Stabilization of Cyclic β-Hairpins by Ugi-Reaction-Derived N-Alkylated Peptides: The Quest for Functionalized β-Turns

Article abstract of DOI:10.1021/acs.orglett.9b02592

A solid-phase approach including on-resin Ugi reactions was developed for the construction of β-hairpins. Various N-alkylated dipeptide fragments proved capable of aligning antiparallel β-sheets in a macrocyclic scaffold, thus serving as β-hairpin templates. Gramicidin S was used as the model β-hairpin to compare the Ugi-derived β-turns with the type-II′ β-turn. The results show that the multicomponent incorporation of such N-alkylated residues allows for the simultaneous stabilization and exo-cyclic functionalization of cyclic β-hairpins.

Full text of DOI:10.1021/acs.orglett.9b02592

Letter
pubs.acs.org/OrgLett
Cite This: Org. Lett. XXXX, XXX, XXX−XXX
Stabilization of Cyclic β‑Hairpins by Ugi-Reaction-Derived
N‑Alkylated Peptides: The Quest for Functionalized β‑Turns
Manuel G. Ricardo,†,‡,§ Aldrin V. Vasco,†,§ Daniel G. Rivera,*,†,‡ and Ludger A. Wessjohann*,†
†Department of Bioorganic Chemistry, Leibniz Institute of Plant Biochemistry, Weinberg 3, 06120 Halle/Saale, Germany
‡Center for Natural Products Research, Faculty of Chemistry, University of Havana, Zapata y G, Havana 10400, Cuba
S
* Supporting Information
ABSTRACT: A solid-phase approach including on-resin Ugi
reactions was developed for the construction of β-hairpins.
Various N-alkylated dipeptide fragments proved capable of
aligning antiparallel β-sheets in a macrocyclic scaffold, thus
serving as β-hairpin templates. Gramicidin S was used as the
model β-hairpin to compare the Ugi-derived β-turns with the
type-II′ β-turn. The results show that the multicomponent
incorporation of such N-alkylated residues allows for the
simultaneous stabilization and exo-cyclic functionalization of cyclic β-hairpins.
acrocyclization approaches based on multicomponent
prototypical β-hairpin compound, and its structure has been
Mreactions (MCRs) have recently emerged as excellent previously used to design novel peptidomimetics10−12 and sugar
amino acid13 turn inducers (Figure 1A). GS is a C2-symmetric
tools for the incorporation of conformational constrains in
peptide sequences.1,2 In particular, Ugi-type macrocycliza-
tions3−6 have proven great success in accessing constrained
cyclic peptides. At the same time, they allow for tunable exo- and
endo-functionalization of the macrocyclic scaffold. Thus the Ugi
macrocyclization has been employed for stapling peptides in α-
helical3 and turn4 conformations, but so far, the Ugi moieties
have not been responsible for imposing a specific conformation
on the peptide backbone. A different scenario shows up in
various MCR macrocyclizations developed by Yudin and
coworkers,5,6 in which either endo-cyclic heterocycles7 or exo-
cyclic amides,8 formed during the MCR ring closure do have a
determinant role in the peptide conformation by participating in
intramolecular hydrogen bonds.
In an endeavor to demonstrate that the N-alkylated peptide
fragment resulting from the Ugi macrocyclization is capable of
enforcing a conformational bias, leading to a stable secondary
structure, herein we describe a strategy for the construction of
Figure 1. (A) Gramicidin S and some β-turn mimics used in its analogs.
(B) Ugi-reaction-derived N-alkylated peptide fragments as novel β-
Ugi-derived cyclic β-hairpins. A β-hairpin is a class of secondary
structure in which a reverse turn or loop is flanked by two
antiparallel β-sheets.9 This class of peptide motif, indeed
comprising diverse conformations, is recognized as one of the
most important protein regular structures and functional
epitopes.9 There are several hairpin-stabilizing templates
known in the literature, such as the dipeptide D-Pro-L-Pro and
combinations of Pro with other amino acids (AAs), for example,
D-AA-L-Pro and Gly-L-Pro.9 However, we are not aware of
approaches based on Ugi or any other type of MCR
macrocyclization targeting peptide mimics of β-hairpins.
hairpin-stabilizing templates.
