Chiral, fully extended helical peptides

Article abstract of DOI:10.1007/s00726-011-0839-9

The synthesis of the N-protected (blocked) homo-peptide esters from the chiral C^α-ethyl, C^α-n-pentylglycine was performed in solution to the hexapeptide level. The conformational propensity exhibited by these oligomers in chloroform solution and in the crystal state was assessed by use of FTIR absorption, NMR, and X-ray diffraction. The results indicated that fully extended helical structures (2.0₅-helices) are overwhelmingly adopted irrespective of the peptide main-chain length. This oligomeric series is of great interest as it is characterized by the longest C _i ^α,H, C _i+1 ^α (per residue) separation achievable in the class of chiral, rigid, helical peptide spacers based on α-amino acids.

Full text of DOI:10.1007/s00726-011-0839-9

Amino Acids (2011) 41:629–641
DOI 10.1007/s00726-011-0839-9
ORIGINAL ARTICLE
Chiral, fully extended helical peptides
•
Marco Crisma Alessandro Moretto Cristina Peggion Lavinia Panella
•
•
•
•
•
•
Bernard Kaptein Quirinus B. Broxterman Fernando Formaggio
Claudio Toniolo
Received: 25 November 2010 / Accepted: 19 January 2011 / Published online: 4 February 2011
ꢀ Springer-Verlag 2011
Abstract The synthesis of the N-protected (blocked)
homo-peptide esters from the chiral Ca-ethyl, Ca-n-
pentylglycine was performed in solution to the hexapeptide
level. The conformational propensity exhibited by these
oligomers in chloroform solution and in the crystal state
was assessed by use of FTIR absorption, NMR, and X-ray
diffraction. The results indicated that fully extended helical
structures (2.05-helices) are overwhelmingly adopted irre-
spective of the peptide main-chain length. This oligomeric
series is of great interest as it is characterized by the lon-
gest Cai ,…, Cia?1 (per residue) separation achievable in
the class of chiral, rigid, helical peptide spacers based on
a-amino acids.
DMAP
DMSO
Dpg
4-(dimethylamino)Pyridine
Dimethylsulphoxide
C
a,a-di-n-Propylglycine
EDC
N-Ethyl,N0-[3-(dimethylamino)
propyl]carbodiimide
Ca-Ethyl, Ca-n-pentylglycine
Ca-Ethylnorvaline or Ca-ethyl,
Ca-n-propylglycine
Ethanol
Epg
Etn
EtOH
Iva
Isovaline or Ca-methyl, Ca-ethylglycine
MeOH
MTBE
OMe
OSu
Methanol
Methyl, tert-butyl ether
Methoxy
Succinimido
Keywords Fully extended helix ꢀ Homo-oligopeptides ꢀ
Infrared absorption ꢀ Nuclear magnetic resonance ꢀ
Ca-Tetrasubstituted a-amino acids ꢀ X-ray diffraction
OtBu
TEMPO
Tfa
tert-Butoxy
2,2,6,6-Tetramethylpiperidinyl-1-oxy
Trifluoroacetyl
Z
Benzyloxycarbonyl
Abbreviations
Ac
Acetyl
Acnc
Aib
1-Aminocycloalkane-1-carboxylic acid
a-Aminoisobutyric acid or Ca-methylalanine
or Ca,a-dimethylglycine
Introduction
It is well recognized that Ca-methylated a-amino acids,
such as the achiral Aib and its chiral higher homologues
(aMe)AAs (Scheme 1) almost exclusively favor the onset
of a (310- or a-) helical structure (Toniolo and Benedetti
1991a) in peptides, even at a very short main-chain length
(Karle and Balaram 1990; Toniolo and Benedetti 1991b;
Toniolo 1993; Toniolo et al. 2001, 2004; Benedetti et al.
2002). An extensive occurrence of Aib and Iva, the latter
being the chiral (aMe)AA with R = CH2CH3, has been
demonstrated in a variety of natural peptaibiotics (Valle
et al. 1989b; Toniolo and Bru¨ckner 2009; De Zotti et al.
2010).
(aMe)AA Ca-Methylated a-amino acid
Beg
Ca-n-Butyl, Ca- ethylglycine
Ca,a-Diethylglycine
Deg
M. Crisma ꢀ A. Moretto ꢀ C. Peggion ꢀ F. Formaggio ꢀ
C. Toniolo (&)
ICB, Padova Unit, CNR, Department of Chemistry,
University of Padova, via Marzolo 1, 35131 Padua, Italy
e-mail: claudio.toniolo@unipd.it
L. Panella ꢀ B. Kaptein ꢀ Q. B. Broxterman
DSM Innovative Synthesis BV, P.O. Box 18,
6160 MD Geleen, The Netherlands
123

630
M. Crisma et al.
form (Toniolo 1980). The relative disposition of the two
dipoles, Ni–Hi and C0i=Oi, is such that there is obviously
some interaction between them. Since these four atoms,
together with the Ca atom, are involved in a pentagonal
i
cyclic structure, this conformation is also called the C5
structure. The C5 structure has been taken into consider-
ation (and often overemphasized) in conformational energy
calculations. Its occurrence in apolar non-interacting sol-
vents has been proposed using mainly IR absorption and 1H
NMR measurements of model peptides (Cung et al. 1972;
Toniolo et al. 1988). Gly derivatives have the highest
population of C5 structure when compared to the deriva-
tives of residues carrying a side chain. The influence of the
bulkiness of the lateral substituent can easily be explained
by considering the intramolecular non-bonded interactions
Scheme 1 Chemical structures of the Ca-tetrasubstituted a-amino
acids discussed in this paper
The same conformational propensity is exhibited by the
large number of Cai ? Cia cyclized residues of the Acnc
family (Scheme 1) investigated so far, including the
smallest member (n = 3), although in this latter case the
helix is somewhat distorted as a significant ring strain
effect is operative (Valle et al. 1989a). It is worth pointing
out that the Ac3c residue has been recently shown to occur
in two groups of peptaibiotics, namely neoefrapeptins
(Fredenhagen et al. 2006) and acretocins (Bru¨ckner and
Kirschbaum 2010). Natural peptides containing other
members of the Acnc family have not been discovered so
far.
between the side group R and the atoms Hi?1 and Oi-1
,
which induce a warping of these nonsymmetric molecules.
