Helices with additional H-bonds: crystallographic conformations of α,γ-hybrid peptides helices composed of β-hydroxy γ-amino acids (statines)

Article abstract of DOI:10.1002/bip.22978

β-Hydroxy-γ-amino acids (Statines) are a class of naturally occurring non-ribosomal amino acids frequently found in many peptide natural products. Peptidomimetics constituted with statines have been used as inhibitors for various aspartic acid proteases. In contrast to the synthetic γ-amino acids, very little is known about the folding behavior of these naturally occurring β-hydroxy γ-amino acids. To understand the folding behavior of statines, three α,γ-hybrid peptides P1 (Ac-Aib-γPhe-Aib-(R, S)Phesta-Aib-γPhe-Aib-CONH₂), P2 (Ac-Aib-γPhe-Aib-(S, S)Phesta-Aib-γPhe-Aib-CONH₂), and P3 (Ac-Aib-γPhe-Aib-(S, S)Phesta-Aib-(S, S)Phesta-Aib-CONH₂) were synthesized on solid phase and their helical conformations in single crystals were studied. Results suggest that both syn and anti diastereoisomers of statines can be accommodated into the helix without deviating overall helical conformation of α,γ-hybrid peptides. In comparison with syn diastereoisomer, the anti diastereoisomer was found to be directly involved in the intramolecular H-bonding with the backbone carbonyl groups (i to i + 3) similar to the backbone amide NHs in the helix.

Full text of DOI:10.1002/bip.22978

Helices with Additional Hꢀbonds: Crystallographic Conformations of α,γꢀ
Hybrid Peptides Helices Composed of βꢀHydroxy γꢀAmino acids (Statines)
Ankita Malik, Mothukuri Ganesh Kumar, Anuapm Bandyopadhyay and Hosahudya N. Gopi*
Department of Chemistry, Indian Institute of Science Education and ResearchꢀPune, Dr. Homi
Bhabha Road, Puneꢀ411 008, India.
*
corresponding author
Correspondence to: Hosahudya N. Gopi; Eꢀmail: hn.gopi@iiserpune.ac.in
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through the copyediting, typesetting, pagination and proofreading process which may lead to
differences between this version and the Version of Record. Please cite this article as an
‘
©
Accepted Article’, doi: 10.1002/bip.22978
2016 Wiley Periodicals, Inc.
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Biopolymers: Peptide Science
Page 2 of 27
ABSTRACT: βꢀHydroxyꢀγꢀamino acids (Statines) are a class of naturally occurring nonꢀ
ribosomal amino acids frequently found in many peptide natural products. Peptidomimetics
constituted with statines have been used as inhibitors for various aspartic acid proteases. In
contrast to the synthetic γꢀamino acids, very little is known about the folding behavior of these
naturally occurring βꢀhydroxy γꢀamino acids. To understand the folding behavior of statines,
three α,γꢀhybrid peptides P1 (BocꢀAibꢀγPheꢀAibꢀ(R, S)PhestaꢀAibꢀγPheꢀAibꢀCONH ), P2 (Bocꢀ
2
AibꢀγPheꢀAibꢀ(S, S)PhestaꢀAibꢀγPheꢀAibꢀCONH ) and P3 (BocꢀAibꢀγPheꢀAibꢀ(S, S)PhestaꢀAibꢀ
2
(
S, S)PhestaꢀAibꢀCONH ) were synthesized on solid phase and their helical conformations in
2
single crystals were studied. Results suggest that both syn and anti diastereoisomers of statines
can be accommodated into the helix without deviating overall helical conformation of α,γꢀhybrid
peptides. In comparison with syn diastereoisomer, the anti diastereoisomer was found to be
directly involved in the intramolecular Hꢀbonding with the backbone carbonyl groups (i to i+3)
similar to the backbone amide NHs in the helix.
