Hydrolysis, polarity, and conformational impact of C-terminal partially fluorinated ethyl esters in peptide models

Article abstract of DOI:10.3762/bjoc.13.241

Fluorinated moieties are highly valuable to chemists due to the sensitive NMR detectability of the 19F nucleus. Fluorination of molecular scaffolds can also selectively influence a molecule's polarity, conformational preferences and chemical reactivity, properties that can be exploited for various chemical applications. A powerful route for incorporating fluorine atoms in biomolecules is last-stage fluorination of peptide scaffolds. One of these methods involves esterification of the C-terminus of peptides using a diazomethane species. Here, we provide an investigation of the physicochemical consequences of peptide esterification with partially fluorinated ethyl groups. Derivatives of N-acetylproline are used to model the effects of fluorination on the lipophilicity, hydrolytic stability and on conformational properties. The conformational impact of the 2,2-difluoromethyl ester on several neutral and charged oligopeptides was also investigated. Our results demonstrate that partially fluorinated esters undergo variable hydrolysis in biologically relevant buffers. The hydrolytic stability can be tailored over a broad pH range by varying the number of fluorine atoms in the ester moiety or by introducing adjacent charges in the peptide sequence.

Full text of DOI:10.3762/bjoc.13.241

Hydrolysis, polarity, and conformational impact of C-terminal
partially fluorinated ethyl esters in peptide models
Vladimir Kubyshkin* and Nediljko Budisa*
Full Research Paper
Open Access
Address:
Beilstein J. Org. Chem. 2017, 13, 2442–2457.
Biocatalysis group, Institute of Chemistry, Technical University of
Berlin, Müller-Breslau-Strasse 10, Berlin 10623, Germany
doi:10.3762/bjoc.13.241
Received: 04 August 2017
Accepted: 19 October 2017
Published: 16 November 2017
Email:
Vladimir Kubyshkin* - kubyshkin@win.tu-berlin.de; Nediljko Budisa* -
nediljko.budisa@tu-berlin.de
This article is part of the Thematic Series "Organo-fluorine chemistry IV".
Guest Editor: D. O'Hagan
* Corresponding author
Keywords:
conformation; ester bond; hydrolysis; peptides; polarity
© 2017 Kubyshkin and Budisa; licensee Beilstein-Institut.
License and terms: see end of document.
Abstract
Fluorinated moieties are highly valuable to chemists due to the sensitive NMR detectability of the 19F nucleus. Fluorination of mo-
lecular scaffolds can also selectively influence a molecule’s polarity, conformational preferences and chemical reactivity, proper-
ties that can be exploited for various chemical applications. A powerful route for incorporating fluorine atoms in biomolecules is
last-stage fluorination of peptide scaffolds. One of these methods involves esterification of the C-terminus of peptides using a
diazomethane species. Here, we provide an investigation of the physicochemical consequences of peptide esterification with
partially fluorinated ethyl groups. Derivatives of N-acetylproline are used to model the effects of fluorination on the lipophilicity,
hydrolytic stability and on conformational properties. The conformational impact of the 2,2-difluoromethyl ester on several neutral
and charged oligopeptides was also investigated. Our results demonstrate that partially fluorinated esters undergo variable hydroly-
sis in biologically relevant buffers. The hydrolytic stability can be tailored over a broad pH range by varying the number of fluo-
rine atoms in the ester moiety or by introducing adjacent charges in the peptide sequence.
Introduction
Fluorine is a rare element in natural biochemical settings [1]. measured by NMR (after 3H and 1H hydrogen isotopes); as a
Notwithstanding several prominent fluoro-organic metabolites result, 19F NMR experiments are remarkably sensitive [4].
in nature [2,3], fluorine is virtually absent from natural biopoly-
mers such as proteins and nucleic acids. Therefore, organofluo- Fluorine-containing groups can be incorporated into biopoly-
rine groups lack a natural background in spectroscopic observa- mers by various approaches, including those that utilize biosyn-
tions of biological samples. This feature is especially beneficial thesis [5-7], enzymatic conversion [8], chemical synthesis
for NMR applications because the sole stable fluorine isotope [9,10], and ligation reactions [11]. Depending on the research
(19F) has the third largest magnetogyric ratio among the nuclei target, 19F NMR measurements can be used to study
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ligand–protein [12] and protein–protein interactions [13]; mem- tial distribution in biochemical compartments and the meta-
brane proteins [14-16] and membrane-associated peptides bolic stability [44,45].
[17,18]; equilibria among conformations of RNA [19], DNA
[20], and peptide nucleic acids (PNA) [21]; and many others.
Particularly recent is the development of peptide-based contrast
agents for 19F imaging [22].
In polypeptides, the incorporation of fluorine can significantly
alter the properties of the native molecule. The hydrophobicity
[23,24], conformational equilibria [25], and the thermodynamic
[26-29] and kinetic [30,31] folding profiles can be altered by
the presence of even a single fluorinated amino acid in the se-
quence. Perhaps the most studied molecules exhibiting such
effects are the proline analogues, with the proline-to-fluoropro-
line exchange providing the first proof-of-principle and experi-
mental basis for a number of subsequent conceptual studies
[32]. For example, these were used to demonstrate
the impact of non-canonical amino acids in proteins [33].
Fluoroproline-containing sequences were also applied
as collagen mimics to dissect collagen-stabilizing forces
[34,35].
However, the impact of fluorine labeling on polypeptides is still
not fully understood and it may appear controversial in the liter-
ature. For example, a donor–acceptor type enhancement of the
face-to-face stacking of phenyl- and pentafluorophenyl groups
has been suggested [36]; though, subsequent studies of α-helical
[37], peptoid [38], and collagen-mimicking models [39] did not
support this suggestion. Though, this was reported later in a
context of a hydrophobic core of model protein structures [40].
Figure 1: A. Dependence of the lipophilicity (logP) on the number of
fluorine atoms in a partially fluorinated terminal alkyl group demon-
strates a characteristic checkmark-shape [41]. B. Selective labeling of
carboxylic acids (C-terminus in peptides) with 2,2-difluorodiazoethane
yields 2,2-difluoroethyl esters [46]. C. Esters of N-acetylproline studied
herein.
Furthermore, the influence of the fluorinated groups on Considering the variety of spectroscopic applications, a labeling
lipophilicity remains uncertain, especially in biochemical litera- strategy that allows specific incorporation of a fluorinated
ture.
moiety into a modular peptide fragment is highly desirable.
Several recent efforts have been made to achieve this goal
The impact of partially fluorinated alkyl groups on the polarity [9,47]. Fluorinated diazoalkanes have shown particularly prom-
of small molecules was recently investigated in a series of ising results in small molecule functionalization [48], and these
model studies by Huchet and others [41-44]. These investiga- have a potential to be used for biomacromolecular functionali-
tions demonstrated the checkmark-shape of the lipophilicity zation as well [49]. As an example, we recently discovered the
(logP) changes upon increasing the number of fluorine atoms in selective formation of 2,2-difluoroethyl esters upon treatment of
the terminal aliphatic alkyl fragment (Figure 1A; see also small molecules and peptide substrates with 2,2-difluorodi-
remark on page S2 in Supporting Information File 1). While a azoethane (Figure 1B) [46]. The transformation was carried out
methyl group is lipophilic, the incorporation of one, two or in chloroform, which certainly limited the substrate scope.
