represent the best catalysts in this series.
When the active site residues of His-3Py-Cit 4T were moved
to the N-terminus or their order was reversed (His-3Py-Cit mid
4T (entry 12) and Cit-3Py-His 4T (entry 13), respectively),
less active catalysts were obtained. The helix C-terminus
thus creates a microenvironment more conducive to catalysis,
perhaps due to differences in fraying and in side chain
orientation.
The pyridyl isomers of His-3Py-Cit 4T were also investi-
gated. When the basic nitrogen was shifted to the ortho-posi-
tion (His-2Py-Cit 4T, entry 10) a catalyst of similar activity was
realized, though moving N to the para-position (His-4Py-Cit
4T, entry 11) significantly impeded catalysis relative to the
parent structure.
Assessment of catalytic efficiency
The compounds identified in the course of screening L3 were
next examined in solution using 4-nitrophenyl 2-methoxyace-
tate in TFE (Scheme 2). The progress of the acyl transfer was
monitored by UV-Vis spectroscopy watching the growth of
4-nitrophenol at 320 nm using 10 mol% of the catalyst. A slow
background rate was observed for the reaction of the ester in
TFE, and this was assigned a relative rate of 1. The initial rates
using His-3Py-Cit 2T and His-2Nal-Gly 2T were 660- (Table 1,
entry 2) and 240-fold (Table 1, entry 3) greater than back-
ground, respectively, verifying that both tagging hits efficiently
catalyze acyl transfer to TFE.
The acyl transfer reaction in Scheme 2 was also followed by
1H NMR spectroscopy. Fig. 5a shows the complete time course
of the reaction catalyzed by 10 mol% His-3Py-Cit 4T. Consump-
tion of ester is roughly first order (see Fig. S4a†) and the
The unique functional motif of His-3Py-Cit 2T was probed
through a variety of methods, including an “alanine scan” to
test peptides lacking one or two of the three key groups: (a) no
3-pyridylalanine (His-Ala-Cit 2T, entry 4, Table 1); (b) no citrul-
line (His-3Py-Ala 2T, entry 5); (c) no imidazole (Ala-3Py-Cit 2T,
1
overall transformation is free of induction periods. In the H
NMR spectrum no shifting of the 3-pyridyl ring resonances
were noted. However, chemical shift changes for the imidazole
CHs indicated that imidazole protonation accompanies the
conversion (to ∼25%, Fig. 5b). A nucleophilic mechanism for
acyl transfer suggests that partial protonation of imidazole
would be deactivating, and might explain the slight slowing
in the first-order plot. Partial protonation of His-containing
peptides could be recapitulated by the sequential addition
Scheme 2 4-Nitrophenyl 2-methoxyacetate (20 mM) and 10 mol%
catalyst in TFE at 25 °C.
Table 1 Initial rate measured for the peptide-catalyzed trans-esterification reaction in TFE-d3 at 25 °C
a See the cartoon representation of the peptide structures in Fig. S3. b Dots are included to align the peptide sequences. c The initial rate for the
uncatalyzed reaction for the acylation of TFE-d3 is 4 × 10−7 mM s−1, and is assigned a relative rate of 1. d R value characterizes the type of helix,
where R = [Θ]222/[Θ]205 (see Peptide structure section).
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Fig. 6 CD spectra of 0.2 mM peptide solutions in TFE at: (a) 4 °C;
Ala8His (black), Ala7HisAib (red); (b) 25 °C; Ala-scaffold 2T (black), Hit 2a
(His-scaffold 2T) (red), Hit 2b (blue).
Fig. 5 Kinetic assay of the peptide-catalyst in TFE-d3 in 20 mM sub-
1
◆
strate and 10 mol% catalyst monitored by H-NMR. (a): ( ) no catalyst
●
(
) His-3Py-Cit 4T; (b) imidazole protonation determined from Δδ of the
δCH protons of imidazole for His-3Py-Cit 4T.
of 4-nitrophenol to the peptide in the absence of ester (see
Fig. S4b†).
Chart 4 Sequence of the Ala-based peptides.
Peptide structures in TFE by CD
To complement the catalytic properties of these peptides in
TFE, their structures were probed by CD spectroscopy. The
right-handed α-helix gives rise to two negative bands at 222
and 208 nm (a superposition of the random coil π–π* tran-
sition at 200 nm and the α-helix π–π* transition at 208 nm) of
almost equal intensities and a strong positive band at about
192 nm.31 The 310-helix has conformational parameters close
to the α-helix, the former being slightly tighter and more
elongated (Fig. 1).32,33 One possible criterion to distinguish
them is by determining the ratio of the bands R = [Θ]222
/
32,33
[Θ]208
.
The α-helical peptides are known to have R values
of ∼1 whereas 310-helices have values near 0.4.32,33 The CD
spectrum of Ala8His, the reference scaffold for L1, exhibits two
negative bands at 222 and 203 nm and a positive band at
190 nm, indicating a helical structure. The ratio, R, of the 222/
203 nm peaks is 0.45 suggesting a dominant 310-helix
(Fig. 6a).32,33 When Ala8His was modified with an Aib residue
in the i + 3 position (Hit1 from L1), the resulting peptide Ala7-
HisAib (Hit 1) shows a greater intensity at 222 and 190 nm,
with R = [Θ]222/[Θ]205 = 0.71 (Fig. 5a) consistent with a better
folded hybrid 310/α-helix.32–34
Fig. 7 CD spectra of 0.2 mM peptide solutions in TFE at 25 °C; His-
scaffold 2T (black), His-3Py-Cit 2T (blue), His-scaffold 4T (red), His-3Py-
Cit 4T (green).