Keywords: Amino acids; Peptides; Conformations: Hꢀbonds; Xꢀray
2
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Biopolymers: Peptide Science
INTRODUCTION
Hydrogen bonds play a significant role in the folding of proteins into well defined, threeꢀ
dimensional structures. The hypothetical dissection of protein structures leads to helices, sheets
and reverse turns along with loosely structured loops. On the basis of continuous Hꢀbonding
interactions between i and i+4 residues in the polypeptide backbone amide groups Pauling
1
proposed the αꢀhelix. The intramolecular hydrogen bonding is the key feature that holds
together the αꢀhelical fold, while the intermolecular Hꢀbonds play crucial role in stabilizing the
2
βꢀsheet structures. Inspired by the nature’s Hꢀbond mediated αꢀpeptide folded structures;
researchers have explored amide Hꢀbonding analogous to the αꢀpeptide helices and sheets to
3
ꢀ6
create well defined, unnatural foldamers. Incredible success has been achieved in this regard
6
ꢀ9
10,11
using nonꢀnatural βꢀ and γꢀpeptide foldamers, initiated by Seebach , Gellman
and
1
2
Hanessian. The oligomers of βꢀ and γꢀamino acids have displayed well defined helical
structures, and similar to the αꢀhelix the major organizing force in folding of these nonꢀnatural
peptides is the chains of intramolecular amide Hꢀbonding. Based on the number of atoms
involved in the Hꢀbonding pseudocycle the βꢀ and γꢀpeptide helices are classified as C ꢀ, C ꢀ,
9
10
1
3ꢀ19
C ꢀ, C ꢀ, C ꢀhelices etc.
Further, the hybrid peptides composed of αꢀ, βꢀ and γꢀamino acids
12
13
14
1
1,14,15,20ꢀ29
displayed a variety of helices with different Hꢀbonding patterns.
Both theoretical and
experimental investigations revealed the stable 12ꢀhelical conformations from the α, γꢀhybrid
3
0ꢀ36
peptides composed of 1:1 alternating αꢀ and γꢀamino acids.
3
7
Schultz and colleagues systematically investigated the contribution of Hꢀbonds to the
stability of αꢀhelix by replacing the amide bond (ꢀCOꢀNHꢀ) with an ester bond (ꢀCOꢀOꢀ). The
substitution of amide by an ester significantly destabilizes the αꢀhelix. Introducing additional Hꢀ
bonds without deviating the overall helical fold may increase the stability of αꢀhelix, however, it
is rather difficult to introduce additional Hꢀbond donors or acceptors on the αꢀpeptide backbone.
3
8ꢀ40
41
Recently Guichard and colleagues
related hybrids which can be described as γꢀpeptide analogues with additional Hꢀbond donor
groups. Seebach and colleagues examined the influence of backbone OH groups on the
conformations of homooligomers of αꢀhydoxy βꢀamino acids as well as βꢀhydroxyꢀγꢀamino
and Clayden et. al reported oligourea foldamers and
4
2
4
3
acids (statines). In addition, Sharma and colleagues showed the intramolecular Hꢀbonds
3
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Page 4 of 27
between the sideꢀchain ꢀOMe and the backbone amide NHs in α,βꢀhybrid peptides containing Cꢀ
44
linked carboꢀβꢀamino acid.
Statines are naturally occurring γꢀamino acids and peptides composed of statines
4
5,46
displayed a variety of biological activities including aspartic acid protease inhibitors,
4
7
48
anticancer, antimalarial properties etc., In continuation of our efforts in understanding the
structural properties of α,γꢀpeptide foldamers, we sought to investigate the influence of backbone
4
9,50
–
OH groups of statine diastereoisomers
on the conformations of hybrid helices. We
anticipate that as the OH group is directly attached to the backbone of γꢀamino acids, it may be
possible to introduce additional intramolecular Hꢀbonds along with the canonical COꢀꢀꢀNH Hꢀ
bonds in α,γꢀhybrid peptide helices. Herein, we are reporting the synthesis and single crystal
conformations of three α,γꢀhybrid peptides (P1ꢀP3) containing statine diastereoisomers. All three
hybrid peptides adopted 12ꢀhelical conformations in single crystals. Further, we studied the
structural analogy of these hybrid statine peptides with recently reported α,γꢀhybrid peptide 12ꢀ
5
1
helix P4. Intriguingly, statine residues with anti stereochemistry (with respect to the amino acid
sideꢀchains) are involved in the intramolecular Hꢀbonding with the backbone CO groups (C=Oꢀꢀꢀ
HꢀO) along with the regular 12ꢀmembered C=OꢀꢀꢀHN Hꢀbonding. In contrast, statine residue
with syn stereochemistry (with respect to the amino acid sideꢀchains) is not involved in the
intramolecular Hꢀbonding with the amide CO, however involved in the intermolecular Hꢀ
bonding with the solvent molecule. The additional Hꢀbonds of statines with anti stereochemistry
can be utilized to stabilize the helical conformations.
