Preparation and determination of X-ray-crystal and NMR-solution structures of γ2,3,4-peptides

Article abstract of DOI:10.1039/b008377l

(R,R,R)-γ-Amino acids with side chains in the 2-, 3-, and 4-positions, prepared by addition of acyloxazolidinones to a nitroolefin and hydrogenation, have been coupled to γ-tetra-, and γ-hexapeptides which are shown to form (M)-2.6₁₄ helices in the crystal state and in MeOH solution.

Full text of DOI:10.1039/b008377l

Preparation and determination of X-ray-crystal and NMR-solution structures
of g2,3,4-peptides
Dieter Seebach,* Meinrad Brenner, Magnus Rueping, Bernd Schweizer and Bernhard Jaun
Laboratorium für Organische Chemie der Eidgenössischen Technischen Hochschule, ETH-Zentrum,
Universitätstrasse 16, CH-8092 Zürich. E-mail: seebach@org.chem.ethz.ch; Fax: +41 1632 1144;
Tel: +41 1632 2990
Received (in Cambridge, UK) 17th October 2000, Accepted 27th November 2000
First published as an Advance Article on the web 4th January 2001
(R,R,R)-g-Amino acids with side chains in the 2-, 3-, and
4-positions, prepared by addition of acyloxazolidinones to
a nitroolefin and hydrogenation, have been coupled to g-
tetra-, and g-hexapeptides which are shown to form (M)-
2.614 helices in the crystal state and in MeOH solution.
residue i and CNO of residue (i 2 3)). The carbamate NH of
residue 1 and the amide NH of residue 2 are involved in
intermolecular H-bonding to the ester CNO and to the CNO of
residue 3 respectively, resulting in a two-dimensional H-bonded
network. Residues 1 to 3 show the typical backbone conforma-
tion found in the (M)-2.614 helix ((2)-sc for the C(2)–C(3) and
the C(3)–C(4) ethane moieties). The side chains at C(2) and
C(4) are in lateral positions, while the C(3)–Me bonds form
angles of approximately 35° with the helix axis. The C-terminal
residue has an extended backbone conformation ((±)-ap for the
C(2)–C(3) and the C(3)–C(4) ethane moieties). Since the C-
terminal ester group has no NH-group which could form an
While there is a lot of activity in the field of b-peptides, their
homologs, the g-peptides, have received much less attention, so
far.1–3 It has been discovered that g-peptides form helical
secondary structures in solution, detectable by NMR spectros-
copy, with chains as short as four residues, and that homologa-
tion of
L
-a-amino acids to
L
-b- and -g-amino acids leads to
L
peptides, folding to helices of alternating polarity and helicity
(a: N ÿ C (P), b: N Ÿ C (M), g: N ÿ C (P)), and of increasing
4
stability. The g-peptides studied hitherto consisted of g -
residues (side chains at C(4))1,2 or of g2,4-residues (two side
chains, one at C(2) and one at C(4)),2,3 rel. configuration l or u.†
Inspection of models leads to the conclusion that g2,3,4-peptides
of type 1/2 (Fig. 1), built of (R,R,R)-amino acid residues, should
be able to form a 2.614-helix without steric interference of the
side chains within the helical backbone.
The required g-amino-acid building blocks were prepared
stereoselectively by Michael addition of the modified Evans
acyloxazolidinones 3 to nitrobutene (? 4),4 reductive cleavage
(? 5), hydrolysis, and N-Boc or C-OBn protection (? 6, 7)
(Scheme 1). Coupling of amino acids 6 and 7 gave dipeptide 8,
which after appropriate deprotection yielded dipeptide building
blocks which were coupled to tetra- and hexapeptides 1 and 2,
respectively.
Of the protected g-tetrapeptide 1 we obtained crystals
suitable for X-ray structure analysis.‡ The quality and size of
the samples allowed only for isotropic refinement and the
determined structure is shown in Fig. 2a. The structure is
characterized by two consecutive 14-membered H-bonded
rings, one between the NH of residue 3 and the CNO of the Boc-
protecting group and the other between the NH of residue 4 and
the CNO of residue 1. Thus, both intramolecular H-bonds fit into
the typical pattern of the 2.614-helix (H-bond between NH of
Scheme 1 Synthesis of the building blocks used for the preparation of
peptides 1 and 2. Hydrolysis of lactam 5a in 6 M HCl and subsequent Boc-
protection yielded amino acid 6. The same procedure was applied with 5b,
followed by benzylation and Boc-deprotection to obtain 7. Compounds 4
and 5 were used as mixtures of diastereoisomers, and separation was
performed at the stage of the corresponding g-amino acid. Coupling of
amino acids 6 and 7 with HATU [O-(7-azabenzotriazol-1-yl)-N,N,NA,NA-
tetramethyluronium hexafluorophosphate] to dipeptide
8 proceeded
Fig. 1 g2,3,4-Tetra- and hexapeptide derivatives 1 and 2 used for the structure
determinations and Fischer representation of the required amino acid
building blocks.
smoothly. Appropriate deprotected derivatives of 8 were fragment coupled
to tetrapeptide 1 and hexapeptide 2 using EDC (1-ethyl-3-[3-(dimethyl-
amino)propyl]carbodiimide·HCl)/DMAP as reagents.
DOI: 10.1039/b008377l
Chem. Commun., 2001, 207–208
This journal is © The Royal Society of Chemistry 2001
207

