Interplay of stereochemistry, conformational rigidity, and ease of synthesis for 13-membered cyclic peptidomimetics containing APC residues

Article abstract of DOI:10.1021/acscombsci.7b00041

As part of a program to design small molecules that bind proteins, we require cyclic peptides (or peptidomimetics) that are severely constrained such that they adopt one predominant conformation in solution. This paper describes syntheses of the 13-membered cyclic tetrapeptides 1 containing aminopyrrolidine carboxyl (APC) residues. A linear precursor was prepared and used to determine optimal conditions for cyclization of that substrate. A special linker was prepared to enable cyclization of similar linear peptidomimetics on a solid phase, and the solution-phase cyclization conditions were shown to be appropriate for this too. Stereochemical variations were then used to determine the ideal APC configuration for cyclization of the linear precursors (on a solid phase, using the conditions identified previously). Consequently, a series of compounds were prepared that are representative of compounds 1. Conformational studies of representative compounds in DMSO solution were performed primarily using (i) NOE studies, (ii) quenched molecular dynamics simulations using no constraints from experiment, and (iii) MacroModel calculations with NMR constraints. All three strategies converged to the same conclusion: the backbone of molecules based on 1 tends to adopt one preferential conformation in solution and that conformation can be predicted from the stereochemistries of the α-amino acids involved.

Full text of DOI:10.1021/acscombsci.7b00041

Research Article
pubs.acs.org/acscombsci
Interplay Of Stereochemistry, Conformational Rigidity, And Ease Of
Synthesis For 13-Membered Cyclic Peptidomimetics Containing APC
Residues
Dongyue Xin, Andrew Jeffries, and Kevin Burgess*
Department of Chemistry, Texas A & M University, Box 30012, College Station, Texas 77842, United States
S
* Supporting Information
ABSTRACT: As part of a program to design small molecules
that bind proteins, we require cyclic peptides (or peptidomi-
metics) that are severely constrained such that they adopt one
predominant conformation in solution. This paper describes
syntheses of the 13-membered cyclic tetrapeptides 1
containing aminopyrrolidine carboxyl (APC) residues. A linear
precursor was prepared and used to determine optimal
conditions for cyclization of that substrate. A special linker was prepared to enable cyclization of similar linear peptidomimetics
on a solid phase, and the solution-phase cyclization conditions were shown to be appropriate for this too. Stereochemical
variations were then used to determine the ideal APC configuration for cyclization of the linear precursors (on a solid phase,
using the conditions identified previously). Consequently, a series of compounds were prepared that are representative of
compounds 1. Conformational studies of representative compounds in DMSO solution were performed primarily using (i) NOE
studies, (ii) quenched molecular dynamics simulations using no constraints from experiment, and (iii) MacroModel calculations
with NMR constraints. All three strategies converged to the same conclusion: the backbone of molecules based on 1 tends to
adopt one preferential conformation in solution and that conformation can be predicted from the stereochemistries of the α-
amino acids involved.
KEYWORDS: peptidomimetics, cyclic peptide, conformational studies, quenched molecular dynamics, macromdel calculations
α- with a cyclic β-amino acid in a linear peptide mimic extends
the main chain by one atom but does not seriously compromise
its rigidity because the original residue and its replacement both
have two significant degrees of freedom.
INTRODUCTION
■
Amide bonds in peptide conformations are presumed to be
planar and s-trans, so a Gly residue may be said to have only
two significant degrees of freedom, corresponding to its ϕ and
ψ dihedral angles. Rotational impediments on ϕ and ψ dihedral
angles imposed by side-chains mean that all the other
genetically encoded residues are more constrained than Gly.
Nevertheless, there is significant flexibility about ϕ and ψ
dihedral angles for all the genetically encoded amino acids
except proline (that is more constrained); hence, they may be
regarded as having two significant degrees of freedom.
