Mimicry of a β-Hairpin Turn by a Nonpeptidic Laterally Flexible Foldamer

Article abstract of DOI:10.1021/acs.orglett.8b01463

The design and characterization of a proteomimetic foldamer that displays lateral flexibility endowed by intramolecular bifurcated hydrogen bonds is reported. The MAMBA scaffold, derived from meta-aminomethylbenzoic acid, adopts a serpentine conformation that mimics the side chain projection of all four residues in a β-hairpin turn.

Full text of DOI:10.1021/acs.orglett.8b01463

Letter
pubs.acs.org/OrgLett
Cite This: Org. Lett. XXXX, XXX, XXX−XXX
Mimicry of a β‑Hairpin Turn by a Nonpeptidic Laterally Flexible
Foldamer
Joseph W. Meisel,* Chunhua T. Hu, and Andrew D. Hamilton*
Department of Chemistry, New York University, New York, New York 10003, United States
S
* Supporting Information
ABSTRACT: The design and characterization of a proteomimetic foldamer that displays lateral flexibility endowed by
intramolecular bifurcated hydrogen bonds is reported. The MAMBA scaffold, derived from meta-aminomethylbenzoic acid,
adopts a serpentine conformation that mimics the side chain projection of all four residues in a β-hairpin turn.
ynthetic structures that emulate the arrangement and
chemical recognition features of peptide and protein
S
surfaces are known as peptidomimetics and proteomimetics,
respectively.1 The development of peptidomimetics to
modulate protein−protein interactions (PPIs) is of particular
interest for the study and treatment of disease, especially where
traditional medicinal chemistry approaches have been un-
successful.2 PPIs are mediated over large surface areas by many
weak interactions, with some “hot-spot” residues contributing
more than others.3 Optimal peptidomimetics and proteomi-
metics should possess large, densely functionalized surfaces that
can mimic and compete with PPI interface motifs.4 Small-
molecule pharmaceuticals typically have a comparatively
reduced surface area and may not contribute the binding
interactions sufficient for high affinity or specificity for a protein
surface.5
A variety of protein structure motifs participate in binding
interactions at the interface of PPIs.6 Therefore, peptidomi-
metic scaffolds have been designed to mimic α-helices, β-
strands, or various turn elements.7 Peptide-based systems often
attempt to mitigate excessive flexibility by using macro-
cyclization8 and rigid template methods.9 Conformational
control in nonpeptidic systems is achieved by restricting
flexibility through conjugation, steric effects, dipolar repulsion,
and hydrogen bonds. We have previously utilized these
approaches in indolinone10 and pyridyl-linked imidazolidin-2-
one11 β-strand mimetics and in terphenyl12 and pyridylamide13
α-helix mimetic foldamer scaffolds (Figure 1). Peptidomimetics
and proteomimetics have successfully been applied to the study
of intrinsically disordered proteins,14 in cancer cell signaling,15
and as antibiotics.16
Figure 1. Peptidomimetic conformational flexibility. (A) Terphenyls
and indolinones twist along the oligomer axis. (B) Pyridylamides and
pyridylimidazolinones curve vertically to form a concave binding
surface. (C) MAMBA oligomers are laterally flexible and form cis (left
schematic) and trans (right schematic) conformers.
that they are generally linear, conjugated systems that tend
toward planarity. These scaffolds possess an axis along which
The arrangement of rigid and flexible components in
peptidomimetics and proteomimetics endows different modes
of folding and therefore defines which protein motifs can be
mimicked. A unifying feature of many nonpeptidic scaffolds is
Received: May 9, 2018
© XXXX American Chemical Society
A
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the oligomer chain extends and a vertical plane from which the
side chains project. Using these directional standards, one can
characterize flexibility in these systems by axial rotation (Figure
1A) and vertical flexion (Figure 1B), which produces twisted
and curved structures, respectively. Acute vertical flexion gives
rise to helical structures.17 The extended conjugation of many
scaffolds derived from aromatic monomers generates rod-like
structures, which have proven effective in reproducing the side
chain orientation of a single face of an α-helix or β-strand.18
However, mimicking broader protein surfaces and nonlinear
motifs with nonpeptidic scaffolds still remains a challenge.
In this work, we report a nonpeptidic scaffold based on
oligoamides of 2,4-dialkoxy-meta-aminomethylbenzoic acid
(MAMBA) that demonstrates lateral flexibility (Figure 1C),
allowing the mimicry of more extended protein surfaces. A
MAMBA trimer mimics the position and orientation of all four
side chains in a β-hairpin turn motif. By combining
conformation-rigidifying hydrogen bonds with flexible methyl-
ene groups, this scaffold can adopt folded conformations that
emulate the three-dimensional array of amino acid side chains
on protein surfaces.
