Conformational properties of peptides incorporating a fluorinated pseudoproline residue

Article abstract of DOI:10.1039/c3nj41084f

We have recently reported the synthesis of enantiomerically pure CF ₃-oxazolidine pseudoprolines (CF₃-ΨPro). Complete NMR studies, together with DFT calculations, have highlighted the marked stereoelectronic effects of the CF₃ group on these new proline surrogates. In this paper, we describe for the first time the conformational features of dipeptides incorporating one CF₃-ΨPro residue. Extensive NMR analyses have been carried out in solution and revealed the presence of a stable type-VI β-turn in a pseudotetrapeptide sequence.

Full text of DOI:10.1039/c3nj41084f

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Conformational properties of peptides incorporating a
fluorinated pseudoproline residue†
Cite this: DOI: 10.1039/c3nj41084f
a
bc
b
b
´
´
Gregory Chaume, Debby Feytens, Gerard Chassaing, Solange Lavielle,
Thierry Brigauda and Emeric Miclet*b
We have recently reported the synthesis of enantiomerically pure CF3-oxazolidine pseudoprolines
(CF3-CPro). Complete NMR studies, together with DFT calculations, have highlighted the marked
stereoelectronic effects of the CF3 group on these new proline surrogates. In this paper, we describe for
the first time the conformational features of dipeptides incorporating one CF3-CPro residue. Extensive
NMR analyses have been carried out in solution and revealed the presence of a stable type-VI b-turn in
a pseudotetrapeptide sequence.
Received (in Montpellier, France)
26th November 2012,
Accepted 22nd January 2013
DOI: 10.1039/c3nj41084f
www.rsc.org/njc
For a few years we have been interested in the stereoselective
synthesis of trifluoromethyl group containing amino acids.10
Introduction
The proline residue is of special significance in the structure
of peptides and proteins. The pyrrolidine ring restricts the
f-dihedral angle to values around À601 and the presence of
the tertiary amide leads to increased cis populations for the
Xaa–Pro peptide bonds (lower free energy difference DGtc).1 These
unique conformational properties of the peptide backbone are
associated with a cis–trans isomerisation rate that is relatively
high compared to the other amino acids (lower activation energy
DG‡tc).2 During the last two decades, it has been shown that
proline analogues can be designed to further tailor the struc-
tural and thermodynamical features of the peptide backbone.
Cd-substituted prolines and pseudoprolines (CPro) have proved to
be very useful as DGtc and DGt‡c can be finely tuned depending on
the nature of the substituent and on the absolute configuration
at Cd.3–6 Replacement of key proline residues by such analogues
in biologically active peptides offers invaluable opportunities for
the modulation of their activity.7–9
Recently, we reported the synthesis of CF3-substituted pseudo-
prolines (CF3-CPro)11 and used both theoretical calculations
and NMR studies to deliver a detailed analysis of the CF3
stereoelectronic effects within these proline surrogates.12
We demonstrated that the trifluoromethyl group (i) was respon-
sible for freezing the puckering of the 5-membered oxazolidine
ring, (ii) lowered the rotational barrier of the cis–trans peptide
bond isomerization and (iii) locally promoted the stabilization
of extended c-dihedral angles. We were then interested in the
conformational studies of peptide oligomers containing such a
CF3-CPro amino acid. Herein, we report the structural proper-
ties of four peptides based on the X–Ala–Ser(CCF ,HPro)–Y
3
sequence, where X is a o-Nbs, Fmoc or Ac group and Y is a
OMe or NHMe group (Scheme 1). For each peptidomimetic
molecule, an extensive NMR study is provided, highlighting
the influence of these terminal groups on the structure
and stability of the backbone of the CF3-CPro containing
peptides 3–6.
a
´
Laboratoire SOSCO, Universite de Cergy-Pontoise, EA 4505, 5 mail Gay Lussac,
95000 Cergy-Pontoise, France
b
´
Laboratoire des BioMolecules, UMR 7203, Ecole Normale Superieure, UPMC Paris
06, 4, Place Jussieu, 75005 Paris, France. E-mail: emeric.miclet@upmc.fr;
Fax: +33 1 44 27 71 50; Tel: +33 1 44 27 31 15
c Organic Chemistry Department, Vrije Universiteit Brussel, Pleinlaan 2, B-1050
Brussels, Belgium
† Electronic supplementary information (ESI) available: NMR spectra of
compounds 3–6, method used for the determination of the stereochemistry of
L-Ser(CCF ,HPro) amino acids, tables with NMR restraints, thermodynamic and
3
kinetic parameters of peptide 6, and X-ray crystal data for peptide 3. CCDC
910329. For ESI and crystallographic data in CIF or other electronic format see
DOI: 10.1039/c3nj41084f
Scheme 1 Synthesis and chemical structures of compounds 3–6.
c
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Table 1 cis : trans ratio calculated for compounds 3–6 at 274 K.a The geometry
of the peptide bond was not clearly established for peptide 3 in CDCl3
Results and discussion
Synthesis and characterization of peptides
Comp.
Solvent
Cis : trans
(2R,4S)-CF3-pseudoproline ester 1 is conveniently synthesized
by condensation of serine esters with trifluoroacetaldehyde
hemiacetal.11 It can subsequently undergo a N-methylamidation
in a good yield leading to compound 2. Both compounds 1 and 2
were used as CF3-CPro-building blocks for the synthesis of
peptides 3–6 (Scheme 1). L-Ala with o-Nbs (3) or Fmoc (4, 5)
N-protecting groups was chosen as an amino acid model for the
extension of the peptide backbone. Coupling reactions have been
carried out using amino acid chlorides in the presence of base in
order to overcome the low nucleophilicity of the CF3-CPro amino
group. Fmoc deprotection of peptide 5 followed by N-acetylation
gave the pseudotetrapeptide 6. Peptides 3, 4 and 5 were obtained,
respectively, from (2R,4S)-CF3-pseudoproline ester 1 and (2R,4S)-
CF3-pseudoproline amide 2 in good yield as a diastereomeric
mixture. Major compounds 3, 4 and 5 (470%) have been isolated
using flash chromatography and were well characterized using 2D
NMR spectroscopy as described in detail in the ESI.† In addition,
we provide the X-ray structure for 3 (Fig. 1)† which nicely con-
firmed the stereochemistry determined by NMR. Although these
major peptides were all found to incorporate a (2S)-alanine and a
(2R,4S)-CF3-pseudoproline, the presence of a minor diastereo-
isomer could be due to the epimerization of the alanine or
the pseudoproline residues during the coupling reactions.
