γ-(S)-Trifluoromethyl proline: Evaluation as a structural substitute of proline for solid state 19F-NMR peptide studies

Article abstract of DOI:10.1039/c5ob00034c

γ-(4S)-Trifluoromethyl proline was synthesised according to a modified literature protocol with improved yield on a multigram scale. Conformational properties of the amide bond formed by the amino acid were characterised using N-acetyl methyl ester model. The amide populations (s-trans vs. s-cis) and thermodynamic parameters of the isomerization were found to be similar to the corresponding values for intact proline. Therefore, the γ-trifluoromethyl proline was suggested as a structurally low-disturbing proline substitution in peptides for their structural studies by ¹⁹F-NMR. Indeed, the exchange of native proline for γ-trifluoromethyl proline in the peptide antibiotic gramicidin S was shown to preserve the overall amphipathic peptide structure. The utility of the amino acid as a selective ¹⁹F-NMR label was demonstrated by observing the re-alignment of the labelled gramicidin S in oriented lipid bilayers. This journal is

Full text of DOI:10.1039/c5ob00034c

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1
9
γ-(S)-Trifluoromethyl proline: Evaluation as a structural substitute of proline for solid state F-
NMR peptide studies
a,b
c
a
b
d,e*
Vladimir Kubyshkin,
Anne S. Ulrich
Sergii Afonin, Sezgin Kara, Nediljko Budisa, Pavel K. Mykhailiuk
and
a,c*
a
Institute of Organic Chemistry, Karlsruhe Institute of Technology (KIT), Fritz-Haber-Weg 6, 76131
Karlsruhe, Germany
b
Institute of Chemistry, Technical University of Berlin, Müller-Breslau-Str. 10, 10623 Berlin, Germany
Institute of Biological Interfaces (IBG-2), KIT, POB 3640, 76021 Karlsruhe, Germany
Faculty of Chemistry, Taras Shevchenko National University of Kyiv, vul. Volodymyrska 62a, 01601
c
d
Kyiv, Ukraine
e
Enamine Ltd., vul. Matrosova 23, 01013 Kyiv, Ukraine
*
Corresponding Authors: pavel.mykhailiuk@gmail.com; pavel.mykhailiuk@mail.enamine.net (PKM),
anne.ulrich@kit.edu (ASU).
Abstract
γ-(4S)-Trifluoromethyl proline was synthesised according to a modified literature protocol with
improved yield on a multigram scale. Сonformational properties of the amide bond formed by the
amino acid were characterised using N-acetyl methyl ester model. The amide populations (s-
trans vs. s-cis) and thermodynamic parameters of the isomerization were found to be similar to
the corresponding values for intact proline. Therefore, the γ-trifluoromethyl proline was
suggested as a structurally low-disturbing proline substitution in peptides for their structural
1
9
studies by F-NMR. Indeed, the exchange of native proline for γ-trifluoromethyl proline in the
peptide antibiotic gramicidin S was shown to preserve the overall amphipathic peptide structure.
1
9
The utility of the amino acid as a selective F-NMR label was demonstrated by observing the re-
alignment of the labelled gramicidin S in oriented lipid bilayers.
Introduction
Proline (Pro) is the only amino acid with a secondary amine function among the canonical
α-amino acids. Locally, in the polypeptide backbone Pro forms unique tertiary X-Pro amide bonds
1
which endows a stable s-cis conformation (Fig. 1A). The Pro residue is conformationally restricted
by the side chain to backbone cyclization, and is therefore the only canonical amino acid with the
rigidly constrained phi angle. Due to these features, prolyl residues often play determining roles in
2
the definition of the three-dimensional structures of proteins and limit the folding kinetics.
Consequently, proline-rich regions in proteins are predominantly located within the solvent-
exposed sections with enigmatic secondary structures such as intra-domain connections, loops,
intrinsically disordered segments. In addition, prolines are ample in the folded domains which
3
constitute the inter-molecular interfaces of the protein-protein and protein-drug recognition sites.
The absence of NH not only precludes participation in the secondary structure-stabilizing
hydrogen bonds, but also makes Pro sites invisible for many routine NMR methods of the polypeptide
structure analysis. Yet, the NMR visibility of Pro can be restored by selective isotope labelling. The
2
13
labelling per se serves enhancing the NMR sensitivity, where among conventional isotope labels, H, C,
15
19
4
19
N, and F, the latter is by far the most effective nucleus. However, introduction of the F isotope, an
unnatural element in the proteinogenic amino acids, requires chemical modification of the side chain, i.e. a
mutation. While for large proteins a single mutation does not necessarily interfere with the native fold, for
19
small peptides such a modification may affect significant percentage of the sequence. For F-NMR
labelling which aims determination of the structure of small peptides, in particular employing anisotropic

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NMR parameters (solid state NMR), the introduction of a fluorine-containing motif is further complicated. In
an ideal label, not only should the physico-chemical nature of the side chain be preserved, but
simultaneously a rigid molecular connectivity between the NMR-reporter group and the aminocaboxylate
5
,6
19
must be introduced.
This challenge is solved in F-NMR by designing the residue type-specific
substitutive amino acids. These have to be close steric surrogates, which possess conformationally rigid
7
,8
9
side chains alongside with the NMR-reporter group - a single fluorine atom, a CF
group
2
-, or the CF
3
-
10,11
(Fig. 1B). In the case of the intrinsically constricted Pro side chain, design of such analogues is
particularly difficult, because the residue steric size always increases, the side chain conformation, unique
amide bond isomerization equilibrium and consequently the preferred near-proline backbone
12,13
conformational angles are strongly influenced by the substituents in the pyrrolidine ring.
Fig. 1. (A) Structure of proline (Pro) and the trans-cis equilibrium in a peptidyl-prolyl fragment; (B) Attachment of a conformationally
19
constrained F-NMR reporter to the polypeptide backbone; (C-F) Known fluorine-substituted α-amino acids which could be
1
9
considered as Pro-replacing F-NMR labels: (C) Monofluorine-substituted γ- and β-fluoroprolines; (D) Perfluoro-tert-butyl-γ-
1
9
22
hydroxyprolines proposed as
thiopseudoprolines; (F) Methanoproline-based difluoro-trans-γ,δ-methanoproline and CF
TfmPro) explored in this study.
a
F-NMR label;
(E) Carriers of the CF
3
-group: α-CF
3
-proline and δ-CF
3
-pseudo and
-(S)-proline
3
-γ,δ-methanoproline; (G) γ-CF
3
(
1
9
There are several potential candidates worth considering for the proline substitutive F-
1
4
15,16,17,18
labelling in polypeptides. For instance, β- and γ-mono-fluoroprolines,
(Fig. 1C) that are
among most explored proline modifications. The utility of these amino acids is toughened by their
1
9,20
compatibility with the natural ribosomal protein expression machinery.
However, mono-
fluoroprolines are particularly known for the over-stabilization of their preferred ring puckers and
both significantly deviate in their trans-cis amide bond equilibria from Pro.
Another beneficial possibility is a straight incorporation of the CF
3
-group. The latter carries
1
9
three times more F spins per residue and allows utilization of the strong homonuclear F-F dipolar
1
9
coupling. In the solid state F-NMR of peptides, the F-F coupling is a structural restraint which is
6
,7,10
superior to the chemical shift anisotropy.
However, the trifluoromethylation not only impose
significant steric demand and potentially modulate electronic configuration of the pyrrolidine ring,
2
1
but is also known to render template structures becoming more lipophilic, which directly affects
function of the membrane-active peptides, for instance. The steric demand and the increase in

