Hybrid peptides: Direct transformation of α/α, β-unsaturated γ-hybrid peptides to α/γ-hybrid peptide 12-helices

Article abstract of DOI:10.1021/ol300987d

A smooth transformation of unusual planar structures of α/vinylogous hybrid peptides to ordered α/γ⁴-hybrid peptide 12-helices and the stereochemical preferences of vinylogous amino acid residues in single crystals are studied.

Full text of DOI:10.1021/ol300987d

ORGANIC
LETTERS
2012
Vol. 14, No. 11
2770–2773
Hybrid Peptides: Direct Transformation of
r/r, β-Unsaturated γ-Hybrid Peptides to
r/γ-Hybrid Peptide 12-Helices
Anupam Bandyopadhyay and Hosahudya N. Gopi*
Department of Chemistry, Indian Institute of Science Education and Research,
Dr. Homi Bhabha Road, Pashan, Pune-411008, India
hn.gopi@iiserpune.ac.in
Received April 17, 2012
ABSTRACT
A smooth transformation of unusual planar structures of R/vinylogous hybrid peptides to ordered r/γ4-hybrid peptide 12-helices and the stereo-
chemical preferences of vinylogous amino acid residues in single crystals are studied.
The design of peptides containing non-natural amino
acids that can mimic natural protein secondary structures
is a challenging task for chemists and biochemists. A good
deal of success has been achieved in this regard using
the oligomers of β- and γ-amino acids or mixed sequences
containing R/β, R/γ, and β/γ-hybrid peptides.1 Various
ordered helical conformations of γ-and mixed R/γ-hybrid
peptides containing 3,3-dialkyl-γ-amino acids (Gpn,
gabapentin),1e,2 cyclic γ-amino acids,3 β-aminooxy acids,4
carbo-γ-amino acids,5 and 2,3,4-substituted γ-amino
acids6 have been reported. However, the progress of
γ4-peptides (oligomers of double homologated R-amino
acids) lags behind that of β- and other γ-peptides, due in
part to the difficulty of obtaining stereochemically pure
γ4-amino acids.7 Nonetheless, Seebach and colleagues8
and Hanessian et al.9 in their pioneering work recognized
the 14-helical conformations of the oligomers of γ4-amino
acids. In addition, recently Hofmann and colleagues pre-
dicted the wide range of helical organizations from the
(1) (a) Seebach, D.; Beck, A. K.; Bierbaum, D. J. Chem. Biodiv. 2004,
1, 1111–1239. (b) Seebach, D.; Gardiner J. Acc. Chem. Res. 2008, 41,
1366–1375. (c) Horne, W. S.; Gellmann, S. H. Acc. Chem. Res. 2008, 41,
1399–1408. (d) Cheng, R. P.; Gellman, S. H.; DeGrado, W. F. Chem.
Rev. 2001, 101, 3219–3232. (e) Vasudev, P. G.; Chatterjee, S.; Shamala,
N.; Balaram, P. Chem. Rev. 2011, 111, 657–687. (f) Price, J. L.; Horne,
W. S.; Gellman, S. H. J. Am. Chem. Soc. 2010, 132, 12378–12387. (g)
Sharma, G. V. M; Nagendar, P.; Jayaprakash, P.; Krishna, P. R.;
Ramakrishna, K. V. S.; Kunwar, A. C. Angew. Chem., Int. Ed. 2005,
44, 5878–5882. (h) Kritzer, J. A.; Hodsdon, M. E.; Schepartz, A. J. Am.
Chem. Soc. 2005, 127, 4118–4119. (i) Bouillere, F.; Thetiot-Laurent, S.;
Kouklovsky, C.; Alezra, V. Amino Acids 2011, 41, 687–707.
(2) (a) Vasudev, P. G.; Chatterjee, S.; Shamala, N.; Balaram, P. Acc.
Chem. Res. 2009, 42, 1628. (b) Chatterjee, S.; Vasudev, P. G.; Raghothama,
S.; Ramakrishnan, C.; Shamala, N; Balaram, P. J. Am. Chem. Soc. 2009,
131, 5956.