Unfortunately, we are not able to assign the absolute configuration
of the minor diastereomers at this stage. Further efforts will be
made for the complete analysis of the epimerization process and
for the optimization of the coupling conditions.
3
4
5
6
6
6
CDCl3
CDCl3
CDCl3
CDCl3
DMSO-d6
45 : 55 or 55 : 45
12 : 88
58 : 42
70 : 30
66 : 34
a
H2O : D2O 90 : 10
58 : 42
a
294 K for 6 in DMSO.
a strong HaAla–HCd Pro cross peak for the major spin system of 4 in
agreement with a trans peptide bond, whereas compounds 5
and 6 displayed an additional HaAla–HCa Pro correlation, indicating
a stabilized cis conformation. We conclude that replacing the
o-Nbs (3) by the bulky Fmoc group (4) strongly destabilized the
Ala CF3-CPro cis peptide bond. This effect was alleviated by
the incorporation of the NHMe C-terminal group (5) leading to
almost equal populations. A further stabilization of the cis
conformation was finally obtained in the pseudotetrapeptide
6, which lacks the Fmoc protecting group.
Interestingly, the population levels of 6 were strongly influ-
enced by the solvent. The highest stabilization of the cis state
was obtained in CDCl3 followed by DMSO-d6 and H2O. The
presence of an intramolecular hydrogen bond involving the NH
carboxamide could be responsible for the relatively high cis
population observed in peptides 5 and 6 in apolar solvent. This
has been assessed by comparing the solvent dependence of the
NH protons chemical shifts (Dd) as well as their temperature
coefficients (Dd/DT).14 When changing the solvent from CDCl3
to DMSO or from CDCl3 to H2O, typical Dd values were 2 ppm
for the NH protons of pseudotetrapeptides 6, except for the
terminal NH methylamide in the cis conformer which displayed
very low values of 0.31 ppm and 0.37 ppm, respectively
(Table 2). The highest temperature coefficient was also
obtained for this proton (À5.8 ppb KÀ1), which was in agree-
ment with a weak hydrogen bond in water.
Analysis of the cis–trans populations
For each compound 3–6, two slowly exchanging conformers
were observed at 274 K in CDCl3, as attested by the presence of
exchange cross peaks in the 1H–1H EXSY spectra.5,13 Integra-
tion of isolated resonances has been used to determine the
corresponding populations. For 3 and 5 the two conformers
were almost equally populated whereas an imbalance of 9 : 1
and 7 : 3 was observed in 4 and 6, respectively (Table 1).
The Ala–CF3-CPro peptide bond conformations were then
established using inter-residue NOE correlations. We observed
To complete the analysis of the cis–trans isomerization
on the pseudotetrapeptide 6, we evaluated the energy barriers
DG‡ct by performing quantitative analyses of the EXSY experi-
ments5 and by measuring the coalescence temperatures.15
The relatively low values obtained in CDCl3, DMSO or in water
(B15 kcal molÀ1, see ESI†) were ascribed to the presence of the
(2R)-CF3 substituent, acting as a stabilizing polar group of the
transition state.12 The DGc‡t values corresponding to the cis to
trans isomerization were not greatly influenced by the solvent.
This could be due to the fact that increasing the solvent polarity
Table 2 Solvent dependence and temperature coefficients of the NH protons
in peptide 6
NH
Ala trans
NH carbox
cis trans
Ala cis
DdCDCl -DMSO (ppm)
1.30
2.17
À8.5
2.33
1.96
À7.8
0.31
0.37
À5.8
2.04
2.09
À8.8
3
Fig. 1 Overview of the X-ray structure obtained for 3. The stereochemistry of the
CF3-CPro residue and backbone dihedral angles (in degree) have been reported.
DdCDCl -H O (ppm)
2
Dd/DT 3(H2O) (ppb KÀ1
)
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destabilized the transition state, but also the cis conformation
by disrupting the H-bond.
Structural analysis of the pseudotetrapeptide 6
We have previously shown that the presence of the CF3 group in the
CF3-CPro residue was also responsible for stereoelectronic effects
that efficiently constrain the ring puckering.12 In Ac–(2R,4S)-CF3-
CPro–NHMe, the ring geometry always adopted an up puckering,
independently of the solvent or the peptide bond conformation.
We have then performed CH2-TROSY experiments to precisely
3
measure the vicinal JHa–Hb couplings in the longer peptide 6
(Fig. 2).16 These constants enable the precise determination of
the 5-membered ring conformation.
Fig. 3 Overlay of the 5-lowest energy structures obtained by molecular
dynamics under NMR restraints. The NHÁ Á ÁOC distance is displayed in Å.
Whereas the trans conformer displayed the characteristic
vicinal coupling values for an up puckering (3JHaHb2 = 7.2 Hz
and 3JHaHb3 = 8.8 Hz), no canonical conformation was assigned
with our previous observations. The cis Ala–CPro peptide bond
conformation together with the backbone dihedral angles
3
to the cis form (3JHaHb2 = 4.6 Hz and JHaHb3 = 8.2 Hz). An
envelope conformation of the 5-membered ring (w1 B 01) or fast
exchanging and equally populated up/down puckerings could
account for these coupling values. This is in strong contrast to
the features observed on the pseudopeptide lacking the Ala
residue (Ac–CF3-CPro–NHMe). First, it was found to adopt the
up puckering, whatever the peptide bond geometry or the
solvent polarity. Second, the C-terminal carboxamide proton
was also involved in an H-bond but for the trans conformer
only, which was consequently the major conformer in CDCl3.