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lipophilicity will be very large when multiple CF -groups are present, e.g. like in the perfluoro-tert-
3
19
22
butyl-γ-hydroxyproline (Fig. 1D) as was recently proposed for F-NMR labelling. Furthermore, in the
solid state a stongly coupled homonuclear 9-spin system will produce NMR spectra with a very complex
multiplicity, which might obscure the data analysis and cancel the initial sensitivity advantage. Therefore
19
an isolated CF -group should be preferred in the solid state F-NMR studies.
3
Synthetically, a sole CF -group can be introduced into proline or the pseudoproline
3
2
3
template in different positions. In particular, it has been described that α-CF proline and δ-CF3
oxazolidine-based pseudoprolines (Fig. 1E) could be incorporated in peptides. Still, in both Pro
3
2
4
surrogates with the CF -substituent adjoining the aminocarboxylate, conformational freedom within
3
the adjacent backbone swerves the parent Pro structure. Besides, incorporation of these
analogues into a peptide chain is still a challenge as the amino acid reactivity in the peptide
2
5
synthesis is predictably lowered. Poor compatibility with the standard solid-phase peptide
synthesis (SPPS) and sophisticated synthesis are the major utilization problem also for the
2
6
11c
recently described difluoro- and trifluoromethyl-γ,δ-methanoprolines
(Fig. 1F). In contrast,
3
-group in the most remote
simple γ-CF -prolines (Fig. 1G) are chemically stable and carry the CF
3
position relative to both amino acid functionalities and correspondingly from the future polypeptide
backbone. Therefore they may be the most balanced candidates for an intact proline substitution.
The conformational impact of a large number of γ-substituents has been addressed within ample
mutagenesis investigations. Curiously, such studies for a single CF -group in the γ-position have been not
3
described so far. We fill this knowledge gap and determine herein conformational and thermodynamic
parameters of the γ-(S)-CF -proline (TfmPro) amide bond in a model compound. Judging by these
3
parameters, we demonstrate TfmPro being close to proline, and further test the amino acid utility as a
19
structural NMR label, in particular in the solid state F-NMR study of the membrane-active peptide
gramicidin S.
Results and Discussion
Synthesis of TfmPro
The synthesis of TfmPro was described in several publications about a decade
2
7,28,29,30
ago.
opinion) synthesis of the N-Boc derivative
hydroxyproline
Instead of using the benzyl ester which was obtained in the original study in a poor 45 %
yield we employed methyl-ester protection and got the protected hydroxyproline in 96 % yield
Scheme 1). In the following, hydroxyl group was converted to the keto-function (leading tot ) with
6 % yield (7 gram) using chromium (VI) oxide – pyridine (Collins reagent). However, for the
to 90
-group and subsequent water abstraction worked well using
the original protocols. This way we obtained in a 94% yield. The compound heated with thionyl
chloride in pyridine for 20 min under reflux gave 20 gram of the alkene in a satisfactory 60 %
yield. The second stereocenter was conventionally generated by hydrogenation of the double
Among these approaches we choose the simplest and the most economic (to our
2
9
1
reported by Qui&Qing,
which starts from
2
.
3
(
7
4
scaled up synthesis we altered to the Parikh-Doering oxidation and increased the yield of
(50 gram). Installation of the CF
4
%
3
5
5
6
bond. Resulting TfmPro derivative
deprotected by saponification giving
7
1
was obtained in 94 % yield. The carboxyl-group was then
(86 % yield, 9 gram). The overall yield of was therefore 39
1
%
, almost twice higher than the 21 % reported by Qui&Qing.
For the following conformational study the model compound Ac-TfmPro-OMe (
prepared (vide infra). In addition, the Boc-protection in was exchanged to Fmoc (
quantitative yield. The Fmoc amino acid was later used for the SPPS.
8
) was
1
9) with
19
NMR spectra, in particular F-NMR of compounds 1, 7, 8, 9 were consistent with only one
diastereomeric form of TfmPro in our synthesis. Furthermore, the [α] value of -77º determined for 1 was
D

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in full agreement with the -77.6° reported by Qui&Qing. We therefore found that in our modification the
induction of the second stereo center under hydrogenation of 6 yields TfmPro of the correct cis relative
stereochemistry despite the induction step was not coupled with the Bn-ester hydrogenolysis as described
in the initial report.
Scheme 1. The synthesis of 4-(S)-trifluoromethylproline, as N-Boc (
conformational analysis, and the gramicidin S analogues used in solid state F-NMR. The X-ray crystal structure of
carbon – blue, oxygen – red, fluorine – green, nitrogen – purple).
1), its derivative Ac-TfmPro-OMe (8) used for the
1
9
8
(
We also obtained crystals of 8 which were then taken for X-ray analysis. To our surprise, the
substance crystallized as racemic mixture in the orthorhombic cell containing four (2S,4S) and four
2R,4R) enantiomeric molecules. The main part of the substance 8 remained liquid and did not crystallize.
(
The fact that the substance was diastereomerically pure strongly indicates that the partial epimerization of
the proline α-(S)-chirality indeed occurred before the induction of the second stereo center. We presume
that partial epimerization happened at the water elimination step (5 to 6). Finally by derivatisation with N-
19
(
2,4-dinitrophenyl)proline and subsequent F-NMR we determined the enantiomeric excess of 1 being 92
%
.