A critical aspect of peptidomimetic design is the scaffold
conformation, which specifies the spacing and orientation of
amino acid side chain isosteres. To investigate these properties
of MAMBA, oligomers were prepared using a solution-phase
Boc/COOMe protection strategy (Figure 2A). The dimer Boc-
The MAMBA scaffold is designed for facile functionalization
and elongation. It is composed of oligomeric meta-amino-
methylbenzoic acid units with alkoxy groups appended in
positions 2 and 4 on the benzene ring. The benzylic methylene
is intended to disrupt conjugation in order to increase scaffold
flexibility. Oligomers of the closely related 2,4-dialkoxy-meta-
aminobenzoic acid are fully conjugated and therefore lack
lateral flexibility. These stuctures form crescent oligoamides and
helices due to vertical flexion.19 Coupling of MAMBA
monomers forms a secondary amide whose proton participates
in bifurcated hydrogen bonds with the phenolic ether oxygen
atoms of adjacent monomers.20 This creates a hydrogen bond
assisted hinge that directs alkoxy groups to one face of the
foldamers, while maintaining flexibility lateral to the extending
chainsimilar to the serpentine form of a snake in motion.
In order to test the scaffold design, we prepared a dimethoxy-
functionalized MAMBA-1 monomer, which we abbreviated as
Mmb1. As MAMBA monomers are δ-amino acids, we chose to
use conventional amino acid nomenclature for describing
carboxy and amino protecting groups. Thus, the tert-
butylcarbamate-protected Mmb1 compound 5 is abbreviated
Boc-Mmb1-OH. Monomer 5 was prepared from commercially
available methyl 2,4-dimethoxybenzoate in five steps (Scheme
1). Methyl ester 1 was formylated regioselectively at the 5-
Figure 2. (A) Chemical structures of MAMBA dimer, trimer, and
truncated trimers. (B) X-ray diffraction structure of dimer 6 as viewed
from the side (left) and the top (right) with methoxy carbon atoms
highlighted in green to illustrate the side chain spatial orientation.
Mmb12-OMe (6) and trimer Boc-Mmb13-OMe (7) were
prepared by amino acid coupling under the standard conditions
(see Supporting Information (SI)). These compounds were
examined by single-crystal X-ray diffraction. The expected
intramolecular bifurcated hydrogen bond was observed in the
crystal structure of dimer 6, causing all methoxy groups to be
displayed on a single face of the structure (Figure 2B). The
benzylic methylene group permits the scaffold to deviate from
planarity, creating a 115° hinge between monomers. A
nonplanar scaffold is advantageous, as it permits mimicry of
nonlinear protein motifs.
The phenolic oxygen atoms of the MAMBA monomer serve
as locations for diverse functionalization and side chain
mimicry. The oxygen atom simulates the α-carbon of an
amino acid side chain with the attached methyl group
simulating the β-carbon (Figure 2B). Interatom distances are
between 3.8 and 4.9 Å for adjacent groups. In the crystal
structure of 6, the terminal tert-butylcarbamate is rotated out of
plane to form an intermolecular hydrogen bond with the
carbonyl oxygen of an adjacent molecule. This was observed for
both the dimer 6 and the trimer 7 (see SI) crystal structures.
The intermolecular contact is mediated by the carbamate
proton, causing the intramolecular hydrogen bond to the 4-
methoxy group to be broken. This suggests that the
benzylamide hydrogen bond to the 4-methoxy (4-OMe)
group is weaker than the benzamide hydrogen bond to the 2-
methoxy (2-OMe) oxygen atom. To minimize the potential for
intermolecular contacts between terminal groups in the solid
state, a truncated trimer 8 was prepared lacking terminal
carboxy, aminomethyl, and methoxy groups. In addition,
truncated trimer analogues 9 and 10 were prepared lacking
an internal methoxy group to investigate the relative hydrogen
bond strengths of the 2-OMe and 4-OMe groups (Figure 2A).
A truncated MAMBA trimer such as 8, with each set of
bifurcated hydrogen bonds intact, can display a surface with
four different functional groups. A nontruncated trimer
Scheme 1. Synthesis of MAMBA-1 Monomers
position under Rieche conditions21 to give aldehyde 2.
Methoxime 3 was prepared using methoxyamine and was
subsequently converted to benzylamine 4 (H-Mmb1-OMe·
HCl) by catalytic hydrogenation under acidic conditions.
