Amidine Functionality As a Conformational Probe of Cyclic Peptides

Article abstract of DOI:10.1021/acs.orglett.0c03369

The amidine functionality switches between hydrogen bond donor and acceptor roles depending on pH. Herein, the amidine was incorporated to select amides in cyclo(d-Ala-Pro-d-Phe-Pro-Gly). The unprotonated amidine-containing macrocyclic conformation resembles its oxoamide counterpart. Upon protonation, minimal alterations in the macrocyclic conformation were observed despite changes to the hydrogen bond network. The amidine disrupts hydrogen bonding at minimal steric cost, making it a useful functionality to study the effect of hydrogen bonding on the macrocyclic conformation.

Full text of DOI:10.1021/acs.orglett.0c03369

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Amidine Functionality As a Conformational Probe of Cyclic Peptides
Sungjoon Huh, Solomon D. Appavoo, and Andrei K. Yudin*
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ABSTRACT: The amidine functionality switches between hydrogen bond donor and acceptor roles depending on pH. Herein, the
amidine was incorporated to select amides in cyclo(D-Ala-Pro-D-Phe-Pro-Gly). The unprotonated amidine-containing macrocyclic
conformation resembles its oxoamide counterpart. Upon protonation, minimal alterations in the macrocyclic conformation were
observed despite changes to the hydrogen bond network. The amidine disrupts hydrogen bonding at minimal steric cost, making it a
useful functionality to study the effect of hydrogen bonding on the macrocyclic conformation.
Stabilization of these conformations is often caused by the
intramolecular hydrogen bond network set by the backbone
amides.3,4 However, the causality in this relationship is still
poorly understood.5
yclic peptides are a promising class of compounds in drug
Cdiscovery owing to their resistance to proteolytic
Replacement of the amide functionality with an isostere alters
the torsional freedom of the backbone and the hydrogen bond
donor/acceptor properties.4,6 Amidines are relatively underex-
plored peptide bond isosteres with many physical properties
similar to amides, particularly when it comes to barrier to
rotation7 due to resonance stabilization.8 Amidines are found in
nature, exemplified by birnbaurmin, pyrostatin, and noformy-
cin.9 They have also been incorporated into known macrocycles
such as vancomycin10,11 and FC131.12,13 In Boger’s study, the
incorporation of amidine into vancomycin resulted in
modulated binding to the precursor peptidoglycan termini, D-
Ala-D-Ala and D-Ala-D-Lac, presumably because removal of the
amide lone pair caused a repulsive interaction between D-Ala-D-
Lac and vancomycin. In its unprotonated state, the amidine lone
pair was shown to act as a hydrogen bond acceptor upon
interaction with the NH amide of the D-Ala-D-Ala fragment.
Upon protonation (pKa ∼ 12.4), the amidine functionality
Figure 1. (a) Cyclo(D-Ala-Pro-D-Phe-Pro-Gly) and (b) its solution
conformation (298 K, DMSO-d6).
Scheme 1. Incorporation of Thioamide to Amidine via
Thiophilic Lewis Acid
Received: October 8, 2020
degradation relative to linear counterparts and capacity to
adopt conformations that can mimic protein binding epitopes.1,2
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Figure 2. (a) ROESY and VT NMR of 1, 3, and 4. (b) Conformation of 3 (green) and 4 (blue) with its hydrogen bonding (red dash). (c) Overlay with
1 (pink), 3 (green), and 4 (blue).
curious counterpart to better known reduced amide bond
isosteres. The latter are also protonated under physiological
conditions yet are wholly different in terms of flexibility and the
resulting conformational properties.4,12
To compare and contrast amidine-containing macrocycles
with their oxoamide counterparts, we turned to a known cyclic
pentapeptide, cyclo(D-Ala-Pro-D-Phe-Pro-Gly) 1.17 This mole-
cule adopts a type-II β-turn between Pro2-Gly3 and a γ-turn
around Pro5 in both the solution conformation and the crystal
structure (Figure 1). As a regioselective handle for amidine
incorporation, thioamide was introduced into the linear peptide
based on procedure by Rapoport et al.18,19 Conversion of
thioamides to amidines is typically accomplished utilizing
thiophilic Lewis acids.11,20 Using Ag2CO3 with ammonia in
methanol solution afforded macrocycle 3 in 2 h with a modest
Figure 3. Amide region of 1H NMR for 3 (below) and 5 (above).