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Conformation analysis of the Ac-TfmPro-OMe model
For detailed conformational characterization of TfmPro in a quasi-peptide context, we
studied the properties of its tertiary amide bond and the pucker in a small derivative. We selected
Ac-TfmPro-OMe model as a well characterised framework in which many Pro analogues were
1
,15
studied.
It is generally known that proline prefers s-trans amide rotamer, and the amide bond trans-
cis interconversion could be conveniently characterized by the Ktrans/cis constant or by
corresponding free energy difference. For instance, for Ac-Pro-OMe model in aqueous solution,
the ∆G300 K was reported being -3.90 kJ/mol, which corresponds to the “native” Pro Ktrans/cis of
1
9
4
.8. It is also known that substituents in the γ-position influence this ratio mainly through
1
2
stabilization of particular pyrrolidine ring γ-puckers. Comparative studies suggest that generally,
an electron withdrawing substituent in the position γ-(S) shifts the equilibrium towards the s-cis
configuration; whereas an electron-donating group alleviates the s-trans rotameric form due to the
gauche-effect. At the same time, a sterically demanding moiety in the γ-(S) configuration should
3
1
also lead to the s-trans stabilization. Thus, the electronegative and sterically demanding
trifluoromethyl group might exhibit two counteracting effects: an influence on the pyrrolidine ring
conformation and on the amide bond configuration in TfmPro. Should these effects be mutually
compensated, TfmPro residue may be rendered as an almost intact proline analogue, i.e. to
possess backbone conformational propensities very close to the ones in Pro.
The electronegative substituent effect was already seen in the pK value for TfmPro which
a
3
2
we determined to be 8.5±0.1 close to that of γ-(S)-fluoroproline (9.2 ). In order to test the
conformational impact, we performed first the van’t Hoff type of analysis for in aqueous medium
8
1
9
employing F-NMR, which experimentally gives more accurate results than could be obtained
from the crowded H-NMR spectra. The chemical shift difference between the two amide bond
rotamers in the F spectra was 0.35 ppm at 25°C and changed to 0.22 ppm at 90°C. In
1
1
9
agreement with our hypothesis, we found (Tab. 1) the equilibrium constant Ktrans/cis for Ac-TfmPro-
OMe being equal 4.0. The γ-(S)-CF -group thus only marginally shifts the overall equilibrium
3
towards the cis-amide bond in the contrast to the large perturbation a single fluorine atom causes
at the same ring position (Ktrans/cis = 2.5 for γ-(S)-fluoroproline). In addition, we have determined
kinetic parameters of the isomerisation process. In particular, the activation energy of the cis-to-
trans rotation process (81.9 kJ/mol) was slightly lower but overall similar to that of proline (84.5
kJ/mol). This effect is potentially related to the higher acidity of the imonium function likewise in
1
9
γ,γ-difluoroproline, where corresponding activation barrier value was even lower, 80.8 kJ/mol.
We also observed cis-trans and trans-cis isomerization processes to be both driven enthalpically
(
amide conjugation), the same way as this is known for proline.
1
The 1D H-NMR spectra of Ac-TfmPro-OMe have also been analysed to assess the
preferred conformation of the pyrrolidine ring. Since in our model, the α-CH couples to both
protons of the adjacent β-CH -group, two characteristic patterns are expected. For the exo-
puckers the two Jαβ couplings should be in the range of 7-11 Hz, whereas for the endo-
2
3
3
3
configuration one coupling should be in the range of 6-10 and the other of 2-3 Hz. For the major
3
s-trans isomer of Ac-TfmPro-OMe we observed two equal Jαβ values of 8.3 Hz, thus confirming its
preference for the exo-pucker. Remarkably, the same α-CH triplet shape for the major s-trans
amide rotamer has been reported for s-trans rotamer of glycosylated γ-(S)-hydroxyproline which
3
4
exhibits the exo-pucker. The splitting of 8.3 Hz also persisted in water, chloroform and DMSO
solutions of . The exo-pucker configuration for the s-trans was also confirmed in the solid state
8
3
(
X-ray structure, Scheme 1). In contrast, the minor s-cis form exhibited two distinct J values of
αβ
3
.8 and 9.8 Hz (in water), having therefore no qualitative pucker preference. Following the same
NMR criteria, proline itself should be classified as preferring an endo-pucker in the s-cis form and
3
5
a mixed pucker in the s-trans form.

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Table 1. The thermodynamic parameters, conformational equilibrium and the tertiary amide bond rotational rates determined in Ac-
TfmPro-OMe (8), compared to proline and γ-fluoroprolines (Flp).
equilibrium
Ac-TfmPro-OMe
Ac-Pro-OMe
Ac-(R)-Flp-OMe
Ac-(S)-Flp-OMe
cis-to-trans
K
trans/cis 300K
∆H, kJ/mol
-4.86 ± 0.11
-5.04 ± 0.05
-7.73 ± 0.26
-3.04 ± 0.03
∆S, J/mol·K
-4.72 ± 0.33
-3.82 ± 0.16
-9.81 ± 0.81
-2.47 ± 0.11
∆G300K, kJ/mol
-3.44 ± 0.21
-3.90 ± 0.10
-4.78 ± 0.50
-2.30 ± 0.07
4.0
4.8
6.8
1
9
1
9
1
9
2.5
-
1
≠
≠
k
300K, s
0.034
0.012
0.026
0.015
300K, s
0.009
0.003
0.004
0.006
∆H , kJ/mol
∆S , J/mol·K
E
a 300K, kJ/mol
81.8 ± 0.6
84.5
Ac-TfmPro-OMe
Ac-Pro-OMe
Ac-(R)-Flp-OMe
Ac-(S)-Flp-OMe
trans-to-cis
78.3 ± 0.3
87.2
84.2
-11.9 ± 1.0
9.00
1
9
1
9
5.37
82.6
84.0
1
9
84.7
2.42
-
1
≠
≠
k
∆H , kJ/mol
∆S , J/mol·K
E
a 300K, kJ/mol
85.2 ± 1.0
88.4
Ac-TfmPro-OMe
Ac-Pro-OMe
Ac-(R)-Flp-OMe
Ac-(S)-Flp-OMe
81.4 ± 0.5
92.3
91.8
-12.6 ± 1.6
12.8
1
9
1
9
14.8
4.17
87.4
86.3
1
9
87.5
TfmPro represents therefore a proline substitute which in summary has the pucker favouring the
exo-conformer, whereas its amide bond thermodynamic preference is slightly shifted towards s-cis. From
the structural labelling perspective, despite differences in the pucker preferences, the two amino acids can
be considered mutual analogues since the overall amide bond configuration in TfmPro is very close to the
one in proline.
TfmPro in gramicidin S
To observe TfmPro in a real peptide and to later evaluate the amino acid as an NMR label
for peptide studies we incorporated TfmPro in the cyclic antimicrobial peptide gramicidin S
D
(
cyclo[Pro-Val-Orn-Leu- Phe] , where Orn stands for ornithine; GS). The two possible peptides
2
1
TfmPro-GS (one TfmPro to Pro substitution, mono-substituted) and 2TfmPro-GS (two TfmPro to
Pro substitutions, doubly-substituted) were produced by conventional Fmoc-SPPS as reported
9
,36
earlier.
The peptides were synthesised in two steps: first a linear sequence was constructed on
the solid support, followed by the cyclisation in solution. Since the linear precursors have to be
pre-organized for the cyclisation, success of the reaction per se indirectly confirms the
compatibility of TfmPro with a turn conformation of the native Pro-containing segment. The
7
peptides were standardly purified using TFA-free RP-HPLC , performing which we observed an
expected stepwise increase in the retention times (compared against GS) as hydrophobicity rose
upon successive introduction the CF -groups (Fig. 2). In the view of GS being the membrane-
3
active peptide, this suggests a potential modulation of the peptide functional activity, but may not
necessarily be a sign of the structural deviation.
Fig. 2. RP-HPLC retention times of GS and its analogues containing TfmPro. The error bars represent deviations of the
values measured in triplicate.