Amino group protection with di-tert-butyl dicarbonate, followed
by saponification of the methyl ester, gave Boc-Mmb1-OH
monomer 5 in good yield.
B
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contains six customizable positions. The benzylic methylene
spacers endow the hydrogen-bonded structure with lateral
flexibility; thus, a trimer can adopt cis or trans conformations,
which are likely in equilibrium. The trans conformation
arranges functional groups in a pleated pattern (Figure 1C,
right), while the cis trimer will adopt a U-shaped turn (Figure
3A). Crystals of truncated trimer 8 suitable for structure
In order to be an effective proteomimetic, the structure must
be capable of adopting the appropriate binding conformation in
solution. Under most biological conditions, this implies a
necessarily polar environment, which may disrupt the intra-
molecular hydrogen bonding of the MAMBA foldamer. To
probe the hydrogen bond in solution, truncated trimers 8−10
1
were characterized by H NMR experiments in various solvent
mixtures. Figure 4 depicts the change in chemical shift for the
Figure 3. (A) Chemical structures (left) and crystal structure (right)
of truncated trimer 8 in a fully hydrogen-bonded cis conformation. (B)
Overlay of 8 (violet) aligned with a canonical type II β-hairpin
(extracted from PDB 1fth). Hydrogen atoms and side chains are
truncated for clarity. An eight-point RMSD of 1.1 Å was calculated
from Cα−Cα and Cβ−Cβ distances.
Figure 4. Amide 1H NMR chemical shift dependence on solvent
polarity for hydrogen-bonded and nonbonded truncated trimers.
two amide protons of 8−10 as a function of increasing the
fraction of dimethyl sulfoxide in chloroform. For the
tetrafunctionalized 8 the chemical shift values for NHa and
NHb are nearly identical in chloroform and shift only slightly
downfield as the solvent polarity increases. Truncated trimer 9
lacks the N-terminal 2-OMe group as a hydrogen bond
acceptor for NHa, which results in a significant upfield shift in
chloroform. As the DMSO fraction increases, this proton is free
to interact with polar DMSO molecules, and the chemical shift
is observed to move downfield to a greater extent than both
amide protons in 8. A similar, albeit less dramatic, trend is
observed for NHb in 10. These data confirm that the
benzamide indeed forms the stronger hydrogen bond and
account for the observation that the hydrogen bond to the 4-
OMe in some crystal structures is preferentially disrupted. We
interpret the lower chemical shifts in 8 compared to 9-NHa and
10-NHb in 100% DMSO to indicate that even in this polar
solvent the hydrogen-bonded structure is a significant
contributor to the overall conformational equilibrium.
Additional conformational analysis was conducted by 2D
NOESY NMR experiments. Truncated trimers 8−10 were
examined in chloroform and dimethyl sulfoxide for interactions
between the amide, benzylic, and aromatic ring hydrogens (see
SI, section 4.2). In chloroform, the signal of an amide with two
adjacent hydrogen bond acceptors (8, 9-NHb, and 10-NHa)
overlaps with the ortho-aryl hydrogen, thus preventing
substantive interpretation. However, in DMSO, these peaks
are well resolved and show strong NOE correlations between
benzylic protons and the ortho-aryl hydrogen and weak or
nonexistant crosspeaks for NH−aryl pairs, indicating the
determination by X-ray diffraction were obtained and showed
the compound in the cis conformation with all four methyl
groups pointing to the same face of the foldamer, similar to a β-
hairpin turn.
The β-hairpin turn motif consists of four amino acids with
each side chain directed to a single face of the β-sheet. The β-
hairpin has been shown to be an important motif in loop-
mediated protein−protein interactions.6c A key structural
aspect of the hairpin is a hydrogen bond between the carbonyl
oxygen of residue i and the N-terminal amide proton of residue
i + 3. β-Hairpin turn mimetics that induce the interaction
between these two residues have been developed using a variety
of scaffolds, including dibenzofuran,22 azobenzene,23 and many
others.24 These structures are often synthetically challenging or
functionally limited. By omitting the side chains of interceding
residues i + 1 and i + 2, many hairpin-templating scaffolds
potentially forsake important binding interactions. To
determine the potential for mimicry of all four turn residues
by the MAMBA scaffold, the cis isomer of 8 was aligned to a
canonical type II β-hairpin turn (Figure 3B). The methoxy
group of the N-terminal benzamide cap mimics the i residue.
The 4-OMe and 2-OMe groups of the central MAMBA unit
overlay with the i + 1 and i + 2 residues, and the C-terminal
benzylamide cap completes the turn to mimic the i + 3 side
chain. An eight-point RMSD of 1.1 Å was determined by using
the methoxy group oxygen and carbon atoms as amino acid Cα
and Cβ isosteres, respectively. A similar overlap was observed
for type I, type I′, and type II′ β-hairpins (see SI, section 6).