β-hairpin in which two antiparallel β-strands are connected by
two type-II′ β-turns induced by the dipeptide D-Phe-Pro. As
depicted in Figure 1B, we envisioned that an N-alkylated peptide
fragment created by the Ugi reaction could serve as a β-hairpin
stabilizing template due to the conformational bias imposed by
the turn-inducing effect of the tertiary amide, in a similar way as
Pro and N-Me-AAs do in reverse turns.9
Our group has gathered previous evidence of the ability of
Ugi-derived N-alkylated residues to fold oligopeptide chains14
We chose the antibacterial cyclic decapeptide Gramicidin S
(GS, cyclo(Pro-Val-Orn-Leu-D-Phe)2) as the model compound
for β-sheet conformational mimicry.10 Whereas our goal is not
to develop novel antibacterials, GS is considered as a
Received: July 24, 2019
© XXXX American Chemical Society
A
DOI: 10.1021/acs.orglett.9b02592
Org. Lett. XXXX, XXX, XXX−XXX

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Letter
a
Scheme 1. (A) Synthesis of Cyclic Peptide β-Hairpins by On-Resin Ugi Macrocyclization, (B) Circular Dichroism Spectra of
b
Ugi-Derived β-Hairpins Compared with Gramicidin S, and (C) Average NMR-Derived Structures of Cyclic Peptides 1 and 2
a
b
In parentheses are the yields of crude products. An analytical sample was purified to >95% purity. Most side chains are omitted for clarity.
and facilitate macrocyclization15 by engaging the two termini. In
addition, a recent study proved that the replacement of Pro by
N-Me-Ala does not affect the activity of the GS analogs.16 As a
result, we initially focused on substituting one of the Pro residues
in GS by an N-alkylated AA generated by the Ugi reaction, which
simultaneously allows an exo-cyclic functional group to be
installed, something that cannot be achieved with the use of Pro
or N-Me-AAs.
Seeking to conduct most steps on resin, including the key Ugi
multicomponent macrocyclization, an orthogonal solid-phase
peptide synthesis (SPPS) strategy was envisioned. For this, it
was important to achieve the previous attachment of the Orn
side chain to a resin, thus leaving the two peptide termini free for
macrocyclization. Albericio and coworkers were the first to
develop an SPPS approach toward cyclic peptides, comprising
the attachment of an amine-containing side chain to a resin.17 In
addition, the same group reported the synthesis of GS analogs by
cyclization of the peptide at the Orn residue, whose side chain
was anchored to a resin through a carbamate functionality.18
However, nowadays it is known that the intrinsic folding of
acyclic GS favors cyclization by residues opposed to one of the β-
turns.19 As a result, we designed the SPPS approach considering
two dissimilar sites for incorporating the N-alkylation at one β-
turn motif.
As depicted in Scheme 1, tripeptide Fmoc-Orn-Leu-D-Phe-
OAll was produced in solution and next anchored to the2-
clorotrityl (2CT) resin by the Orn side chain. Subsequent
growth of the peptide sequence included the incorporation of
either Ala or 2-aminoisobutyric acid (Aib) at the N-terminus,
followed by the deprotection of the two termini. Thus the on-
resin Ugi macrocyclization of the main-chain amino and
carboxylic acid groups was conducted following a procedure
recently developed for cyclizing peptide side chains.3 The
protocol comprises an initial transimination step using 4 equiv of
paraformaldehyde and pyrrolidine, followed by washing of the
resin-linked iminopeptide and a subsequent reaction with 4
equiv of the isocyanide in trifluoroethanol (TFE)/CH2Cl2 1:1
(v/v). Mini-cleavages and high-performance liquid chromatog-
raphy (HPLC) analysis were carried out after 12 h of reaction,
usually showing complete consumption of the linear peptide. In
cases of incomplete macrocyclization after 12 h, a second cycle
of imine formation and reaction with isocyanide is required (see
the Supporting Information (SI)). It is worth mentioning that
cleavage of the peptide from the 2CT resin was not detected
during the course of the entire SPPS protocol, not even during
the macrocyclization conducted in TFE/CH2Cl2.
Cyclic peptides 1 and 2 were produced by conducting the Ugi
reaction ring closure with Ala and Aib, respectively, as the amino
component and D-Phe as the carboxylic acid, thus forming an N-
alkylated β-turn inducer opposed to the natural D-Phe-Pro
sequence. The fact that even a peptide bearing a bulky Aib
residue at the N-terminus undergoes, with two cycles, the Ugi
macrocyclization in excellent yield corroborates the strength of
the method. In parallel, cyclic peptides 3, 4, and 5 were prepared
by choosing the other site recommended for cyclization, that is,
between Leu and D-Phe. Once more, high crude purities were
obtained for the three peptides, in which not only Pro (5) but
also Gly (4) and Aib (3) were installed as preceding residues. GS
was produced in a similar way in 67% crude purity using a final
macrolactamization instead of the Ugi reaction ring closure.