Unequivocal verification of the occurrence of the C5 form
has been obtained in the crystal state by the X-ray dif-
fraction analyses of a few favorable compounds, i.e. Gly-
rich short peptides (Toniolo 1980; Birkedal et al. 2002).
In globular proteins, a repeating C5 motif (2.05-helix)
has so far been authenticated only in the X-ray diffraction
analysis of the -(Gly)4- sequence of His-tRNA-synthetase
˚
(Aberg et al. 1977). In addition, a recent statistical analysis
¨
of proteins (Hovmoller et al. 2002) has shown that more
The 3D-structural tendencies of a-amino acids are quite
different when both side chains of a Ca,a-disubstituted Gly
residue are the same, longer than a methyl group and not
interconnected in a cyclic system (Toniolo and Benedetti
1991b, c; Toniolo 1993; Imawaka et al. 2000; Toniolo et al.
2001, 2004; Tanaka 2007), as in the achiral Deg shown in
Scheme 1. Other examples of this class of residues include
Ca,a-di-n-propyl, diphenyl and dibenzyl a-amino acids. All
of them overwhelmingly induce the formation of the
multiple fully extended C5-conformation (2.05-helix).
The fully extended peptide conformation was proposed
at an early stage in structural studies of proteins. In this
form H-bonding takes place between the N–H groups of
one chain and the C=O groups of the chains on either side,
thus making a planar sheet held together by intermolecular
H-bonds directed approximately perpendicular to the main-
chain axis. Pauling and Corey (1951) investigated the
possibility of small contractions of the peptide chains and
proposed precise conformations for parallel and antiparal-
lel, pleated, b-sheet forms which better satisfy stereo-
chemical and H-bonding requirements and have main-
chain repeat lengths nearer those found experimentally.
These authors were also able to show that steric hindrance
between adjacent main chains prevents the onset of the
planar sheet in case the side chain is anything but a
hydrogen, that is, it could be formed only by -(Gly)n-
homopeptides.
than 99% of the residues populating the C5 region of the
/,w map are Gly.
Conformational energy computations indicated that the
/,w space explorable by the ‘‘monopeptides’’ from
Ca-tetrasubstituted, achiral (but not Ca,a-dimethylated)
a-amino acids is severely restricted and the minimum
energy conformation corresponds to the C5 structure
(Benedetti et al. 1988; Toniolo and Benedetti 1991c).
On the basis of the aforementioned results, for many years
the general view held by structural peptide chemists was that
most of the achiral a-amino acids could represent useful
building blocks for the production of the 2.05-helix structure.
However, more recently, in a paper published by Imawaka
et al. (2000), it was clearly shown that the essential pre-
requisite for the peptide C5-structure formation is not side-
chain symmetry (i.e. amino acid achirality), but rather any
kind of substitution at both Cb atoms (i.e. even chiral Ca-
tetrasubstituted a-amino acids can be involved). In particu-
lar, their most exciting result was the X-ray diffraction
structure of the 2.05-helical, terminally protected, homo-
tetramer from (S)-Beg (Scheme 1). This finding changed the
picture of the conformational preferences of Ca-tetrasubsti-
tuted a-amino acids to a relevant extent, in the sense that it
paved the way to the current belief that a sequence of
a-amino acid residues with both side chains longer than a
methyl group into a linear peptide might result in a marked
stabilization of the uncommon 2.05-helix.
The repeating motif of the fully extended polypeptide
conformation is the 2 ? 2 intramolecularly H-bonded
123

Fully extended peptides
631
The principal aim of the present work is to corroborate
the pioneering data of the Japanese authors (Imawaka et al.
2000) by extending the conformational investigation to
homo-peptides based on other, chiral, Ca-ethylated a-
amino acids. To this end, we have synthesized and studied
then filtered in order to remove (NH4)2SO4 and the pH was
adjusted to 8.7 by slow addition of concentrated ammonia.
The precipitated salts were filtered off and the aqueous
layer was extracted with CHCl3 (3 9 400 ml) giving the
title compound (138.3 g, 0.80 mol; yield, 86.0%). As,
1
a
set of terminally protected (blocked) [(S)-Epg]n
according to H-NMR data, a small amount of ketone was
(Scheme 1) homo-oligomers to the hexamer level. A pre-
liminary report of part of this work has been recently
published (Toniolo et al. 2009). Interestingly, the presence
of a chiral, Ca-ethylated a-amino acid (Etn), the lower
homologue of Beg, has been reported in a peptaibiotic
(Tsantrizos et al. 1996; Rainaldi et al. 2003).
still present from the first step, an HCl solution (3 N, 1 eq.)
was added and the ketone was extracted with MTBE
(300 ml). The pH was then again adjusted to 8.7 with
concentrated ammonia and the product extracted from the
aqueous layer with CHCl3 to afford the pure amide as an
oil (132.3 g, 0.77 mol; yield 83%).
1H-NMR (CDCl3, 200 MHz): d 7.38 (bs, 1H, CONH2),
5.71 (bs, 1H, CONH2), 2.00–1.65 (m, 2H, CH2), and
1.65–1.10 ppm [m, 11H, (CH2)4 and NH2]. 13C-NMR
(CDCl3, 50 MHz): d 179.47, 61.11, 40.54, 33.41, 32.17,
23.50, 22.58, 14.02, and 8.06 ppm.
Materials and methods
a-Amino acid synthesis
(R,S)-2-Amino-2-ethylheptanenitrile
Enzymatic resolution of (R,S)-2-amino-2-ethylheptanoic
amide
NaCN (49.5 g, 1.01 mol) was dissolved in aqueous
ammonia (25%, 700 ml, 5 mol) at room temperature, fol-
lowed by a slow addition of AcOH (60.9 g, 1.01 mol)
while the temperature increased to 38ꢁC. After cooling to
room temperature, 3-octanone (128.2 g, 1 mol) was slowly
added to the solution, giving rise to a biphasic reaction
mixture. MeOH (50 ml) was added to the mixture to
increase the solubility of the ketone and, therefore, the rate
of the reaction (in fact, after the MeOH addition the tem-
perature rose from 28 to 32ꢁC). The mixture was stirred
overnight, after which time the conversion was 56%
The racemic amide (101 g, 0.59 mol) was mixed with H2O
(915 g, 10% w/w) to a final concentration of 0.56 mol/kg.