C
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bifurcated hydrogen bonds maintain the predicted conforma-
tion. The amides with a single hydrogen bond acceptor (9-NHa
and 10-NHb) show stronger NH−aryl crosspeaks, especially in
the absence of the 2-OMe group in 9-NHa. This evidence
confirms that the planar 2-OMe hydrogen bond is stronger
than the 4-OMe bond and that the MAMBA scaffold is flexible,
yet retains its hydrogen-bonded character in polar solvents.
In conclusion, a proteomimetic scaffold MAMBA was
designed with lateral flexibility provided by a tripartite
bifurcated hydrogen bond hinge. The foldamer is extended
through amide bonds to form the conformation-directing
hydrogen bond motif, which is maintained in polar solvents.
MAMBA foldamers display a broad and densely functionalized
proteomimetic surface and are synthetically accessible, and the
cis conformation was shown to replicate the position and
orientation of all four residues in a β-hairpin turn. Synthetic
methods are currently being developed in our laboratory to
incorporate chemically diverse side chains and to accommodate
solid-phase synthetic methods. The development of MAMBA
oligomers as customizable macromolecular recognition tools
and as modulators of biomedically relevant PPIs is underway.
REFERENCES
■
(1) (a) Jayatunga, M. K. P.; Thompson, S.; Hamilton, A. D. Bioorg.
Med. Chem. Lett. 2014, 24, 717−724. (b) Fletcher, S.; Hamilton, A. D.
Curr. Opin. Chem. Biol. 2005, 9, 632−638.
(2) (a) Ivanov, A. A.; Khuri, F. R.; Fu, H. Trends Pharmacol. Sci.
2013, 34, 393−400. (b) Vagner, J.; Qu, H.; Hruby, V. J. Curr. Opin.
Chem. Biol. 2008, 12, 292−296.
(3) Thangudu, R. R.; Bryant, S. H.; Panchenko, A. R.; Madej, T. J.
Mol. Biol. 2012, 415, 443−453.
(4) Corbi-Verge, C.; Kim, P. M. Cell Commun. Signaling 2016, 14, 8.
(5) Scott, D. E.; Bayly, A. R.; Abell, C.; Skidmore, J. Nat. Rev. Drug
Discovery 2016, 15, 533−550.
(6) (a) Watkins, A. M.; Wuo, M. G.; Arora, P. S. J. Am. Chem. Soc.
2015, 137, 11622−11630. (b) Watkins, A. M.; Arora, P. S. ACS Chem.
Biol. 2014, 9, 1747−1754. (c) Gavenonis, J.; Sheneman, B. A.; Siegert,
T. R.; Eshelman, M. R.; Kritzer, J. A. Nat. Chem. Biol. 2014, 10, 716−
722.
(7) Pelay-Gimeno, M.; Glas, A.; Koch, O.; Grossmann, T. N. Angew.
Chem., Int. Ed. 2015, 54, 8896−8927.
(8) (a) Patgiri, A.; Jochim, A. L.; Arora, P. S. Acc. Chem. Res. 2008,
41, 1289−1300. (b) Walensky, L. D.; Bird, G. H. J. Med. Chem. 2014,
57, 6275−6288. (c) Favre, M.; Moehle, K.; Jiang, L.; Pfeiffer, B.;
Robinson, J. A. J. Am. Chem. Soc. 1999, 121, 2679−2685.
(9) Nowick, J. S.; Lam, K. S.; Khasanova, T. V.; Kemnitzer, W. E.;
Maitra, S.; Mee, H. T.; Liu, R. J. Am. Chem. Soc. 2002, 124, 4972−
4973.
ASSOCIATED CONTENT
* Supporting Information
■
S
(10) Wyrembak, P. N.; Hamilton, A. D. J. Am. Chem. Soc. 2009, 131,
4566−4567.
The Supporting Information is available free of charge on the
ACS Publications website at DOI: 10.1021/acs.or-
glett.8b01463.
(11) German, E. A.; Ross, J. E.; Knipe, P. C.; Don, M. F.; Thompson,
S.; Hamilton, A. D. Angew. Chem., Int. Ed. 2015, 54, 2649−2652.
(12) Orner, B. P.; Ernst, J. T.; Hamilton, A. D. J. Am. Chem. Soc.
2001, 123, 5382−5383.
Experimental details, synthesis and characterization of all
new compounds, 1D and 2D NMR spectra, and
crystallographic information (PDF)
(13) Ernst, J. T.; Becerril, J.; Park, H. S.; Yin, H.; Hamilton, A. D.