An isocyanide bearing a protected amine was employed to
introduce an additional exo-cyclic amino (cationic) group at the
GS scaffold. In principle, any functionalization can be installed
here in such a cyclo-ligation process. To address the effect of
such structural variations on the stability of the β-hairpin,
circular dichroism (CD) spectra were recorded and compared
with those of GS. As shown in Scheme 1B, cyclic peptides 3, 4,
and 5 exhibit CD spectra almost identical to those of GS,
suggesting that the N-functionalization of D-Phe does not
modify the type-II′ β-turn motif and keeps the stability of the β-
hairpin. This result is reasonable for compounds 4 and 5, in
which the N-alkylated D-Phe-Gly and D-Phe-L-Pro fragments can
be seen as mimetics of other type-II′ β-turns such as D-Pro-L-Pro
and D-Pro-Gly. However, the fact that the N-alkylated D-Phe-Aib
motif in 3 also induces this type of β-turn reproducing the β-
hairpin conformation of GS is noteworthy.
However, in the case of cyclic peptides 1 and 2, there is a small
increase in the minimum at 205 nm as compared with the CD
B
DOI: 10.1021/acs.orglett.9b02592
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a
Scheme 2. (A) Synthesis of Cyclic β-Hairpins by an SPPS Approach Comprising Two Ugi Reactions, (B) Circular Dichroism
Spectra of Double Ugi-Derived β-Hairpins Compared with Gramicidin S, and (C) Average NMR-Derived Structure of Cyclic
b
Peptide 9
a
b
In parentheses are the yields of crude products. An analytical sample was purified to >95% purity. Most side chains are omitted for clarity.
spectrum of GS, proving a slight deviation from the model GS β-
hairpin structure. To better understand this conformational
change, the solution-phase structures of 1 and 2 were
determined by means of nuclear magnetic resonance (NMR)
and molecular dynamics (MD) simulations (see the SI). The
NMR spectra of peptide 1 demonstrate an extended
conformation for Val, Orn, and Leu residues (i.e., coupling
shown in Scheme 1C, the NMR structure of cyclic peptide 2
shows a distorted reverse turn at the D-Phe-N-(Alkyl)Aib corner,
which certainly destabilizes the β-hairpin conformation.
We next wondered if the double substitution of the D-Phe-Pro
fragments by the Ugi-derived D-Phe-N-(Alkyl)Ala would result
in a stable β-hairpin conformation. As depicted in Scheme 2, an
SPPS approach was employed in which an initial on-resin Ugi
reaction enabled the introduction of the first N-alkylated Ala
residue using Fmoc-D-Phe-OH as the carboxylic acid
component and a variety of isocyanides. After the consecutive
incorporation of the following AAs, a final Ugi macrocyclization
was employed, as previously described, again coupling the
terminal D-Phe and Ala with the introduction of a second amide
N-substitution. The overall yields and purities of the crude
peptides were acceptable considering the complexity of this
SPPS sequence, which involved several coupling steps and two
on-resin Ugi reactions, one of them for the ring closure. Thus
cyclic peptides bearing long exo-cyclic aliphatic chains, phenyl
rings, as well as anionic or cationic groups were readily
produced.
The CD spectra of cyclic peptides 6−9 show a much more
intense minimum around 205 nm for all compounds as
compared with GS. This suggests a deviation from the native
GS conformation, likely due to the presence of β-turn
conformations different from that of GS. To get a deeper
insight into this finding, we chose cyclic peptide 9 to determine
the solution-phase structure based on NMR and MD
simulations. Hence, all NMR evidence confirms that this type
of cyclic peptide occurs in a β-hairpin conformation, albeit
featuring β-turns different from the native type II′. The 1H NMR
spectrum of 9 proves the conformational symmetry expected for
a cyclic C2-symmetric β-hairpin, in which there are only five
amide protons (see the SI). Moreover, as expected for extended
conformations, coupling constants 3JNH-Hα higher than 8 Hz and
strong αiNi+1 NOEs were detected for Val, Orn, and Leu
residues (i.e., the β-sheet region), and NiNi+3 NOEs between the
amide protons of Val and Leu again confirmed the β-turn
conformation at the two corners of the β-hairpin. The
temperature coefficients of the amide protons were also
determined (see the SI), showing low values for Leu and Val
3
constants JNH-Hα higher than 8 Hz and strong αiNi+1 nuclear
Overhauser effects (NOEs)) and two β-turns comprising the
Leu-D-Phe-Pro-Val and Leu-D-Phe-N-(Alkyl)Ala-Val fragments,
as confirmed by the NiNi+3 NOEs between the amide protons of
residues Val and Leu. In addition, cross-peaks indicative of the
spatial proximity between the Hα of both Orn residues were
observed.