(3) (a) Guo, L.; Chi, Y.; Almeida, A. M.; Guzei, I. A.; Parker, B. K.;
Gellman, S. H. J. Am. Chem. Soc. 2009, 131, 16018–16020. (b) Guo, L.;
Almeida, A. M.; Zhng, W.; Reidenbach, A. G.; Choi, S. H.; Guzei, I. A.;
Gellman, S. H. J. Am. Chem. Soc. 2010, 132, 7868–7869. (c) Guo, L.;
Zhang, W.; Reidenbach, A. G.; Giuliano, M. W.; Guzei, I. A.; Spencer,
L. C.; Gellman, S. H. Angew. Chem., Int. Ed. 2011, 50, 5843–5846.
(4) (a) Yang, D.; Zhang, Y. H.; Li, B.; Zhang, D. W.; Chan, J. C. Y.;
Zhu, N. Y.; Luo, S. W.; Wu, Y. D. J. Am. Chem. Soc. 2004, 126, 6956–
6966. (b) Yang, D.; Zhang, Y. H.; Zhu, N. Y. J. Am. Chem. Soc. 2002,
124, 9966–9967.
(5) Sharma, G. V. M; Jadhav, V. B.; Ramakrishna, K. V. S.;
Jayaprakash, P.; Narsimulu, K.; Subash, V.; Kunwar, A. C. J. Am.
Chem. Soc. 2006, 128, 14657–14668.
(6) (a) Seebach, D.; Brenner, M.; Rueping, M.; Schweizer, B.; Jaun,
B. Chem. Commun. 2001, 207–208.
(7) In γ4-amino acids, 4 indicates the position of the side-chain
substitution as described by Seebach et al. in ref 1a.
(8) Hintermann, T.; Gademann, K.; Jaun, B.; Seebach, D. Helv.
Chim. Acta 1998, 81, 893–1002.
(9) Hanessian, S.; Luo, X.; Schaum, R.; Michnick, S. J. Am. Chem.
Soc. 1998, 120, 8569–8570.
r
10.1021/ol300987d
Published on Web 05/18/2012
2012 American Chemical Society

Vinylogous amino acids (insertion of ꢀCHdCHꢀ be-
tween CRH and CO, R,β-unsaturated-γ-amino acids) have
been frequently found in many peptide natural products.11
The structures of R,β-unsaturated γ-amino acids and their
oligomers have little explored. Nevertheless, Schreiber and
colleagues12 and others13 reported the β-sheet, β-turn, and
unusual helical type of structures for the peptides contain-
ing vinylogous amino acids. Recently, we reported the
utility of E-vinylogous amino acids in the construction of a
stable and functionalizable hybrid β-hairpin.14 In conti-
nuation of our studies in the design of hybrid peptides
containing E-vinylogous amino acids, we designed the
peptides D1 and D2 to understand the behavior of vinylo-
gous amino acids in the presence of conformationally
constrained, helixfavoringAib residues15 and to transform
into their saturated γ-peptide analogues (Scheme 1). Further,
this approach also provides an unprecedented opportunity
to analyze and understand the conformational preferences
of both R,β-unsaturated and saturated γ4-amino acids in
oligopeptides. The E-vinylogous amino acid dgF [(S,E)-4-
amino-5-phenylpent-2-enoic acid] was synthesized using
the Wittig reaction starting from N-Boc-(S)-phenylalanal.16
Peptides D1 and D2 were synthesized in the solution-phase
method using Boc chemistry.
Scheme 1. Direct Transformation of R/Vinylogous Hybrid
Peptides (D) to R/γ4-Hybrid Peptides (G)a
a The sequences of R/vinylogous hybrid peptides (D1 and D2) and
R/γ4-hybrid peptides (G1 and G2) are shown.
We began our analysis with D1; the 1H NMR shows the
wide dispersion of NH and vinylic protons. Surprisingly,
the 2D NMR (ROESY) analysis reveals that no character-
istic intramolecular NOEs corresponding to either the
helix, sheet or reverse turn conformations (see the Sup-
porting Information), indicating no secondary structure in
the hybrid peptide. Similarly, the solution structure analysis
of D2 suggests no regular secondary structures. Further,
we were able to get single crystals of D1 from the slow
evaporation of methanol/toluene solution, and its X-ray
structure is shown in Figure 1. Interestingly, D1 adopted
an unusual planar structure in crystalline state and as
anticipated did not show any protein secondary structural
properties. No intramolecular H-bonding is observed in
the crystal structure. Examination of the torsional angles
of helix favoring Aib residues reveal that both residues
adopted opposite right and left handed helical conforma-
tions with the φ and ψ values ꢀ51, ꢀ48, and 64 and 52,
respectively. The local conformations of the vinylogous
residues were determined by introducing additional torsional
variables θ1 (NꢀCγꢀCβdCR) and θ2 (CγꢀCβdCRꢀC) as
shown in Figure 1. The vinylogous residue dgF2 adopted a
fully extended conformation by having the torsional angles
φ=ꢀ139, θ1 =121,θ2 = 178, and ψ=ꢀ161ꢀ. Interestingly,
another vinylogous residue, dgF4 adopted NꢀCγꢀCβdCR
Figure 1. X-ray structures of hybrid R-vinylogous peptide (D1)
and R/γ4-hybrid peptide (G1). Local torsional variables of γ-
residues are shown at the top.