Fig. 2 (M)-2.614 Helical structures of g-peptides 1 and 2. a, Structure of g-tetrapeptide 1 in the crystal state determined by X-ray structure analysis. b, Bundle
of 20 conformers of hexapeptide 2 in MeOH obtained by simulated annealing calculations using restraints from NMR data. c, Superposition of the peptide
backbones from the X-ray diffraction structure (blue) and NMR structure (red).
intramolecular H-bond the conformation of residue 4 may by
determined by crystal packing factors.
Notes and references
† Peptides built of g2,4-amino acids with rel. configuration l form 2.614
helical structures, while a tetrapeptide consisting of the corresponding u
residues was found to form a turn motif.
‡ Crystal data for C46H80N4O7 1: M = 801.14, monoclinic, space group
P21, a = 9.462(2), b = 20.472(6), c = 13.866(4) Å, b = 106.14(2)°, V =
2580.1(12) Å3, Z = 2, Dc = 1.031 g cm23, m(Cu-Ka) = 0.543 mm21
crystal size 0.30 3 0.20 3 0.02 mm. Data were collected on an Enraf
Nonius CAD-4 diffractometer using graphite-monochromatized Cu-Ka
radiation. A total of 4479 unique reflections (3.32 < 2Q < 66.23°) were
processed of which 1140 were considered significant with Inet > 3s(Inet).
Part of the structure was solved by direct method with SIR97,5 the
remaining non-H-atoms were found from a difference Fourier map. The
non-H atoms were refined isotropically with SHELXL97.6 The number of
observed reflections did not allow anisotropic refinement. H-atoms were
calculated at idealized positions and included in the structure factor
calculation with fixed isotropic displacement parameters. Final residuals
were R = 0.0898 and Rw = 0.1961 (GOF = 1.525) for 243 parameters.
CCDC 182/1874.
The observation that g-peptides with just four residues form
a helical structure in the crystal state led us to examining g-
hexapeptide 2 by means of high-resolution NMR techniques.
2D-NMR Studies were carried out on a 500 MHz spectrometer
with solutions in CD3OH. We used DQF-COSY and TOCSY
,
1
techniques to assign all H resonances in their respective spin
systems. HSQC and HMBC experiments led to the assignment
of the amino acid sequence. ROESY spectra of 2 at different
mixing times were acquired and NOEs were extracted from
spectra with a mixing time of 300 ms.
The NOEs were classified according to their relative volume
in three distance categories with the following upper bound
distance limits: strong < 3.0, medium < 3.5 and weak < 4.5 Å.
A total of 83 NOEs were used as distance restraints in simulated
annealing, following the XPLOR protocol. This calculation
yielded a set of 20 structures with low restraint violation and
minimum energy (Fig. 2b). The structures show a well-defined
left-handed helix with three 14-membered hydrogen-bonded
rings from CNO of residue i to NH of residue i + 3. The helix has
a pitch of ca. 5.0 Å and has ca. 2.6 residues per turn. An overlay
of the backbone of one of the NMR-derived structures of 2 and
the backbone from the crystal-structure of tetrapeptide 1 is
shown in Fig. 2c. This superposition shows a good agreement
between the central residues of the two molecules.
The structure was determined by B. Schweizer of our X-ray service.
1 T. Hintermann, K. Gademann, B. Jaun and D. Seebach, Helv. Chim. Acta,
1998, 81, 983.
2 S. Hanessian, X. Luo, R. Schaum and S. Michnick, J. Am. Chem. Soc.,
1998, 120, 8569.
3 S. Hanessian, X. Luo and R. Schaum, Tetrahedron Lett., 1999, 40,
4925.
4 M. Brenner and D. Seebach, Helv. Chim. Acta, 1999, 82, 2365.
5 A. Altomare, B. Carrozzini, G. L. Cascarano, C. Giacovazzo, A.
Guagliardi, A. G. Moliterni and R. Rizzi, SIR97, A Package for Crystal
Structure Solution by Direct Methods and Refinement, 1997, Istituto di
Ricerca per lo Sviluppo di Metodologie Cristallografiche, CNR, Campus
Universitario, Via Orabona 4, 70125 Bari, Italia.
This study shows that g2,3,4-peptides constructed from
(R,R,R)-trisubstituted g-amino acid residues adopt well
defined (M)-2.614-helices without steric interferences of the
side chains, and allowed for the first time the characterization of
this secondary structural motif by X-ray crystal structure
analysis.
6 G. M. Sheldrick, SHELXL97, Program for the Refinement of Crystal
Structures, 1993, University of Göttingen, Germany.
208
Chem. Commun., 2001, 207–208