Peptidomimetics from acyclic β-amino acids will almost
invariably be more flexible than the corresponding ones in
genetically encoded peptides because they have three significant
degrees of freedom. However, like proline, cyclic β-amino acids
are less flexible than their acyclic analogs: they have only two
significant degrees of freedom. Consequently, substitution of an
Received: March 1, 2017
Revised: April 10, 2017
© XXXX American Chemical Society
A
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On the basis of the generalities outlined above, it is to be
expected that substitution of α-amino acids in peptides with
cyclic β-amino acids tends to extend them without significantly
increasing their flexibility. However, syntheses of substituted
carbocyclic β-amino acids are nontrivial so it is difficult to
preserve side-chain functionalities when α-amino acids are
substituted with cyclic β-amino acids.
favoring closure to 13-membered systems. Consequently, the
research described here features the first reported syntheses of
cyclic peptidomimetics containing APC residues, specifically
the 13-membered ring derivatives 1.
Aminopyrrolidine carboxyl (APC) residues like A and B are
special because they are relatively rigid and have an endocyclic
N-atom that could be alkylated with groups to mimic side-
chains or serve other purposes. Gellman’s group was first to use
APC residues A and ent-A in linear peptidomimetics.1,2 To
facilitate this, they devised a synthesis of A and ent-A that
involved an inexpensive chiral auxiliary (1-phenylethylamine,
available as both enantiomers) and purification of diaster-
eomers via recrystallization.3,4 Consequently, there are at least
16 papers in the literature that report applications of A and ent-
A in foldamers.5−7 For instance, these residues have been
incorporated into peptidomimetics intended to disrupt
protein−protein interactions (PPIs, e.g., in BH3 domains,8−10
glucagon-like peptide 1,11 vascular endothelial growth factor12),
as scaffolds for presenting carbohydrates13 or peptide nucleic
acid residues,14 and as mimics of nonhelical secondary
structures.15
Targets 1 were selected because they have several attractive
features. We hypothesized macrocycles 1 may adopt single
conformational states in solution; that rigidity is useful when
attempting to understand how preferred solution state
conformations relate to binding. Unpublished molecular
dynamics calculations from our group that suggest 12-
membered ring cyclic tetrapeptides also should be conforma-
tionally rigid provided they do not contain Gly and Pro
residues, but these are hard to prepare. Conversely, we
suspected compounds 1 might be far more accessible via
solid phase syntheses than cyclic tetrapeptides composed of
only α-amino acids. These putative attributes, and the fact that
the pyrrolidine NH could be functionalized, are valuable to our
broader research program that features conformationally rigid
compounds that can present three amino acid side-chains in
disruption of protein−protein interactions.34,35 Consequently,
we decided to study synthetic accessibilities and the conforma-
tional biases of the APC-containing, cyclic tetrapeptides 1.
It emerges here that the featured compounds 1 are (i)
accessible via solid phase syntheses, (ii) constrained to single
conformers in DMSO solution, and unexpectedly, (iii) are
inclined to adopt conformations that correlate with the
stereochemistries of their constituent amino acids in a
predictable way.
The cis-APC stereomers B are more time-consuming to
prepare in enantiomerically pure form than their trans-isomers
A,16−18 hence it is unsurprising that they have been used less
frequently. Most of the literature on cis-APCs B and ent-B
concerns specific applications in medicinal chemistry,19−24 but
there are few in peptidomimetics.
A long-term project in our laboratories requires conforma-
tionally rigid cyclic peptidomimetics that can be prepared
expeditiously via solid phase methods. To our surprise, we were
unable to find examples of cyclic peptidomimetics containing
either trans- or cis-APC residues. Broadening the search to
include peptidomimetics containing carbocyclic analogs of
APCs like C and D also revealed little information. There is one
report of a cyclic peptidomimetic based on C25 and one on
D;26 in each case these are larger, and presumably more
conformationally flexible, macrocycles than the ones in this
manuscript (see below).
RESULTS AND DISCUSSION
■
Syntheses. Samples of the trans-APC compounds required
in this study were prepared via Gellman’s method.3 Syntheses
of their cis-isomers were achieved via a small modification of a
literature procedure by Confalone et al.;16 this change enabled
access to the hitherto unknown FMOC-protected compounds
3 and 4 (Scheme 1).
Microwave (mw) assisted solid phase syntheses of the linear
APC-containing peptidomimetics 5 were performed as outlined
in Scheme 2. Use of chlorotrityl resin (Chem-Impex) gave
relatively hindered esters that we hypothesized would protect
the APC residues from epimerization.36 In the event, the overall
procedure worked well to give the desired linear products in
>95% crude purity by HPLC (UV detection at 254 nm), and
no epimerization was detected.