oligomers of unsubstituted γ-amino acids, R,β-unsatu-
rated γ-amino acids and 1:1 heterooligomers of R/γ-amino
acids using ab initio theoretical calculations.10 Here we
report the unusual planar structures of the R/vinylogous
hybrid peptides Boc-Aib-dgF-Aib-dgF-OEt (D1, dgF =
R,β- dehydro γ4-phenylalanine) and Boc-Ala-dgF-Aib-
dgF- OEt (D2) and their direct transformation to ordered
R/γ4-hybrid peptide 12-helices, Boc-Aib-γ4Phe-Aib-γ4Phe-
OEt (G1) and Boc-Ala-γ4Phe-Aib-γ4Phe-OEt (G2), respec-
tively, using catalytic hydrogenation.
(12) Hagihara, M.; Anthony, N. J.; Stout, T. J.; Clardy, J.; Schreiber,
S. L. J. Am. Chem. Soc. 1992, 114, 6568–6570.
(13) (a) Grison, C.; Coutrot, P.; Geneve, S.; Didierjean, C.; Marraud,
M. J. Org. Chem. 2005, 70, 10753–10764. (b) Chakraborty, T. K.;
Ghosh, A.; Kumar, S. K.; Kunwar, A. C. J. Org. Chem. 2003, 68,
6459–6462.
(10) (a) Baldauf, C.; Gunther, R.; Hofmann, H.-J. Helv. Chim. Acta
2003, 86, 2573–2588. (b) Baldauf, C.; Gunther, R.; Hofmann, H.-J.
J. Org. Chem. 2005, 70, 5351–5361. (c) Baldauf, C.; Gunther, R.;
Hofmann, H.-J. J. Org. Chem. 2006, 71, 1200–1208.
(14) Bandyopadhyay, A.; Mali, S. M.; Lunawat, P.; Raja, K. M. P.;
Gopi, H. N. Org. Lett. 2011, 13, 4482–4485.
(15) (a) Toniolo, C.; Benedetti, E. Trends Biochem. Sci. 1991, 16, 350–
353. (b) Karle, I. L.; Balaram, P. Biochemistry 1990, 29, 6747–6756.
(16) Mali, S. M.; Bandyopadhyay, A.; Jadhav, S. V.; Ganesh Kumar,
M.; Gopi, H. N. Org. Biomol. Chem. 2011, 9, 6566–6574.
(11) (a) Linington, R. G.; Clark, B. R.; Trimble, E. E.; Almanza, A.;
Uren, L. D.; Kyle, D. E.; Gerwick, W. H. J. Nat. Prod. 2009, 72, 14–17.
(b) Coleman, J. E.; de Silva, E. D.; Kong, F.; Andersen, R. J.; Allen,
T. M. Tetrahedron 1995, 51, 10653–10662.
Org. Lett., Vol. 14, No. 11, 2012
2771

Table 1. Backbone Torsional Variables of R/E-Vinylogous and
R/γ4-Hybrid Peptides
peptide residue
φ
θ1
θ2
ψ
D1
G1
G2
G3
dgF2
dgF4
ꢀ139
ꢀ81
121
178
180
ꢀ161
12
ꢀ168
γPhe2 ꢀ127 ( 6 50 ( 3 62.5 ( 0.5
ꢀ117 ( 7
γPhe4 ꢀ104 ( 3 61 ( 2
ꢀ175 ( 3 ꢀ155 ( 7
ꢀ123
γPhe2 ꢀ123
γPhe4 ꢀ113
γPhe2 ꢀ117
γPhe4 ꢀ111
51
57
49
43
63
ꢀ177
ꢀ130
ꢀ125
ꢀ137
68
56
Scheme 2. Stable Eclipsed Conformers of Vinylogous Residues
Observed in the Crystal Structures
Figure 2. (A) X-ray structures of G2 and G3 (Boc-Ala-γ4Phe-
Aib-γ4Phe-CONHMe). (B) Backward 12-membered H-bonds
observed in the crystal structures is depicted in the extended
model G3.
