Exploring β-hydroxy γ-amino acids (statines) in the design of hybrid peptide foldamers

Article abstract of DOI:10.1021/ol403290h

The synthesis and characterization of syn and anti β-hydroxy γ-amino acid (statine) diastereoisomers, their utilization in the design of hybrid peptide foldamers, and their single crystal conformations are studied.

Full text of DOI:10.1021/ol403290h

Letter
pubs.acs.org/OrgLett
Exploring β‑Hydroxy γ‑Amino Acids (Statines) in the Design of
Hybrid Peptide Foldamers
Anupam Bandyopadhyay, Ankita Malik, Mothukuri Ganesh Kumar, and Hosahudya N. Gopi*
Department of Chemistry, Indian Institute of Science Education and Research, Dr. Homi Bhabha Road, Pune-411 008, India
S
* Supporting Information
ABSTRACT: The synthesis and characterization of syn and anti β-hydroxy
γ-amino acid (statine) diastereoisomers, their utilization in the design of
hybrid peptide foldamers, and their single crystal conformations are studied.
γ-Amino-β-hydroxy acids (statines) are naturally occurring
nonribosomal amino acids widely present in many peptide
natural products. Peptides constituted with statines have been
used as protease inhibitors. For example, pepstatin,1 a naturally
occurring peptide, has shown broad inhibitory activities against
various aspartic acid proteases,2 such as pepsin, cathespin D and
E, rennin, HIV-1 protease, β-secretase, plasmepsin I and II of
the malarial parasite Plasmodium falciparum, etc. In addition,
several natural peptides containing statines or modified statines,
such as didemnins,3 dolastatins,4 hapalosin,5 tamandarins,6 etc.,
displayed promising anticancer properties. Inspired by the
remarkable biological applications of these peptide natural
products, various synthetic strategies have also been developed
for the synthesis of statines.7 Although the peptides containing
statines have been extensively investigated for their biological
properties, very little is known regarding their conformational
properties. The natural occurrence and their excellent biological
activities inspired us to investigate the conformational behavior
of these amino acids in hybrid peptides.
The conformational properties of γ- and hybrid γ-peptides
with various types of γ-amino acids such as γ4-amino acids,8 3,3-
dialkyl γ-amino acids, cyclic γ-amino acids,10 and 2,3,4-alkyl
substituted γ-amino acids have been thoroughly investigated.
Results suggest that most of these γ- and hybrid γ-peptides
prefer to adopt helical conformations. However, peptides with
α,β-unsaturated γ-amino acids12 and 4,4-dimethyl substituted γ-
amino acids13 prefer to adopt an extended sheet type of
structure. We have been interested in the conformational
analysis of various γ- and α,γ-hybrid peptides and sought to
investigate the influence of β-hydroxyl groups of statine
residues in the folding of hybrid γ-peptides. As we are
interested in understanding the conformational behavior of
both the syn and anti (with respect to side chains of the amino
acids) diastereoisomers of statines (Scheme 1), β-keto-γ-amino
esters were chosen as starting materials for the synthesis.
Scheme 1. Syn and Anti β-Hydroxy-γ-amino Acids
a
(Statines)
a
Local torsional variables of statines are also shown.
Recently, we reported the mild protocol for the synthesis of β-
keto-γ-amino esters14 starting from the N-protected amino
aldehydes by adopting the Roskamp’s procedure.15 Rich et al.
reported the synthesis of syn and anti diastereoisomers of
statines through the mild reduction of β-keto γ-amino esters
using NaBH4 in various sovlent combinations.16 We chose to
utilize THF as a sovlent in the reduction of β-keto esters to
reduce the diastereoselectivity in the reaction. The schematic
representation of the synthesis of statines is shown in Scheme
2. By utilizing the reported protocol,14 various N-Boc-β-keto-γ-
amino esters (Table 1, 1a−e) were synthesized starting from
the corresponding amino aldehyde and ethyl diazoacetate in the
Received: November 14, 2013
Published: December 9, 2013
© 2013 American Chemical Society
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Letter
amino acids were measured by introducing two additional
variables θ1 (N−Cγ−Cβ−Cα) and θ2 (Cγ−Cβ−Cα−C′) along
with ϕ (C′−N−Cγ−Cβ) and ψ (Cβ−Cα−C′−N/O) (Scheme
1). Instructively, the crystal structure of 1a reveals that the
carbonyls (CO) of keto and ester functional groups oriented
∼90° perpendicular to one another. In contrast to the
vinylogous amino acids8d where the side chain is almost
perpendicular to the double bond, the amino acid side chain
adopted a skew conformation (−18°) with the keto functional
group. The analysis of the statine 2a suggested that the
molecule adopted gauche+ (g+) and anti (t) conformations
along Cγ−Cβ and Cβ−Cα bonds, respectively. Further, the β-
hydroxyl group was involved in the network of bifurcated
intermolecular H-bonding with the other hydroxyl groups (see
Supporting Information (SI)). Encouraged by these results, we
subjected other β-keto-γ-amino esters (1) in Table 1 to the
mild NaBH4 reduction in THF. Both major (2, syn) and minor
(3, anti) diastereoisomers were separated using column
chromatography. The yield (2 + 3) and the diastereomeric
ratio of the statines are given in the Table 1. Interestingly, all
syn diastereoisomers (2a−d) displayed a more downfield amide
NH shift over their anti diastereoisomers (3a−d).