After adjusting the pH to 7.1 with H2SO4 (96%), a cell sus-
pension containing the amidase from Ochrobactrum
anthropi [expressed in E. coli (Sonke et al. 2005)] (10 g,
2,000 units/g) was added and the mixture was shaken at
55ꢁC for 48 h to reach a conversion of 40% according to
ammoniadetermination. ThepH was adjusted from 7.5to 2.0
with 6 M H2SO4 to dissolve the precipitated amino acid. The
mixture was centrifuged (1,3009g, 20ꢁC, 20 min) to remove
the cell mass. The supernatant was partially concentrated to
isolate the (R)-amide and the (S)-acid by ion-exchange
chromatography on the strongly basic Amberlyst A-26 resin.
The (R)-amide was recovered by eluting with water, fol-
lowed by extraction with CHCl3 (3 9 400 ml). After drying
(Na2SO4) and concentration under reduced pressure, 50.6 g
(50%) of (R)-2-amino-2-ethylheptanoic amide was isolated
as an oil. e.e. 74% (R). 1H-NMR (CDCl3, 200 MHz): d 7.38
(bs, 1H, CONH2), 5.71 (bs, 1H, CONH2), 2.00–1.65 (m, 2H,
CH2), and 1.65–1.10 ppm [m, 11H, (CH2)4 and NH2]. 13C-
NMR (CDCl3, 50 MHz): d 179.47, 61.11, 40.54, 33.41,
32.17, 23.50, 22.58, 14.02, and 8.06 ppm.
1
(according to an H-NMR measurement). The conversion
increased to 72% by stirring for further 24 h. Additional
NaCN (24.5 g, 0.5 mol) and NH4Cl (26.5 g, 0.5 mol) were
added and the reaction was stirred for another 24 h. At
approximately 90% conversion, the reaction was worked
up. The aqueous solution was extracted with CHCl3
(3 9 400 ml), the organic solution was dried (Na2SO4),
and the solvent completely removed under reduced pres-
sure. This procedure afforded the title compound as an oil
(144.6 g, 0.94 mol; yield: 93.1%).
1H-NMR (CDCl3, 200 MHz): d 2.08–1.20 [m, 12H,
(CH2)5 and NH2], 1.20–1.03 (t, 3H, CH2CH3), and
0.98–0.82 ppm [t, 3H, (CH2)4CH3].
The (S)-amino acid was obtained by eluting the ion-
exchange column with 1 N AcOH and concentrating to
dryness. This procedure gave 31.3 g (31%) of (S)-2-amino-
2-ethylheptanoic acid as a solid. e.e. [ 99%. 1H-NMR
(D2O, 200 MHz): d 2.10–1.55 (m, 4H, aCH2), 1.55–1.04
(m, 6H, bcdCH2), and 1.04–0.40 ppm (m, 6H, CH3). 13C-
NMR (D2O, 50 MHz): d 175.00. 64.55, 35.07, 30.84,
28.90, 22.40, 21.58, 13.27, and 7.22 ppm.
(R,S)-2-Amino-2-ethylheptanoic amide
The nitrile prepared as above discussed (0.93 mol, 143.5 g)
was added dropwise to H2SO4 (96%, 500 ml) at 6ꢁC (ice/
water bath). During the addition, the temperature increased
to 39ꢁC. The solution was stirred for 18 h, after which
water (16.8 g, 1 eq.) was added and stirring was continued
for an additional hour. The resulting yellow solution was
Repeating the enzymatic resolution on a 5 g substrate
scale under identical conditions, already resulted in 50%
123

632
M. Crisma et al.
conversion after 20 h, most likely as a result of better
mixing in the presence of the solid acid formed during the
hydrolysis. This experiment resulted in (S)-acid and (R)-
amide with 95 and 98% e.e., respectively.
disappearance of the starting material. Then, the solvent
was evaporated to dryness and the resulting product
(without any purification) was subject to the coupling step.
Characterization
Chemical hydrolysis of (R,S)-2-amino-2-ethylheptanoic
amide
The various peptides and their synthetic intermediates were
characterized by melting point determination, TLC (in
three solvent systems), solid-state IR absorption spectros-
The racemic amide (1.0 g, 5.8 mmol) was dissolved in
H2O (10 ml), 12 N HCl solution (10 ml) was added and the
solution was stirred for 3 h. After completion of the reac-
tion according to TLC, the solvent was completely
removed under reduced pressure, affording the racemic
2-amino-2-ethylheptanoic acid hydrochloride, together
with 1 eq. of NH4Cl. This material was used as such as
reference compound for the e.e. determination by HPLC.
1
copy (Table 1) and H NMR spectrometry (the latter data
not reported).
IR absorption
The solution IR absorption spectra were recorded using a
Perkin Elmer model 1720X FTIR spectrophotometer,
nitrogen-flushed, equipped with a sample shuttle device, at
2 cm-1 nominal resolution, averaging 100 scans. Cells
with path lengths of 0.1, 1.0, and 10 mm (with CaF2
windows) were used. Spectrograde deuterochloroform
(99.8% d) was purchased from Aldrich (Milwaukee, WI).
Solvent (baseline) spectra were recorded under the same
conditions.
Determination of e.e. by HPLC of (R)-2-amino-2-ethyl-
heptanoic amide and (S)-2-amino-2-ethylheptanoic acid
The following method was used for the e.e. determination
of the amide and the acid.
Column: Nucleosil, 250/4 mm, dd 5 lm; column tem-
perature: 40ꢁC; eluant: aqueous NaOAc buffer (pH = 5.3)
containing either 48% MeOH (for the amide) or 40%
MeOH (for the acid); flow: 1.0 ml/min; detection: UV,
254 nm; sample derivatization: (2S,3S)-dibenzoyltartaric
acid anhydride; injection volume: 100 ll.
Alternatively, the e.e.’s of the acid and amide were
determined by the following conditions using a chiral
HPLC column.