MATERIALS AND METHODS
General Experimental Details: All amino acids, Weinreb amine hydrochloride salt, DCC,
LAH, DIPEA, SnCl , Ethyl diazoacetate, NaBH , HOBt, HBTU, THF, DCM and DMF were
2
4
used as commercially available. THF was dried over sodium and distilled prior to use. Column
1
chromatography was performed on silica gel (100ꢀ200 mesh). H NMR spectra were recorded on
1
3
4
7
00 MHz instrument (100 MHz for C) using residual solvent as internal standard (CDCl δ ,
3 H
.24 ppm, δ 77.0 ppm). The chemical shifts (δ) were reported in ppm and coupling constant (Ј)
c
in Hz. High resolution mass spectra were obtained from ESIꢀTOF MS spectrometer and MALDIꢀ
4
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Biopolymers: Peptide Science
TOF/TOF spectrometer. The synthesis, CD and single crystal conformation (CCDC NO.
5
1
9
01428) of P4 were reported earlier.
General procedure for the synthesis of NꢀBocꢀβꢀketoꢀγꢀamino esters: The Nꢀprotected amino
o
aldehyde (2.0 mmol) was dissolved in 15 mL of DCM at room temperature (20ꢀ25 C) and then
0
.0756 g (20 mol %) of tin (II) chloride was added followed by 0.239 g (2.1 mmol) of ethyl
diazoacetate. Immediate gas evolution was observed. The reaction mixture was stirred and the
progress of the reaction was monitored by TLC. After completion of the reaction, it was
quenched with 10 mL of 0.5N HCl and the reaction mixture was extracted with DCM (30 mL x
3
). The combined organic layer was washed with 20 mL of brine, dried over anhydrous
sodium sulfate and concentrated under reduced pressure to get greenish oily crude product
which was purified on silica gel column chromatography.
(S)ꢀethyl 4ꢀ(tertꢀbutoxycarbonylamino)ꢀ3ꢀ oxoꢀ5ꢀphenylpentanoate (1): White
crystal
0
25
1
(
0.521 g, 78% ); Melting Point = 61.4 C; [α]D = ꢀ 54.5 ( c = 0.6, MeOH); H NMR (400
MHz, CDCl ): δ 12.16 (s, 1H, enolic 17%), 7.24 –7.15 (m, 5H, C H ), 5.03ꢀ5.01 (d, J= 7.3 Hz,
3
6
5
1
H, NH), 4.57ꢀ4.52 (q, J =6.4 Hz, 1H, CH), 4.18ꢀ4.12(q, J = 7.2 Hz, 2H, ꢀOCH2), 3.51ꢀ3.40
(
dd, J=16 Hz, J=11.4 Hz, 2H, CH2, AB coupling), 3.15ꢀ2.95 (m, 2H, CH Ph), 1.38(s, 9H,
2
13
C(CH ) ), 1.26ꢀ1.22 (t, J=7.2 Hz, 3H, CH );
C NMR (100 MHz, CDCl ): δ 201.96,
3
3
3
3
1
1
66.86, 155.18, 136.04, 129.24, 128.67, 127.00, 80.21, 61.45, 60.43, 46.86, 36.89, 28.20,
+
4.02; HRꢀMS m/z Calcd. for C H NO [M+Na] 358.1630, obsrvd 358.1633.
1
8
25
5
General procedure for the synthesis of synꢀ and antiꢀβꢀhydroxy γꢀamino esters: The Nꢀ
protected βꢀketo γꢀamino ester (1) (1 mmol) was dissolved in 10 mL of dry THF under N2
o
atmosphere, cooled to ꢀ78 C, and then NaBH (1.4 mmol, 0.142 g) was added in one portion.
4
The reaction mixture was stirred for further 3 hrs to complete the reaction. The progress of the
reaction was monitored by TLC. After completion of the reaction, it was quenched by pouring
the mixture into iceꢀcold 1 N hydrochloric acid (10 mL). The aqueous phase was extracted with
ethyl acetate (3 × 20 mL). Then the combined organic layers was washed with brine (60 mL) and
dried over anhydrous Na SO , and evaporated under reduced pressure. The diastereoisomer
2
4
mixtures (Table 4) were separated passing through normal silica gel column chromatography
o
using petꢀ ether (60ꢀ80 C)–ethyl acetate solvent system.