three fluorine atoms produces a non-additive increase in the Nonetheless, due to the high acidity of the C-terminal
molecular dipole. On the other hand, the linear increase in the carboxylic group [50], a number of C-terminally modified
molecular volume due to the hydrogen-to-fluorine exchange in- peptides were readily prepared by this method with full conver-
creases the lipophilicity. As the result of these two opposite ten- sion. The availability of the C-terminal 2,2-difluoroethanol-
dencies, the logP value decreases with incorporation of each esterified peptides also enables the investigation of the impact
moiety in the following order: RCH3 > RCF3 > RCHF2 ≥ of partial fluorination on target peptides. Of particular impor-
RCH2F. The polarity and lipophilicity are among the most im- tance are investigations of the hydrolytic stability of the
portant effects of fluorination, as these parameters strongly partially fluorinated esters and the possible conformational
impact other important biological properties, such as the poten- impact on the peptide. Thus, we herein report results from
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Beilstein J. Org. Chem. 2017, 13, 2442–2457.
studies of N-acetylproline esters, which were used as models for an acetonitrile/water (1:1) mixture. However, only a very low
C-terminally modified peptides (Figure 1C). In addition, the yield (15%) was obtained with 2,2-difluorodiazoethane gener-
conformational impact is examined using several oligopeptides. ated directly in the solvent mixture. Therefore, we tried gener-
ating 2,2-difluorodiazoethane in acetonitrile with subsequent
Results and Discussion
addition of this solution to an aqueous solution of the substrate.
Synthesis
Although the yield increased, it remained low (20%). The poor
The synthesis of the C-terminal esters was performed starting yields in the aqueous medium are explained by the high reactiv-
from N-acetylproline (6), which was prepared as described pre- ity of the diazomethane species, which favors nonspecific reac-
viously [50]. The reference methyl and ethyl esters 1 and 2 were tions in water [52]. Very recently, more specific diazomethane
prepared by stirring of 6 in acidic alcohol (methanol or ethanol, reagents for water-tolerant esterification have also been de-
respectively) at room temperature overnight (Scheme 1A). We veloped [53]. During the revision of this paper Peng et al. re-
attempted to employ the same procedure for esterification of 6 ported on esterification of carboxylic acids using 2,2-difluorodi-
in trifluoroethanol. However, this resulted in a very low yield of azoethane, which was performed in a number of aprotic organic
the desired product 5 (4%). The monofluoro- and trifluoroethyl solvents, including acetonitrile [54].
esters 3 and 5 were then prepared via the corresponding chlo-
ranhydride, which was generated as described (Scheme 1B) Hydrolytic stability
[51]. The drawback of this method is that generation of chloran- The kinetic stability of the ester linkage in the aqueous medium
hydride from N-acetylated proline leads to partial epimerization is of critical importance, as it further defines the time window
of the residue.
for the reactions with the corresponding esters under the biolog-
ically relevant conditions of aqueous buffers. Hydrolysis of the
C-terminal esters has been fairly well described in the literature
[55-57], indicating the hydrolysis occurs via a pseudo-first
order reaction in a buffered medium assuming that [HO−] is
constant (Equations 1–3):
(1)
(2)
(3)
Particularly interesting is the dependence of the hydrolysis rate
on the nature of the alkyl group of the ester; it has been demon-
strated that ethyl esters hydrolyze approximately 2–3 times
slower compared with the methyl esters [58], whereas methyl
esters hydrolyze approximately 30 times slower compared with
the 3’-tRNA esters in aminoacyl-tRNA [59]. Clearly, the hydro-
lysis rate depends on the electron donating/withdrawing effect
of the alkyl moiety R, indicating a significantly compromised
kinetic stability is likely for the partially fluorinated ester
groups.
Scheme 1: Synthesis of the model compounds.
Finally, ester 4 was prepared by treatment of 6 with 3 equiva-
lents of the 2,2-difluorodiazoethane (Scheme 1C). The reaction
was performed smoothly using chloroform as a solvent giving a
good yield (94%). We also attempted to perform the reaction in
Next, we determined the half-life values following the above
mentioned kinetic model for esters 1–5 in aqueous medium at
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pH 11 and 298 K (Figure 2). In accordance with previous mark-shape: the lipophilicity decreased with the introduction of
reports, we found that the ethyl ester hydrolyzed 3 times slower the first polar C–F bond, and with the introduction of subse-
compared with the methyl ester, whereas the introduction of the quent fluorine atoms, the lipophilicity increased due to the
first fluorine atom dropped the half-life by a factor of approxi- increase in molecular volume. The lipophilicity of 2,2-difluo-
mately 8. For the subsequent fluorine atoms, the hydrolysis rate roethyl derivative 4 is nearly identical to that of parent ethyl
increased by a factor of 3–4 for each appended fluorine atom. ester 2, and the relative difference in the logP from the increase
The trifluoroethyl ester 5 delivered the fastest hydrolysis rate (5 vs 4, ΔlogP = +0.53 ± 0.13) and decrease (3 vs 4, ΔlogP =
with a half-life of only 6.4 ± 1.7 min. Extrapolation to pH 8.0 −0.45 ± 0.13) in the number of fluorine atoms are equivalent to
gives a value of 107 ± 28 hours. (This procedure requires multi- one methyl group difference (2 vs 1, ΔlogP = +0.44 ± 0.08).
plication by 1000.) We experimentally determined the half-life The amplitude of the change is similar to that reported recently
of 5 at pH 8 to be 102 ± 2 hours, which agrees well with the for the corresponding fluorinated ethanols [63]. Importantly,
extrapolation. The mutual consistency of the values determined C-terminal esterification maintains a much higher lipophilicity
with different kinetic modes (minutes at pH 11, days at pH 8) compared with the C-terminal amide 7 (Table 1).
provides a good indication of their accuracy.
Figure 2: Kinetics of the C-terminal ester hydrolysis.
These data show that chemical modification with the terminally
fluorinated ethyl esters 3–5 is possible over the course of days
at physiologically relevant pH < 8. In fact, this stability enables
further determination of several physicochemical properties for
the examined compounds in water; for example, partitioning
can be safely measured. It is also notable that the hydrolysis rate
Figure 3: Partitioning of the esters 1–5 between octan-1-ol and water.
Insert: comparison with the other partially fluorinated propoxy and me-
thoxy groups from references [41,44].
of the C-terminal trifluoroethyl or difluoroethyl esters may The observed checkmark shape differs from that previously re-
suggest a mechanism of self-cleavage for a potential drug mole- ported for an isolated partially fluorinated methyl group at the
cule under physiological conditions, in addition to the most end of a saturated aliphatic chain. In the latter case, the methyl
common path mediated by the esterases [60]. Though, the group is the most lipophilic, more than a trifluoromethyl group;
susceptibility of the fluorine-bearing esters towards natural and the same has been reported for the partially fluorinated
esterases is still unknown, and systematic investigation of this propyl ethers OCH2CH2CFnH3−n (logP: CH3 > CF3 > CHF2 ≥
issue is lacking in the literature. Notably, pH-programmed de- CH2F) [41,44] (see insert on Figure 3). In contrast, among the
composition of fluoro-organic molecules has been recently re- partially fluorinated methyl ethers OCFnH3−n, the lipophilicity
ported for few other cases [61,62].
of the methoxy group is the lowest, while the fluoromethoxy
group is the most lipophilic (logP: CF3 > CHF2 > CH2F ≈ CH3)
[44,64]. These differences were explained by the mutual intra-
Lipophilicity
To characterize the lipophilicity, esters 1–5 were subjected to molecular compensation of the C–O dipole and the dipole from
24 hours of partitioning between octan-1-ol and water at 298 K. the partial fluorination [65]. Thus, the polar effect from the
The resulting partition is illustrated in Figure 3. In accordance fluorine-bearing region becomes less prominent as the number
with previous observations, the logP values exhibit the check- of the C–C bonds to the polar fragments decreases. The
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Beilstein J. Org. Chem. 2017, 13, 2442–2457.
served identical parameters for the amide rotation around the
glycyl–prolyl amide bond in AcGlyGlyProGlyGlyNH2 [71] and
AcGlyProOMe [68] compounds when measured in deuterium
oxide.