NMR
The NMR spectra were recorded with a Bruker (Karlsruhe,
Germany) model AM 400 spectrometer. Measurements
were carried out in deuterochloroform (99.96% d; Aldrich)
and deuterated DMSO (99.96% d; Acros Organics, Geel,
Belgium) with tetramethylsilane as the internal standard.
The free-radical TEMPO was purchased from Sigma-
Aldrich (St. Louis, MO).
Column: Chiralpak AD, 250/46 mm; column tempera-
ture: room temperature; eluant: hexane/EtOH/MeOH 93.5/
3.8/2.7% v/v; flow: 1.0 ml/min; pre-column reaction:
o-phthaldialdehyde/3-mercapto-D-isobutyric acid (for the
a-amino acid) (Duchateau et al. 1992), while o-phthaldi-
aldehyde/N-acetyl-L-cysteine (for the a-amino amide);
detection: UV, 210 nm; injection volume: 20 ll.
X-ray diffraction
Colorless crystals of Tfa-[(S)-Epg]2-OtBu and Tfa-[(S)-
Epg]3-OtBu were grown from methanol by slow evapo-
ration at room temperature and from hot acetonitrile,
respectively. Data collections were performed on a Phi-
lips PW1100 four-circle diffractometer in the h–2h scan
mode using graphite-monochromated CuKa radiation.
Cell parameters were obtained by least-squares refine-
ment of the angular settings of 48 carefully centred
reflections in the 12 7 20ꢁ h range. Three standard
reflections, periodically monitored, did not show any
significant intensity variation. This finding implies sta-
bility of the crystals and the electronics. Intensities were
corrected for Lorentz and polarization effects, not for
absorption. Both structures were solved by direct meth-
ods of the SHELXS 97 program (Sheldrick 2008) and
refined by full-matrix block least-squares procedures on
F2, using all data, by application of the SHELXL 97
Peptide synthesis
General procedure
For the in situ pre-formation of the 5(4H)-oxazolone, Tfa-
(S)-Epg-OH was dissolved in anhydrous CH3CN (15 ml)
and 1 eq of EDC 9 HCl was added. After 10 min, 1 eq of
H-[(S)-Epg]n-OtBu (n = 1–5) was added and the reaction
mixture was refluxed for 36 h under stirring. The product
was isolated by flash chromatography.
For the Tfa Na-deprotection step, a weighted amount of
Na-trifluoroacetylated amino acid derivative or peptide was
dissolved in EtOH and treated with NaBH4 under reflux.
The reaction was periodically monitored by TLC until
123

Fully extended peptides
633
Table 1 Physical properties and analytical data for the Epg derivatives and peptides synthesized in this work
Compound
Mp/ꢁCa
Recryst. solventb
TLCc
V/cm-1 d
Rf(I)
Rf(II)
Rf(III)
Z-(S)-Epg-OH
Oil
–
0.70
0.45
0.90
0.95
0.95
0.95
0.95
0.95
0.95
0.95
0.95
0.85
0.95
0.95
0.95
0.95
0.95
0.95
0.90
0.95
0.95
0.85
0.95
0.95
0.95
0.95
0.95
0.95
0.80
0.95
0.95
0.90
0.90
0.90
0.50
0.25
0.90
0.70
0.85
0.90
0.90
0.60
0.60
0.60
0.55
0.40
0.85
0.90
0.90
0.90
0.90
3,408, 1,708
Tfa-(S)-Epg-OH
Oil
–
3,356, 1,709
Z-(S)-Epg-OtBu
Oil
–
3,419, 1,732, 1,717
3,410, 3,308, 1,731, 1,681, 1,658
3,386, 1,724
Ac-(S)-Epg-OtBu
Tfa-(S)-Epg-OMe
Z-[(S)-Epg]2-OtBu
Z-[(S)-Epg]3-OtBu
Ac-[(S)-Epg]2-OtBu
Ac-[(S)-Epg]3-OtBu
Ac-[(S)-Epg]4-OtBu
Ac-[(S)-Epg]5-OtBu
Ac-[(S)-Epg]6-OtBu
Tfa-[(S)-Epg]2-OtBu
Tfa-[(S)-Epg]3-OtBu
Tfa-[(S)-Epg]4-OtBu
Tfa-[(S)-Epg]5-OtBu
Tfa-[(S)-Epg]6-OtBu
a
Oil
–
Oil
–
Oil
–
3,400, 1,725, 1,673
3,385, 3,353, 1,729, 1,674
3,392, 3,306, 1,724, 1,676, 1,660
Oil
–
104–106
135–136
141–142
148–149
189–191
72–74
136–138
184–186
215–217
223–225
EtOAc/LP
EtOAc/LP
EtOAc/LP
EtOAc/LP
EtOAc/LP
MeCN
MeCN
MeCN
MeCN
MeCN
3,403, 3,335, 3,316, 1,722, 1,674, 1,653
3,397, 3,360, 3,332, 1,721, 1,675, 1,652
3,419, 3,360, 3,309, 1,726, 1,667, 1,643
3,408, 3,293, 1,724, 1,667, 1,661
3,386, 3,319, 1,728, 1,671
3,391, 3,348, 1,731, 1,663
3,393, 3,345, 1,730, 1,678, 1,659
3,394, 3,346, 1,730, 1,678, 1,656
3,436, 3,394, 3,346, 1,723, 1,678, 1,655
Determined on a Leitz model Laborlux 12 apparatus (Wetzlar, Germany)
b
c
EtOAc ethyl acetate, LP light petroleum (bp 40–60ꢁC), MeCN acetonitrile
Silica gel plates (60F-254 Merck, Darmstadt, Germany) using the following solvent systems: (I) chloroform–ethanol 9:1; (II) butan-1-ol–acetic
acid–water 6:2:2; (III) toluene–ethanol 7:1. The compounds were revealed either with the aid of a UV lamp or with the hypochlorite–starch–
iodide chromatic reaction. A single spot was observed in each case
d
Determined in KBr pellets (or as a film between KBr disks in the case of oils) on a Perkin Elmer model 1720X FTIR spectrophotometer
program (Sheldrick 2008) with all non-H atom aniso-
tropic, and allowing the positional parameters and the
anisotropic displacement parameters of the non-H atoms
to refine at alternate cycles.