The peculiar features of the peptide 6 are then clearly related to
the presence of the preceding Ala residue. The unusual geometry
observed for the oxazolidine ring may be of importance for the
establishment of the H-bond in the cis conformer.
To fully characterize the structural properties of the cis
conformation of the pseudotetrapeptide 6, we have conducted
structure calculations using a restrained dynamic simulated
annealing. A total of 11 proton–proton distance restraints have
been extracted from the NOESY spectrum in CDCl3 and 3 coupling
constants have been measured (see ESI†). Despite the limited
number of NMR constraints, the 5-lowest energy structures were
well superimposed as shown in Fig. 3. We observed an H-bond
between the NH carboxamide proton and the oxygen of the
N-terminal acetyl carbonyl, which was in perfect agreement
of the Ala and the CF3-CPro residues (fAla = À661; cAla
=
1401; o = À11; fCPro = À761 and cCPro = 21) defined a nice
type-VI a1 b-turn (canonical dihedral angles are fi+1 = À601;
ci+1 = 1201; o = 01, fi+2 = À901 and ci+2 = 01). Similar dihedral
angles were also measured in the crystal structure of 3 for the
Ala–CF3-CPro pair (Fig. 1), suggesting that the X-ray peptide
scaffold was maintained in solution. Similarly, the w1 angle of
the CPro residue measured on the calculated structures of 6
was ca. (5 Æ 3)1, which corresponds to a slightly down-puckered
envelope and is in fair agreement with the X-ray structure of 3
(w1 = +151).
It should be stressed that both the relatively high population
of the cis conformer in water (58%) and the temperature
coefficient for the NH carboxamide (À5.8 ppb KÀ1) indicate
the persistence of an H-bond in this solvent, although partly
destabilized. Therefore, the Ala–CF3-CPro unit appears to be
very valuable for inducing type-VI b-turn in solution. This
original secondary structure has attracted considerable interest
because it is implicated in the function of important protein
and is associated with the high potency of numerous peptide
hormone analogues.9,17–22 However, the design of new mole-
cules mimicking the type-VI b-turn remains a challenge since it
requires the stabilization of the cis peptide bond. Several
attempts have been made in the past fifteen years. Direct
cyclization of the peptide backbone has been considered and
led to several templates such as 8-membered ring lactam,18
functionalized piperidinone,19 aminopyroglutamate,20 or bicyclic
systems.21 Alternatively, proline surrogates that favor the cis
peptide bond conformation have been assessed for their pro-
pensity to induce type-VI b-turns. This included Cd-substituted
prolines,14,22 pseudoprolines9,23 or azaprolines.24 It has been
predicted that the combination of these different modifications
would enhance the cis preference for the peptide bond, by
taking advantage of the steric and the electronic effects.25
Herein, we used the (2R,4S)-CF3-pseudoproline that bears the highly
electronegative and bulky CF3 group in addition to the oxazolidine
ring oxygen. We demonstrated that the (2R,4S)-CF3-CPro induces
a 58% cis peptide bond conformation in water for a minimal
Fig. 2 (a) CbH2 region of the CH2-TROSY experiment recorded on compound 6
in CDCl3, 274 K, 500 MHz. (b) Schematic representation of the up and down
3
puckerings with the corresponding JHaHb vicinal couplings.
c
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pseudotetrapeptide sequence. The MD calculations matched Mate II or using a Waters (LC) ESI/TOF spectrometer. Melting
the X-ray structure and revealed a preference for a type VI b-turn points were obtained on a Bu¨chi apparatus and are uncorrected.
o-Nbs-Ala-Ser(WCF ,HPro)-OMe (3)11. To a solution of the
3
conformation in solution. Further structural studies on longer
peptides incorporating the CF3-CPro residue are currently o-Nbs-alanine26 (1.30 g, 4.74 mmol, 7 equiv.) suspended in
under investigation in our laboratory.
dichloromethane (4.8 mL), under argon, was added 1-chloro-
N,N-2-trimethyl-1-propenylamine (628 mL, 4.74 mmol, 7 equiv.)