As depicted in the NMR structure of 1 (Scheme 1C), this
cyclic backbone occurs in a β-hairpin conformation, in which the
exo-cyclic aminopeptidic moiety inserted as an amide N-
substitution points to the opposite face of the Orn side chains.
In this regard, Wishart and coworkers have shown that for type-
II′ β-turns,20 the side chains of residues i+1 and i+2 within the
turn have equatorial and axial orientations, respectively, that is,
D-Phe (i+1) and Pro (i+2) in GS. However, in cyclic peptide 1,
both the D-Phe (i+1) and N-(Alkyl)Ala (i+2) side chains have an
equatorial disposition with respect to peptide backbone (see the
side view in the SI), whereas it is actually the exo-cyclic N-
substituent that is axially oriented. This analysis suggests that the
Ugi-derived β-turn centered at D-Phe-N-(Alkyl)Ala is not of type
II′. Nonetheless, this fact does not have a detrimental influence
on the stability of the β-hairpin conformation; therefore, the
combination of D-Phe with Ugi-derived N-alkylated L-AA could
be considered as a new type of β-hairpin stabilizing template.
On the contrary, NMR evidence proves that the installation of
an N-alkylated Aib does have a negative influence on the β-
hairpin stability because the typical β-sheet characteristics are
missing in cyclic peptide 2. In this case, only the two Leu
3
residues and one Val residue show coupling constants JNH-Hα
higher than 8 Hz. Furthermore, the NOEs between Leu and Val
amide protons are observed only for the residues enclosing the
β-turn centered at D-Phe-Pro, whereas the dipeptide fragment D-
Phe-N-(Alkyl)Aib does not induce a typical β-turn motif. As
C
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residues that further confirm the occurrence of closed β-turns.20
Scheme 2C depicts the NMR structure of peptide 9, featuring a
β-hairpin conformation with the two amide N-substituents
pointing toward the opposite faces of Orn side chains.
́
(4) Vasco, A. V.; Perez, C. S.; Morales, F. E.; Garay, H. E.; Vasilev, D.;
Gavín, J. A.; Wessjohann, L. A.; Rivera, D. G. Macrocyclization of
peptide side chains by the Ugi reaction: achieving peptide folding and
exocyclic N-functionalization in one shot. J. Org. Chem. 2015, 80,
6697−6707.
In conclusion, we have developed a new type of cyclic β-
hairpin peptide by proving that the incorporation of N-alkylated
AA residues by Ugi reactions may induce β-turn conformations
and thereby stabilize the β-hairpin architecture. Our results
suggest that the combination of N-alkylated D-Phe at i+1 with
Pro, Gly, and even Aib at i+2 enables us to mimic type-II′ β-turn
motifs, as found in GS. Thus the incorporation of these
fragments opposite to the native D-Phe-Pro keeps the overall β-
hairpin conformation of GS. The substitution of Pro (i+2) by N-
alkylated Ala (but not Aib) also leads to a β-hairpin
conformation, in which the D-Phe-N-(Alkyl)Ala sequence
induces a β-turn, but not of type II′. Nonetheless, the Ugi-
derived β-turn equally imposes the antiparallel alignment of the
two β-strands in a β-sheet structure, thus forming a cyclic β-
hairpin. In this sense, the Ugi reaction-based approach allows for
the stabilization of the β-hairpin and the simultaneous
functionalization of the β-turn motifs. This exo-cyclic
modification can be used not only to modulate the bioactivity
by adding additional cationic or hydrophobic tails but also to
install bioconjugation handles, fluorescent and affinity tags, and
so on.
(5) Frost, J. R.; Scully, C. C. G.; Yudin, A. K. Oxadiazole grafts in
peptide macrocycles. Nat. Chem. 2016, 8, 1105−1111.
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enabled by amphoteric molecules. J. Am. Chem. Soc. 2010, 132, 2889−
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biological activities. Acc. Chem. Res. 2008, 41, 1278−1288.
(10) Pal, S.; Ghosh, U.; Ampapathi, R. S.; Chakraborty, T. K. Recent
Studies on Gramicidin S Analog Structure and Antimicrobial Activity.