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Since we address only structural aspect, in the following, we only focused on the studies of
potentially more perturbed doubly-substituted analogue 2TfmPro-GS.
As it is usually done in the label-validation studies to verify global conformational impact of a
substitute, the new analogue was compared against GS by circular dichroism spectroscopy (CD) in
membrane-mimicking environment (Fig. 3). Practically the same spectral shapes for both molecules
suggest convergence of the modified backbone conformation to the parent GS structure in a membrane-
mimicking environment. Formally, since we have demonstrated TfmPro possesses proline-like intrinsic
amide conformational preferences and not likely to influence overall geometry of the GS skeleton, we
19
6,11c
therefore could call TfmPro a reasonable F-label to substitute proline.
Fig. 3. Circular dichroism spectra of GS and its analogue 2TfmPro-GS in aqueous buffer–trifluoroethanol mixture (phosphate buffer
(
PB), 10 mM, pH 7.4; TFE - 30%) at 25 °C.
To validate the use of TfmPro in the solid state NMR structural studies of peptides, we used the
GS system, solid state NMR behaviour of which is comprehensively studied. In particular, we
benefited from the knowledge of the conditions in which the peptide changes its membrane-bound
1
9
state. Using conventional F-labels it was earlier shown that in the oriented lipid bilayers
molecules of GS change their alignment from the surfacially-bound basic state (“S-state”, Fig. 4A)
to an upright orientation (“I-state”), the latter being attributed to the formation of homooligomeric
9
,37
pores relevant to the biological activity.
We knew that the amount of molecules in the I-state increases: i) with the increase of the
concentration, (ii) is more pronounced at temperatures close to the gel-to-fluid phase transition
3
8
(
T ) of the bilayers and (iii) in the short-chain saturated glycerophospholipids. To satisfy these
m
conditions we prepared 2TfmPro-GS–containing oriented proteobilayers using 1,2-didodecanoyl-
sn-glycero-3-phosphocholine (DLPC) as a lipid matrix and 1/40 as a peptide/lipid ration. From
1
9
these samples a series of solid state F-NMR spectra were collected as a function of
temperature. The temperature range was chosen to include the T of DLPC. Notably, the gel-to-
fluid transition in DLPC is a complex process, characterised by conversions of several
m
3
9
polymorphic mesophases and is rather broad (spans between -2°C and +7°C). As can be seen
1
9
in the Fig. 4C, the oriented peptide in fluid lipids revealed single F-NMR signal (F-F dipolar
coupling of +2.3 kHz), which was no longer resolved in the more rigid gel-phase bilayers. Such
behaviour was expected and in both cases the peptide molecules should reside in the same
orientation (the S-state), but should have different mobilities. The re-orientation to the I-state was
indeed observed upon cooling at around Tm (the triplet with ca. -6.0 kHz splitting). Thus we
reproduced temperature-dependent re-alignment of GS and demonstrated a qualitative application

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of TfmPro to monitor this process, but also showed this process for the first time by using F-F
3
7,40
dipolar couplings, not the chemical shift.
Fig. 4. (A) Schematic representation of GS in its membrane-bound S-state viewed from the bilayer plane; definition of the membrane
4
1
normal (n), and rotational axis of C
2
symmetry in GS molecule. Backbone structure as determined by Xu et al. The side-chains are
taken as statistically most probable rotamers, protons are not shown for clarity, the prolines are highlighted in red. (B) Definition of
1
9
the angle Θ (pucker is arbitrarily selected) which can be determined from solid state F-NMR from the dipolar splitting F-F according
2
to the relation F-Fobs = F-Fmax·Smol·(3cos Θ-1)/2. (C) Temperature-dependent re-alignment of 2TfmPro-GS in DLPC as followed by
1
9
solid state F-NMR. The sample is oriented with the membrane normal parallel to the magnetic field, temperatures and characteristic
F-F values are indicated.
Based upon the above confirmation that 2TfmPro-GS has the same membrane alignment as GS,
we attempted to validate the membrane-bound structure of the molecule. Intrinsically symmetric GS
molecules in the S-state (Fig. 4A) should have both Pro residues identically oriented with respect to the
membrane normal. We indeed observed only one signal, thus confirmed the C
2
symmetry of the 2TfmPro-
GS in the lipid-bound situation. The angle Θ (Fig. 4B) between the F-reporter group (the vector which
connects the Cγ and the carbon of the CF3,) and the external magnetic field (B ) can be directly
determined from the value of the orientation-dependent F-F dipolar coupling. The observed F-F splitting
19
0
11
37
(
+2.3 kHz), considering the order parameter Smol of 0.34 and the maximum F-F of the rotating CF group
3
of 15.8 kHz, gives Θ of ~37°. When we docked the TfmPro crystal geometry (exo-pucker, s-trans) onto
41
NMR-determined backbone structure of GS and align the model in the S-state as it was done in the
3
7,38
earlier studies
, to our dissatisfaction, we obtain Θ of 45-50°. However, when we do the same using
42
one of the reported crystal GS structures, Θ assumes the range of 25-30°. Remarkably, our