at 0 1C. The resulting solution was stirred at 0 1C until the
disappearance of the precipitate (usually 20 min). The total
conversion of the acid to chloride was checked by TLC after
Experimental
Synthesis and characterization of compounds 3–6
Unless otherwise mentioned, all the reagents were purchased quenching with methanol. The o-Nbs-alanine chloride solution
from a commercial source. All glassware was dried in an oven was added via cannula to a neat mixture of pseudoprolines
at 150 1C prior to use. CH2Cl2 was distilled under nitrogen (R,S)-1 (135 mg, 0.68 mmol, 1.0 equiv.) and collidine (90 mL,
from CaH2 prior to use. 1H NMR (400.00 MHz), 13C NMR 0.68 mmol, 1.0 equiv.) at 0 1C. The temperature was allowed to
(100.50 MHz) and 19F NMR (376.20 MHz) were measured on a warm to room temperature and the solution was concentrated
JEOL ECX400 spectrometer, 1H NMR (500.00 MHz) and 13C NMR twice using a stream of argon. After 24 h, the resulting mixture
(125.75 MHz) were measured on a Bruker Avance III spectro- was diluted with dichloromethane, and quenched with a satu-
meter operating at a 1H frequency of 500 MHz and equipped rated aqueous solution of NaHCO3. The layers were separated,
with a triple resonance, z-axis pulsed-field-gradient cryogenic and the aqueous layer was extracted with dichloromethane. The
probehead, optimized for 1H detection. Complete proton combined organic layers were washed with water, dried over
assignments were obtained from the analysis of 2D total MgSO4, filtered, and evaporated under reduced pressure. The
correlation spectroscopy (TOCSY) experiments using 80 ms crude 73 : 27 mixture of diastereomers was purified by flash
DIPSI-2 mixtime and 2D nuclear Overhauser effect spectroscopy chromatography (70 : 30 cyclohexane–ethyl acetate) to give
(NOESY) experiments (typically 500 ms mixing time). Homo- 70 mg (23%) of minor diastereomer 3min and 210 mg (68%)
nuclear experiments were typically performed using 512 (t1) of major diastereomer (R,S)-3 as a 45 : 55 inseparable mixture of
and 4096 (t2) time-domain matrices over a spectral width of cis–trans rotational isomers in CDCl3 at 274 K. (R,S)-3 major
10 ppm, with 8 scans per t1 increment. Carbon assignment was diastereomer: white solid; mp 182–183 1C; Rf = 0.30 (60 : 40
1
1
deduced from heteronuclear 2D H–13C HSQC and 2D H–13C cyclohexane–ethyl acetate); [a]D24 À122.5 (c 1.8, CHCl3); IR (neat)
CH2-TROSY16 experiments, using 256 (t1) Â 1024 (t2) time- 3300, 3107, 1730, 1677, 1535, 1440, 1154 cmÀ1
;
1H NMR
domain matrices, with 32 scans per t1 increment. Data were (500 MHz, CDCl3, 274 K) (major rotamer) d 1.44 (d, J = 5.5
processed using the TOPSPIN 2.0 software (Bruker). Shifted Hz, 3H, Hb Ala-H), 3.71 (s, 3H, OMe), 4.30 (t, J = 6.4 Hz, 1H, Hb
sine-bell window functions were applied in both indirect and CPro-Ha), 4.39 (m, 1H, Ha Ala-H), 4.45 (m, 2H, Ha and Hb CPro-
direct detected dimensions and extensive zero-filling prior to Hb), 5.97 (q, J = 5.2 Hz, 1H, Hd CPro-H), 6.22 (d, J = 9.5 Hz, 1H,
Fourier transformation was used to yield high digital resolu- NH Ala); 7.73–7.85 (m, 2H, o-Nbs arom.), 7.95–8.05 (m, 2H,
tion. Chemical shifts of 1H NMR are expressed in parts per o-Nbs arom.); (minor rotamer) d 1.46 (d, J = 5.5 Hz, 3H, Hb Ala-H),
million downfield from tetramethylsilane (d = 0) in CDCl3. 3.95 (s, 3H, OMe), 4.49 (m, 1H, Ha Ala-H), 4.52 (d, J = 7.1 Hz, 2H,
Chemical shifts of 13C NMR are expressed in parts per million Hb CPro-H), 4.63 (t, J = 6.9 Hz, 1H, Ha CPro-H), 5.61 (q, J = 4.9 Hz,
downfield from CDCl3 as internal standard (d = 77.0). Chemical 1H, Hd CPro-H), 6.57 (d, J = 10.1 Hz, 1H, NH Ala), 7.73–7.85 (m,
shifts of 19F NMR are expressed in parts per million downfield 2H, o-Nbs arom.), 8.03 (m, 1H, o-Nbs arom.), 8.25 (m, 1H, o-Nbs
from C6F6 as an internal standard (d = À164.9). Coupling arom.); 13C NMR (125.75 MHz, DMSO-d6, 298 K) (major rotamer)
constants are reported in Hertz. Column chromatography was d 18.8 (CH3, Cb Ala), 51.5 (CH, Ca Ala), 53.1 (CH3, OMe), 56.3 (CH,
performed on SDS 60 Å (40–63 mm) silica gel, employing Ca CPro), 68.9 (CH2, Cb CPro), 84.5 (q, J = 36.4 Hz, CH, Cd CPro),
a mixture of the specified solvent as an eluent. Thin-layer 122.3 (q, J = 279.9 Hz, CF3), 124.3 (CH, o-Nbs arom.), 129.5 (CH,