The sequence LLL-5aay′ was used for optimization studies
(throughout this paper lower case one-letter codes relate the
side-chains R1−R3 to the closest amino acid, and primed letters
denote protection, e.g., y′ for −CH2C6H4−4-OtBu and a for
−CH3 of Ala). In fact, the calculations described below were
performed for the sequence LLL-5aaa but it was impractical to
focus on that motif for experimental studies because Ala-Ala-
Ala has no conspicuous chromophore; consequently, we settled
on LLL-5aay′ for optimization. Approximately 20 sets of
conditions were tested for cyclization of (R,S)-APC-LLL-5aay′
12-Membered cyclic tetrapeptides are notoriously difficult to
prepare,27−33 but we suspected replacement of a genetically
encoded amino acid with an APC residue should enhance the
conformational rigidity (amino acid with 2 significant degrees
of freedom replaced by another having the same, see above)
while the extra atom could reduce ring strain effects thus
B
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(Figure 1) and the conditions shown in Figure 1a were the best
Scheme 1. Synthesis of New, FMOC-Protected, cis-APC
Derivatives
identified for this cyclization step.
Figure 1. Cyclizations of LLL- and DDD-stereoisomers of the linear
peptidomimetics to give protected forms of the featured product 6.
Scheme 2. Solid-Phase Syntheses of Linear Peptidomimetics
5
Having found suitable conditions for cyclization of (R,S)-
APC-LLL-5aay′, we set out to determine the APC stereoisomer
that would best facilitate cyclization of LLL-5aay′. Compound
availability issues made it more convenient to test (R,R)-APC-
DDD-5aay′ than (S,S)-APC-LLL-5aay′ and (R,S)-APC-DDD-
5aay′ than (S,R)-APC-LLL-5aay′, but in each case the result
would be the same since these are pairs of enantiomers. Thus,
the compounds in Figure 1b represent one enantiomeric form
of every energetically distinct cyclization via the disconnection
implied in Figure 1. In the event, the cis-APC, (R,R)-APC-
DDD-6aay′ gave a higher yield of its corresponding cyclization
product than any of the other reactions. Undesired cyclic dimer
formed in all four reactions (LC-MS), but least for the cis-
isomer (R,R)-APC-DDD-6aay′. These observations indicate
cyclization to the enantiomer of (R,R)-APC-DDD-6aay′, and
(S,S)-APC-LLL-6aay′, would be similarly favored. Thus, cis-
(S,S)-APC was more conducive to efficient cyclization than
either of the trans-isomers when the α-amino acids were in the
L-configuration. It is for that reason that only cis-(S,S)-APC was
used in subsequent studies.
Cyclization of all isomers of (S,S)-APC-6aay′ were studied to
explore if these reactions can be as efficient as those described
above but when other α-amino acid stereomers are used. Figure
2b indicates that the least efficient cyclization to the desired
monomeric cyclic peptidomimetic was for the (S,S)-APC
combination with DDD-6aay′. Close examination of the data in
Figure 2b reveals that the cyclization reactions are more
efficient when the N-terminal residue AA1 has the L-
configuration than in comparable reactions in which it has D-
stereochemistry. This assertion is true for all the examples
shown in Figure 2b (consider the pairs connected by a bracket
on the right side of that graphic).
Figure 2c summarizes isolated yields (based on resin loading)
for formation of products 1 after solid-phase syntheses of the
C
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Scheme 3. Synthesis of a cis-APC-Loaded Linker for On-
Bead Cyclization
conditions (Scheme 4). A problem was encountered for the
FMOC-deprotection step; specifically, 2% DBU in dioxane
(conditions we had used previously to remove FMOC without
cleavage of the nosyl-like linker)37,38 caused epimerization
adjacent the APC-allyl ester. In response, 20% piperidine in
DMF at room temperature for 3 min was used for the FMOC
deprotection. When this reaction was performed at elevated
temperatures or for longer times then partial cleavage of the
nosyl-like linker was observed.
Scheme 4 showed Pd-mediated cleavage of the allyl ester then
the final FMOC deprotection before the critical on-bead
cyclization. If the order of these steps was reversed then
impurities formed, probably because of allylation of the N-
terminal amine. The cyclization step was performed using
similar conditions to those that had been developed for
solution (Figures 1 and 2); the only changes that were made
were to use a less expensive carbodiimide, and CH2Cl2/DMF
because the reaction on TentaGel was faster and gave higher
purities in that medium.