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DOI: 10.1039/C5OB00034C
experimental value of ~37° lays in-between these two values, but this appears to be a pure coincidence as
clearly the major uncertainty in membrane-bound structure determination is the assumed backbone
conformation of GS.
1
15
Fig. 5. H N sofastHMQC spectra on the GS and its analogue 2TfmPro-GS in: (A) aqueous buffer-trifluoroethanol and (B) DMSO.
The ornithine δ-NH resonance is a folded signal.
The uncertainty in the peptide backbone urged us to re-evaluate the degree of conformational
perturbance TfmPro provides on the GS backbone. From CD analysis above we have concluded
2
TfmPro-GS does not interfere with the GS backbone in the rough approximation only. To have a more
detailed conformational insight we have additionally characterized the structure of 2TfmPro-GS by NMR.
19
The F-NMR solution spectra of the 2TfmPro-GS showed single resonances in DMSO, water-
trifluoroethanol and aqueous SDS micelles at -69.5, -71.4 and -70.8 ppm, respectively, confirming the
1
15
overall C symmetry in solution. Conformation of backbone amides was examined by H N single bond
2
correlation NMR in 30% trifluoroethanol (Fig. 5A), i.e. exactly at conditions of CD (Fig. 3). In addition we
43
inspected 2TfmPro-GS in DMSO environment (Fig. 5B), which is a better membrane mimic , but a
D
condition inaccessible to CD spectroscopy. In the first case the Phe NH signals of were affected only in
1
the H dimension implying a different solvent exposure, which may come from the deviations within the β-
turn atom arrangements. The Val NH, which is a part of the Val-TfmPro peptide bond, exhibited in contrast
1
5
a prominent shift in the N dimension which persisted in both solvents. This is a potential indication of a
local conformational effect of the γ-CF -substituent in the proline analogue. Alternatively, an electrophilic
3
influence of the CF -substituent to the proline carbonyl could cause such a shift. Nevertheless, the rest of
3
the signals remained in the modified peptide at same or close positions as they were in the wild type
peptide. NMR data therefore corroborates the CD spectra-based conclusion that the TfmPro is compatible
with the global secondary structure of GS and could affect only the local arrangement. The former finding
19
is critically important to conclude solid state F-NMR data consistency with conditions-dependent states
of the native peptide in membrane environment. Whereas the correspondence of the local atomic
arrangements is a technical prerequisite for correct conversion of the experimental anisotropic parameters
(
F-F dipolar couplings) into alignment angles for the wild type peptide molecule.
In the DMSO solution we found also, that the Pro ring conformation in GS (α-CH as d, J = 7.2 Hz)
was different to the TfmPro containing analogue (α-CH as dd, J = 9.8 and 4.5 Hz). In 2TfmPro-GS, the α-
CH multiplicity of the imino acid is no longer consistent with the preference for the exo-pucker seen in the
trans-amide of the Ac-TfmPro-OMe. This observation alludes additional reason to the uncertainties in the
2
TfmPro-GS structure analysis from the solid state NMR (vide supra). Besides, it demonstrates that the

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substituted prolines may not necessarily maintain their “free” conformations (Ac-TfmPro-OMe model) in
the restrained context of a structured polypeptide. Just as in the recent reports for γ-mono-fluoroprolines in
44
45
the thiredoxin fold and γ-hydroxyprolines in the cyclotide kalata B1 .
Conclusions
In summary we describe herein the properties of a peptide bond formed by the γ-(S)-CF3-
proline as an analogue of proline. The CF
minimal influence on both the trans-cis amide equilibrium ratio (slightly shifted towards cis) and the
rotational barrier (minimally reduced). The energy parameters of the γ-(S)-CF -proline amide were
described in details. In the β-turn of gramicidin S, the proline surrogate did not change the overall
3
-group in the γ-position of the pyrrolidine ring showed
3
1
9
3
peptide structure. Used as a solid state F-NMR label, γ-(S)-CF -proline confirmed the changes of
the peptide alignment in lipid membranes in a qualitative way. Straightforward synthesis, marginal
perturbation of the amide bond conformation, remote position from the peptide bond, and excellent
1
9
NMR properties make γ-(S)-CF -proline a good choice for a broad range of F-NMR proline
3
labelling studies in peptides and proteins.
Experimental part
The chemical shifts are given in δ scale according to the conventional internal deuterium
referencing. The standards are TMS (Me Si) and Freon-11 (CCl F).
4
3
Methyl esther N-Boc hydroxyproline (
to the literature protocol (19.0 g, 96 % yield). 4-ketoproline (
50 g) was dissolved in dichloromethane (700 ml), triethylamine (91 ml) was added and the
3
) was obtained from hydroxyproline (
2
, 10.5 g) according
4
6
4) was then prepared as following.
3
(
solution was cooled down in an ice bath under argon atmosphere. Suspension of pyridine·SO3
complex (63.5 g, 2 eq.) in DMSO (250 ml) was added portionwise within 8 min. The mixture was
stirred for 18 hours at ambient temperature. It was then poured in 1 kg of ice. Organic layer was
separated, washed with citric acid solution (10 %, 2 x 500 ml), hydrochloric acid (4 %, 1 x 500 ml),
sodium hydrogencarbonate (saturated, 1 x 500 ml), citric acid solution (10 %, 1 x 500 ml) and
sodium hydrogencarbonate (saturated, 1 x 500 ml), dried over sodium sulphate, filtered and
1
concentrated under reduced pressure.
NMR data was consistent with the literature.
Compound was prepared as following.
4
was obtained as yellowish oil (46.9 g, 90 % yield). H
4
7
5
4 (44.72 g, 184 mmol) was dissolved in THF (300 ml)
under argon atmosphere. The solution was cooled down in an ice bath. CF TMS (28.5 ml, 193
3
mmol) was added within 5 min followed by TBAF (1M in THF, 1 ml). The ice was allowed to melt
down, and the mixture was stirred at ambient temperature for 22 hours. Saturated ammonium
chloride (300 ml) was added, the mixture was stirred for 20 min, then TBAF solution (1M in THF,
1
90 ml) was added. The mixture was stirred for 1 hour. Organic layer was separated, and
aqueous layer was extracted with diethyl ether (2 x 150 ml). Combined organic fractions were
washed with water (1 x 300 ml), brine (1 x 300 ml), dried over sodium sulphate, filtered and
1
concentrated under reduced pressure.
5
(54.1 g, 94 %) was obtained as glassy oil. H-NMR
(
CDCl , 400 MHz), δ, ppm (two rotamers): 4.52 and 4.43 (two d, J = 4.5 Hz, 1H), 4.37 and 4.22
3
(
1
(
two br s, 1H), 3.77 and 3.75 (two s, 3H), 3.75-3.62 (m, 2H), 2.53 (m, 1H), 2.19 (t, 1H, J = 13 Hz),
1
9
.44 and 1.39 (two s, 9H). F-NMR (CDCl
).
3
, 376 MHz), δ, ppm (two rotamers 1:1): -81.1 and -81.2
two s, CF
3
2
-tert-butyl 2-methyl (2S)-trifluoromethyl-3-pyrrolin-1,2-dicarboxylate (6)
In a 2 l reactor (33.9 g, 108 mmol) and dry pyridine (1 l) were placed under argon. Thionyl
5
chloride (100 ml) was added and the mixture was heated and refluxed. The reflux was continued
for 20 min and then the mixture was allowed to cool down to ambient temperature. The mixture
was poured into iced water (0.5 kg). Aqueous layer was extracted with diethyl ether (4 x 300 ml)