was achieved within six hours and the pure R/γ4-hybrid
peptide was isolated in 95% yield. Fully assigned 1H NMR
(amide and vinylic region) before and after the catalytic
hydrogenation of peptide D1 is shown in the Supporting
Information. Single crystals of R/γ4-hybrid peptide G1
obtained from the slow evaporation of ethanol solution
yield the structure shown in Figure 1. Four molecules of
peptide G1 were observed in the asymmetric unit with
slight variation in the torsional values. Instructively, the
peptide adopted a right-handed helical conformation in
the crystalline state. Moreover, Hofmann and co-workers
have predicted the formation of 12-helix [12-atom ring
eclipsed conformation by having θ1 close to 0ꢀ. Further,
the torsional angle φ adopted a semiextended conforma-
tion with the value ꢀ81ꢀ and the other torsional variables
θ2 and ψ of dgF4 showed the extended conformations. The
torsional angles of vinylogous residues are tabulated in
Table 1. In addition, the local s-cis conformations (ψ =
∼180ꢀ) of the conjugated ester (dgF4) and the amide
(dgF2), favoring the extended planar structure.
Surprised by the unusual planar crystal structure of D1,
we sought to analyze the conformations adopted by the
vinylogous residues in crystal structures. A careful analysis
of the crystal structures of E-vinylogous residues reported
earlier12,14,16 and from the present work revealing very
intriguing results. From the analysis of total twenty one
units of vinylogous residues in the single crystals of both
monomers and peptides (see ESI), two lowest energy
eclipsed conformations are observed (Scheme 2). The
NꢀCγꢀCβdCR eclipsed conformation A (θ1 = 0ꢀ) is
normally observed in the vinylogous esters, while
HꢀCγꢀCβdCR eclipsed conformation B (θ1 = ( 120ꢀ)
is observed in the vinylogous amides. In all the cases, θ1
takes the value either close to 0 or (120ꢀ. The conjugated
carbonyl functional group mostly adapted local s-cis con-
formation (ψ = ∼(180ꢀ) compared to the s-trans con-
formation (ψ = ∼(0ꢀ). Results from the analysis reveal
that vinylogous residues (amides) prefer the extended
conformations and may serve as ideal candidates for the
construction of β-sheet structures.14
H-bonds of CdO(i) HꢀN(iþ3)] by 1:1 alternative R
3 3 3
and unsubstituted γ-residues using ab initio MO theory.10c
In complementary work, Balaram and colleagues2 and
Gellman et al.3 showed the formation of 12-helix in the
R/γ-hybrid peptides containing 3, 3-dialkyl γ-amino acids
and cyclic γ-amino acids, respectively. Indeed peptide
G1 adopted a 12-helix conformation in single crystals.
The analysis of the crystal structure reveals that the two
cooperative twelve membered H-bonds CdO(Boc)
˚
3 3 3
NH(3) [CdO HꢀN dist. 2.064 A, O N dist. 2.920
3 3 3
3 3 3
˚
A
and —O HꢀN 172ꢀ] and CdO(1) NH(4)
3 3 3 3 3 3
˚ ˚
[CdO HꢀN dist. 1.984 A, O N dist. 2.819 A and
3 3 3
3 3 3
—O HꢀN 163ꢀ] stabilizing the helical conformation.