5
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Page 6 of 27
(3R, 4S)ꢀethyl 4ꢀ((tertꢀbutoxycarbonyl)amino)ꢀ3ꢀhydroxyꢀ5ꢀphenylpentanoate (2): White
2
0
1
powder (0. 196 g, 60%); [α]D
:
–14.3 (c = 1, MeOH); H NMR (400 MHz, CDCl :
δH
3
7
3
.32ꢀ7.22 (m, 5H, ꢀPh), 4.56ꢀ4.54 (d, J = 9.8, 1H, NH), 4.21ꢀ4.16 (q, J = 7.2, 2H, ꢀOCH ), 4.00ꢀ
2
.99 (d, J = 6.5, 1H,ꢀCHꢀOH), 3.90ꢀ3.84 (m, 1H, ꢀCHꢀ), 3.61 (b, 1H, ꢀOH), 3.01ꢀ2.82 (m, 2H, ꢀ
CH ꢀPh), 2.61ꢀ2.47 (m, 2H, ꢀCH CO), 1.36 (s, 9H, Boc ꢀ(CH ) ) ,1.30ꢀ1.26 (t, J = 7.2, 3H, ꢀ
2
2
3 3
13
CH ); C NMR (100 MHz, CDCl ):
δC 173.01, 155.71, 137.60, 129.47, 128.45, 126.43,
3
3
7
9.60, 70.06, 60.89, 55.11, 38.12, 35.79, 28.24, 14.13; ESI m/z for molecular formula
+
C H NO Calcd. [M+Na] 360.1786, observed 360.1789.
18
27
5
(3S, 4S)ꢀethyl 4ꢀ((tertꢀbutoxycarbonyl)amino)ꢀ3ꢀhydroxyꢀ5ꢀphenylpentanoate (3): White
2
0
1
powder (0.134 g, 40%); [α]D : – 36.2 (c = 1, MeOH); H NMR (400 MHz, CDCl :
δH
3
7
3
.31ꢀ7.22 (m, 5H, ꢀPh), 4.97ꢀ4.95 (d, J = 9.8, 1H, NH), 4.16ꢀ4.11 (q, J = 7.2, 2H, ꢀOCH ), 4.00ꢀ
2
.97 (d, J = 8, 1H,ꢀCHꢀOH), 3.76ꢀ3.70 ( m, 1H, ꢀCHꢀ), 3.52 (b, 1H, ꢀOH), 2.93ꢀ2.91 (m, 2H, ꢀ
CH ꢀPh), 2.63ꢀ2.35 (m, 2H, ꢀCH CO), 1.42 (s, 9H, Boc ꢀ(CH ) ) ,1.27ꢀ1.25 (t, J = 7.0, 3H, ꢀ
2
2
3 3
13
CH ); C NMR (100 MHz, CDCl ) : δ 173.61, 155.81, 138.14, 129.41, 128.45, 126.36, 79.40,
3
3
C
6
6.94, 60.85, 55.34, 38.53, 29.68, 28.33, 14.09; ESI m/z for molecular formula C H NO
18 27 5
+
Calcd. [M+Na] 360.1786, observed 360.1792.
General procedure for the synthesis of Fmocꢀβꢀhydroxy γꢀamino acid: The pure
diastereomers of BocꢀNHꢀγPhe(βꢀOH)OEt (3, 1.0 g, 3 mmol) was dissolved in MeOH (5 mL)
and add 1 N NaOH (2 eq.) and stir the reaction for 4 hr. After the completion of reaction
evaporate the solvent methanol and acidified with 10% HCl and extract the compound using
EtOAc (3 × 30 mL). The combined organic layer was washed with brine solution (1 × 30 mL).
Then BocꢀNHꢀγPhe(βꢀOH)OH was dissolved in DCM (3 mL). The solution was cooled to 0 °C
and to this added TFA (3 mL). The reaction mixture was stirred for another 1 hr at RT. After
completion of the reaction, solvent DCM and TFA were evaporated under reduced pressure and
residue was coꢀevaporated with DCM (three times). Then the residue was dissolved in 10 %
aqueous Na CO (12 mL) and added THF (3 mL). To this reaction mixture FmocꢀOSu (0.91 g,
2
3
2
.7 mmol) in THF (8 mL) was added. The reaction mixture was stirred for another 8 hr. After the
completion of reaction, solvent THF was evaporated and residue was treated with 5 % aqueous
HCl to make pH ~2. Then the aqueous layer was extracted with ethyl acetate (50 mL × 3) and the
6
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Biopolymers: Peptide Science
combined organic layer was washed with brine solution (50 mL). The combined organic layer
was dried over anhydrous Na SO and concentrated under reduced pressure to get FmocꢀNHꢀ
2
4
γPhe(βꢀOH)ꢀOH (4, 1.04 g) in 90 % yield, which was used for solid phase peptide synthesis
without further purification.