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DOI: 10.1039/C5OB00034C
and ethyl acetate (1 x 1 l). Combined organic fractions were concentrated under reduced pressure
to have the volume of ~ 300 ml. Resulting organic solution was washed with hydrochloric acid (5
%
, 2 x 150 ml), sodium hydrogenecarbonate (saturated, 1 x 150 ml) and brine (1 x 150 ml), dried
over sodium sulphate and concentrated in vacuum. The black crude material was filtered through
a short silicagel (70 g) column in hexane – ethyl acetate 2:1 mixture. Final product (19.2 g, 60
) was obtained as yellowish oil.
6
%
1
H-NMR (CDCl
3
, 400 MHz), δ, ppm (two rotamers): 6.19 (td, J = 19 and 2 Hz, 1H, CH=C), 5.08
Me), 4.33 (m, 2H, N-CH ), 3.71 (two s, 3H, OCH ), 1.42 and 1.37 (two s,
). C-NMR (CDCl , 101 MHz), δ, ppm (two rotamers): 169.2 and 168.8 (two s,
Me), 153.4 and 152.8 (two s, N-C(=O)-O), 132.8 and 132.6 (two q, J = 36 Hz, C=CH), 128.3
and 128.1 (two q, J = 5 Hz, C=CH), 120.7 (two q, J = 270 Hz, CF ), 81.1 (two s, O-C(CH ), 66.5
and 66.3 (two s, CH-CO Me), 52.8 and 52.6 (two s, OCH ), 51.0 and 50.8 (two s, N-CH ), 28.3
(
9
two m, 1H, N-CH-CO
2
2
3
1
3
H, C(CH
3
)
3
3
CO
2
3
3 3
)
2
3
2
1
9
and 28.2 (two s, C(CH ) ). F-NMR (CDCl , 282 MHz), δ, ppm: -65.3 (d, J = 19 Hz, CF ). IR
3
3
3
F-H
3
-
1
bands: 3093, 2983, 2876, 1748, 1703, 1667 rest of the peaks below 1460 cm . Mass-spectrum
EI), m/z: 236 [M-Bu ] , 195 [M-Boc] . [α]
t +
+
20
(
= -202 (CHCl , c = 0.55). CHN, found. C, 49.03; H,
3
D
5
.30; N 4.99. C H F NO requires C, 48.81; H, 5.46; N 4.47.
12 16 3 4
Methyl (2S,4S)-N-tert-buthoxycarbonyl-4-trifluoromethylprolinate (7)
(13 g; 44 mmol) and Pd/C (5 %, 3 g) in methanol (100 ml) were stirred under hydrogen (1
atm) for 5 hours. The mixture was filtered and concentrated under reduced pressure to give
6
7
(
12.3 g, 94 %) as white solid.
1
H-NMR (CDCl
3
, 400 MHz), δ, ppm (two rotamers): 4.42 and 4.33 (2t, J = 8 Hz, 1H), 3.92 and
3
1
.84 (2t, J = 10 Hz, 1H), 3.77 (s, 3H), 3.51 (t, J = 10 Hz), 2.96 (m, 1H), 2.56 (m, 1H), 2.14 (m, 1H),
1
3
.49 and 1.43 (2s, 9H). C-NMR (CDCl , 101 MHz), δ, ppm (two rotamers): 172.3 and 172.0 (2s,
3
CO Me), 153.8 and 153.2 (2s, N-C(=O)-O), 126.0 (q, J = 277 Hz, CF ), 80.8 (s, O-C(CH ) ), 58.5
2
3
3 3
and 58.2 (2s, N-CH-CO Me), 52.4 and 52.2 (2s, OCH ), 45.8 and 45.7 (2m, N-CH ), 41.9 and 41.1
2
3
2
1
9
(
q, J = 30 Hz, CH-CF ), 30.1 and 29.1 (2s, CH -CH-CO Me), 28.3 and 28.2 (2s, C(CH ) ). F-
3 2 2 3 3
NMR (CDCl , 282 MHz), δ, ppm: -70.4 (d, J = 8 Hz, CF ). IR bands: 2987, 2870, 1747, 1695,
3
H-F
3
-
1
+
+
+
further peaks below 1481 cm . Mass-spectrum (EI), m/z: 297 [M] , 240 [M-But] , 239 [M-CO Me] ,
24, 196 [M-Boc] . T
2
+
20
2
4
= 50 °C, [α]D = -70 (CHCl , c = 2.1). CHN, found. C, 48.59; H, 6.05; N
melt
3
.85. C H F NO requires C, 48.48; H, 6.10; N 4.71.
1
2
18
3
4
The compound
1 was prepared as following. To 6 (10.88 g, 36.6 mmol) in methanol (100 ml)
sodium hydroxide (1M in methanol, 40 ml) was added. The mixture was stirred for 2 hours at the
room temperature and then concentrated in vacuum (temperature in bath ≤ 40 °C). The residue
was dissolved in water (200 ml) and washed with diethyl ether (2 x 40 ml) and organic fractions
were discarded. The water fraction was then acidified with hydrochloric acid (13 %, 17 ml) until
acidic pH was reached (~ 1-2). Aqueous layer was extracted with dichloromethane (4 x 60 ml), the
organic fractions were dried over sodium sulphate, filtered and concentrated under reduced
1
pressure.
1
(8.98 g, 86 %) was obtained as white solid. H-NMR (CDCl , 500 MHz), δ, ppm (two
3
rotamers): 6.75 (br s, 1H), 4.43 and 4.36 (2m, 1H), 3.93 and 3.85 (2m, 1H), 3.50 (m, 1H), 2.97 (m,
H), 2.61 and 2.54 (2m, 1H), 2.41 and 2.22 (2m, 1H), 1.49 and 1.43 (2s, 9H). Spectral data was
1
2
9
consisted with the one published by Qiu&Qing. Also, the [α] values varied in different repetitions
D
of the full synthesis from -72 to -77 indicating different levels of racemization on the previous
steps. In this particular approach described in this experimental section we obtained the product
with [α] -77 (c = 1.0, CHCl ).
D
3
(
2S,4S)-N-(9-fluorenylmethoxycarbonyl)-4-trifluoromethylproline (9)
To a solution of (569 mg, 2.0 mmol) in dichloromethane (10 ml) trifluoroacetic acid (3 ml) was
1
added, the solution was stirred at the room temperature for 2 hours. Liquids were removed under
reduced pressure (temperature in bath 30 °C), then additional dichloromethane (10 ml) was added