3 3 3
The transformation of D1 to G1 represents an interest-
ing example of nonfolded to H-bond guided folded state of
peptides. Inspired by the 12-helical conformation of G1,
we further subjected D2 to the catalytic hydrogenation to
give saturated peptide G2. The crystal conformations of
G2 is shown in Figure 2A. The crystal structure analysis
suggests that similar to G1, peptide G2 also adopted 12-
helical conformation in single crystals. The stereochemical
analysis of γ4-residues in both G1 and G2 reveal that
γ4Phe2 adopted gaucheþ, gaucheþ (gþ, gþ, θ1 ≈ θ2 ≈
60ꢀ) local conformation about the CβꢀCγ and CRꢀCβ
We further investigated the possibilities of transforming
a hybrid unsaturated peptide to its saturated analogue
using catalytic hydrogenation. The pure peptide D1 was
subjected to catalytic hydrogenation using 20% Pd/C in
ethanol. The reaction was monitored by MALDI-TOF
and HPLC. The complete conversion of peptide D1 to G1
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Org. Lett., Vol. 14, No. 11, 2012

bonds, while the C-terminal γ4Phe4 residues displayed
gaucheþ (CβꢀCγ), anti (CRꢀCβ) conformation due to the
lack of terminal H-bond donor (NH). However, a weak 10
membered CꢀH O interaction between the acidic R-
3 3 3
hydrogens of γ4Phe4 and the CdO of γ4Phe2 (1r3) is
observed. We anticipate that helix favoring gþ, gþ con-
formation can be induced in the terminal γ4-amino acids
by introducing H-bond donor (NH) at the C-terminus.
Peptide G3 (Boc-Ala-γ4Phe-Aib-γ4Phe-CONHMe) was
synthesized from G2 to evaluate the hypothesis. The treat-
ment of aqueous methyl amine to the N-hydroxy succini-
midyl ester of G2leads totheformation of G3and its X-ray
structure is shown in Figure 2A. As anticipated, the ter-
minal γ4-residue adopted gþ, gþconformation and accom-
modatedinto thehelix. Thehelicalstructure isstabilized by
three consecutive intramolecular backward C12 H-bonds
(Figure 2B). The H-bonding between the 1r4 residues in
all R/γ4 hybrid peptides indicating the backbone expanded
version of a 310-helix. The H-bond parameters of all the
peptides (G1-G3) are tabulated in the Supporting Informa-
tion. In contrast to peptide D1, R-residues in G1-G3 showed
the right handed helical conformations by having average
φ and ψ values ꢀ58 ( 3 and ꢀ39 ( 5ꢀ, respectively.
The dihedral angles of γ4-residues were measured by
introducing two additional variables θ1 (NꢀCγꢀCβꢀCR)
and θ2 (CγꢀCβꢀCRꢀC) (Figure 1) and are given in the
Table 1. The circular dichroism spectra of all C12-helical
peptides (G1ꢀG3) along with their unsaturated analogues
D1 and D2 are shown in Figure 3. Similar to the β-peptide
12-helices, R/γ4-hybrid peptide 12-helices also showed CD
maxima at ∼205 nm and a weak minima at 218 nm.17 The
facile transformation and the structural analysis of R/γ-
hybrid peptides containing backbone homologated γ4-
amino acids presented here may be useful in the design of
functional foldamers similar to the R/β-hybrid peptides.1c
In conclusion, we presented the stereochemical analysis
of hybrid R/R,β-unsaturated γ-peptides and their smooth
transformation to R/γ4-hybrid peptides. The crystal structure
Figure 3. CD spectra of R/vinylogous hybrid peptides and R/γ4
hybrid peptide 12-helices in MeOH (c = 200 μM).
analyses of vinylogous residues reveal the two lowest
energy eclipsed conformations, which restrict the rotations
around CγꢀCβ and CRꢀCdO bonds. The presence of
geometrical constrains of the double bonds in the hybrid
vinylogous peptides forced the molecules to adopt unusual
planar conformations. After the geometrical constrains of
the double bonds were released, the H-bond strength
overrode the conformational flexibility of the saturated
γ4-amino acids to accommodate into the helical conforma-
tions. The conformational analysis of the hybrid peptides
presented here may provide basic information for the
structure-based designs with specific functions.
Acknowledgment. We thank the Department of Science
and Technology, Government of India, for financial support.
A.B. is thankful to CSIR for a Senior Research Fellowship.
Supporting Information Available. Experimental pro-
cedures, compound characterization, and crystallographic
information. This material is available free of charge via
the Internet at http://pubs.acs.org.
(17) (a) Park, J. S.; Lee, H. S.; Lai, J. R.; Kim, B. M; Gellman, S. H.
J. Am. Chem. Soc. 2003, 125, 8539–8545. (b) Porter, E. A.; Wang, X.;
Schmitt, M. A.; Gellman, S. H. Org. Lett. 2002, 4, 3317–3319.
The authors declare no competing financial interest.
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