chromatography (TLC) was performed on Merck silica gel o-Nbs arom.), 132.6 (CH, o-Nbs arom.), 133.1 (CH, o-Nbs arom.),
(Merck 60 PF254) plates. Silica TLC plates were visualized under 134.2 (C, o-Nbs arom.), 147.3 (C, o-Nbs arom.), 168.8 (CQO),
UV light, by a 10% solution of phosphomolybdic acid in 170.8 (CQO); (minor rotamer) d 19.3 (CH3, Cb Ala), 51.9 (CH, Ca
ethanol followed by heating. Mass spectra (MS) were obtained Ala), 53.8 (CH3, OMe), 56.6 (CH, Ca CPro), 70.4 (CH2, Cb CPro),
on a GC/MS apparatus HP 5973 MSD with an HP 6890 Series 84.4 (q, J = 36.4 Hz, CH, Cd CPro), 122.3 (q, J = 279.9 Hz, CF3),
GC. Ionization was obtained by electronic impact (EI 70 eV). 124.3 (CH, o-Nbs arom.), 129.5 (CH, o-Nbs arom.), 132.6 (CH,
Infrared spectra (IR) were obtained by Fourier-transformation o-Nbs arom.), 133.5 (CH, o-Nbs arom.), 133.8 (C, o-Nbs arom.),
on Bruker Tensor 27, wavenumbers are given in cmÀ1. Liquid 147.3 (C, o-Nbs arom.), 168.7 (CQO), 170.8 (CQO); 19F NMR
Chromatography-Mass Spectrometry (LC-MS) analyses were (376.2 MHz, DMSO-d6, 298 K) (major rotamer) d À80.9 (s, CF3);
done on a Waters (LC) ESI/TOF spectrometer and MS spectrum (minor rotamer) d À81.7 (s, CF3); LCMS (ES+) m/z = 456.46
was recorded using an ESI ionization and positive ion mode. [M + H]+; anal. calcd for C15H16F3N3O8S (455.06): C, 39.56;
Elemental analyses were performed on a Perkin–Elmer CHN H, 3.54; N, 9.23. Found: C, 39.45; H, 3.47; N, 8.99%. Minor
2400. Optical rotations were determined using a JASCO P1010 diastereomer 3min: colorless oil; Rf = 0.41 (50 : 50 cyclohexane–
polarimeter. HRMS analyses were performed on a Jeol JMS-GC ethyl acetate); 1H NMR (400 MHz, CDCl3, 323 K) (single rotamer):
c
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d 1.43 (d, J = 6.9 Hz, 3H, Hb Ala-H), 3.82 (s, 3H, OMe), 4.43 (dq, J = (t, J = 7.5 Hz, 2H, Fmoc arom.), 7.58 (t, J = 7.4 Hz, 2H, Fmoc
8.7, 6.9 Hz, 1H, Ha Ala-H), 4.57 (d, J = 6.4 Hz, 2H, Hb CPro-H), arom.), 7.79 (d, J = 7.5 Hz, 2H, Fmoc arom.); 13C NMR
5.16 (t, J = 6.4 Hz, 1H, Ha CPro-H), 5.56 (q, J = 5.0 Hz, 1H, Hd (125.75 MHz, CDCl3, 274 K): (trans rotamer) d 17.8 (CH3, Cb
CPro-H), 6.15 (d, J = 8.7 Hz, 1H, NH Ala), 7.70–7.75 (m, 2H, o-Nbs Ala), 46.8 (CH, Fmoc CH), 48.7 (CH, Ca Ala), 53.1 (CH3, OMe),
arom.), 7.88–7.92 (m, 2H, o-Nbs arom.); 13C NMR (100.5 MHz, 56.6 (CH, Ca CPro), 67.2 (CH2, Fmoc CH2), 69.0 (CH2, Cb CPro),
CDCl3, 323 K) (single rotamer): d 18.7 (CH3, Cb Ala), 51.3 (CH, Ca 84.9 (q, J = 36.0 Hz, CH, Cd CPro), 120.0 (2 Â CH, Fmoc arom.),
Ala), 53.4 (CH3, OMe), 57.2 (CH, Ca CPro), 70.4 (CH2, Cb CPro), 121.8 (q, J = 286.6 Hz, CF3), 125.0 (2 Â CH, Fmoc arom.), 127.6
84.0 (q, J = 36.4 Hz, CH, Cd CPro), 122.4 (q, J = 286.6 Hz, CF3), (2 Â CH, Fmoc arom.), 127.7 (2 Â CH, Fmoc arom.), 140.6
125.8 (CH, o-Nbs arom.), 129.6 (CH, o-Nbs arom.), 133.0 (CH, (2 Â C, Fmoc arom.), 143.0 (2 Â C, Fmoc arom.), 155.6
o-Nbs arom.), 134.1 (CH, o-Nbs arom.), 134.3 (C, o-Nbs arom.), (C, CQO), 167.8 (C, CQO), 167.9 (C, CQO); (cis rotamer) d
147.4 (C, o-Nbs arom.), 168.7 (C, CQO), 171.2 (C, CQO); 19F NMR 19.1 (CH3, Cb Ala), 46.8 (CH, Fmoc CH), 48.5 (CH, Ca Ala), 53.7
(376.2 MHz, CDCl3, 323 K) (single rotamer): d À82.3 (d, J = 5.0 Hz, (CH3, OMe), 56.8 (CH, Ca CPro), 67.2 (CH2, Fmoc CH2), 70.4
CF3); MSMS (ES+) m/z = 456.15 [M + H]+, 478.14 [M + Na]+.
(CH2, Cb CPro), 84.5 (q, J = 34.0 Hz, CH, Cd CPro), 120.0
Representative procedure for the preparation of Fmoc- (2 Â CH, Fmoc arom.), 121.8 (q, J = 286.6 Hz, CF3), 125.0
alanine chloride assisted by ultrasonication27. To a 0.2 M (2 Â CH, Fmoc arom.), 127.6 (2 Â CH, Fmoc arom.), 127.7
solution of the Fmoc-alanine (1.0 equiv.) suspended in dichloro- (2 Â CH, Fmoc arom.), 140.6 (2 Â C, Fmoc arom.), 143.0 (2 Â C,
methane under argon was added freshly distilled SOCl2 Fmoc arom.), 155.7 (C, CQO), 167.2 (C, CQO), 167.9 (C, CQO);
(13.8 equiv.). The mixture was sonicated until the complete 19F NMR (376.2 MHz, CDCl3, 298 K): (trans rotamer) d À82.0
disappearance of the precipitate (usually 30 min), then solvent (s, CF3); (cis rotamer) d À81.8 (s, CF3); MSMS (ES+) m/z = 493.27
and excess of SOCl2 were removed in vacuo to give the Fmoc- [M + H]+, 515.23 [M + Na]+, 531.23 [M + K]+; HRMS (EI) calcd for
alanine chloride as a white solid directly used in the next step
C24H23F3N2O6 492.1508, found: 492.1590. Minor diastereomer
without further purification.