1
yield of 59% (Scheme 1). H NMR of 3 in DMSO-d6 showed
sharp, distinguishable peaks, consistent with a well-defined
conformation on the NMR time scale.21
switched to the hydrogen bond donor and bound to the
modified D-Ala-D-Lac fragment. Despite these advances, little is
known about the conformational preferences of cyclic peptides
Using variable-temperature (VT) NMR, backbone NHs were
determined to be either solvent exposed or undergoing H-
bonding based on temperature coefficient values (<−4.6 ppb/K
suggests solvent exposed, ≥−4.6 ppb/K suggests H-bonding).16
The amidine macrocycle 3 featured two protons (D-Ala4-NH
and amidine-NH) that were significantly above the −4.6 ppb/K
threshold, indicating involvement in intramolecular hydrogen
bonds. Additionally, temperature coefficients of Gly3-NH (−4.2
ppb/K) and D-Phe1-NH (−3.93 ppb/K) suggest that these
protons are on the cutoff for intramolecular hydrogen bonds.
The ROESY spectrum of compound 3 showed strong NOE
coupling from Gly3-NH to CαH-Pro2 and D-Phe1-NH to CαH-
Pro5 (Figure 2a).
that bear amidines. The switch between the hydrogen bond
acceptor to donor may impart significant changes to the
backbone conformation of cyclic peptides. pH-dependent
conformational switching has been shown to increase oral
bioavailability in certain small molecules owing to their ability to
adopt a more hydrophobic conformation,14 which provides
additional motivation to consider the “amidine effect” in
peptides.
In this study, amidines were incorporated into carbonyl sites
that act as hydrogen bond acceptors in cyclo(D-Ala-Pro-D-Phe-
Pro-Gly) 1 via the thioamidated macrocycles upon treatment
with a Lewis acid and ammonia. Using NMR analysis (1H, 13C,
ROESY, variable temperature),15,16 the effect of the protonated
and unprotonated amidine functionality on the peptide
conformation has been evaluated. We find that amino acid
side chains appear to be the main conformational determinants,
with intramolecular hydrogen bonding playing a relatively
insignificant role. Overall, the amidine functionality offers a
To further elucidate the conformation of compound 3, a low
energy structure was generated using a Monte Carlo conforma-
tional search (OPLS3e) using distances derived from the
1
ROESY experiment and dihedral angles from the H NMR. A
type-II β-turn was observed between Pro2-Gly3, where
hydrogen bonding was evident between D-Ala4-NH and D-
Phe1-CO. A γ-turn was observed around Pro5 where
B
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Figure 4. (a) ROESY and VT NMR of 5 and 6. (b) Conformation of 5 (green) and 6 (blue) with its hydrogen bonding (red dash). (c) Overlay with 1
(pink), 5 (green), and 6 (blue).
hydrogen bonding occurred between D-Phe1-NH and Pro5-C
O (Figure 2b). These findings matched the solution
conformation and the crystal structure of compound 1 and
can be verified by the overlay of the solution structures of 1 and 3
(Figure 2 c). The macrocycle 4, which featured amidine at the D-
Ala residue, showed a similar conformation to 1 and 3 (Figure
2c). The incorporation of an unprotonated amidine did not
perturb the conformation of 1 and maintained the hydrogen
bonding pattern. This suggests that the amidine in its
unprotonated state has similar hydrogen bond acceptor capacity
as its carbonyl counterpart.
signals of D-Phe1-NH to amidine-NH1 and CαH-Pro5 and
amidine-NH1 in compound 6 were also observed.
The conformations of 5 and 6 given by the ROESY-restrained
search (Figure 4b) showed similarity to macrocycle 1, as seen in
the overlay (Figure 4c). Slight deviation of the hydrogen
bonding pattern from compound 1 was seen in compound 5 as
D-Phe1-NH was solvent-exposed and not involved in hydrogen
bonding. Additionally, no hydrogen bonding to D-Ala4-NH was
observed, and a possible hydrogen bond between amidine-NH2
and D-Ala4-CO was seen in compound 5. The conformation
of 6 showed only D-Phe1-CO and D-Ala4-NH participating in
hydrogen bonding to form a type-II β-turn. This observation
deviates from what the VT NMR showed for residue D-Phe1-
NH (−0.33 ppb/K). The 20 minimized energy conformations
for 5 and 6 showed retention of the β-turn portion of the
molecule (see Figures S1 and S2). However, D-Phe-NH areas
showed some flexibility, which explains the observed NH
temperature coefficient. Alternatively, this discrepancy may also
be due to the deshielding effects caused by the adjacent aromatic
ring.22
Disruption of intramolecular hydrogen bonding in cyclic
peptides has been explored in Snyder’s work on roseotoxin B,5
where hydrogen-bonded amides were substituted to esters, and
in Robinson’s work where amides undergoing hydrogen
bonding were replaced with N-methyl amide.23 The alteration
of hydrogen bonds was done at the hydrogen bond donor site. In
the case of the amidine, hydrogen bonding was disrupted by
altering the hydrogen bond acceptor site upon protonation. This
provides a complementary method to interrogate the conforma-
tional consequences of intramolecular hydrogen bonding. For
macrocycles 5 and 6, protonation of the amidine blocked off the
lone pair that originally participated in an intramolecular
hydrogen bond, switching the amidine role from the hydrogen
Upon protonating compound 3, the 1H NMR spectrum of 3
and 5 showed significant changes in chemical shifts, especially
for the amidine-NH (Figure 3). The amidine proton of 3 (∼6.1
ppm) shifted downfield, and two peaks (∼8.6 ppm and ∼9.5
ppm) corresponding to the two protons of amidine of 5 were
observed.