Solid Phase Peptide Synthesis: All peptides were synthesized at 0.2 mmol scales on Rink
amide resin using standard Fmocꢀchemistry. All peptide coupling reactions were carried out
using HBTU/HOBt as coupling agents. Fmoc deprotections were facilitated using 20%
piperidine in DMF. The coupling reactions were monitored by Kaiser Test. Double couplings
were used in the case statine and Aib residues. Final coupling was performed using AcꢀAib to
avoid acetylation of free OH groups in statines. After completion of the synthesis, peptides were
cleaved from resin using TFA. After cleavage, the resin was filtered and washed with TFA. The
filtrate was evaporated under reduced pressure to give gummy product. The gummy product was
triturated with cold diethyl ether to give crude peptides. These crude peptides were further
purified using reverse phase HPLC on C column using methanol/water gradient system. The
1
8
mass of the pure peptides was confirmed by MALDIꢀTOF/TOF.
Crystal Structure Analysis
Crystal data analysis of AcꢀAibꢀγPheꢀAibꢀ(3R, 4S)ꢀβꢀhydroxyꢀγPheꢀAibꢀγPheꢀAibꢀCONH2
(P1): Crystals were grown by slow evaporation from a solution of trifluoroethanol. A single
crystal (0.1 × 0.08 × 0.02 mm) was mounted on loop with a small amount of the paraffin oil. The
Xꢀray data were collected at 100 K temperature on a Bruker APEX DUO CCD diffractometer
using Mo K radiation (λ = 0.71073 Ǻ), ωꢀscans (2θ = 50.48), for a total of 24569 independent
α
reflections. Space group P 21, a = 12.60 (2), b = 17.38 (3), c = 14.02 (2), β = 107.54 (2), V =
3
2
928 (8) Ǻ , Monoclinic, Z = 2 for chemical formula C H N O 2 (C H F O), with one
51
72
8
9
2
3
3
ꢀ3
ꢀ1
molecule in asymmetric unit; ρ calcd. = 1.295 gcm , ꢀ = 0.103 mm , F (000) = 1212, R =
int
0
.2381. The final R value was 0.0998 (wR2 = 0.2834) 7413 observed reflections (F ≥ 4σ (|F |))
0 0
5
2
and 733 variables, S = 1.016. The structure was obtained by direct methods using SHELXSꢀ97.
All nonꢀhydrogen atoms were refined anisotropically. The hydrogen atoms were fixed
geometrically in the idealized position and refined in the final cycle of refinement as riding over
the atoms to which they are bonded. The largest difference peak and hole were 0.331 and ꢀ0.309
3
eǺ , respectively.
7
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Crystal data analysis of AcꢀAibꢀγPheꢀAibꢀ(3S, 4S)ꢀβꢀhydroxyꢀγPheꢀAibꢀγPheꢀAibꢀCONH2
(P2): Crystals were grown by slow evaporation from a solution of trifluoroethanol. A single
crystal (0.18 × 0.09 × 0.01 mm) was mounted on loop with a small amount of the paraffin oil.
The Xꢀray data were collected at 100 K temperature on a Bruker APEX DUO CCD
diffractometer using Mo K radiation (λ = 0.71073 Ǻ), ωꢀscans (2θ = 50.996), for a total of
α
4
0794 independent reflections. Space group P 21, a = 10.722 (11), b = 17.315 (18), c = 15.790
3
(
17), β = 108.633 (17), V = 2791(5) Ǻ , Monoclinic, Z = 2 for chemical formula C H N O
5
1
72
8
9
ꢀ3
ꢀ1
1
(C H F O), with one molecule in asymmetric unit; ρ calcd. = 1.239 gcm , ꢀ = 0.093 mm , F
2
3
3
(
000) = 1112, R = 0.406. The final R value was 0.0885 (wR2 = 0.2549) 10129 observed
int
reflections (F ≥ 4σ (|F |)) and 678 variables, S = 0.911. The structure was obtained by direct
0
0
5
2
methods using SHELXSꢀ97. All nonꢀhydrogen atoms were refined anisotropically. The
hydrogen atoms were fixed geometrically in the idealized position and refined in the final cycle
of refinement as riding over the atoms to which they are bonded. The largest difference peak and
3
hole were 0.297 and ꢀ0.330 eǺ , respectively. After thorough crystallization process, we are able
to get small size crystals in trifluoroethanol solvent, which results in moderate quality data leads
to R value above 0.25.