4min: white solid; mp 64–66 1C; Rf = 0.40 (70 : 30 cyclohexane–
Fmoc-Ala-Ser(WCF ,HPro)-OMe (4). To a solution of pseudo- ethyl acetate); [a]D23 +42.8 (c 1.0, CHCl3); IR (neat): 3326, 2956,
3
proline (R,S)-1 (60 mg, 0.3 mmol, 1.0 equiv.) in dichloro- 1752, 1686, 1524, 1153, 742 cmÀ1; H NMR (400 MHz, CDCl3,
1
methane (300 mL) was added DIEA (50 mL, 0.3 mmol, 302 K): d 1.47 (d, J = 6.5 Hz, 3H, Hb Ala-H), 3.83 (s, 3H, OMe),
1.0 equiv.). The resulting mixture was added via cannula to 4.19 (t, J = 6.9 Hz, 1H, Fmoc CH); 4.32–4.42 (m, 3H, Ha Ala-H,
the freshly prepared Fmoc-alanine acid chloride solid (109 mg, Fmoc CH2), 4.46 (t, J = 8.2 Hz, 1H, Hb CPro-Ha), 4.52–4.60
0.33 mmol, 1.1 equiv.). The reaction mixture was stirred for (m, 1H, Hb CPro-Hb), 5.30 (d, J = 6.4 Hz, 1H, NH Ala), 5.47
24 h, diluted with dichloromethane, and washed with 1 M (dd, J = 7.6, 4.4 Hz, 1H, Ha CPro-H), 5.98 (q, J = 5.1 Hz, 1H,
aqueous solution of HCl. The organic layer was dried over Hd CPro-H), 7.31 (t, J = 7.5 Hz, 2H, Fmoc arom.), 7.41 (t, J =
MgSO4, filtered, and evaporated under reduced pressure. The 7.5 Hz, 2H, Fmoc arom.), 7.56 (t, J = 7.5 Hz, 2H, Fmoc arom.),
crude 89 : 11 mixture of diastereomers was purified by flash 7.79 (d, J = 7.5 Hz, 2H, Fmoc arom.); 13C NMR (100.5 MHz,
chromatography (80 : 20 cyclohexane–ethyl acetate) to give CDCl3, 323 K) d 18.0 (CH3, Cb Ala), 47.2 (CH, Fmoc CH), 48.6
26 mg (18%) of pure minor diastereomer 4min, 44 mg (30%) (CH, Ca Ala), 53.0 (CH3, OMe), 57.8 (CH, Ca CPro), 67.3
of a mixture of both diastereomers and 62 mg (42%) of pure (CH2, Fmoc CH2), 70.3 (CH2, Cb CPro), 84.2 (q, J = 35.5 Hz,
major diastereomer (R,S)-4 as a 12/88 inseparable mixture of CH, Cd CPro), 120.0 (2 Â CH, Fmoc arom.), 122.6 (q, J =
cis–trans rotational isomers in CDCl3 at 274 K. (R,S)-4 major 285.6 Hz, CF3), 124.9 (2 Â CH, Fmoc arom.), 127.0 (2 Â CH,
diastereomer: white solid; mp 56–60 1C; Rf = 0.29 (70 : 30 Fmoc arom.), 127.8 (2 Â CH, Fmoc arom.), 141.3 (2 Â C, Fmoc
cyclohexane–ethyl acetate); [a]2D4 À81.2 (c 0.95, CHCl3); IR (neat) arom.), 143.6 (2 Â C, Fmoc arom.), 156.3 (C, CQO), 169.2
3324, 3015, 2955, 1743, 1684, 1523, 1154, 757, 743 cmÀ1
;
(C, CQO), 173.8 (C, CQO); 19F NMR (376.2 MHz, CDCl3,
1H NMR (500 MHz, CDCl3, 274 K): (trans rotamer) d 1.45 (d, 298 K): d À82.6 (s, CF3); MSMS (ES+) m/z = 493.26 [M + H]+,
J = 6.7 Hz, 3H, Hb Ala-H), 3.76 (s, 3H, OMe), 4.17 (t, J = 7.3 Hz, 515.22 [M + Na]+, 531.22 [M + K]+.
1H, Fmoc CH); 4.30 (dd, J = 10.5, 7.3 Hz, 1H, Fmoc CH2-Ha),
Fmoc-Ala-Ser(WCF ,HPro)-NHMe (5). To a solution of pseudo-
3
4.31–4.35 (m, 1H, Hb CPro-Ha), 4.31–4.37 (m, 1H, Ha Ala-H), proline (R,S)-2 (110 mg, 0.55 mmol, 1.0 equiv.) in dichloro-
4.42 (dd, J = 10.5, 7.1 Hz, 1H, Fmoc CH2-Hb), 4.47 (t, J = 8.9 Hz, methane (1 mL) was added DIEA (91 mL, 0.55 mmol, 1.0 equiv.).
1H, Hb CPro-Hb), 5.04 (t, J = 8.2 Hz, 1H, Ha CPro-H), 5.48 The resulting mixture was added via cannula to the freshly
(d, J = 7.6 Hz, 1H, NH Ala), 6.28 (q, J = 4.8 Hz, 1H, Hd CPro-H), prepared Fmoc-alanine chloride solid (0.61 mmol, 1.1 equiv.).