Scheme 2. Synthesis of Statines Starting from Boc-Amino
Aldehydes
Table 1. Diastereomeric Ratio of Statines Obtained after
Column Purification
To understand the conformational properties of statines in
peptide sequences, we designed two hybrid peptides P1and P2.
The sequences of these peptides are shown in Scheme 3. In the
Scheme 3. Sequences of Hybrid Peptides P1 and P2
Containing Anti (3S,4S) and Syn (3R,4S) Diastereoisomers
of β-Hydroxy-γ-phenylalanine, Respectively
presence of anhydrous tin chloride. Out of all the β-keto-γ-
amino esters in Table 1, we were able to obtain single crystals
for 1a and its X-ray structure is shown in Figure 1. To
case of P1, we incorporated the minor diastereoisomer (3S,4S)
3a, and in the case of P2, the major diastereoisomer (3R,4S) 2a
was used along with an alternating α-amino acid, Aib. Both P1
and P2 were synthesized in solution phase through a 2 + 2
convergent strategy using Boc chemistry (see SI). Statines were
used in the coupling reaction without protection of the β-
hydroxyl group. All coupling reactions were performed using
HBTU/HOBt coupling conditions, and peptides were isolated
in moderate to good yields (see SI) after silica gel column
chromatography. Instructively, all coupling reactions were
found to be clean and efficient, suggesting that unprotected
hydroxyl groups experienced no interference during the peptide
synthesis.
Figure 1. X-ray structures of β-keto-γ-phenylalanine and syn-(3R,4S)-
β-hydroxy γ-phenylalanine (major product).
Single crystals of peptides P1 and P2 were obtained from the
solution of aqueous methanol after slow evaporation, and their
X-ray structures are shown in Figure 2. The analysis of the
crystal structure of P1 reveals that the presence of two
molecules in the asymmetric unit which are interconnected by
two water molecules. Both molecules in the asymmetric unit
adopted a helical conformation with a slight variation in their
torsional variables. The helical conformation of P1 is stabilized
by two intramolecular 12-membered H-bonds between the
amide NH and CO of i and i+3 residues similar to the α,γ4-
understand the compatibility and the stereochemical output, we
initially subjected 1a to the NaBH4 reduction in dry THF at
−78 °C. The two diastereoisomers 2a (major) and 3a (minor)
obtained after the mild NaBH4 reduction were separated using
column chromatography. The major isomer (2a) gave single
crystals after slow evaporation of a methanol solution, and its
X-ray structure is shown in Figure 1. Similar to the other γ-
amino acids, the torsional values of β-keto and β-hydroxy γ-
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Organic Letters
Letter
and the structure is stabilized by two intramolecular H-bonds
between the residues i and i+3. The Phe-sta2 is accommodated
nicely into the 12-helical conformation by adopting the g+, g+
conformation along θ1 and θ2. Further, Phe-sta4 in P2 adopted
an extended conformation by having the torsional values θ2 and
ψ −170° and 160°, respectively, similar to P1. Due to the
stereochemical requirement, the β-OH group of (3R,4S) β-
hydroxy-γ-phenylalanine in P2 pointed toward the C-terminus
of the helix similar to the amide CO groups. In contrast to the
β-hydroxyl group of anti (3S,4S)Phe-sta2 in P1, the β-OH
group of the Phe-sta2 in P2 is involved in the six-membered
intramolecular H-bonding with CO of the same residue with a
CO···H distance of 2.09 Å and CO···H−O angle of 142°.
This intraresidue H-bonding is not observed in the terminal
Phe-sta4, as it does not participate in the helix due to the lack of
C-terminal amide NH; however, it is involved in the strong
intermolecular H-bonding with the solvent methanol. The
structural analysis of P1 and P2 reveal that, irrespective of the
stereochemistry at the β-position and the projection of the
hydroxyl groups, both peptides adopted the right-handed
helical conformation. Further, the superposition of the hybrid
statine peptide P2 with the known α,γ4-hybrid tetrapeptide 12-
helix8d (Boc-Aib-γ4Phe-Aib-γ4Phe-OEt) reveals a very nice
backbone correlation (Figure 2D) suggesting that statines can
be utilized as surrogates for other γ-amino acids in the design of
functional foldamers. Overall, these results suggest that syn and
anti diastereoisomers of β-hydroxy γ-amino acids can be
accommodated nicely into the 12-helical conformation without
much deviation from the α,γ4-hybrid peptide 12-helix. Although
both syn and anti diastereoisomers adopted the required g+, g+
to accommodate into the helix similar to the γ4-residues, anti
diastereoisomers may stabilize the helical folds through
additional inter-residue H-bonding compared to the syn
diastereoisomers and γ4-residues.