2T (Table 1, Fig. S5†). Thus, while 3Py increases helicity, it
does not alone correlate with catalytic activity.
Increasing the length of His-scaffold 2T from 2- to 4-turns
results in an increase in α-helical content, with R = [Θ]222
/
[Θ]205 increasing from 0.54 to 0.66 (Fig. 6b and Table 1, entries
1 and 8).32–34 The 15-residue peptide-hit, His-3Py-Cit 4T, shows
a smaller increase in α-helical content with respect to His-3Py-
Cit 2T, with R increasing from a two-turn value of 0.66 to 0.72
(Fig. 7 and Table 1, entries 2 and 9). However, only small
differences in R-values were observed for His-3Py-Cit 4T rela-
tive to the control peptides (His-2Py-Cit 4T, His-4Py-Cit 4T,
His-3Py-Cit mid 4T, and Cit-3Py-His 4T, Fig. S6a† and Table 1,
entries 9–13). As with the 2T peptides, the R value alone does
not correlate with catalytic activity in the 4T series. The CD
spectra for the His-2Nal-Gly 2T hit and His-2Nal-Gly 4T indi-
cate a helical structure but naphthyl absorbance artifacts at
222 nm band make it difficult to estimate the type of helix
(Fig. S6b†).
The scaffolds identified in the screening of L2 (Hit 2a and
b) showed nearly identical CD spectra corresponding to a
hybrid 310/α-helix (R = [Θ]222/[Θ]205 = 0.54, Fig. 6b).32–34 Repla-
cing His with Ala in His-scaffold 2T (Hit 2a) to give Ala-scaffold
2T (Chart 4) did not change the peptide conformation, sup-
porting the notion that helicity is primarily nucleated by the
Aib residue (Fig. 6b). Replacing two Ala residues with 3-pyridyl-
alanine and citrulline to give His-3Py-Cit 2T results in higher
α-helix content, as judged by a shift in the middle band from
204 to 206 nm, increased intensity at 222 nm and R222/205
=
0.66 (Fig. 7).32–34 The same feature was observed for all pep-
tides containing 3-pyridylalanine (Table 1, Fig. S5†), indepen-
dent of catalytic activity. When 3-pyridylalanine was removed
(His-Ala-Cit 2T), the spectrum was similar to the His-scaffold
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efficiency than 4-pyridylalanine. Thus, based on mutation
studies, 3Py and Cit both appear to be critical elements in the
catalytic triad, along with His.
These studies clearly indicate the subtle influences of
structure on reactivity, including the length of the helix, place-
ment of functionality within the helix and the preference for
310 or alpha-helicity. These factors would be difficult to
predict, reinforcing the benefits of the screening methodology
used here. While the exact catalytic roles of the 3Py and Cit
sidechains have yet to be determined, this work clearly demon-
strates the opportunities for discovering novel catalytic moi-
eties using reactive tagging of helical peptide libraries and the
subtle features that can influence catalytic activity.
Fig. 8 Computationally determined low energy conformation for the
peptide Ala8His highlighting a possible contact between the imidazole
and the N-acyl terminus.
Acknowledgements
Computational modeling
This work was supported with funding from the Defense
Threat Reduction Agency (HDTRA1-10-1-0030). M.M. acknowl-
edges a fellowship from the NRC.
To better understand how the length of the peptide and/or
position of the His in the helix may influence reactivity, a
quantum mechanical calculation (M06-2X geometry optimi-
zation in TFE; see ESI† for details) on a model alpha-helical
scaffold (Ala8His, Chart 4) was performed (Fig. 8). A minimum
energy structure was identified in which the imidazole group
is in a gauche orientation (N–Cα–Cβ–Cγ dihedral angle =
−54.5°) and the δCH proton is approximately 2.4 Å away from
the capping acyl methyl group. This contact is not possible in
the His-scaffold 4T and His-3Py-Cit 4T peptides, as the spacing
of an extra turn between the imidazole and the N-terminus
brings them outside of interaction range. Moreover, the gauche
orientation found for Ala8His in contrast to the lowest energy
conformation in an aqueous alpha-helix, which has been
shown to be the trans conformation.35 Interestingly, the trans
conformation is required to bring His in close proximity to the
i + 3 and i + 4 residues, suggesting that the 4T peptides may
populate a more favorable conformation for catalysis than do
the 2T peptides, in which His is close to the N-terminus. This
is also consistent with the fact that placing 3Py and Cit at the
i − 3 and i − 4 positions, respectively, (Cit-3Py-His 4T) results
in a 4-fold decrease in reactivity (compare Table 1, entries 9
and 13). Thus, the conformation of His may be influenced by
its location in the helix, which influences its catalytic activity.
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