In Peptidomimetics II. Topics in Heterocyclic Chemistry; Lubell, W., Eds.;
Springer: Cham, Switzerland, 2015; Vol. 49, pp 159−202.
(11) Xiao, J.; Weisblum, B.; Wipf, P. Electrostatic versus steric effect in
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(12) Yamada, K.; Kodaira, M.; Shinoda, S.; Komagoe, K.; Oku, H.;
Katakai, R.; Katsu, T.; Matsuo, I. Structure−activity relationships of
gramicidin S analogs containing (β-3-pyridyl)-α,β-dehydroalanine
residues on membrane permeability. MedChemComm 2011, 2, 644−
649.
ASSOCIATED CONTENT
* Supporting Information
■
S
(13) Grotenbreg, G. M.; Timmer, M. S.; Llamas-Saiz, A. L.; Verdoes,
M.; van der Marel, G. A.; van Raaij, M. J.; Overkleeft, H. S.; Overhand,
M. An Unusual Reverse Turn Structure Adopted by a Furanoid Sugar
Amino Acid Incorporated in Gramicidin S. J. Am. Chem. Soc. 2004, 126,
3444−3446.
The Supporting Information is available free of charge on the
ACS Publications website at DOI: 10.1021/acs.orglett.9b02592.
Experimental procedures, RP-HPLC chromatograms,
NMR, and HR-ESI-MS spectra of cyclic peptides
(PDF)
́
(14) Rivera, D. G.; Vasco, A. V.; Echemendía, R.; Concepcion, O.;
́
Perez, C. S.; Gavín, J. A.; Wessjohann, L. A. A Multicomponent
Conjugation Strategy to Unique N-Steroidal Peptides: First Evidence of
the Steroidal Nucleus as a β-Turn Inducer in Acyclic Peptides. Chem. -
Eur. J. 2014, 20, 13150−13161.
AUTHOR INFORMATION
Corresponding Authors
■
́
(15) Puentes, A. R.; Morejon, M. C.; Rivera, D. G.; Wessjohann, L. A.
*E-mail: dgr@fq.uh.cu (D.G.R.).
*E-mail: wessjohann@ipb-halle.de (L.A.W.).
ORCID
Peptide macrocyclization assisted by traceless turn inducers derived
from Ugi peptide ligation with cleavable and resin-linked amines. Org.
Lett. 2017, 19, 4022−4025.
(16) Li, Y.; Bionda, N.; Yongye, A.; Geer, P.; Stawikowski, M.; Cudic,
P.; Martinez, K.; Houghten, R. A. Dissociation of Antimicrobial and
Hemolytic Activities of Gramicidin S through N - Methylation
Modification. ChemMedChem 2013, 8, 1865−1872.
Daniel G. Rivera: 0000-0002-5538-1555
Author Contributions
§M.G.R. and A.V.V. contributed equally.
(17) Alsina, J.; Rabanal, F.; Giralt, E.; Albericio, F. Solid-Phase
Synthesis of “Head-to-Tail” Cyclic Peptides via Lysine Side-Chain
Anchoring. Tetrahedron Lett. 1994, 35, 9633−9636.
Notes
The authors declare no competing financial interest.
(18) Andreu, D.; Ruiz, S.; Carreno, C.; Alsina, J.; Albericio, F.;
̃
́
́
́
Jimenez, M. A.; de la Figuera, N.; Herranz, R.; García-Lopez, M. T.;
ACKNOWLEDGMENTS
■
́
Gonzalez-Muniz, R. IBTM-Containing Gramicidin S Analogues :
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Evidence for IBTM as a Suitable Type II′ β-Turn Mimetic. J. Am.
Chem. Soc. 1997, 119, 10579−10586.
A.V.V. and D.G.R. are grateful to DAAD, Germany for Ph.D. and
University Academics fellowships, respectively. Dedicated to
Prof. Dr. Armin de Meijere on the occasion of his 80th birthday.
(19) Wadhwani, P.; Afonin, S.; Ieronimo, M.; Buerck, J.; Ulrich, A. S.
Optimized Protocol for Synthesis of Cyclic Gramicidin S: Starting
Amino Acid Is Key to High Yield. J. Org. Chem. 2006, 71, 55−61.
(20) Gibbs, A. C.; Bjorndahl, T. C.; Hodges, R. S.; Wishart, D. S.
Probing the Structural Determinants of Type II ′ β-Turn Formation in
Peptides and Proteins. J. Am. Chem. Soc. 2002, 124, 1203−1213.
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