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DOI: 10.1039/C5OB00034C
and evaporation was repeated. Water (10 ml) and sodium carbonate solution (10%, 7 ml) was
added to reach pH ≈ 8-9 and acetone (5 ml) was subsequently added until clear solution was
afforded. Resulting mixture was cooled down in an ice bath. Suspension of Fmoc-OSu (745 mg,
2
.2 mmol) in acetone (2 ml) was added within 1 min upon stirring, and then about 10 ml of acetone
was added to the reaction mixture. After 4 hours the ice bath was removed and stirring was
continued for 25 hours at ambient temperature. Acetone was removed under reduced pressure
(
temperature in bath ≤ 31 °C), the transparent solution was poured into water (100 ml), and
resulting suspension was washed by diethyl ether (4 x 30 ml). The aqueous layer was acidified by
hydrogen chloride solution (1M) until pH ≈ 1;, resulting aqueous fraction was extracted with ethyl
acetate (4 x 50 ml). The ethyl acetate fractions were dried over magnesium sulphate, filtered and
concentrated under reduced pressure. The residue was dissolved in acetonitrile-water mixture, the
solution was frozen in liquid nitrogen and lyophilized overnight to give
9 (813 mg, 100%) as beige
powder (amorphous).
1
H-NMR (CDCl , 700 MHz), δ, ppm (two rotamers 3:2): 7.89 (br s, 1H, CO H), 7.79 (major) and
3
2
7
7
.73 (minor) (2d, J = 7 Hz, 2H, aromatic CH), 7.58 (major) and 7.54 (minor) (m, 2H, aromatic CH),
.43 (major) and 7.37 (minor) (m, 2H, aromatic CH), 7.34 (major) and 7.30 (minor) (m, 2H,
aromatic CH), 4.55-4.40 (m, 2H, CH -O), 4.50 (major) and 4.31 (minor) (2t, J = 8, 1H, α-CH), 4.27
2
(
(
major) and 4.15 (minor) (2t, J = 7 Hz, CH-CH -O), 3.54 (m, 1H, δ-CH), 2.98 (major) and 2.94
2
minor) (m, 1H, γ-CH), 2.61 (m, 1H, β-CH), 2.28 (major) and 2.21 (minor) (2m, 1H, β-CH). C-
1
3
NMR (CDCl , 176 MHz), δ, ppm (two rotamers): 176.1 and 175.2 (CO H), 155.0 and 154.1 (N-
3
2
C(=O)-O), 143.8, 143.5, 141.4 and 141.3 (aromatic C), 127.9, 127.7, 127.2, 127.1, 125.0, 124.8,
1
20.04 and 120.0 (aromatic CH), 125.8 (CF ), 68.2 and 67.8 (CH -O), 58.5 and 57.7 (α-CH), 47.13
3
2
and 47.10 (CH-CH
2
-O), 46.2 and 45.8 (δ-CH
2
), 41.9 (major) and 41.0 (minor) (q, J = 30 Hz, CH-
1
9
3 2 3
CF ), 30.1 and 28.8 (β-CΗ ). F-NMR (CDCl , 659 MHz), δ, ppm, (two rotamers): -70.34 (major)
and -70.38 (minor) (d, J = 8 Hz). IR bands: 3600-2150 broad peak with a maximum at 2957, 1705
-
1
+
+
20
broad peak, further peaks below 1430 cm . Mass-spectrum (EI), m/z: 405 [M] , 179 [Fmoc] . [α]
-50.7 (CHCl , c = 1.04). CHN, found. C, 62.02; H, 4.40; N 3.60. C21 NO requires C, 62.22;
D
=
3
H
18
F
3
4
H, 4.48; N 3.46.
Methyl (2S,4S)-N-acetyl-4-trifluoromethylprolinate (8)
Acetyl chloride (0.45 ml, 6.3 mmol) was mixed to dry methanol (25 ml) and resulting acidic
methanol was added to (1.57 g, 5.3 mmol) in methanol (75 ml). The mixture was stirred at the
7
room temperature for 2 hours and then refluxed for the next 4 hours. The mixture was
concentrated under reduced pressure. Dichloromethane (70 ml) and triethylamine (1.62 ml, 11.6
mmol) were added to the residue and the mixture was stirred for 5 min, then acetyl chloride (0.41
ml; 5.8 mmol) was added and the mixture was stirred at the room temperature for 3 days. The
mixture was then concentrated under reduced pressure, the obtained residue was rinsed with
diethyl ether (2 x 50 ml, 2 x 20 ml) and the organic fractions were concentrated in vacuum to give
pure matter. This product was additionally purified on a silica gel column using ethyl acetate
elution to give
8 (1.03 g, 82 %) as colourless oil.
1
H-NMR (D O, 700 MHz), δ, ppm (two rotamers): 4.81 (dd, J = 9.7 and 3.7 Hz, minor) and 4.50
2
(
t, J = 8.2 Hz, major, 1H, CH-CO Me), 4.00 (dd, J = 8.7 and 9.7 Hz, 1H major, the minor rotamer
2
resonance lays underneath, 1H, N-CHH), 3.75 (s, minor) and 3.71 (s, major, 3H, OCH ), 3.69 (t, J
3
=
10.0 Hz, major) and 3.42 (dd, J = 12.7 and 6.3 Hz, minor, 1H, N-CHH), 3.28 (m, major) and 3.18
(
m, minor, 1H, CH-CF ), 2.75 (m, minor) and 2.63 (m, major, 1H, CHH-CH-CO Me), 2.42 (m,
3
2
minor) and 2.11 (m, major, 1H, CHH-CH-CO Me), 2.07 (s, major) and 1.98 (s, minor, 3H, CH -
2
3
1
3
C=O). C-NMR (CDCl , 176 MHz), δ, ppm (two rotamers): 173.62 (s, major, CO Me), 173.60 (s,
3
2
minor, N-C=O), 173.3 (s, minor, CO Me), 172.9 (s, major, N-C=O), 126.7 (q, J = 277 Hz, minor)
2
and 126.0 (q, J = 276 Hz, major, CF ), 59.7 (s, minor) and 58.6 (s, major, CH-CO Me), 53.4 (s,
3
2
minor) and 53.1 (s, major, OCH ), 47.2 (q, J = 3 Hz, major) and 45.6 (q, J = 3 Hz, minor, N-CH ),
3
2