Results and discussion
a-Amino acid synthesis and optical resolution
In both structures the N-terminal Tfa group shows
rotational disorder. It was refined on two sets of posi-
tions [atoms F1, F2, and F3 in one conformer, while F10,
F20, and F30 in the other (the latter is rotated by about
60ꢁ with respect to the former)], with occupancies
0.70:0.30 in the dipeptide and 0.50:0.50 in the tripeptide.
In the structure of the tripeptide the Cf atom of Epg(2) is
also disordered. It was refined over two positions (atoms
C2Z and C2Z0) with occupancy of 0.65 and 0.35,
respectively. Restraints were applied to the bond dis-
tances and bond angles involving the disordered groups
and side-chain atoms. H-atoms were calculated at ide-
alized positions and refined using a riding model. Rele-
vant crystal data and structure refinement parameters are
listed in Table 2.
Enantiomerically pure H-(R)-Epg-NH2 and H-(S)-Epg-OH
were prepared by the chemo-enzymatic process described
in Scheme 2 (Sonke et al. 2000). Strecker reaction of
3-octanone in MeOH/water resulted in a 93% yield of
crude a-aminonitrile after 3 days. This latter compound
was hydrolyzed in concentrated sulfuric acid to the racemic
2-amino-2-ethylheptanoic amide. Enzymatic resolution
using the amidase from Ochrobactrum anthropi NCIMB
40321, overexpressed in E. coli (Sonke et al. 2005), gave
the (S)-acid and (R)-amide in high enantiomeric excess
(e.e.) of [99% and [98%, respectively (corresponding
to an E-ratio of 180–220 for the enzymatic reaction)
(Chen et al. 1982).
Peptide synthesis
CCDC 795268 and 795269 contain the supplementary
crystallographic data for this paper. These data can be
obtained from The Cambridge Crystallographic Data
Centre via http://www.ccdc.cam.ac.uk/data_request/cif.
The Z-protected H-(S)-Epg-OH was prepared using Z-OSu
(Paquet 1982) in CH3CN/H2O in the presence of triethyl-
amine. The Tfa-protected amino acid was synthesized using
123

634
M. Crisma et al.
Table 2 Crystal and refinement data for Tfa-[(S)-Epg]2-OtBu and
Tfa-[(S)-Epg]3-OtBu
Parameter
Tfa-[(S)-Epg]2-OtBu Tfa-[(S)-Epg]3-OtBu
Empirical formula
C24 H43 F3 N2 O4
480.60
C33 H60 F3 N3 O5
635.84
Mr
T (K)
˚
293(2)
293(2)
k (A)
1.54178
Monoclinic
P21
1.54178
Monoclinic
P21
Crystal system
Space group
˚
a (A)
8.428(2)
16.525(3)
10.325(2)
95.08(8)
1,432.3(5)
2
6.612(2)
30.544(5)
9.847(3)
97.86(8)
1,970.0(9)
2
˚
b (A)
˚
c (A)
Fig. 1 FTIR absorption spectra (N–H stretching region) of the Tfa-
[(S)-Epg]n-OtBu (n = 2–6) homo-peptides in CDCl3 solution. Peptide
concentration: 1 mM
b (ꢁ)
Volume (A )
3
˚
Z
qcalc (Mg m-3
)
1.114
1.072
of Z-(S)-Epg-OtBu was achieved by H2SO4-catalyzed
treatment of the Z-protected amino acid with isobutylene in
anhydrous CH2Cl2. Ac-(S)-Epg-OtBu was synthesized from
H-(S)-Epg-OtBu (prepared in turn by catalytic hydrogena-
tion of the corresponding Z-derivative) by reaction with a
50% solution of acetic anhydride in CH2Cl2.
l (mm-1
)
0.732
0.664
F(000)
Crystal size (mm3)
520
692
0.40 9 0.20 9 0.07 0.40 9 0.35 9 0.30
h range for data
collection (ꢁ)
Index ranges
5.07–60.10
4.76–60.01
For the synthesis of the Z-[(S)-Epg]n-OtBu homo-pep-
tide series, we first used the acylfluoride C-activation
method (Carpino et al. 1990). Since the coupling yields in
the formation of dimer and trimer were poor (10–30%), we
decided to follow the procedure described by Imawaka
et al. (2000) for the synthesis of the (S)-Beg homo-oligo-
mers. It involves Tfa Na-protection (see above) and sub-
sequent, selective, reductive Tfa removal by NaBH4 in
EtOH. Peptide bond formation was achieved in anhydrous
CH3CN using the in situ pre-formed 5(4H)-oxazolone from
the Tfa-protected amino acid. The reaction yields were
acceptable (35–65%), although decreasing with peptide
main-chain length elongation. The 5(4H)-oxazolone was
synthesized by treating Tfa-(S)-Epg-OH with EDC. The
electronegative trifluoromethyl group is probably the main
responsible for the good electrophilicity of the carbonyl
moiety of the otherwise modestly reactive 5(4H)-oxazo-
lone. Peptide Na-acetylation was obtained in excellent
yields by treating the Na-deprotected homo-peptides with
an excess of acetic anhydride.
-9 B h B 9
0 B k B 18
0 B l B 11
-7 B h B 7
0 B k B 34
0 B l B 11
Reflections collected/ 2355/2,212
unique
3,169/2,991
(Rint = 0.0297)
(Rint = 0.0782)
Data/restraints/
parameters
Goodness-of-fit on F2 0.891
2,212/67/325
2,991/108/434
0.908
Final R indices
[I C 2r(I)]
R1 = 0.0912,
wR2 = 0.2207
R1 = 0.0827,
wR2 = 0.2163
R indices (all data)
R1 = 0.1487,
wR2 = 0.2492
R1 = 0.1056,
wR2 = 0.2370
-3
)
˚
Residuals (eꢀ A
0.354/-0.290
0.334/-0.203
Solution conformational analysis
The solution conformational preferences of the Tfa (or Ac)
Na-protected (S)-Epg homo-peptide tert-butylester series
from dimer through hexamer were examined in a solvent of
low polarity (CDCl3) at varying concentrations in the range
Scheme 2 Chemo-enzymatic synthesis of H-(R)-Epg-NH2 and
H-(S)-Epg-OH
1
10–0.1 mM by using FTIR absorption and H NMR.