int
Crystal data analysis of AcꢀAibꢀγPheꢀAibꢀ(3S, 4S)ꢀβꢀhydroxyꢀγPheꢀAibꢀ(3S, 4S)ꢀβꢀhydroxyꢀ
γPheꢀAibꢀCONH (P3): Crystals were grown by slow evaporation from a solution of
2
trifluoroethanol. A single crystal (0.1 × 0.08 × 0.1 mm) was mounted on loop with a small
amount of the paraffin oil. The Xꢀray data were collected at 100 K temperature on a Bruker
APEX DUO CCD diffractometer using Mo K radiation (λ = 0.71073 Ǻ), ωꢀscans (2θ = 50.996),
α
for a total of 40794 independent reflections. Space group P 21, a = 10.612 (2), b = 17.651 (4), c
3
=
15.805 (3), β = 108.640 (5), V = 2805.3 (10) Ǻ , Monoclinic, Z = 4 for chemical formula C
51
ꢀ3
H N O 1(C H O), with one molecule in asymmetric unit; ρ calcd. = 1.16 gcm , ꢀ = 0.084
7
2
8
10
3
8
ꢀ1
mm , F (000) = 1042, R = 0.1933. The final R value was 0.0778 (wR2 = 0.2170) 13646
int
observed reflections (F ≥ 4σ (|F |)) and 672 variables, S = 0.893. The structure was obtained by
0
0
5
2
direct methods using SHELXSꢀ97. All nonꢀhydrogen atoms were refined anisotropically. The
hydrogen atoms were fixed geometrically in the idealized position and refined in the final cycle
of refinement as riding over the atoms to which they are bonded. The largest difference peak and
3
hole were 0.582 and ꢀ0.591 eǺ , respectively.
8
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Biopolymers: Peptide Science
Details of data collection, coordinates, bond lengths and angles, anisotropic thermal parameters,
and hydrogen coordinates all three peptides (P1ꢀP3) are deposited in the Cambridge
Crystallographic Data Centre, Cambridge CB2 1EZ, UK, ref CCDC No. 1036533ꢀ35.
RESULTS AND DISCUSSION
The sequences of the peptides P1ꢀP3 are shown in Scheme 1. The stereochemically constrained
helix favoring αꢀamino isobutyric acid (Aib) was used as an αꢀamino acid component in all
5
3,54
hybrid peptide sequences.
we selected recently reported, well characterized α, γꢀhybrid peptide P4 as a model peptide.
The γꢀPhe was synthesized using Witting reaction followed by the catalytic hydrogenation as
To have a direct comparison to the new statine peptides (P1ꢀP3)
5
1
5
5
reported earlier.
The phenylalanine statines were synthesized from the mild NaBH reduction
4
of the ethyl ester of NꢀBocꢀβꢀketo γꢀPhe (1) and the syn (2) and anti (3) diastereoisomers were
4
9,50
separated using column chromatography.
Ethyl ester of NꢀBocꢀβꢀketoꢀγꢀPhe was synthesized
5
6
from Roskamp’s procedure using Bocꢀphenylalaninal and ethyl diazoacetate as reported
5
7
earlier.
The solid phase compatible NꢀFmocꢀprotected statine diastereoisomers were
synthesized after the saponification, NꢀBoc deprotection followed by the Fmocꢀ protection of the
free amine. The schematic representation of the synthesis of statines is shown in Scheme 2. The
syn diastereoisomer was used to synthesize P1 and the anti diastereoisomer was used to construct
P2 and P3. Heptapeptide P3 was synthesized by replacing the two Cꢀterminal γꢀPhe residues of
P4 and γꢀPhe was retained at the Nꢀterminus, because similar to the Nꢀterminal amide NHs in the
helix there is no Hꢀbond acceptor for the Nꢀterminal βꢀhydroxyl group.
All three peptides, P1ꢀP3 were synthesized using solid phase method on Rink Amide resin using
standard Fmocꢀ chemistry. All peptide couplings were performed using standard HBTU/HOBt
coupling conditions. After the synthesis, peptides were cleaved from the resin and purified using
reverse phase HPLC on C column.