In conclusion, we demonstrated the facile synthesis of
various β-hydroxy γ-amino acids starting from β-keto-γ-amino
esters and studied their single crystal conformations. The
stereochemically pure anti (3S,4S) and syn (3R,4S) β-hydroxy
γ-phenylalanine were utilized in the synthesis of hybrid peptides
with alternating α-amino acids. Results suggest that irrespective
of the stereochemistry at the β-carbon center, both the α,γ-
hybrid peptides adopted 12-helical conformations. However,
the β-hydroxyl groups in anti diasteroisomers projected toward
the N-terminus of the helix similar to the amide NHs, while in
syn diastereoisomers the β-hydroxyl group projected toward the
C-terminus of the helix similar to the amide carbonyl. These
stereochemical requirement enabled the β-hydroxyl groups to
be involved in the 6-membered intraresidue H-bonds in the syn
diasteroisomers and 10-membered interresidue H-bonds in the
anti diastereoisomers, respectively. The influence of these
additional −OH group interactions with the backbone carbonyl
groups in the higher ordered helical peptides is yet to be
investigated. Overall, the facile synthesis of statines and
stereochemical analysis of monomers as well as hybrid peptides
of statines reported here may provide realistic information
regarding the structure-based design of peptides containing β-
hydroxy γ-amino acids.
Figure 2. X-ray structures of (A) P1, (B) P2, (C) Superposition of P1
and P2, and (D) Overlay of P2 and α,γ4-hybrid tetrapeptide 12-helix.
Side-chain hydrogens are not shown for clarity.
hybrid peptides.8c,d Interestingly, the β-hydroxyl groups are
pointed toward the N-terminus of the helix similar to the amide
NHs. Instructively, as a result of this unique projection of the β-
OH toward the N-terminus, the Phe-sta4 β-OH is involved in
the 10-membered intramolecular H-bonding with the CO
group of the Phe-sta2. Further, β-OH of the Phe-sta2 is
involved in the strong intermolecular H-bonding with the water
molecule which connects the other helix through intermolec-
ular H-bonding with the penultimate Aib3 CO group. Further,
analysis of the torsional variables reveals that Phe-sta2 adopted
g+, g+ conformations along Cγ−Cβ and Cβ−Cα bonds and
anticlinal conformations along N−Cγ and Cα−C(O) bonds.
In contrast, the Phe-sta4 adopted a fully extended conformation
due to the lack of terminal amide NH to participate in the
intramolecular H-bonding. The torsional values of Phe-sta
residues are given in Table 2. The H-bond parameters are
tabulated in the Supporting Information. Analysis of the crystal
structure of P2 with (3R,4S) β-hydroxy-γ-phenylalanine (syn)
suggests the presence of a single molecule in the asymmetric
unit. Similar to P1, P2 also adopted a 12-helical conformation
Table 2. Torsional Variables (deg) of β-Keto and β-Hydroxy
γ-Amino Acid Residues
no.
residue
ϕ
θ1
θ2
162
ψ
AAs 1a
2a
−135
35
59
53
94
−131
−123
−105
−126
−105
−169
60
−172
−122
154 (−57)
−121
ASSOCIATED CONTENT
■
P1
Phe-sta2
3
3
3
3
1
S
* Supporting Information
Phe-sta4
Phe-sta2
Phe-sta4
173
51
3
168
63
9
P2
Experimental procedures, compound characterization, and
crystallographic information for 1a, 2a, P1, and P2. This
56
−170
−158
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Organic Letters
Letter
L.; Zhang, W.; Guzei, I. A.; Spencer, L. C.; Gellman, S. H. Org. Lett.
2012, 14, 2582.
material is available free of charge via the Internet at http://
pubs.acs.org.
(11) Seebach, D.; Brenner, M.; Rueping, M.; Jaun, B. Chem.Eur. J.
2002, 8, 573.
AUTHOR INFORMATION
■
(12) (a) Hagihara, M.; Anthony, N. J.; Stout, T. J.; Clardy, J.;
Schreiber, S. L. J. Am. Chem. Soc. 1992, 114, 6568. (b) Bandyopadhyay,
A.; Mali, S. M.; Lunawat, P.; Raja, K. M. P.; Gopi, H. N. Org. Lett.
2011, 13, 4482.
(13) Jadhav, S. V.; Gopi, H. N. Chem. Commun. 2013, 49, 9179.
(14) Bandyopadhyay, A.; Agarwal, N.; Mali, S. M.; Jadhav, S. V.;
Gopi, H. N. Org. Biomol. Chem. 2010, 8, 4855.
Corresponding Author
*E-mail: hn.gopi@iiserpune.ac.in.
Notes
The authors declare no competing financial interest.
(15) Holmquist, C. R.; Roskamp, E. J. J. Org. Chem. 1989, 54, 3258.
(16) Maibaum, J.; Rich, D. H. J. Org. Chem. 1988, 53, 869.
ACKNOWLEDGMENTS
■
A.B. and M.G.K. are thankful to CSIR for the Senior Research
Fellowship.
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