The FTIR absorption spectra in the N–H stretching
region (3,500–3,200 cm-1) of the Tfa-[(S)-Epg]n-OtBu
(n = 2–6) homo-peptide series are reported in Fig. 1. In
trifluoroacetic acid anhydride. Tfa-(S)-Epg-OMe was
obtained from Tfa-(S)-Epg-OH and MeOH in anhydrous
CH3CN in the presence of EDC.HCl and DMAP. Preparation
123

Fully extended peptides
635
H
N
O
H
N
O
F
H
N
O
series, where the absorption maximum in the difference
spectra is only slightly shifted (3,359 cm-1). In the con-
centration range examined (10–0.1 mM) the FTIR
absorption spectra of the (S)-Epg homo-peptides changes
only slightly in the 3,500–3,200 cm-1 region. This finding
indicates that the observed N–HꢀꢀꢀO = C H-bonds are
almost exclusively of the intramolecular type.
F
O
N
F
N
R
R
R
O
O
O
I
II
III
Scheme 3 Intraresidue, intramolecularly H-bonded conformers
occurring in Na-trifluoroacetylated peptides based on Ca-ethylated
a-amino acids
In the C=O stretching region, a quite intense band is
visible for all oligomers at 1,720 cm-1, due to overlapping
of the carbonyl vibrators of the trifluoroacetamido and tert-
butylester moieties (Toniolo et al. 1988). As for the peptide
C=O groups, a single amide I band at 1,666 cm-1 is found
for the tripeptide, whereas in the spectra of the higher
oligomers two bands are visible near 1,675 cm-1 and at
1,654–1,648 cm-1 (not shown). The stronger absorption
(&1,650 cm-1) can be associated with H-bonded carbon-
yls, while the position of the weaker absorption is in the
range usually attributed to free carbonyls (Toniolo et al.
1988; Kennedy et al. 1991). However, the very low amount
of free N–H groups (see above) makes the occurrence of a
significant amount of free carbonyls unlikely. It is
worth recalling that peptides in the antiparallel b-sheet
conformation give rise to a strong C=O absorption at
about 1,630 cm-1 accompanied by a weak band at
1,680–1,690 cm-1 (Miyazawa 1967). In that case, the
splitting of the amide I band has been ascribed to coupling
of neighboring vibrators. Interestingly, the split character
of the IR amide I band is observed also in spectra computed
for single-stranded peptides in the b-sheet conformation
(Kubelka and Keiderling 2001; Kubelka et al. 2006). In the
experimental spectra of our Epg homo-oligomers, the
splitting of the amide I band arises at the level of the tri-
peptide, in which two peptide carbonyls are present (beside
the trifluoroacetyl and ester C=O groups). In the higher
homologues, the intensity ratio between the two bands does
not change dramatically if compared to that of the tripep-
tide. On the basis of these observations, we are inclined to
hypothesize that the occurrence of two amide I bands at
about 1,675 cm-1 and 1,650 cm-1 might be the result of
the coupling of consecutive peptide carbonyls each
involved in an intraresidue (i, i or C5) H-bond, as com-
putationally assessed for the Deg homo-peptides (Maekawa
et al. 2010, in press).
Fig. 2 FTIR absorption difference spectra (N–H stretching region)
between each of the Tfa-[(S)-Epg]n-OtBu (n = 3–6) homo-peptides
and that of the corresponding homo-dipeptide in CDCl3 solution.
Peptide concentration: 1 mM
the dipeptide the two bands at about 3,335 and 3,385 cm-1
are assigned to N–H vibrators involved in intramolecular
H-bonds of the types FꢀꢀꢀH(N)ꢀꢀꢀO=C (peptide) (conformer
I) and (peptide) N–HꢀꢀꢀO=C (ester) (conformer II)
(Scheme 3), at the N- and C-termini of the main chain
(Cung et al. 1972; Toniolo et al. 1988). Notably, in both
conformers the N–HꢀꢀꢀO=C H-bonding is of the intraresi-
due (i, i or C5) type. The amount of free (non-H-bonded)
N–H stretching vibrators (very weak band near
3,440 cm-1) (Pysh and Toniolo 1977; Toniolo et al. 1988)
is low. Upon backbone elongation to tri-, tetra-, penta- and
hexapeptides, the absorption maximum of the band at
lower frequency (3,335 cm-1) significantly shifts to higher
wavenumbers and its relative intensity increases linearly
and markedly. The ‘‘internal’’ C5 conformer III
(Scheme 3), (peptide) Ni-HiꢀꢀꢀOi = Ci (peptide), appears to
provide an ever increasing contribution to the changes
observed in the spectra of the four latter peptides. This
effect is particularly evident from the FTIR absorption
difference spectra shown in Fig. 2 (see also Toniolo et al.
2009), where the contributions of conformers I and II (both
present in the dipeptide) are subtracted from the spectra of
the higher homo-oligomers. The resulting maximum, per-
fectly aligned in all oligomers, of ever increasing intensity
along the peptide series and attributable to conformer III, is
seen at 3,356 cm-1. Very similar data (not shown) were
also found for the Na-acetylated (S)-Epg homo-peptide
All of the results of our FTIR absorption investigation fit
beautifully with those already published for the Deg
(Toniolo et al. 1988) and Beg (Imawaka et al. 2000) homo-
peptides found to adopt consecutive C5 conformations
(2.05-helices) (Toniolo and Benedetti 1991c).