The VT NMR of compound 5 suggests a strong hydrogen
bonding at amidine-NH1 (−2.39 ppb/K). Weaker hydrogen
bonds were observed for D-Ala4-NH (−3.76 ppb/K) and
amidine-NH2 (4.02 ppb/K). This hydrogen bonding pattern
differed from compound 1 as no hydrogen bonding was
observed for D-Phe1-NH in compound 5 (Figure 4a). In
compound 6, temperature coefficients of D-Phe1-NH (−0.33
ppb/K) and D-Ala4-NH (−0.95 ppb/K) indicated that these
two residues were undergoing hydrogen bonding, while Gly3-
NH (−7.59 ppb/K) did not. Additionally, hydrogen bonding
appeared to occur for the two amidine protons (NH1 = −1.43
ppb/K and NH2 = −2.69 ppb/K). Similar NOE couplings were
observed for 1, 5, and 6 where NOE signals of both Gly3-NH to
CαH-Pro2 and D-Phe1-NH to CαH-Pro5 were observed. In
compound 5, additional NOE signals of D-Ala4-NH to amidine-
NH1 and CαH-Pro5 and amidine-NH2 were observed. NOE
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bond acceptor to the hydrogen bond donor and changing the
intramolecular hydrogen bonding pattern. However, the general
backbone conformation of both macrocycles was retained.
Conformational rigidity was previously recorded in Snyder’s
work on roseotoxin B, where hydrogen bonds displayed a
considerably smaller effect on the conformation of small cyclic
peptides.5 Unlike proteins, where there are many cooperative
hydrogen bonds that are stronger than a single hydrogen bond
by up to 25%,24 small cyclic peptides can have only a few
hydrogen bonds. In addition, the partial double bond character-
istics of the amide backbone, the rigidifying proline residue, and
sterically bulky side chains may be determining the structure of
small peptides. These features stabilize the conformation, which
sets the intramolecular hydrogen bond network. With the parent
cyclic pentapeptide 1, stabilizing structural elements such as the
proline side chains and the potentially sterically bulky D-Phe are
present. Although the amidine functionality disturbs the original
hydrogen bonding interaction of the cyclic peptide backbone, its
steric contribution is insignificant and has a minimal role in
setting the conformation of the cyclic peptide. These results
highlight the dominance steric effects have over intramolecular
hydrogen bonding when it comes to the conformation of cyclic
peptides.
In summary, the conformational influence of the amidine
functionality on cyclic pentapeptides has been investigated.
When an amidine was substituted for a carbonyl group that acts
as a hydrogen bond acceptor, the retention of both the backbone
conformation and intramolecular hydrogen bonding was
observed in unprotonated form of amidine. Upon protonation
of the amidine, changes in the intramolecular hydrogen bonding
interactions were recorded. However, minimal consequences to
the overall conformation of the macrocycle have taken place.
This sheds additional light on the balance between the
conformational preferences induced by side chains and
transannular hydrogen bonds in cyclic peptides. The replace-
ment of hydrogen bond accepting carbonyls with amidines in
flexible cyclic peptides may have a more significant effect on the
overall backbone conformation. This is a topic of an ongoing
investigation.
Complete contact information is available at:
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Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
We thank D. Burns, J. Sheng, and Karl Z. Demmans from the
University of Toronto CSICOMP NMR facility for their
assistance with NMR spectroscopic experiments. This work
was supported by the NSERC (Discovery Grant and Canada
Research Chairs Programs).
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The Supporting Information is available free of charge at
https://pubs.acs.org/doi/10.1021/acs.orglett.0c03369.
Experimental details, including synthesis, HRMS data,
NMR spectra, VT-NMR, and solution structure determi-
nation (PDF)
AUTHOR INFORMATION
Corresponding Author
■
Andrei K. Yudin − Davenport Research Laboratories,
Department of Chemistry, University of Toronto, Toronto,
Ontario M5S 3H6, Canada; orcid.org/0000-0003-3170-
9103; Email: ayudin@chem.utoronto.ca
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ester equivalents in peptide and peptidomimetics synthesis. Org.
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et al. Use of a Conformational-Switching Mechanism to Modulate
Exposed Polarity: Discovery of CCR2 Antagonist BMS-741672. ACS
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Authors
Sungjoon Huh − Davenport Research Laboratories,
Department of Chemistry, University of Toronto, Toronto,
Ontario M5S 3H6, Canada
Solomon D. Appavoo − Davenport Research Laboratories,
Department of Chemistry, University of Toronto, Toronto,
Ontario M5S 3H6, Canada
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