Table 1: Summarized properties of the analyzed compounds as deter-
mined by NMR at 298 K.
compound
τ½, min
logP
(aq buffer) (octan-1-ol/water)
224 ± 20
1
−0.44 ± 0.05
(pH 11.0)
603 ± 58
0.00 ± 0.03
(pH 11.0)
2
3
4
5
Scheme 2: Amide isomerism in the N-acetylprolyl fragment.
80 ± 14
For 1–5, we found the trans/cis ratios ≈ 5 for all five esters
when measured in aqueous medium; the kinetic parameters of
the amide rotation were also nearly identical (Table 2). We then
noticed that the trans/cis ratios measured for the fluorine-
labeled esters 3–5 in nonpolar solvents (such as benzene) were
systematically higher relative to the reference compounds 1 and
2. We tested the relative increase in the trans-amide preference
ΔΔG as a function of solvent (Equation 4) and found a depen-
dence of this parameter on the dielectric constant, as would be
expected from an electrostatic interaction between the polar
alkoxy group and the amide moiety (Figure 4).
−0.43 ± 0.07
(pH 11.0)
19.7 ± 2.5
+0.02 ± 0.06
(pH 11.0)
6.4 ± 1.7
(pH 11.0)
+0.55 ± 0.07
6112 ± 70
(pH 8.0)
7
–
−1.57 ± 0.10
(4)
partially fluorinated ethoxy moieties reported here represent an
intermediate situation between the partially fluorinated me- A somewhat similar situation has been recently reported by
thoxy and propoxy/higher alkoxy groups (logP: CF3 > CHF2 ≈ Siebler et al. for the C-terminal amide in dimethylamido
CH3 > CH2F).
N-acetylproline, where the conformational equilibrium rendered
the s-cis conformation remarkably more polar compared with
the s-trans counterpart (Scheme 3A), which leads to an elevated
Amide isomerism
The tertiary amide bond in N-acylprolyl can exist in two confor- trans/cis ratio in nonpolar solvents such as dioxane and chloro-
mational states, s-trans and s-cis (Scheme 2), with the former form [72]. In contrast to this situation, in methyl ester 1, the de-
being thermodynamically preferred in most cases [66,67]. The pendence of the trans/cis ratio on the solvent is known to be
intrinsic contextual preference in the amide isomerism around marginal [73,74].
the proline residue is usually characterized using small molecu-
lar models such as esters of N-acetylproline, similar to those in To explain the elevated trans/cis ratio observed with partially
this study [35,68,69]. Within this model, the trans-amide preva- fluorinated esters 3–5, we simulated the dipolar moment of
lence is not large, and both rotameric states are readily ob- compound 5 by DFT modelling. The dipolar moment for the
served in NMR spectra. In the N-acylprolyl models, the nature lowest energy structure predicted a strong preference in favor of
of the terminal groups can influence the amide isomerism. The the less polar s-cis conformation (Ψ = +101, μ = 2.3 D) over the
most important factor is the presence of the C-terminal charge, s-trans (Ψ = +157, μ = 5.8 D; Scheme 3B). Semi-empirical
which reduces the trans-amide stability [50,70]. Nonetheless, simulation of the four-state model (see Scheme 3A) also pre-
when the charge is eliminated, the influence of the C-terminal dicted a higher polarity for the s-trans conformational states
moiety may become negligible. For instance, we recently ob- (μ = || 4.6, ┴ 7.1 D) compared to the s-cis (μ = || 4.7, ┴ 3.7 D)
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Table 2: Summarized conformational properties of the compounds 1–5 as measured by NMR.
structure amide isomerism
α-CH multiplicity:a
dd, JHH = Hz
in D2O
k∙103, s−1 (310 K)b
in C6D6
in D2O
in C6D6
Ktrans/cisa
Ktrans/cisa
k∙103, s−1 (298 K)b
trans→
cis
cis→
trans
trans→
cis
cis→
trans
trans
cis
trans
cis
4.94 ±
0.05
7.0 ±
0.5c
5.10 ±
0.10
8.5,
4.7
8.7,
2.5
8.1,
3.7
8.6,
2.6
1
2
3
4
5
33 ± 2c
20 ± 5
30 ± 2
32 ± 4
35 ± 5
67 ± 2
67 ± 2
52 ± 2
47 ± 2
48 ± 2
342 ± 10
309 ± 14
348 ± 11
430 ± 17
477 ± 13
4.60 ±
0.08
4.67 ±
0.03
8.7,
4.5
8.8,
2.4
8.3,
3.6
8.5,
2.7
6 ± 1
7 ± 1
7 ± 1
6 ± 1
4.74 ±
0.04
6.80 ±
0.04
9.1,
4.6
8.9,
2.7
8.2,
4.2
8.4,
2.6
4.95 ±
0.05
9.06 ±
0.26
9.3,
4.7
8.8,
2.5
8.0,
3.9
8.7,
2.5
5.48 ±
0.14
10.04 ±
0.15
9.2,
4.7
8.8,
2.4
8.0,
4.1
8.7,
2.6
aDetermined in 1H (and 19F) one-dimensional NMR spectra, analyte 50 ± 10 mM, 298 K; bmeasured by 1H and 19F{1H} EXSY NMR at 50 ± 10 mM
analyte concentration; cas reported in [69].
Figure 4: Enhancement of the trans/cis thermodynamic preferences in the ester models as a function of the solvent dielectric constant, ε. The trans/
cis ratios were determined from the 1H and 19F NMR spectra at 298 K. Solvent set: C6D6, CDCl3, CD2Cl2, CD3OD, CD3CN, D2O. For details see
Table S1 in Supporting Information File 1.
counterpart due to the conformational contribution of the termi- this moiety may be rather flexible, similar to the parent esters 1
nal fluorinated moiety. Both predictions clearly contradict the and 2. This conclusion is suggested by the logP differences be-
experimentally observed tendencies.
tween esters 3–5, which are nearly identical to those between
the parent alcohols (mono, di- and trifluoroethanols) [63,75]. It
A possible problem with the existing models is that they assume is therefore apparent that in contrast to 1 and 2, a more com-
a defined orientation for the fluorine-bearing moiety. However, plex equilibrium should be considered for compounds 3–5 that
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Beilstein J. Org. Chem. 2017, 13, 2442–2457.
An alternative explanation for the elevated trans/cis ratio is
strengthening of the n→π* interaction between the carbonyl
groups. For example, Hodges and Raines reported elevated
trans/cis ratios in phenyl esters of N-formylproline containing
electron withdrawing substituents (X in Scheme 4A) when
measured in chloroform [76]. The trans-amide preference was
enhanced by 2.2 kJ mol−1 for X = NO2 relative to X = N(CH3)2.
Thus, it is possible that in the case of the esters 3–5, the elec-
tron-withdrawing effect of fluorine atoms leads to a similar en-
hancement of the n→π* interaction between the carbonyl
groups due to the higher electrophilicity of the carboxyl carbon
atom (Scheme 4B). Nonetheless, this explanation does not
explain why, despite the electrophilicity of the carboxyl group
indicated by the hydrolysis experiments (Figure 2), the
exchange of the ethyl group by the methyl group enhances the
hydrolysis rate while only marginally impacts the trans/cis
ratio. Furthermore, it is unclear why a polar solvent quenches
the enhancement of the electrophilicity of the C-terminal car-
bonyl, which requires the differential solvation of two rotamers.