18
As crystal structures provide unambiguous structural information, we subjected all three peptides
to the crystallization in various solvent combinations. The Xꢀray quality crystals of all three
peptides were obtained from isopropanol/trifluoroethanol solvent combination. The crystal
structures of the peptides, P1ꢀP3, are shown in Figure 1. All three peptides adopted 12ꢀhelical
5
1
conformations similar to the P4 (Figure 2). The 12ꢀhelix adopted by the P1 is stabilized by six
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4
consecutive intramolecular Hꢀbonds. Similar to the other two γ ꢀPhe residues, the (R, S) Phesta
+
+
γ
β
β
α
also adopted g , g conformation along the C ꢀC and C ꢀC bonds to accommodate into the 12ꢀ
helix. The βꢀhydroxyl group is not involved in the intramolecular Hꢀbonding with the backbone
amide carbonyl groups, however is involved in the intermolecular Hꢀbonding with the solvent
trifluoroethanol. The insertion of syn hydroxyl group did not affect the overall 12ꢀhelical
conformation of α, γꢀhybrid peptide, suggesting that small functional groups can be
accommodated at the βꢀposition of γꢀamino acids without deviating the overall helical fold. The
φ and ψ dihedral angles of γꢀresidues are defined by comparison with αꢀresidues, and two
additional torsion angles along C –C and C –C bonds are designated as θ and θ respectively
γ
β
β
α
1
2,
(Figure 1B). The torsion angles of P1 are shown in Table 1. The Hꢀbond distances and angles of
P1 are tabulated and given in the supporting information.
Crystal structure analysis of P2 reveals that the peptide adopted a 12ꢀhelical conformation. In
contrast to P1, the helical structure of P2 is stabilized by seven intramolecular Hꢀbonds. Both (S,
+
+
S) Phesta and the γꢀPhe adopted g , g conformation. Detailed structure analysis reveals that
both anti βꢀOH and the amide NH of Phesta4 are involved in the intramolecular bifurcated Hꢀ
bonding with the Aib1 carbonyl group (i → i+3). In addition to the regular 12ꢀmembered Hꢀ
bonding existed between the amide groups of i and i+3 residues, an additional 15ꢀmembered Hꢀ
bond existed between the C=O and βꢀOH group. The C=OꢀꢀꢀHꢀO and OꢀꢀꢀO distances were
found to be 2.13 Å and 2.94 Å, respectively and the C=OꢀꢀꢀH bond angle was found to be 145°.
It has been observed that the hydroxyl and carboxylate amino acid sideꢀchains are often part of
58
capping motifs in stabilizing αꢀhelices in proteins. In addition, the frequent occurrence of
asparagine at the Nꢀterminus of the helices has also been believed that it may supply the Hꢀbond
partners for the unpaired backbone amide NHs to induce the stability of the helix through
5
9ꢀ61
backbone sideꢀchain interactions.
The single crystal analysis of P3 reveals a helical conformation similar to the P2 structure. The
+
+
anti Phesta residues and γPhe adopted g , g conformations to accommodate nicely into the
helix. Careful structure analysis suggests that in contrast to the six Hꢀbonds between i and i+3
residues in P1, or a regular α,γꢀhybrid heptapeptide helix (P4), the helical structure of P3 is
stabilized by eight intramolecular Hꢀbonds (i and i+3 residues). The anti βꢀOH groups of both
1
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Biopolymers: Peptide Science
Phesta4 and Phesta6 are involved in the 15ꢀmembered intramolecular Hꢀbonding with the
carbonyl groups of Aib1 and Aib3, respectively along with the six regular 12ꢀmembered Hꢀ
bonds. The carbonyl groups of Aib1 and Aib3 are involved in the bifurcated Hꢀbonding with the
βꢀOH groups as well as amide NHs of statines. The bond angle and bond distances of C=OꢀꢀꢀOꢀ
H Hꢀbonds are found to be very similar to that of COꢀꢀꢀHꢀN Hꢀbonds (Figure 2). The torsional
angels of γꢀresidues are given in the Table 1. The Hꢀbond parameters are tabulated in the
supporting information. The overlay of α,γꢀhybrid peptide 12ꢀhelix P4 and P3 (Figure 2)
confirms the very close similarity between the two backbones, which only differ by the presence
of additional Hꢀbonds in P3. Overall, the crystallographic analyses of all three peptides reveal
that statines with anti stereochemistry with respect to the amino acid sideꢀchains may stabilize
the hybrid helix with additional Hꢀbonds along with the regular backbone amide Hꢀbonds. We
further subjected all these peptides for the CD analysis to understand whether the insertion of
additional hydroxyl groups may influence CD signature of the 12ꢀhelix. All peptides showed CD
6
2ꢀ64
negative minima at 220 nm and positive maxima at 205 nm similar to the βꢀpeptide
as well
5
1,65
as α,γꢀhybrid peptide 12ꢀhelices.
The CD spectra of hybrid peptides are shown in Figure 3.