7.31 (t, J = 7.3 Hz, 2H, Fmoc arom.), 7.40 (t, J = 7.5 Hz, 2H, Fmoc The reaction mixture was stirred for 24 h, diluted with dichloro-
arom.), 7.55 (t, J = 7.4 Hz, 2H, Fmoc arom.), 7.76 (d, J = 7.5 Hz, methane, and washed successively with 1 M aqueous solution
2H, Fmoc arom.); (cis rotamer) d 1.38 (d, J = 6.4 Hz, 3H, Hb Ala- of HCl and brine. The organic layer was dried over MgSO4,
H), 3.76 (s, 3H, OMe), 4.20 (t, J = 7.1 Hz, 1H, Fmoc CH); filtered, and evaporated under reduced pressure. Purification
4.30–4.36 (m, 1H, Fmoc CH2-Ha), 4.38–4.44 (m, 1H, Fmoc by flash chromatography (40 : 60 cyclohexane–ethyl acetate)
CH2-Hb), 4.52–4.56 (m, 1H, Hb CPro-Ha), 4.58–4.61 (m, 1H, gave 102 mg (37%) of a mixture of both diastereomers and
Hb CPro-Hb), 4.58–4.66 (m, 1H, Ha Ala-H), 4.71–4.78 (m, 1H, 77 mg (28%) of pure major diastereomer (R,S)-5 as a 58/42
Ha CPro-H), 5.69 (d, J = 7.8 Hz, 1H, NH Ala), 5.88 (q, J = 4.3 Hz, inseparable mixture of cis–trans rotational isomers in CDCl3
1H, Hd CPro-H), 7.33 (t, J = 7.3 Hz, 2H, Fmoc arom.), 7.43 at 298 K. (R,S)-5 major diastereomer: white solid; Rf = 0.34
c
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(40 : 60 cyclohexane–ethyl acetate); [a]2D4 À60.5 (c 1.1, CHCl3); IR (s, 3H, CH3 Ac), 2.84 (d, J = 4.6 Hz, 3H, CH3 NHMe), 4.43 (t, J =
(neat) 3322, 3066, 2947, 1667, 1530, 1252, 1152, 731 cmÀ1
;
8.5 Hz, 1H, Hb CPro-Ha), 4.47–4.51 (m, 1H, Ha Ala-H), 4.72 (t, J =
1H NMR (500 MHz, CDCl3, 274 K): (trans rotamer) d 1.43 8.5 Hz, 1H, Hb CPro-Hb), 4.85 (t, J = 8.5 Hz, 1H, Ha CPro-H),
(d, J = 6.3 Hz, 3H, Hb Ala-H), 2.83 (d, J = 4.7 Hz, 3H, NHMe), 6.23 (d, J = 6.0 Hz, 1H, NH Ala), 6.47 (q, J = 5.3 Hz, 1H, Hd CPro-
4.20 (t, J = 6.6 Hz, 1H, Fmoc CH), 4.39 (m, 1H, Ha Ala-H), 4.40 H), 6.51–6.55 (m, 1H, NHMe); (cis rotamer) d 1.41 (d, J = 7.0 Hz,
(m, 3H, Fmoc CH2 and Hb CPro-Ha), 4.75 (t, J = 7.6 Hz, 1H, Hb 3H, Hb Ala-H), 2.04 (s, 3H, CH3 Ac), 2.89 (d, J = 4.6 Hz, 3H, CH3
CPro-Hb), 4.85 (t, J = 7.6 Hz, 1H, Ha CPro-H), 5.37 (d, J = 6.9 Hz, NHMe), 4.31 (quint, J = 6.3 Hz, 1H, Ha Ala-H), 4.54 (t, J = 8.3 Hz,
1H, NH Ala), 6.27–6.30 (m, 1H, Hd CPro-H), 6.52–6.56 (m, 1H, 1H, Hb CPro-Ha), 4.59 (dd, J = 8.3, 4.3 Hz, 1H, Ha CPro-H), 4.69
NHMe), 7.32 (t, J = 7.3 Hz, 2H, Fmoc arom.), 7.42 (t, J = 7.0 Hz, (dd, J = 8.3, 4.3 Hz, 1H, Hb CPro-Hb), 5.96 (q, J = 5.1 Hz, 1H, Hd
2H, Fmoc arom.), 7.61 (d, J = 7.8 Hz, 2H, Fmoc arom.), 7.77 CPro-H), 6.63 (d, J = 4.9 Hz, 1H, NH Ala), 8.38 (q, J = 4.6 Hz, 1H,
(d, J = 7.3 Hz, 2H, Fmoc arom.); (cis rotamer) d 1.39 (d, J = NHMe); 13C NMR (125.75 MHz, CDCl3, 274 K): (trans rotamer)
6.9 Hz, 3H, Hb Ala-H), 2.83 (d, J = 4.7 Hz, 3H, NHMe), 4.20 (t, J = d 17.5 (CH3, Cb Ala), 22.5 (CH3, Ac), 26.6 (CH3, NHMe), 47.5
6.6 Hz, 2H, Fmoc CH and Ha Ala-H), 4.40 (m, 2H, Fmoc CH2), (CH, Ca Ala), 57.8 (CH, Ca CPro), 68.8 (CH2, Cb CPro), 84.8
4.54–4.63 (m, 3H, Hb CPro-H, Ha CPro-H), 5.43 (d, J = 6.1 Hz, (q, J = 38.5 Hz, CH, Cd CPro), 122.5 (q, J = 284.5 Hz, CF3), 168.3
1H, NH Ala), 5.96 (q, J = 4.9 Hz, 1H, Hd CPro-H), 7.32 (t, J = (C, CQO), 172.1 (C, CQO), 173.2 (C, CQO); (cis rotamer) d 15.7
7.3 Hz, 2H, Fmoc arom.), 7.42 (t, J = 7.0 Hz, 2H, Fmoc arom.), (CH3, Cb Ala), 22.3 (CH3, Ac), 26.7 (CH3, NHMe), 48.7 (CH, Ca
7.57 (d, J = 7.5 Hz, 2H, Fmoc arom.), 7.77 (d, J = 7.3 Hz, 2H, Ala), 58.6 (CH, Ca CPro), 71.5 (CH2, Cb CPro), 84.9 (q, J =
Fmoc arom.), 7.93–7.97 (m, 1H, NHMe); 13C NMR (125.75 MHz, 38.5 Hz, CH, Cd CPro), 122.6 (q, J = 284.5 Hz, CF3), 168.3
CDCl3): (trans rotamer) d 18.0 (CH3, Cb Ala), 26.6 (CH3, NHMe), (C, CQO), 171.0 (C, CQO), 175.1 (C, CQO); 19F NMR
46.8 (CH, Fmoc CH), 48.5 (CH, Ca Ala), 57.7 (CH, Ca CPro), 67.4 (376.2 MHz, CDCl3, 298 K): (single rotamer) d À81.9 (s, CF3);
(CH2, Fmoc CH2), 68.7 (CH2, Cb CPro), 85.0 (q, J = 32.6 Hz, CH, MSMS (ES+) m/z = 312.1 [M + H]+, 334.1 [M + Na]+; HRMS (ES+)
Cd CPro), 120.2 (2 Â CH, Fmoc arom.), 122.4 (q, J = 285.9 Hz, calcd for C11H17F3N3O4: 312.1171, found 312.1172.