This differential solvation may be explained by the polarity of
the two rotamers; thus, the polarity model expressed in
Scheme 3 cannot be dismissed.
Scheme 3: A. Four-state conformational equilibrium model used by
Siebler et al. [72] for explanations of the elevated trans/cis ratio in non-
polar solvents, which occurs for X = N(CH3)2 and does not occur for
X = OCH3. B. The two-state lowest energy model. C. The simplistic ex-
planation of the C-terminal polarity effect observed for the partially fluo-
rinated esters 3–5.
Scheme 4: Elevation of the trans/cis ratio in derivatives of N-acyl
proline may result from the enhanced n→π* interaction between the
carbonyl-groups. EWG = electron-withdrawing group, EDG = electron-
donating group.
involves at least three mutually orienting dipoles in a complex
energy landscape. This may be very challenging for
computational analysis since the amplitude of the effect is Additional factors, such as multipolar interactions [77] and
only ≤2 kJ mol−1. At this point, we can propose a simplistic especially the polar surface exposure [78], may also be consid-
perception of this complex interaction based on the fact that in ered. Further understanding of the relevant interactions requires
the s-trans conformation, the amide carbonyl group is moved more detailed studies and experimental models. Nonetheless,
towards the carboxyalkyl moiety, which increases the interac- the experimental observations and the simple description pro-
tion between the mutually orienting dipoles within a more posed herein (Scheme 3C) suggest that the amide bond of the
compact structure (Scheme 3C). As the result, the gradual N-acetylproline fragment can function as an intramolecular
increase in the polarity from the most distant partially fluori- probe of the C-terminal ester group polarity. Notably, the pre-
nated moiety leads to an increase in the effect in the order 3 < 4 dicted flexibility of the partially fluorinated alkoxy groups in
< 5 (Figure 4).
the presence of other strong dipoles is inconsistent with the
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Beilstein J. Org. Chem. 2017, 13, 2442–2457.
concept of conformational adaptors elaborated by Müller et al.
quite recently [44,65].
Side chain conformation
The side-chain conformations of proline are restricted by the
pyrrolidine ring structure. The two main envelope conforma-
tions are exo- and endo- (alternatively designated as up-/down-,
respectively), in which by the C4-ring atom is oriented toward
or away from the carboxyl group orientation, respectively
[79,80]. This conformational equilibrium manifests in the
χ angles, which is reflected by the J-coupling observed be-
tween the α-CH and β-CH2 groups in the 1H NMR spectra (Jαβ)
[81,82]. A recent analysis of the Jαβ values by Braga et al. was
used to quantify the pucker equilibrium in compound 1 [74].
We examined the values of Jαβ for compounds 1–5 in a polar
(deuterium oxide) and nonpolar (benzene-d6) solvents. The ob-
served Jαβ values are consistent with those reported previously
[74,83], and the value differences likely reflect a change of only
a few percent due to the contribution from the two conforma-
tions. This result indicates that the C-terminal partially fluori-
nated ester has a negligible influence on the side chain confor-
mation of the prolyl moiety.
Scheme 5: Synthesis of the model peptides.
Conformation of short oligopeptides
We then examined how the properties observed this far impact
the conformational properties of a peptide chain. As described
above, the C-terminal charge has a large effect on the stability
of the trans-amide alignment of the peptide bond. It has been
demonstrated that removing the terminal charge enhances the
stability of the polyproline-II helical fold in collagen-mimicking
[84] or polyproline sequences [70] and significantly shifts the
equilibrium of short model sequences in favor of the polypro-
line-II over the β-structure conformation [85]. Reaction with
2,2-difluorodiazoethane esterifies and thus eliminates the
charge of the C-terminal carboxyl group, thereby enhancing the
polyproline-II stability. This effect contrasts with the effects of
other terminal modifications, such as aromatic amino acids,
which reduce the polyproline-II fold stability [86].
We then prepared hexaproline peptide 8a, which was subse-
quently esterified with the diazomethane reagent to give 8b.
Methyl ester 8c was also prepared for comparison (Scheme 5A).
Measured circular dichroism (CD) spectra for the hexapeptides
8a–c (Figure 5, top) demonstrate strong negative bands at
204 nm and weak positive bands at 227 nm, which indicate a
polyproline-II fold. Remarkably, this spectral shape was ob-
served for all three hexaproline peptides 8a–c, while some
increase of the negative band amplitude was only observed for
the methanol samples. Overall, this model suggests a small con-
Figure 5: Mean residue molar circular dichroism (Δε) of peptides 8–10
in methanol (left) and aqueous phosphate buffer (right, 50 mM buffer,
pH 7.0) samples. Recorded at 298 K and 100 μM peptide.
formational impact from the C-terminal 2,2-difluoroethylation These findings motivated assessment of a model system with a
or methylation as compared to the parent peptide 8a.
more dynamic structure. For example, it has been shown that
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Beilstein J. Org. Chem. 2017, 13, 2442–2457.
oligolysine sequences favor a polyproline-II fold in short
stretches [87]. We prepared tri- and hexalysine peptides with-
out (9a, 10a) and with (9b, 10b) the C-terminal difluoroethyl
ester, as well as the methyl esters (9c, 10c) as shown in
Scheme 5B. The CD spectra of these peptides (Figure 5)
revealed a negative band at ≈195 nm and a band at ≈216 nm,
thus enabling measurement of the conformational impact. This
band was clearly positive for the esterified peptides 9b,c and
10b,c in contrast to the parent non-esterified peptides 9a and
10a. This is especially seen in the shortest trilysine sequence,
where the esterification significantly increases the charge densi-
ty, and thus the extended polyproline-II helix can be stabilized
via the charge repulsion forces. Longer hexalysine sequences
exhibited the same effect, albeit to a lower extent. Predictably,
the C-terminal charge has a decreasing effect as the length of
the peptide increases.
Figure 6: Conformational analysis of the peptides by 1H DOSY NMR
(D2O, 298 K). The theoretical values were calculated according to
Equation 5 assuming deuterium oxide viscosity and 298 K.
An independent refinement of these conclusions can be found in
diffusion measurements conducted by 1H DOSY (diffusion
ordered spectroscopy). We previously applied a simple set of The conformational impact of the C-terminal esterification de-
equations for estimation of the diffusion coefficients of an scribed here is also expected to be generic, as the polyproline-II
oligomeric hydrophobic polyproline-II helix [71]. Here, we structure is preferred by various oligopeptide sequences [89,90]
combined these equations in order to derive Equation 5, which and is highly abundant in the unfolded protein states [91].
describes diffusion as a function of molecular weight assuming
pure deuterium oxide solution at 298 K. This equation Hydrolytic stability in peptides
considers molecules to be rigid spheres, as it is based on the Finally, we tested hydrolytic stability in esterified peptides 8b,
Stokes–Einstein relationship. The ‘coil’ state significantly con- 9b and 10b by observing hydrolysis of the ester using 19F NMR
tributes to the conformational flexibility, which results in a in buffered deuterium oxide (pH 7). We expected pseudo-first
diffusion deceleration [88]. We used the simple criterion of the order kinetics as for the hydrolysis of esters 1–5 at pH 11.
difference between the experimental logD and the ‘theoretical’ Nonetheless, for the peptide esters 8b, 9b and 10b, the experi-
logD derived from Equation 5 (MW – molecular weight in Da, mental decay (Figure 7) of the ester concentration resembled
logD in log m2 s−1) (Figure 6). The increase in these values in- pseudo-zero order kinetics, which was also observed for another
dicates a ‘coil’ contribution in the molecular conformation. amino acid ester hydrolysis not shown here.