CONCLUSION
In conclusion, we are reporting the conformational properties of α,γꢀhybrid peptides composed
of naturally occurring βꢀhydroxy γꢀamino acids. Single crystal analysis suggests that both syn
and anti diastereoisomers of statines can be accommodated into the 12ꢀhelix without deviating
the overall helical conformation of α,γꢀhybrid peptides. In contrast to the syn diastereoisomer,
the anti diastereoisomers were found to be directly involved in the intramolecular Hꢀbonding
with the backbone carbonyl group (i
→ i+3) similar to the amide NHs. As helices are generally
held together by the intramolecular Hꢀbonds, we speculated that the utilization of anti
diastereoisomers of statines in the design of helices may increase the stability through additional
Hꢀbonds. As statines have been recognized as protease inhibitors for various bacterial and viral
infections as well as other diseases, the remarkable structural properties of statines reported here
can be utilized further in the design of functional foldamers and peptidomimetics.
1
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Page 12 of 27
ACKNOWLEDGEMENTS
M. G. K is thankful to CSIR for research fellowship. We thank DST, Govt. of India and IISER
Pune for financial support
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6
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SCHEMES AND FIGURE LEGENDS
SCHEME 1: Sequences of α,γꢀhybrid peptides composed of γꢀ and βꢀhydroxy γꢀamino acids.
Chemical structures of γꢀPhe and βꢀhydroxy γꢀPhe diastereoisomers are shown below the peptide
sequences.
SCHEME 2: Synthesis of statines starting from NꢀBocꢀamino aldehydes
FIGURE 1: a) Xꢀray structures of P1, P2 and P3. b) Local torsion angles of γꢀresidues. c) Two
different types of intramolecular Hꢀbonds observed in P2 and P3 crystal structures are
highlighted in red [regular CO(i)ꢀꢀꢀNH (i+3)12ꢀmembered Hꢀbonds] and green [15ꢀmembered Hꢀ
bonds exists between CO(i)ꢀꢀꢀꢀHꢀO (i+3) residues].
4
52
FIGURE 2: a) Xꢀray structure of α,γ ꢀhybrid peptide P4. B) Superposition of P4 and P3. C)
Parameters of bifurcated Hꢀbonds between i and i+3 residues in P3 are highlighted.
FIGURE 3: Circular dichroism spectra of statine contained peptides P1, P2 and P3 peptides (0.2
mM) in methanol at 20 °C.
1
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TABLES
Table 1: Torsion angles(°) of peptides P1, P2 and P3
Pept.
Resd.
Aib1
φ
θ1
ꢀ
θ2
ꢀ
ψ
ω
ꢀ59
ꢀ50
ꢀ175
ꢀ168
ꢀ175
ꢀ172
ꢀ170
ꢀ173
ꢀ
γPhe2
Aib3
ꢀ117
ꢀ60
52
ꢀ
64
ꢀ
ꢀ135
ꢀ33
P1
(
R, S)γPhesta4
ꢀ126
ꢀ53
48
ꢀ
60
ꢀ
ꢀ120
ꢀ43
Aib5
γPhe6
Aib7
ꢀ130
ꢀ54
55
ꢀ
58
ꢀ
ꢀ118
ꢀ40
Aib1
ꢀ58
ꢀ
ꢀ
ꢀ41
ꢀ178
ꢀ169
ꢀ175
ꢀ165
ꢀ176
ꢀ175
ꢀ
γPhe2
Aib3
ꢀ116
ꢀ62
49
ꢀ
66
ꢀ
ꢀ137
ꢀ36
P2
(
S, S)γPhesta4
ꢀ122
ꢀ64
46
ꢀ
64
ꢀ
ꢀ124
ꢀ35
Aib5
γPhe6
ꢀ127
ꢀ54
51
ꢀ
60
ꢀ
ꢀ116
ꢀ43
Aib7
Aib1
ꢀ59
ꢀ
ꢀ
ꢀ41
ꢀ178
ꢀ169
ꢀ174
ꢀ165
ꢀ175
ꢀ176
ꢀ
γPhe2
ꢀ116
ꢀ63
49
ꢀ
66
ꢀ
ꢀ135
ꢀ34
Aib3
P3
(
S, S)γPhesta4
Aib5
ꢀ123
ꢀ61
44
ꢀ
63
ꢀ
ꢀ127
ꢀ38
(
S, S)γPhesta6
Aib7
ꢀ124
ꢀ54
47
ꢀ
65
ꢀ
ꢀ117
ꢀ44
1
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Scheme 1
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Scheme 2
2
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Figure 1
2
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Figure 2
2
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Figure 3
2
3
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Table of Contents
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