To obtain further information on the 3D-structural ten-
dency of the Na-trifluoacetylated (S)-Epg homo-peptide
esters in CDCl3 solution, the NH chemical shifts in their 1H
NMR spectra were investigated as a function of addition of
DMSO and TEMPO. The polar solvent DMSO is expected
123

636
M. Crisma et al.
to interact strongly with the exposed amide NH protons via
N–HꢀꢀꢀO = S H-bonds, thus inducing a downfield shift in
their resonances (Kopple et al. 1969; Martin and Hauthal
1975). The paramagnetic free-radical TEMPO, on the other
internal NH signal of the tripeptide (at 7.4 ppm) was
assigned by elimination. These assignments for the tri-
peptide were confirmed by a combination of 2D-NMR
HMBC and ROESY spectral experiments. It is clear that
the NH proton at &8.0 ppm, which gives a cross-peak with
the quartet signal of the Tfa carbonyl carbon at about
195 ppm (Fig. 3a) is that of the N-terminal residue. By
using the ROESY spectra (Fig. 3b), it was then possible to
assign the N(2)H proton (at approximately 7.4 ppm) and
the N(3)H proton (near 6.85 ppm) from the known N(1)H
proton. Interestingly, the observation of the occurrence
of the internal NH signals of all homo-oligomers in
the narrow range 7.40–7.45 ppm (see, for example,
Fig. 4a) highlights their closely related 3D-structural
environment.
1
hand, is known to perturb the H NMR spectra of com-
pounds containing exposed –CONH– groups by broaden-
ing the resonances of their NH groups by virtue of
nitroxide radical–amide association of the N–HꢀꢀꢀO–N=
type (Kopple and Schamper 1972).
The trifluoroacetamido NH signal of the (S)-Epg pep-
tides was identified by virtue of its low-field position
(&8.0 ppm) (Toniolo et al. 1988). The C-terminal NH
signal of the dimer was found near 6.85 ppm. By analogy,
we related the 6.8–6.9 ppm signal in the spectra of the
higher homologues to their C-terminal NH group. The
Fig. 3 Section of the HMBC spectrum (a) and the ROESY spectrum (b) of Tfa-[(S)-Epg]3-OtBu in CDCl3 solution. Peptide concentration:
10 mM
Fig. 4 a Plot of the NH proton
1
chemical shifts in the H NMR
spectra of Tfa-[(S)-Epg]6-OtBu
as a function of increasing
percentages of DMSO (v/v)
added to the CDCl3 solution.
b Plot of bandwidths of the NH
1
proton signals in the H NMR
spectra of the same homo-
hexapeptide as a function of
increasing percentages of
TEMPO (w/v) added to the
CDCl3 solution. Peptide
concentration: 1 mM
123

Fully extended peptides
637
trifluoroacetyl group in the stabilization of the N-terminal
C5 conformer.
Crystal-state conformational analysis
Numerous attempts to grow single crystals appropriate for
an X-ray diffraction analysis allowed us to solve the 3D-
structures of the homo-dipeptide Tfa-[(S)-Epg]2-OtBu
(Toniolo et al. 2009) and the homo-tripeptide Tfa-[(S)-
Epg]3-OtBu (Figs. 5, 6, respectively). Lists of the most
relevant conformational and intramolecular H-bond
parameters for the two oligomers are reported in Tables 3
and 4, respectively.
The s (N–Ca–C0) bond angle (Touw and Vriend 2010)
ranges from 103.9(3)ꢁ to 104.6(5)ꢁ. These values, charac-
teristic of the internal angles of a pentagonal structure
(Toniolo and Benedetti 1991c), are markedly narrower than
those of a regular tetrahedral sp3 atom (109.5ꢁ). This
finding represents a clear evidence for the presence of the
strain introduced by an intramolecularly H-bonded struc-
ture of the C5-pseudoring type.
Fig. 5 X-ray diffraction structure of Tfa-[(S)-Epg]2-OtBu with atom
numbering. Only the major-occupancy conformer of the disordered
Tfa group is shown. The two intramolecular N–HꢀꢀꢀO=C H-bonds are
represented by dashed lines (the intramolecular N–HꢀꢀꢀF H-bond is
not shown)
In all five (S)-Epg residues investigated (in particular in
the three residues of the trimer) the sets of /,w backbone
torsion angles show values close to planarity (180ꢁ, 180ꢁ),
the largest deviation observed being 3.1ꢁ. This is the
strongest indication for the occurrence of consecutive, fully
extended (C5) conformers (Toniolo and Benedetti 1991c).
An additional, although indirect, proof to this assumption is
based on the observation that all N–H and C=O groups,
which generate the C5 conformers, are not involved in any
intermolecular H-bond interaction. On the average, the
The study of the solvent accessibilities of the NH pro-
tons of the five homo-peptides, indicative of their partici-
pation as donors to peptide—additive or intramolecular
peptide—peptide H-bonds and tested by using the DMSO
and TEMPO acceptors, strongly support the view that
almost all of the NH protons are totally insensitive to the
presence of the two perturbing agents. The results for the
longest homo-peptide (the hexamer) are reported in Fig. 4.
Indeed, according to the TEMPO experiments, the NH
proton of the C-terminal residue of each of the tri-, tetra-,
penta- and hexapeptides (for the latter, see Fig. 4b) exhibits
a (modest) sensitivity, that increases slightly upon main-
chain elongation. This finding suggests a limited fraying of
the peptide C-termini. In this connection, it is worth
recalling that the C-terminal carbonyl ester is a poorer
H-bond acceptor if compared to the internal peptide car-
bonyl groups, as indicated by the results of our FTIR
absorption analysis (see above). Again, the data of these
conformational investigations in solution agree well with
those of the Na-trifluoacetylated Deg (Toniolo et al. 1988)
and Beg (Imawaka et al. 2000) homo-oligomers. Interest-
ingly, our corresponding TEMPO titrations with the Na-
acetylated (S)-Epg homo-peptides (not shown) are strongly
in favor of the conclusion that in this series the N-terminal
proton is significantly more exposed to the perturbing agent
than its corresponding C-terminal proton. These data
underscore the important role played by the Na-
˚
intramolecular NꢀꢀꢀO distance in each residue is 2.578 A.
Also, the x (peptide and ester) torsion angles show values
in the proximity of 180ꢁ, the largest deviation being noted
for the (ester) x2 angle of the dipeptide, -174.6(6)ꢁ. The
trifluoroacetamido moieties of the two peptides are close to
planarity, the related x torsion angles being -176.7(6)ꢁ
and -173.9(5)ꢁ, respectively. In both structures, the
N-terminal NH group is also within H-bonding distance
from one of the fluorine atoms of one of the conformers of
the rotationally disordered Tfa group. This finding supports
the occurrence of an intramolecular, bifurcated H-bond of
the type FꢀꢀꢀH(N)ꢀꢀꢀO=C (peptide) (conformer
I in
Scheme 3). All these crystal-state results agree very well
with those already published for the Deg (Toniolo et al.