However, it should be kept in mind that the diffusion data de-
scribes the overall disorder of the peptide body, including the
side chain conformations; in contrast, CD represents only the
backbone.
(5)
The diffusion data (Figure 6) indicated that the hexaproline
peptides 8a–c are rather rigid, the trilysine peptides 9a–c are
somewhat more flexible, and the hexalysine peptides 10a–c are
characterized by remarkable molecular disorder. In all cases, the
C-terminal esterification reduced the level of disorder, although,
the effect is hardly distinguishable from experimental error
values. Overall, these results demonstrate that the side-chain
Figure 7: Hydrolysis of the C-terminal 2,2-difluoroethyl esters of the
disorder may play a more significant role in the peptide diffu-
sion properties, than the backbone rigidity induced by an elimi-
nated terminal charge.
oligopeptides in buffered deuterium oxide at 298 K and pH 7. Dashed
lines reflect the pseudo zero-order kinetic behavior of hydrolysis during
the initial stages of this process.
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Beilstein J. Org. Chem. 2017, 13, 2442–2457.
Both fittings to the zero (Equations 6 and 7) and first order equilibrium between the two conformers was shifted toward a
(Equations 1–3) kinetic models demonstrated fairly good agree- more compact arrangement of the polar groups in the s-trans
ment with each other (see Experimental).
rotamer.
In oligopeptides, the C-terminal 2,2-difluoroethylation
increased the polyproline-II structural contribution. The
effect was seen most prominently in a short oligolysine
peptide. The hydrolytic stability of the ester bond in the
peptide depends on the charge of the peptide, as it was
impaired in the positively charged oligolysine peptides
compared with the neutral oligoproline. These results
(6)
(7)
Notably, the half-life (Equation 7) of oligoproline 8b suggest the potential application of late-stage C-terminal
(152 ± 14 days) demonstrated a good agreement with the esterification with partially fluorinated groups as a tool in
half-life of monoproline peptide 4 extrapolated to pH 7.0 peptide and therapeutic design, as well as in 19F NMR applica-
(137 ± 17 days). The half-life values for the oligolysine tions.
peptides 9b and 10b were much shorter at 5.0 ± 0.5 and
Experimental
4.0 ± 0.3 days, respectively. These results can be explained by
the increased local concentration of negatively charged hydrox- Synthesis of model compounds
ide ions resulting from the positive charge, which causes ester Esterification in acidic alcohols
hydrolysis. The accelerated hydrolysis of oligolysine esters can N-Acetylproline (150 mg, 0.95 mmol) was dissolved in absolute
potentially lead to partial 2,2-difluoroethyl ester hydrolysis alcohol (methanol, ethanol or 2,2,2-trifluoroethanol, 2 mL) and
during the CD sample handling, and thus explain somewhat trimethylsilyl chloride (0.1 mL, 0.8 mmol) was added. The mix-
reduces CD intensity compared to the methyl ester samples ture was stirred for 21 hours at room temperature, solvent was
(Figure 5).
removed under reduced pressure, and the crude material was
purified on a short silica gel (≈11 g) column using an ethyl
This result also suggests that the hydrolysis rate of a C-terminal acetate/methanol 39:1 as the eluent. 143 mg of 1 was obtained
ester in peptides can be well tailored by installing proximal as a clear oil (yield 88%), Rf = 0.53 (ethyl acetate/methanol
charges in the molecule. This conclusion is of particular impor- 39:1). 167 mg of 2 was obtained as a clear oil (yield 94%),
tance due to growing interest in modifying potential peptide Rf = 0.61 (ethyl acetate/methanol 39:1). 10 mg of 5 was ob-
therapeutics with positively charged residues for improved cell tained as a clear oil (yield 4%), Rf = 0.74 (ethyl acetate/metha-
permeability [92]. Though, the charge-induced hydrolysis accel- nol 39:1).
eration from the proximal lysine charge (9b, 10b vs 8b,
factor ≈ 30) is lower than the α-NH2/NH3+ hydrolysis accelera- Esterification via chloranhydrides
tion in the classical studies of α-amino acid esters Compound 3: N-acetylproline (101 mg, 0.64 mmol) was dis-
(factor ≈ 100–150) [58]. The hydrolysis rate can thus be further solved in anhydrous dichloromethane (3 mL) and the resulting
tuned by changing the proximity of the positive charge to the solution was cooled down in an ice bath. Thionyl chloride
ester group. Further investigations of the peptide ester hydroly- (0.1 mL, 1.38 mmol, 2.2 equiv) was added, followed by 2-fluo-
sis catalyzed by the proximal charges may be very beneficial for roethanol (80 μl, 1.38 mmol, 2.2 equiv). The mixture was
understanding of the role of ribosome electrostatics during stirred at ambient temperature for 14 hours. The solvent was re-
translation termination [93,94], which is an ester hydrolysis moved under reduced pressure, and the resulting crude material
reaction performed by bulk water [95].
was subjected to a silica gel (20 g) column purification using an
ethyl acetate/methanol 39:1 eluent. 55 mg of 3 was obtained
as a clear oil (yield 42%), Rf = 0.63 (ethyl acetate/methanol
Conclusion
We have described the properties of C-terminal partially fluori- 39:1).
nated ethyl esters of N-acetylproline. The hydrolytic stability
decreases up to two orders of magnitude as the number of fluo- Compound 5 was obtained in an analogous procedure to 3
rine atoms in the ester group increases. Furthermore, the starting from N-acetylproline (100 mg, 0.64 mmol), thionyl
measured lipophilicity values follow the characteristic check- chloride (0.1 mL, 1.4 mmol, 2.2 equiv) and trifluoroethanol
mark-shape, while the side chain conformation was not (0.12 mL, 1.6 mmol, 2.5 equiv). 141 mg of the product was ob-
affected. The amide isomerism remained unchanged in polar tained as a clear oil (yield 93%), Rf = 0.73 (ethyl acetate/metha-
aqueous medium. Conversely, in the nonpolar solvents, the nol 39:1).
2451

Beilstein J. Org. Chem. 2017, 13, 2442–2457.
Esterification with diazoalkanes
half-life time (squared correlation coefficient > 0.90). The ex-
No special precautions were applied when working with 2,2- periments were performed in triplicate.
difluorodiazoethane except for performing all work in a fume
hood and maintaining conventional lab protection.