Conformational Analyses. The following approach was
used to investigate conformations of the target cyclic
peptidomimetics 1. First a molecular dynamics technique
(quenched molecular dynamics, QMD39,40) was used to
ascertain (i) the most favorable conformation that could be
found and (ii) the diversity of simulated conformations in a
continuous medium of dielectric 46.7 (corresponding to
DMSO). As mentioned above, these calculations were
performed on (S,S)-APC-LLL-1aaa; this gives the calculated,
preferred conformations of the backbone with only the simplest
possible side-chains.
Figure 2. Cyclization a complete set of stereoisomers to afford the
protected macrocycles 6. (a) Reaction conditions, (b) HPLC yield vs
time plots, and (c) summary of isolated yields for cyclic products 1
based on loading of the resin and after rpHPLC purification.
linear precursors, cleavage from the resin, cyclization in solution
t
under dilute conditions, then deprotection of Bu/BOC side-
chain and the pyrrolidine-N. The sequence involves many steps
(four coupling/deprotection reactions, cleavage, cyclization,
and deprotection) so these yields indicate the process is highly
efficient overall.
In the experiments described above, it was advantageous to
break the synthesis down into on-resin preparation of linear
peptides then cyclization. However, once the process had been
optimized, an approach using on-bead cyclization was desired
for accelerated production of the peptidomimetics 1, hence a
linker that was stable to acid and base was necessary to realize
that approach. Scheme 3 shows how a nosyl-based system
developed in our laboratories37,38 was modified to fulfill these
needs. The known compound E was hydrolyzed to 7 then
coupled with Gly. Oxidation of sulfide 8 to the sulfonyl chloride
9 was achieved using a chlorination reagent (1,3-dichloro-5,5-
dimethylhydantoin) then the featured FMOC-protected cis-
(S,S)-APC 4 was added and the tBu group was removed to give
the completed “handle” 10.
Solid-phase syntheses of the cyclic products 1 began with
coupling handle 10 to TentaGel-NH2 resin under microwave
D
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using Macromodel41 with NMR constraints (distance limitations
from NOESY). Using two simulation approaches in this way is
advantageous because they are complementary: confidence in
the calculations is increased if the conformations deduced
without using experimental data (QMD) compare well with
those that employ NMR constraints (Macromodel). Moreover,
strategies using NMR constraints are not necessarily more
realistic than unconstrained calculations because (i) NOE/ROE
effects depends on 1/r6 distance relationships,42 hence close
contacts are accentuated and distal ones are less conspicuous
and (ii) conformations deduced from molecular dynamics
calculations constrained in accord with NMR data are less
reliable for small molecules (compared with proteins for
example) because relatively few data points are involved.
Figure 3 shows conformations for one enantiomeric series of
compounds (S,S)-APC-LLL-1aaa in black (from QMD)
overlaid with the corresponding conformations of (S,S)-APC-
LLL-1aay in silver (from Macromodel with NOE constraints).
There is a maximum RMSD of 0.56 Å (based on Cα−Cβ)
indicating a good correspondence between both strategies. This
observation indicates a high level of confidence overall in the
deduced conformations.
Scheme 4. Solid-Phase Syntheses of the Featured
Compounds 1 via On-Bead Cyclization
One of the strengths of the QMD approach is that it
indicates the diversity of conformers that are energetically
accessible. Flexible molecules will afford multiple families, each
consisting of a cluster of conformations. However, QMD
analyses of all the stereoisomers shown in Figure 3 each gave
one predominant family. Only some of the stereomers gave two
families, and in the cases where there were two, the second
family was formed from far fewer conformations, and the lowest
energy conformer in that group was higher than the
corresponding one in the primary family. These observations
indicate the APC-containing molecules 1 are almost conforma-
tionally homogeneous in solution. This assertion is supported
by the proton NMR data that shows single sets of resonances
for each stereoisomer with sharp, resolved peaks for the side-
chains and for the amide protons.
Review of the structures in Figure 3 shows there are no H-
bonded NH atoms in the preferred conformers, and the degree
of solvent shielding by the ring is difficult to access. Consistent
with this temperature coefficient data43,44 (Table 1) gave no
discernible trend.