1988) and Beg (Imawaka et al. 2000) homo-peptides. The
ethyl and n-pentyl side chains of the five (S)-Epg residues
adopt a fully extended conformation and point outwards
orthogonally from the peptide backbone. Such a side-chain
disposition allows the residues to release unfavorable
intramolecular interactions.
The packing mode of both structures (for that of the
tripeptide, see Fig. 7) is characterized by the lack of any
123

638
M. Crisma et al.
Fig. 6 X-ray diffraction
structure of Tfa-[(S)-Epg]3-
OtBu with atom numbering.
Only the major-occupancy
conformers of the disordered
Tfa group and of Epg(2)
n-pentyl side chain are shown.
The three intramolecular
N–HꢀꢀꢀO=C H-bonds are
represented by dashed lines (the
intramolecular N–H … F
H-bond is not shown)
Table 3 Relevant conformational parameters (ꢁ) observed for the (S)-Epg residues in the crystal state in Tfa-[(S)-Epg]2-OtBu and Tfa-[(S)-
Epg]3-OtBu
Peptide
Residue
s
/
w
x
Tfa-[(S)-Epg]2-OtBu
1
2
1
2
3
104.6(5)
104.4(5)
104.4(3)
104.6(3)
103.9(3)
176.4(7)
-176.9(5)
-176.9(5)a
-178.9(4)
179.7(4)
179.1(6)
-178.8(6)
179.9(5)
-174.6(6)b
-179.7(4)
178.5(4)
Tfa-[(S)-Epg]3-OtBu
179.7(4)
-179.9(4)
-179.9(4)c
177.9(5)d
a
N2-C2A-C2-OT
b
C2A-C2-OT-CT1
c
N3-C3A-C3-OT
d
C3A-C3-OT-CT1
Table 4 Intramolecular H-bond parameters for Tfa-[(S)-Epg]2-OtBu and Tfa-[(S)-Epg]3-OtBu
˚
˚
Peptide
Donor D–H Acceptor A Symmetry equiv. of A Distance (A) DꢀꢀꢀA Distance (A) HꢀꢀꢀA Angle (ꢁ) D–HꢀꢀꢀA
Tfa-[(S)-Epg]2-OtBu N1–H1
F2
x, y, z
x, y, z
x, y, z
x, y, z
x, y, z
x, y, z
x, y, z
2.643(8)
2.536(7)
2.585(7)
2.630(7)
2.529(5)
2.558(4)
2.573(4)
2.21
2.08
2.14
2.20
2.07
2.11
2.13
111
112
111
111
112
112
112
N1–H1
O1
O2
F2
N2–H2
Tfa-[(S)-Epg]3-OtBu N1–H1
N1–H1
N2–H2
N3–H3
O1
O2
O3
˚
˚
intermolecular N–HꢀꢀꢀO=C H-bond. In the structure of the
dipeptide a relatively short C–HꢀꢀꢀO contact is observed
between the C2Z methyl group and the O1 carbonyl oxy-
gen atom of a (-1 ? x, y, z) symmetry related molecule
[CꢀꢀꢀO distance 3.580(14) A, HꢀꢀꢀO distance 2.69 A,
C–HꢀꢀꢀO angle 155ꢁ]. In addition, a C–HꢀꢀꢀF contact is
found between the C-terminal CT4 methyl group and a (x,
y, 1 ? z) equivalent of the F20 atom belonging to the minor
123

Fully extended peptides
639
Fig. 7 Packing mode of the
Tfa-[(S)-Epg]3-OtBu molecules
in the crystal as viewed along
the a direction
conformer of the disordered N-terminal Tfa group [CꢀꢀꢀF
chiroptical analyses. The Tfa/OtBu protected (S)-Epg
homo-oligopeptides synthesized in this work dissolve
readily in CDCl3, an appropriate solvent to record vibra-
tional CD spectra. However, CDCl3 is not transparent for
use in the far-UV region where peptide electronic transi-
tions are known to absorb (Beychock 1967). On the other
hand, solvents suitable for electronic CD studies of pep-
tides (e.g., alcohols) tend to destabilize, at least partially,
the 2.05-helix (unpublished results). To this end, we are
planning to synthesize a longer (S)-Epg homo-oligomer
series carrying N- and/or C-terminal blocking groups
suitable for increasing peptide solubility in far-UV trans-
parent solvents of low polarity.
˚
˚
distance 3.42(3) A, HꢀꢀꢀF distance 2.58 A, C–HꢀꢀꢀO angle
146ꢁ]. These two interactions connect molecules along the
a and c directions, respectively.
Weak C–HꢀꢀꢀO and C–HꢀꢀꢀF interactions characterize
also the packing mode of the tripeptide. Specifically, the
shortest intermolecular C–HꢀꢀꢀO contacts [with CꢀꢀꢀO dis-
˚
tances ranging from 3.228(18) to 3.310(6) A, HꢀꢀꢀO dis-
˚
tances within 2.62–2.70 A and C–HꢀꢀꢀO angles between
124 and 121ꢁ] are observed between C2Z and O2 (sym-
metry equivalence: 1 ? x, y, -1 ? z), C3Z and O3 (x, y,
1 ? z), and between C2G2 and O2 (-1 ? x, y, z).
A C–HꢀꢀꢀF contact occursbetween the C-terminalCT3methyl
group and a (-x, -1/2 ? y, 1 - z) symmetry equivalent of
the F2 atom of the disordered N-terminal Tfa group [CꢀꢀꢀF
Acknowledgments A.M. and F.F. are grateful to the University of
Padova for financial support through the PRAT 2007 funding
initiative.
˚
˚
distance 3.379(15) A, HꢀꢀꢀF distance 2.55 A, C–HꢀꢀꢀO angle
144ꢁ]. This latter interaction connects molecules related by
the twofold screw axis parallel to the b direction.
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