Partitioning
To 4–5 mg analyte, octan-1-ol (1.00 mL) and deionized water
The procedure in chloroform involved mixing 2,2-difluoro- (1.00 mL) were added. The mixture was shaken gently at
ethylamine (160 mg, 1.97 mmol, 3 equiv) in chloroform (5 mL), ambient temperature (298 ± 2 K) for 24 hours. Each phase
tert-butyl nitrite (90% purity, 260 mg, 2.27 mmol, 3.5 equiv) (200 μL) was sampled using identical NMR tubes, and aceto-
and acetic acid (2.5 μL, 0.044 mmol, 7 mol %). The resulting nitrile-d3 (300 μL) was added to each sample. The NMR mea-
yellowish mixture was refluxed for 10 min. The reflux was surements were performed by 1H or 19F detection at 298 K
stopped, and the mixture was allowed to stand at room tempera- using calibrated 90-degree pulses and one-scan experiments in
ture for 2 min. Then, a solution of N-acetylproline (100 mg, order to ensure complete pre-relaxation. The same acquisition
0.64 mmol) in chloroform (1.5 mL) was added. The mixture parameters were used for the octan-1-ol and water samples; for
was stirred at room temperature for the next 12 hours. Volatiles processing, only the zero phase was adjusted. The spectra were
were removed under reduced pressure; the crude material was baseline corrected, and equivalent resonances were integrated.
purified on a short (21 g) silica gel column using ethyl acetate/ The ratio between the signal intensities was considered as the
methanol 39:1 mixture as the eluent. 132 mg of 4 was obtained partitioning constant. The partitioning experiments were per-
as a clear oil (yield 94%), Rf = 0.69 (ethyl acetate/methanol formed in triplicate. Subsequent addition of water and octan-1-
39:1).
ol was performed in forward and reverse manner, and the result-
ing partitioning constants were identical within experimental
The procedure in acetonitrile/water was performed using same error.
proportions as those in the chloroform reaction. The amine and
nitrite were mixed in acetonitrile (2.5 mL), followed by the ad- Conformational analysis
dition of N-acetylproline (2 mg) for activation. The mixture was The amide rotational thermodynamic and kinetic measurements
refluxed for 10 min, and after the heating was stopped, this were performed by NMR as described in [50,68]. The equilib-
solution was added to a solution of N-acetylproline in water rium ratio between the rotameric states was obtained from the
(2.5 mL). The mixture was stirred at room temperature for 1H, 19F and 19F{1H} (inverse-gated decoupling) spectra at
16 hours. Then, trifluoroacetic acid (0.1 mL) was added, and the 298 K by integration. The kinetics was measured in 1H and/or
mixture was stirred for an additional hour to quench residual 19F{1H} z-cross-relaxation experiments at either 310 K (for
diazo compound. The solution was then freeze-dried. Purifica- deuterium oxide samples) or 298 K (for benzene samples). The
tion of the crude material on a silica gel column afforded 30 mg Jαβ-coupling values were obtained by visual inspection of the
of 4 as a clear oil (yield 20%). When the diazo compound was α-CH resonances in the 1H NMR spectra, and the accuracy was
generated in an acetonitrile/water (1:1) mixture (4.5 mL), no defined by the length of the time domain spectrum at approxi-
heating was applied, and N-acetylproline was added to the mately 0.3 Hz. The semi-empirical calculations were per-
yellowish mixture 5 min after mixing the amine with the nitrite. formed by using the PM6 algorithm from the MOPAC package.
From this run, 21 mg of 4 was isolated after silica gel purifica- DFT geometry optimization of the 5 amide rotamers was per-
tion (yield 15%).
formed using B88-PW91 GGA with the DZVP basis set.
Physical chemistry
Characterization of model compounds
Hydrolytic stability
1H and 13C NMR spectra were assigned using 1H{19F} inverse-
Aqueous potassium phosphate buffer (100 mM) at pH 11.0 or gated decoupled, 1H{13C} dept45, 1H{13C} HSQC,
8.0 was mixed with 1:10 (v/v) deuterium oxide to give 550 μL 1H-13C HMBC, 1H NOESY/EXSY and 19F{1H} HOESY ex-
of 91 mM buffer. The analyte 1–5 was added from a 200 mM periments. For the minor s-cis conformation, only chemical
stock solution in deuterium oxide to give 0.5 mM analyte. The shifts and, when possible, multiplicities are given. The enan-
first NMR spectra were collected within 4 min following addi- tiomeric ratio (er) was measured in 19F{1H} inverse-gated
tion of the analyte. The monitoring was continued by recording decoupled NMR spectra of the dichloromethane-d2 solutions
1H and 19F NMR spectra at regular time points until >50% containing 40 mM analyte (550 μL) recorded upon addition of
hydrolysis. Ambient temperature was maintained at 298 ± 1 K. an equivalent amount of europium(III) tris-[3-(heptafluoro-
The decay of the integral intensity of the analyte was quantified propylhydroxymethylene)-D-camphorate] in the same solvent
by integration. Plotting the logarithm of the intensity versus (200 μL) as the shifting reagent. The spectra were measured at
time delivered the kinetic constant, which was converted to the 298 K.
2452

Beilstein J. Org. Chem. 2017, 13, 2442–2457.
Methyl N-acetylprolinate (1): 1H NMR (D2O, 700 MHz) = 47, 30 Hz, s-cis); HRMS (ESI-orbitrap): [M + H]+ calcd for
δ s-trans, 4.37 (dd, JHH = 8.5, 4.7 Hz, α-CH), 3.69 (s, 3H, C9H15FNO3, 204.1030; found, 204.1028; [α]D25 −45 (c 2.0,
OCH3), 3.60 and 3.56 (two m, 2H, δ-CH2), 2.23 and 1.95 (two CHCl3), er 74:26.
m, β-CH2), 2.05 (s, 3H, CH3C=O), 1.94 (m, 2H, γ-CH2); s-cis,
4.63 (dd, JHH = 8.7, 2.5 Hz, α-CH), 3.73 (s, OCH3), 3.47 (ddd, 2,2-Difluoroethyl N-acetylprolinate (4): 1H NMR (D2O,
JHH = 11.6, 8.5, 3.5 Hz, δ-CH), 3.41 (dt, JHH = 11.4, 8.4 Hz, 500 MHz) δ s-trans, 6.07 (tt, JHH = 3.4 Hz, JHF = 54 Hz, 1H,
δ-CH), 2.27 and 2.15 (two m, β-CH2), 1.93 (s, CH3C=O), 1.89 CHF2), 4.43 (dd, JHH = 9.3, 4.7 Hz, 1H, α-CH), 4.38 (ddt, JHH
and 1.79 (two m, γ-CH2); 13C{1H} NMR (D2O, 126 MHz) = 3.4, 2.1 Hz, JHF = 15 Hz, 2H, OCH2), 3.61 and 3.58 (two m,
δ s-trans, 175.0 (s, CO2), 173.0 (s, N-C=O), 59.0 (s, α-CH), 2H, δ-CH2), 2.27 and 1.99 (two m, 2H, β-CH2), 2.05 (s, 3H,
52.9 (s, OCH3), 48.4 (s, δ-CH2), 29.2 (s, β-CH2), 24.2 (s, CH3C=O), 1.96 (m, 2H, γ-CH2); s-cis, 6.11 (tt, JHH = 3.2 Hz,
γ-CH2), 21.2 (s, CH3); s-cis, 174.6 (s, CO2), 173.4 (s, N-C=O), JHF = 54 Hz, CHF2), 4.74 (dd, JHH = 8.8, 2.5 Hz, α-CH), 4.44
60.7 (s, α-CH), 53.2 (s, OCH3), 46.6 (s, δ-CH2), 30.6 (s, (m, OCH2), 3.50 (ddd, JHH = 11.6, 8.6, 3.4 Hz, δ-CH), 3.43 (dt,
β-CH2), 22.3 (s, γ-CH2), 21.2 (s, CH3); HRMS (ESI-orbitrap): JHH = 11.5, 8.3 Hz, δ-CH), 2.32 and 2.20 (two m, β-CH2), 1.95
[M + H]+ calcd for C8H14NO3, 172.0968; found, 172.0968; (s, CH3C=O), 1.92 and 1.80 (two m, γ-CH2); 13C{1H} NMR
[α]D25 −83 (c 2.0, CHCl3).