The series of structures featured in Table 1 and Figure 3 have
a fixed configuration at the (S,S)-APC residue hence an
enantiomer exists for each of the compounds illustrated.
Consistent with this, the CD spectra recorded for the set of
isomers shown in Figure 4 does not vary in a regular stepwise
pattern between the molar elipticity extremes. However, one
crude trend is evident: the isomers predominantly consisting of
all L-α-amino acids have negative molar elipticity maxima, while
those from D-α-amino acids tend to have positive molar
ellipticities.
CONCLUSION
■
Unlike cyclic tetrapeptides composed of α-amino acids, the 13-
membered ring systems featured in this work can be prepared
efficiently via solution or solid phase methods. Conformational
analyses by molecular dynamics showed that the stereo-
chemistry of the APC residues do not have a profound impact
on the conformations of the cyclic systems 1. Our experimental
data shows that the cyclization efficiencies were significantly
influenced by the APC stereochemistry. It is a little unfortunate
that the cis-APC isomers are favored because these are more
Data from the QMD experiments was then compared with
calculations and experimental NMR data obtained for (S,S)-
APC-LLL-1aay. This comparison is informative because QMD
does not use any constraints from experimental data. In parallel,
conformations of the cyclic compounds 1 were also determined
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Table 1. Temperature Coefficient Data for Isomers of (S,S)-
APC-1aay in DMSO
−Δδ/ΔT (PPB/K in DMSO-d6)
diasteromer (S,S)-APC-???-1aay APC-NH Ala1-NH Ala2-NH Tyr-NH
LLL
1.5
2.3
5.8
3.4
4.2
2.0
3.6
3.6
1.4
0.8
3.6
2.3
3.1
3.3
1.4
2.7
1.7
3.9
5.0
6.6
4.6
4.6
2.0
4.0
2.0
4.0
3.1
0.0
2.7
5.9
1.4
2.1
DLL
LDL
LLD
DDL
DLD
LDD
DDD
Figure 4. CD spectra of (S,S)-APC-LLL-1aay in acetonitrile.
time-consuming than the trans-ones to prepare, but overall, this
is not a major factor.
The CD spectra shown in Figure 4 are informative insofar as
they show a steady gradation between extremes (even though
the stereochemistry of the APC residue is constant
throughout). This implies that the conformations of these
molecules vary in a stepwise fashion as the stereochemistry of the
α-amino acids is changed. We have observed exactly the same
phenomena in a study of 13-membered ring compounds
containing anthranilic acid,45 and explain this in a similar way.
Calculated structures in Figure 5 indicate the carbonyl groups
point downward around the equator of the LLL-isomer, and all
upward in the DDD-structure. Isomers representing transitions
between these extremes have carbonyl groups that follow this
trend, that is, ones that are part of L-amino acids point down,
while those on D-amino acids point up.
Overall, this paper indicates that it is possible to prepare the
targeted 13-membered ring compounds via an efficient ring
closure step, yet the products retain a high degree of
conformational rigidity. It is an advantage that both solution
and solid phase methods have been developed because the
former allows for scalable preparation of select compounds
while the latter facilitates parallel syntheses of libraries.
Figure 3. Conformations of (S,S)-APC-LLL-1aaa deduced without
NMR constraints overlaid with conformations of (S,S)-APC-LLL-1aay
from the strategy described with NMR constraints.
F
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ASSOCIATED CONTENT
* Supporting Information
The Supporting Information is available free of charge on the
ACS Publications website at DOI: 10.1021/acscombs-
ci.7b00041.
■
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Experimental procedures and spectroscopic data for all
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AUTHOR INFORMATION
Corresponding Author
*E-mail: burgess@tamu.edu.
ORCID
Kevin Burgess: 0000-0001-6597-1842
Notes
The authors declare no competing financial interest.
■
ACKNOWLEDGMENTS
■
Financial support for this project was provided by the National
Science Foundation (CHE-1608009), National Institutes of
Health (HL126346), The Robert A. Welch Foundation (A-
1121), DoD BCRP Breakthrough Award (BC141561), and
CPRIT (RP150559 and RP170144). We thank Dr. Lisa M.
Perez for useful discussions. NMR instrumentation at Texas
A&M University was supported by a grant from the National
Science Foundation (DBI-9970232) and the Texas A&M
University System.
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DOI: 10.1021/acscombsci.7b00041
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