CF3), 125.1 (2 Â CH, Fmoc arom.), 127.2 (2 Â CH, Fmoc arom.),
Structure calculations
127.9 (2 Â CH, Fmoc arom.), 141.3 (2 Â C, Fmoc arom.), 143.0
(2 Â C, Fmoc arom.), 156.3 (C, CQO), 168.2 (C, CQO), 175.1
Simulated annealing (SA) calculations were carried out with the
program DYNAMO 2.128 and consisted of three stages. The first
(C, CQO); (cis rotamer) d 16.1 (CH3, Cb Ala), 26.6 (CH3, NHMe),
46.8 (CH, Fmoc CH), 49.4 (CH, Ca Ala), 58.5 (CH, Ca CPro), 67.4
(CH2, Fmoc CH2), 71.6 (CH, Cb CPro), 84.8 (q, J = 36.7 Hz, CH,
Cd CPro), 120.2 (2 Â CH, Fmoc arom.), 122.4 (q, J = 285.9 Hz,
CF3), 125.1 (2 Â CH, Fmoc arom.), 127.2 (2 Â CH, Fmoc arom.),
127.9 (2 Â CH, Fmoc arom.), 141.3 (2 Â C, Fmoc arom.), 143.0
(2 Â C, Fmoc arom.), 156.8 (C, CQO), 168.2 (C, CQO), 173.2
(C, CQO); 19F NMR (376.2 MHz, CDCl3, 298 K): (trans rotamer)
d À82.0 (s, CF3); (cis rotamer) d À81.8 (s, CF3); MSMS (ES+)
m/z = 492.2 [M + H]+, 514.2 [M + Na]+; HRMS (ES+) calcd for
stage comprised an initialization period of 1000 steps (3 fs
long) of molecular dynamics at 4000 K and very tight tempera-
ture control. In the second stage, the coordinates were allowed
to vary at 4000 K with loose temperature control for 4000 steps.
In the final stage, the structure was annealed by slowly reducing
the temperature from 4000 K to 0 K over the space of 20 000
molecular dynamic steps (3 fs long). In the first two stagÀes2,
force constants were set as follows: 1000 kcal molÀ1
Å ,
250 kcal molÀ1 radÀ2, 50 kcal molÀ1 radÀ2, 2 kcal molÀ1 ÅÀ2
for bonds, angles, impropers and NOE constraints, respectively.
No van der Waals interactions were operative. During the final
C
24H25F3N3O5: 492.1746, found 492.1755.
Ac-Ala-Ser(WCF ,HPro)-NHMe (6). To a solution of the Fmoc-
3
stage, bonds force constant was maintained at 1000 kcal molÀ1 ÅÀ2
,
dipeptide (R,S)-5 (300 mg, 0.61 mmol, 1.0 equiv.) in dichlor-
omethane (12 mL) under argon was added dropwise piperidine
(600 mL, 6.10 mmol, 10.0 equiv.) at room temperature. The
reaction mixture was stirred until it was completed as mon-
itored by TLC (approximately 80 min), then solvent was
removed in vacuo. Purification by flash chromatography
(90 : 10 ethyl acetate–methanol) gave 164 mg (98%) of pure
deprotected dipeptide which was then diluted in dichloro-
methane (18 mL). After the addition of acetic anhydride
(62 mL, 0.66 mmol, 1.1 equiv.), the reaction mixture was stirred
for 1 h then solvent was removed under reduced pressure. The
solid residue was washed with diethyl ether, diluted in dichlor-
omethane, filtered and evaporated under reduced pressure to
afford 184 mg (97%) of pure tetrapeptide mimic 6 as a 70/30
inseparable mixture of cis–trans rotational isomers in CDCl3 at
274 K. (R,S)-6: white solid; mp 145–155 1C; Rf = 0.51 (90 : 10 ethyl
both angles and impropers at 500 kcal molÀ1 radÀ2, the NOE
term was increased exponentially from 2 to 30 kcal molÀ1 ÅÀ2
.
The van der Waals interactions and J-coupling restraints were
slowly introduced during cooling to reach the final values of
4 kcal molÀ1 ÅÀ2 and 1 kcal molÀ1 radÀ2, respectively. NMR
constraints consisted of 3 1H–1H vicinal J-coupling constants
and 11 NOE (see ESI†). For each oligomer 100 structures have
been calculated starting from an extended fold. For the 5
lowest energy structures, typical final energies (kcal molÀ1) were
0.39 Æ 0.04; 1.13 Æ 0.07; 0.12 Æ 0.01; 2.77 Æ 0.02; 6.79 Æ 0.10;
and 1.31 Æ 0.05 for J-coupling, bond, impropers, angle, NOE and
van der Waals force constants, respectively.
Acknowledgements
acetate–methanol); [a]D26 À113.6 (c 1.0, CHCl3); IR (neat) 3390, Dr D. Feytens is a post-doctoral researcher of the Fund for
2989, 1652, 1544, 1151 cmÀ1; 1H NMR (500 MHz, CDCl3, 274 K): Scientific Research Flanders (FWO-Vlaanderen). T. Brigaud
(trans rotamer) d 1.44 (d, J = 7.1 Hz, 3H, Hb Ala-H), 2.02 thanks Central Glass Company for financial support. G. Chaume
c
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Paper
12 D. Feytens, G. Chaume, G. Chassaing, S. Lavielle,
T. Brigaud, B. J. Byun, Y. K. Kang and E. Miclet, J. Phys.
Chem. B, 2012, 116, 4069.
thanks the Agence Nationale de la Recherche for financial
support (No. ANR-09-JCJC-0060-01). E. Miclet thanks the Agence
Nationale de la Recherche for financial support (No. ANR-12-
JS08-0005-01). This work has benefited from the facilities and
expertise of the Small Molecule Mass Spectrometry platform of
IMAGIF (Centre de Recherche de Gif – www.imagif.cnrs.fr).
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16 G. Guichard, A. Violette, G. Chassaing and E. Miclet, Magn.
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c
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