(D2O, 126 MHz) δ s-trans, 173.4 (s, CO2), 173.0 (s, N-C=O),
113.0 (t, JCF = 240 Hz, CHF2), 63.0 (t, JCF = 27 Hz, OCH2),
Ethyl N-acetylprolinate (2): 1H NMR (D2O, 700 MHz) 58.9 (s, α-CH), 48.4 (s, δ-CH2), 29.2 (s, β-CH2), 24.3 (s,
δ s-trans, 4.34 (dd, JHH = 8.7, 4.5 Hz, α-CH), 4.15 (m, 2H, γ-CH2), 21.1 (s, CH3); s-cis, 173.4 (s, N-C=O), 173.0 (s, CO2),
OCH2), 3.59 and 3.56 (two m, 2H, δ-CH2), 2.24 and 1.95 (two 113.0 (m, CHF2), 63.3 (d, JCF = 27 Hz, OCH2), 60.6 (s, α-CH),
m, β-CH2), 2.04 (s, 3H, CH3C=O), 1.94 (m, 2H, γ-CH2), 1.19 46.6 (s, δ-CH2), 30.6 (s, β-CH2), 22.3 (s, γ-CH2), 21.2 (s, CH3);
(t, JHH = 7.2 Hz, 3H, CH3); s-cis, 4.61 (dd, JHH = 8.8, 2.4 Hz, 19F NMR (D2O, 471 MHz) δ −126.92 (dtd, JFF = 292 Hz, JFH =
α-CH), 4.20 (q, JHH = 7.2 Hz, OCH2), 3.47 (ddd, JHH = 11.5, 53, 15 Hz, 1F, s-trans), −126.93 (dtd, JFF = 292 Hz, JFH = 53,
8.7, 3.7 Hz, δ-CH), 3.41 (dt, JHH = 11.5, 8.3 Hz, δ-CH), 2.28 15 Hz, 1F, s-trans), −127.20 (dt, JFH = 54, 15 Hz, s-cis); HRMS
and 2.15 (two m, β-CH2), 1.93 (s, CH3C=O), 1.90 and 1.80 (ESI-orbitrap): [M + H]+ calcd for C9H14F2NO3, 222.0936;
(two m, γ-CH2); 13C{1H} NMR (D2O, 176 MHz) δ s-trans, found, 222.0933; [α]D25 −76 (c 2.0, CHCl3).
174.7 (s, CO2), 173.0 (s, N-C=O), 62.6 (s, OCH2), 59.3 (s,
α-CH), 48.5 (s, δ-CH2), 29.3 (s, β-CH2), 24.3 (s, γ-CH2), 21.2 2,2,2-Trifluoroethyl N-acetylprolinate (5): 1H NMR (D2O,
(s, CH3), 13.2 (s, CH3); s-cis, 174.2 (s, CO2), 173.3 (s, N-C=O), 700 MHz) δ s-trans, 4.78 (q, JHF = 8.7 Hz, 2H, ΟCH2), 4.59
63.0 (s, OCH2), 60.8 (s, α-CH), 46.7 (s, δ-CH2), 30.7 (s, (dd, JHH = 9.2, 4.7 Hz, 1H, α-CH), 3.63 and 3.59 (two m, 2H,
β-CH2 ), 22.3 (s, γ-CH2 ), 21.2 (s, CH3 ), 13.2 δ-CH2), 2.29 and 2.01 (two m, 2H, β-CH2), 2.06 (s, 3H,
(s, CH3); HRMS (ESI-orbitrap): [M + H]+ calcd for CH3C=O), 1.98 (m, 2H, γ-CH2); s-cis, 4.78 (dd, JHH = 8.8, 2.4
C9H16NO3, 186.1125; found, 186.1120; [α]D25 −81 (c 2.0, Hz, α-CH), 4.71 (m, OCH2), 3.50 (ddd, JHH = 11.6, 8.8, 3.3 Hz,
CHCl3).
δ-CH), 3.43 (dt, JHH = 11.5, 8.1 Hz, δ-CH), 2.33 and 2.22 (two
m, β-CH2), 1.97 (s, CH3C=O), 1.94 and 1.82 (two m, γ-CH2);
2-Fluoroethyl N-acetylprolinate (3): 1H NMR (D2O, 13C{1H} NMR (D2O, 176 MHz) δ s-trans, 173.4 (s, N-C=O),
500 MHz) δ s-trans, 4.63 (dm, JHF = 47 Hz, 2H, CH2F), 4.41 172.6 (s, CO2), 122.9 (q, JCF = 277 Hz, CF3), 60.9 (q, JCF = 36
(dd, JHH = 9.1, 4.6 Hz, 1H, α-CH), 4.37 (ddd, JHH = 5.1, 3.0 Hz, OCH2), 58.8 (s, α-CH), 48.3 (s, δ-CH2), 29.1 (s, β-CH2),
Hz, JHF = 30 Hz, 2H, OCH2), 3.62 and 3.59 (two m, 2H, 24.3 (s, γ-CH2), 21.1 (s, CH3); s-cis, 173.4 (s, N-C=O), 172.2
δ-CH2), 2.26 and 1.98 (two m, 2H, β-CH2), 2.05 (s, 3H, (s, CO2), 122.9 (q, JCF = 276 Hz, CF3), 61.3 (d, JCF = 36 Hz,
CH3C=O), 1.96 (m, 2H, γ-CH2); s-cis, 4.69 (dd, JHH = 8.9, 2.7 OCH2), 60.5 (s, α-CH), 46.6 (s, δ-CH2), 30.6 (s, β-CH2), 22.3
Hz, α-CH), 4.65 (m, CH2F), 4.43 (m, OCH2), 3.49 (ddd, JHH = (s, γ-CH2), 21.2 (s, CH3); 19F NMR (D2O, 471 MHz) δ −73.6
11.6, 8.6, 3.5 Hz, δ-CH), 3.42 (dt, JHH = 11.4, 8.2 Hz, δ-CH), (t, JFH = 9 Hz, s-trans), −73.7 (t, JFH = 8 Hz, s-cis); HRMS
2.31 and 2.20 (two m, β-CH2), 1.95 (s, CH3C=O), 1.91 and 1.81 (ESI-orbitrap): [M + H]+ calcd for C9H13F3NO3, 240.0842;
(two m, γ-CH2); 13C{1H} NMR (D2O, 126 MHz) δ s-trans, found, 240.0837; [α]D25 −13 (c 2.0, CHCl3), er 59:41.
174.2 (s, CO2), 173.0 (s, N-C=O), 81.9 (d, JCF = 165 Hz,
CH2F), 64.9 (d, JCF = 19 Hz, OCH2), 59.1 (s, α-CH), 48.4 (s, Synthesis of the peptides
δ-CH2), 29.2 (s, β-CH2), 24.3 (s, γ-CH2), 21.2 (s, CH3); s-cis, Ac(Pro)6OH (8a) and Ac(εBocLys)nOH (n = 3, 6) peptides
173.8 (s, CO2), 173.4 (s, N-C=O), 82.3 (m, CH2F), 65.2 (d, JCF were synthesized on pre-loaded 2-chlorotrityl resin convention-
= 19 Hz, OCH2), 60.7 (s, α-CH), 46.6 (s, δ-CH2), 30.6 (s, ally. The esterification with 2,2-difluorodiazoethane was per-
β-CH2), 22.3 (s, γ-CH2), 21.2 (s, CH3); 19F NMR (D2O, formed as described in [46]. Alternatively, the procedure was
471 MHz) δ −224.3 (tt, JFH = 47, 30 Hz, s-trans), −224.6 (tt, J performed as follows: tert-Butyl nitrite (≈ 6 μL) and 2,2-
2453

Beilstein J. Org. Chem. 2017, 13, 2442–2457.
difluoroethylamine (≈ 11 μL) were mixed in chloroform projection peak. Experimental logD values were compared to
(100 μL), and this mixture was shaken at room temperature for the calculated ones (Equation 5).