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DOI: 10.1039/C5OB00034C
4
2
7
1.3 (q, J = 29 Hz, major) and 39.4 (q, J = 29 Hz, minor, CH-CF ), 29.4 (q, J = 2 Hz, minor) and
3
8.3 (q, J = 3 Hz, major, CH -CH-CO Me). F-NMR (470 MHz), δ, ppm (two rotamers): in D O -
2 2 2
1.0 (major, JF-H = 9 Hz) and -71.3 (minor, JF-H = 10 Hz); in DMSO-d6 -69.4 (major, JF-H = 9 Hz)
1
9
and -69.9 (minor, JF-H = 10 Hz); in CDCl -70.7 (major, J = 8 Hz) and -71.1 (minor, J = 9 Hz).
3
F-H
F-H
-
1
IR bands: 2959, 2892, 1743, 1652, further peaks below 1438 cm . Mass-spectrum (EI), m/z: 239
+
20
[
M ], 180, 139. [α]
D
3 9 12 3 3
= 78 (CHCl , c = 0.56). CHN, found. C, 45.34; H, 5.30; N 6.05. C H F NO
requires C, 45.19; H, 5.06; N 5.86. X-ray crystall structure can be found in the Cambridge
Crystallographic Data Center under ID CCDC 1042476.
19
F-NMR parametrization of the amide rotation
1
9
The F-NMR spectra were measured on Bruker Avance III 500 spectrometer (470.7 MHz)
equipped with a BBFO probe. The variable temperature unit was calibrated using sample with
acidified glycerol. A temperature series of conventional 1-pulse spectra was run in the range 30-90
°
C with solution of Ac-TfmPro-OMe (8, 75 mg) in deuterium oxide as 12 experiments with the
th
temperature pre-equilibration delay of 5 min. Resulting spectra were baseline corrected (5 order)
and integrated. Corresponding rotameric ratios (Ktrans/cis) were calculated and then converted to
∆
G according to ∆G = -RTlnK. The ∆G values were plotted against the temperature, ∆H and ∆S
values were then extracted using ∆G = ∆H - T∆S equation.
1
9
The amide rotation rate constants were determined in F cross-relaxation experiments (EXSY)
with the following setup: “noesygpph” experiment from the standard Bruker library, with the mixing
time of 1 s and 20 ms for referencing. 18 experiments (9 for exchange and 9 referencing) were
acquired at 30-55 °C. Resulting 2D spectra were baseline corrected in both dimensions and
integrated. The exchange rates (exchange rate matrices) were calculated with EXSYCalc®
(
Mestrec) freeware. In particular, detected at 55 °C the exchange rates were k
= 0.617 and
cis-trans
-
1
ktrans-cis = 0.182 s . Standard linearisation according to the Eyring equation (in ln(k/T) – 1/T
coordinates) delivered the values of ∆H and ∆S.
Peptide synthesis
Linear peptides were synthesised on a Val-preloaded 2-chlorotrytyl resin. The amino acids
were taken as N-Fmoc (ornithine side chain amine was Boc-protected) in 4 eq. along with 4 eq. of
6
Cl-HOBt, 3.9 eq. of HCTU, 8 eq. of DIPEA premixed in 2 ml of DMF before adding to the resin for
a coupling step which was performed for 2 hours. The Fmoc-removal was done with 22 %
piperidine in DMF for 20-30 min. The linear peptides were cleaved from the resin by treatment with
2
5 % hexafluoroisopropanol in dichloromethane for 15 min. Cyclisation was performed under high
dilution conditions in dichloromethane (0.15 mmol / 1 l) with 3 eq. of HOBt and 3 eq. PyBOP and 6
eq. of DIPEA, which were pre-mixed in DMF (1.5 ml) before addition. The reaction was continued
for 12 hours. The Boc-protection groups from the ornithine side chains were removed by treatment
with TFA:TIS:water 92.5:5:2.5 cocktail for > 15 min. The crude matters were then purified on the
semi-preparative RP-HPLC C18 column (10 x 250 mm) with the water-acetonitrile gradient elution.
5
mM hydrogen chloride concentration in the eluent was used as an ion-paring agent. Analytical
2
6
RP-HPLC was done as described on a biphenyl analytical column (4.6 x 250 mm).
1
9
1
TfmPro-GS
:
F-NMR (1:1, vol/vol, acetonitrile/water), 282 MHz), δ, ppm: -70.5 (d, JF-H = 10
Hz, CF ). Mass-spectrum (MALDI-TOF), m/z, found/calcd.: 1211.2/1209.5.
3
1
9
2
TfmPro-GS
:
F-NMR (1:1, vol/vol, acetonitrile/water), 282 MHz), δ, ppm: -70.5 (d, JF-H = 9
Hz, CF ). Mass-spectrum (MALDI-TOF), m/z, found/calcd.: 1278.3 /1277.5.
3
Solution NMR of peptides
1
15
The spectra were recorded on Bruker Avance III 700 spectrometer ( H 700.2 MHz; N 71.0
MHz) equipped with a TXI probe at 25 °C. The phosphate buffer was of pH 6.0 (22 °C) and 15 mM
concentration containing 10 vol % D O for deuterium lock. The spectra in DMSO-d6 were
2

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DOI: 10.1039/C5OB00034C
referenced using deuterium lock signal, while the spectra in PB-TFE were referenced using TPS
1
internal standard. Assignment of the proton spectra was done using H TOCSY experiments
1
15
(
dipsi2 spin lock of 60 ms). The H N single bond correlations were detected in the sofast-HMQC
experiments with the recycling delay of 100 ms.
1
15
D
Found HN resonances ( H/ N). GS in DMSO-d6: 9.08/128.1 ( Phe), 8.72/125.8 (Leu),
D
8
.34/124.0 (Orn), 7.24/113.3 (Val); 2TfmPro-GS in DMSO-d6: 9.12/128.0 ( Phe), 8.70/125.8
D
(
Leu), 8.32/123.4 (Orn), 7.34/110.5 (Val); GS in PB-TFE: 8.76/129.6 (Leu), 8.48/127.5 ( Phe),
D
8
8
.12/125.8 (Orn), 7.83/121.2 (Val); 2TfmPro-GS in PB-TFE: 8.77/129.4 (Leu), 8.66/127.7 ( Phe),
.13/125.8 (Orn), 7.91/117.4 (Val).
CD spectra of the peptides
The spectra were measured on Jasco J-720 spectropolarimeter. The spectra were measured at
2
5 °C in the same buffer as was taken for NMR at 90 µM peptide concentrations.
Solid state NMR
1
9
The solid state F-NMR spectra were recorded on Bruker Avance III 500 spectrometer (470.6
MHz) equipped with wide bore magnet and the home-built HF lowE flat-coil probe.
An oriented sample was prepared from 0.6 mg 2TfmPro-GS and 11.7 mg of DLPC
(
(12:0/12:0)PC) at the peptide-to-lipid molar ratio of 1/40. Dry mixtures were co-dissolved in
methanol, spread over 16 rectangular glass plates (18 x 7.5 mm; Marienfeld, Germany), were
dried in vacuum (> 4 hours), stacked and hydrated at 96 % relative humidity (saturated potassium
sulfate at 48°C) for 26 hours. A fresh sample was wrapped in Nescofilm® and Sarogold® films for
3
1
prevention of drying. The proper orientation of the lipid bilayers was checked by P-NMR to have
at least 80% bilayer lipids being coplanar with the slide surface.
1
9
The F-NMR spectra in oriented samples were measured using aring composite pulse
sequence (for background suppression) with proton decoupling during acquisition. Accurate
temperature series (in particular, 1 °C step in the range +5 ÷ -5 °C) was performed with 10 min
temperature pre-equilibration time at each temperature. The temperature was calibrated using
acidified methanol sample. The spectra were processed with Lorentzian window function (LB 250
Hz).
Acknowledgements
V.K acknowledges Dr. Andi Mainz (Berlin, Germany) for provision of the sofast-HMQC setup.
Enamine Ltd. and Prof. Andrey Tolmachev (Kyiv, Ukraine) are acknowledged for financial support.
Prof. Igor V. Komarov (Kyiv) and Dr. Patrick Durkin (Berlin) are acknowledged for revision of the
manuscript.
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