Angew. Chem., Int. Ed. 2003, 42, 535−539.
(14) (a) Kumar, S.; Hamilton, A. D. J. Am. Chem. Soc. 2017, 139,
5744−5755. (b) Oh, M.; Lee, J. H.; Wang, W.; Lee, H. S.; Lee, W. S.;
Burlak, C.; Im, W.; Hoang, Q. Q.; Lim, H.-S. Proc. Natl. Acad. Sci. U. S.
A. 2014, 111, 11007−11012.
Accession Codes
CCDC 1828445−1828448 contain the supplementary crystal-
lographic data for this paper. These data can be obtained free of
charge via www.ccdc.cam.ac.uk/data_request/cif, or by email-
ing data_request@ccdc.cam.ac.uk, or by contacting The
Cambridge Crystallographic Data Centre, 12 Union Road,
Cambridge CB2 1EZ, UK; fax: +44 1223 336033.
(15) Chang, Y. S.; Graves, B.; Guerlavais, V.; Tovar, C.; Packman, K.;
To, K.-H.; Olson, K. A.; Kesavan, K.; Gangurde, P.; Mukherjee, A.;
Baker, T.; Darlak, K.; Elkin, C.; Filipovic, Z.; Qureshi, F. Z.; Cai, H.;
Berry, P.; Feyfant, E.; Shi, X. E.; Horstick, J.; Annis, D. A.; Manning, A.
M.; Fotouhi, N.; Nash, H.; Vassilev, L. T.; Sawyer, T. K. Proc. Natl.
Acad. Sci. U. S. A. 2013, 110, E3445−E3454.
AUTHOR INFORMATION
Corresponding Authors
■
(16) Zerbe, K.; Moehle, K.; Robinson, J. A. Acc. Chem. Res. 2017, 50,
1323−1331.
(17) Jiang, H.; Leg
3448−3449.
́
er, J.-M.; Huc, I. J. Am. Chem. Soc. 2003, 125,
*Joseph W. Meisel: joe.meisel@nyu.edu.
*Andrew D. Hamilton: andrew.hamilton@nyu.edu.
ORCID
(18) (a) Azzarito, V.; Prabhakaran, P.; Bartlett, A. I.; Murphy, N. S.;
Hardie, M. J.; Kilner, C. A.; Edwards, T. A.; Warriner, S. L.; Wilson, A.
J. Org. Biomol. Chem. 2012, 10, 6469−6472. (b) Volonterio, A.;
Moisan, L.; Rebek, J. Org. Lett. 2007, 9, 3733−3736. (c) Li, X.;
Joseph W. Meisel: 0000-0003-3348-0242
Chunhua T. Hu: 0000-0002-8172-2202
Author Contributions
Miltschitzky, S.; Konig, B. Bioorg. Med. Chem. 2006, 14, 6075−6084.
̈
(19) Gong, B. Acc. Chem. Res. 2008, 41, 1376−1386.
(20) Du, P.; Jiang, X.-K.; Li, Z.-T. Tetrahedron Lett. 2009, 50, 316−
All authors have given approval to the final version of the
manuscript.
319.
(21) Rieche, A.; Gross, H.; Hoft, E. Chem. Ber. 1960, 93, 88−94.
̈
Notes
(22) Diaz, H.; Espina, J. R.; Kelly, J. W. J. Am. Chem. Soc. 1992, 114,
8316−8318.
The authors declare no competing financial interest.
(23) Aemissegger, A.; Krautler, V.; van Gunsteren, W. F.; Hilvert, D.
̈
J. Am. Chem. Soc. 2005, 127, 2929−2936.
ACKNOWLEDGMENTS
■
(24) (a) Eckhardt, B.; Grosse, W.; Essen, L.; Geyer, A. Proc. Natl.
Acad. Sci. U. S. A. 2010, 107, 18336−18341. (b) Burgess, K. Acc. Chem.
Res. 2001, 34, 826−835. (c) Whitby, L. R.; Boger, D. L. Acc. Chem. Res.
2012, 45, 1698−1709.
The authors thank NYU for funding to A.D.H. and the
National Institute of General Medical Sciences of the National
Institutes of Health (F32GM126851) for funding to J.W.M. We
are thankful for the support of the X-ray facility from the
Materials Research Science and Engineering Center (MRSEC)
program of the National Science Foundation (NSF) under
Award Numbers DMR-0820341 and DMR-1420073.
D
DOI: 10.1021/acs.orglett.8b01463
Org. Lett. XXXX, XXX, XXX−XXX