10 min. It was then added to the peptide (25 μmol; in this exam-
ple 18.4 mg of Ac(εBocLys)3OH) soaked in chloroform Circular dichroism spectra were recorded in a 1 mm quartz cell
(100 μL). The resulting mixture was shaken at room tempera- at 298 K in methanol (HPLC grade) or aqueous potassium phos-
ture for approximately 10 hours. Volatiles were removed with phate buffer (50 mM, pH 7.01 at 297 K). The peptide concen-
nitrogen gas, and the residue was freeze-dried from an aceto- tration was 100 μM as determined gravimetrically. The mean
nitrile/water mixture to afford the target peptides. The Boc-pro- residue absorption difference Δε was calculated assuming
tected peptides were purified on a silica gel column using a 6 residues for peptides 8a–c, 10a–c and 3 residues for peptides
dichloromethane/methanol 9:1 → 1:0 gradient elution. The Boc 9a–c. Hydrolysis was measured in deuterium oxide solution of a
deprotection was performed as follows. The peptide (3–4 mg) 150 mM potassium phosphate buffer. The buffer pH 7.01 was
was mixed with 4 M hydrogen chloride in dioxane (100 μL), adjusted in water at 298 K. It was then lyophilized, then dis-
and the turbid mixture was shaken at room temperature for solved in deuterium oxide. The peptides were dissolved in the
20 min. Water (100 μL) was added, resulting a clear solution, buffer, and the resulting samples were kept at 298 ± 2 K, while
which was shaken for an additional 20 min. The mixture was the 19F NMR measurements were conducted at several time
then freeze-dried from water to afford target peptides 9a,b or points. The starting concentrations of the analytes were 8 mM
10a,b.
for 8b, 5 mM for 9b and 2.5 mM for 10b. The half-life was
calculated using the first (zero) order kinetic model.
The esterification of 8a with methanol was performed as
follows. Ac(Pro)6OH (8a, 5.6 mg) was dissolved in methanol Ac(Pro)6OH, 8a. HRMS: calcd for [M + H]+ 643.3450, for
(0.5 mL), and trimethylsilyl chloride (0.08 mL) was added. The [M + Na]+ 665.3269; found, 643.3443 and 665.3257; logD,
mixture was shaken at room temperature for 24 hours. Vola- calcd for [M] −9.462; found, −9.468 ± 0.026.
tiles were removed with nitrogen gas, and the residue
was freeze-dried from water. No HPLC purification Ac(Pro)6OCH2CHF2, 8b. HRMS: calcd for [M + H]+ 707.3574,
has been applied in order to avoid ion-pairing agent for [M + Na]+ 729.3394; found, 707.3566 and 729.3377; logD,
contaminations and hydrolysis of the esters. Additionally, calcd for [M] −9.475; found, −9.477 ± 0.011; τ½ = 152 ± 14
full conversion was observed in the synthesis of peptides 8b and (139 ± 20) days (buffered deuterium oxide, pH 7.0).
8c.
Ac(Pro)6OCH3, 8c. HRMS: calcd for [M + H]+ 657.3606, for
Oligolysine peptides were esterified as follows. A peptide [M + Na]+ 679.3426; found, 657.3599 and 679.3416; logD,
Ac(εBocLys)nOH (n = 3, 6; 3 mg) was dissolved in methanol calcd for [M] −9.465; found, −9.467 ± 0.027.
(1 mL), and trimethylsilyl chloride (0.08 mL) was added. The
mixture was shaken at ambient temperature for 20 hours. Sol- Ac(Lys)3OH∙3HCl, 9a. HRMS: calcd for [M + 3H]3+ 149.1093;
vent was removed with nitrogen gas, and the residues were found, 149.1096; MALDI–TOF: calcd for [M + H]+ 445.3;
freeze-dried from deuterium oxide (0.2 mL) to afford target found, 445.2; logD, calcd for [M + 3H+] −9.410; found, −9.442
peptides 9c, 10c.
± 0.025.
Characterization of the peptides
Ac(Lys)6OH∙6HCl, 10a. MALDI–TOF: calcd for [M + H]+
Mass spectra were recorded using high-resolution electrospray- 829.6; found, 829.5; logD, calcd for [M + 6H+] −9.499; found,
orbitrap mass spectrometry ionization (HRMS) for all peptides −9.607 ± 0.038.
except hexalysine peptides 10a–c, which produced no distin-
guishable molecular ions. For cationic peptides 9a–c and 10a–c, Ac(Lys)3OCH2CHF2∙3HCl, 9b. HRMS: calcd for [M + 3H]3+
additional MALDI–TOF analysis confirmed the molecular as- 170.4468; found, 170.4474; MALDI–TOF: calcd for [M + H]+
signment.
509.3; found, 509.2; logD, calcd for [M + 3H+] −9.429; found,
−9.439 ± 0.021; τ½ = 5.0 ± 0.3 (4.5 ± 0.5) days (buffered
Diffusion coefficients were determined in a 1H stimulated echo deuterium oxide, pH 7.0).
experiment with bipolar gradients and a spoil pulse. The
settings were as follows: 298 K, deuterium oxide, 700 MHz, Ac(Lys)6OCH2CHF2∙6HCl, 10b. MALDI–TOF: calcd for
Δ = 30 ms, δ/2 = 2 ms, spoil pulse 0.5 ms, 128 array experi- [M + H]+ 893.6; found, 893.6; logD, calcd for [M + 6H+]
ments with linear gradient increase. The reported error value is −9.510; found, −9.583 ± 0.025; τ½ = 4.0 ± 0.3 (3.7 ± 0.3) days
the distance between the midpoint and the top of the logD (buffered deuterium oxide, pH 7.0).
2454

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Supporting Information
17.Wadhwani, P.; Strandberg, E.; van der Berg, J.; Mink, C.; Bürck, J.;
Ciriello, R. A. M.; Ulrich, A. S. Biochim. Biophys. Acta, Biomembr.
2014, 1838, 940–949. doi:10.1016/j.bbamem.2013.11.001
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Ulrich, A. S. Biochim. Biophys. Acta, Biomembr. 2014, 1838,
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Supporting Information File 1
Amide equilibrium constants (Table S1) and copies of the
NMR and CD spectra.
[http://www.beilstein-journals.org/bjoc/content/
supplementary/1860-5397-13-241-S1.pdf]
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Acknowledgements
VK acknowledges DFG research group 1805 for a postdoctoral
position. Enamine Ltd. (Kyiv, Ukraine) is thanked for provi-
sion of 2,2-difluoroethylamine. Dr Oleg Babii (KIT, Karlsruhe)
is gratefully acknowledged for MALDI-TOF analysis.
22.Kirberger, S. E.; Maltseva, S. D.; Manulik, J. C.; Einstein, S. A.;
Weegman, B. P.; Garwood, M.; Pomerantz, W. C. K.
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ORCID® iDs
23.Salwiczek, M.; Nyakatura, E. K.; Gerling, U. I. M.; Ye, S.; Koksch, B.
Chem. Soc. Rev. 2012, 41, 2135–2171. doi:10.1039/C1CS15241F
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Ulrich, A. S. Org. Biomol. Chem. 2015, 13, 3171–3181.
doi:10.1039/C5OB00034C
Vladimir Kubyshkin - https://orcid.org/0000-0002-4467-4205
Nediljko Budisa - https://orcid.org/0000-0001-8437-7304
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