The use of 4,4,4-trifluorothreonine to stabilize extended peptide structures and mimic β-strands

Article abstract of DOI:10.3762/bjoc.13.276

Pentapeptides having the sequence R-HN-Ala-Val-X-Val-Leu-OMe, where the central residue X is L-serine, L-threonine, (2S, 3R)-L-CF₃-threonine and (2S, 3S)-L-CF₃-threonine were prepared. The capacity of (2S, 3S)- and (2S, 3R)-CF_3<

Full text of DOI:10.3762/bjoc.13.276

The use of 4,4,4-trifluorothreonine to stabilize extended
peptide structures and mimic β-strands
Yaochun Xu1, Isabelle Correia2, Tap Ha-Duong1, Nadjib Kihal1, Jean-Louis Soulier1,
Julia Kaffy1, Benoît Crousse1, Olivier Lequin*2 and Sandrine Ongeri*1
Full Research Paper
Open Access
Address:
Beilstein J. Org. Chem. 2017, 13, 2842–2853.
1Molécules Fluorées et Chimie Médicinale, BioCIS, Univ. Paris-Sud,
CNRS, Université Paris Saclay, 5 rue Jean-Baptiste Clément, 92296
Châtenay-Malabry Cedex, France and 2Sorbonne Universités, UPMC
Univ Paris 06, Ecole Normale Supérieure, PSL Research University,
CNRS, Laboratoire des Biomolécules, 4 place Jussieu, 75252 Paris
Cedex 05, France
doi:10.3762/bjoc.13.276
Received: 28 September 2017
Accepted: 13 December 2017
Published: 21 December 2017
This article is part of the Thematic Series "Organo-fluorine chemistry IV".
Guest Editor: D. O'Hagan
Email:
Olivier Lequin* - olivier.lequin@upmc.fr; Sandrine Ongeri* -
Sandrine.ongeri@u-psud.fr
© 2017 Xu et al.; licensee Beilstein-Institut.
License and terms: see end of document.
* Corresponding author
Keywords:
aggregation; beta-sheet; fluorine; peptide; unnatural amino acid
Abstract
Pentapeptides having the sequence R-HN-Ala-Val-X-Val-Leu-OMe, where the central residue X is L-serine, L-threonine, (2S,3R)-
L-CF3-threonine and (2S,3S)-L-CF3-threonine were prepared. The capacity of (2S,3S)- and (2S,3R)-CF3-threonine analogues to
stabilize an extended structure when introduced in the central position of pentapeptides is demonstrated by NMR conformational
studies and molecular dynamics simulations. CF3-threonine containing pentapeptides are more prone to mimic β-strands than their
natural Ser and Thr pentapeptide analogues. The proof of concept that these fluorinated β-strand mimics are able to disrupt
protein–protein interactions involving β-sheet structures is provided. The CF3-threonine containing pentapeptides interact with the
amyloid peptide Aβ1-42 in order to reduce the protein–protein interactions mediating its aggregation process.
Introduction
It is estimated that 20% of administered drugs contain fluorine hance the stability against enzymatic, chemical and thermal
atoms or fluoroalkyl groups, representing 150 fluorinated mole- denaturation while generally retaining the original structure and
cules, and this trend is expected to increase to about 30% in the biological activity [3,4]. Fluorinated amino acids can also
early future as a new generation of fluorinated compounds is be used as powerful 19F NMR probes for the study of
currently in Phase II−III clinical trials [1]. In parallel, pharma- protein–ligand interactions and enzymatic activities [5-8]. How-
ceutical peptides are attracting increasing interest as around 100 ever, the development of fluorinated peptides as drug candi-
peptides are on the pharmaceutical market [2]. Peptide fluori- dates seems to be largely under-exploited. Investigation on the
nation has appeared as a general and effective strategy to en- influence of a fluorinated substituent incorporated in the side-
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Beilstein J. Org. Chem. 2017, 13, 2842–2853.
chain of amino acids on peptide conformations has recently natural hexapeptide adopted a folded conformation while for the
raised attention [9]. While the effect of fluorinated analogs of trifluoromethylated analogue an extended backbone conforma-
hydrophobic aliphatic and aromatic amino acids has been tion predominated.
prominently studied, the influence of fluorinated polar amino
acids has been rarely explored. To our knowledge, only one ex- In the present study, our objective was to evaluate the capacity
ample of conformational studies of a peptide containing a of both (2S,3S)- and (2S,3R)-CF3-threonine analogues (the
(2S,3S)-CF3-threonine has been conducted by Kitamoto et al. (2S,3S)- analogue being the exact analogue of the natural threo-
[7,10]. These authors reported a significant conformational nine residue, see Figure 1A) to stabilize an extended structure
difference between an enkephalin-related hexapeptide deriva- when introduced in the central position of pentapeptides, with
tive and its fluorinated analogue containing a (2S,3S)-CF3-thre- the intent of designing inducer or stabilizer of β-strand mimics.
onine at its C-terminus. NMR studies demonstrated that the Indeed, β-strand mimics have a particular interest as ligand of
Figure 1: A) Natural threonine and its trifluoromethyl analogues sawhorse projections. B) Structure of Boc-protected pentapeptides 1a–4a and free
amine pentapeptides 1b–4b.
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β-sheet structures and as potential inhibitors of protein–protein the key intermediate 6 was obtained, as a mixture of two dia-
interactions involving β-sheet structures [11-13]. For example, stereoisomers (9:1, evaluated by 19F NMR) via a nucleophilic
β-strand mimics have been successfully introduced in inhibitors trifluoromethylation reaction of Ruppert’s reagent on the (R)-
of amyloid proteins aggregation characterized by ordered Garner’s aldehyde 5 in THF and in the presence of a catalytic
β-sheet structure assemblies [14,15]. In this context, we synthe- amount of TBAF. Benzylation of the alcohol of 6 was then per-
sized and analyzed, by NMR and molecular modeling, the con- formed to obtain the desired intermediate as two diastereoiso-
formational preferences of eight pentapeptides, containing a mers 7a and 7b that were easily separated at this stage by
L-serine, a L-threonine, a (2S,3R)-L-allo-CF3-threonine or a column chromatography. The major diastereomer 7a was used
(2S,3S)-L-CF3-threonine in the third position. Both N-Boc pro- in the following steps. Hydrolysis of the oxazolidine, followed
tected (compounds 1a–4a) and N-deprotected pentapeptides by Jones oxidation of the alcohol 8, allowed us to recover the
(1b–4b) were studied.
desired acid 9 in good yield (90%). The optical rotation of a
solution of the product 9 (2S,3R), dissolved in MeOH was
measured at 25 °C. The value obtained was equal to −13° and
Results and Discussion
Synthesis. First, we synthesized the two (2S,3R)- and (2S,3S)- opposite to the value (+13°) described by Zeng et al. [17] for
CF3-Thr analogues. An enantioselective synthesis of (2S,3R)- the enantiomer (2R,3S).
Boc-CF3-Thr was proposed in 2003 [16] from propargylic
alcohol in ten steps, based on the trifluoromethylation key step The synthesis of (2S,3S)-CF3-threonine has been described in
of 1-(((E)-3-bromoallyloxy)methyl)benzene to obtain (E)-1- several publications [7,10,19-22]. Among these approaches, we
benzyloxy-4,4,4-trifluoro-2-butene. The sequence then involved followed a general procedure to access to (2S,3S)-CF3-threo-
Sharpless asymmetric dihydroxylation, nucleophilic opening of nine through an aldol reaction of CF3CHO with the Ni(II) com-
cyclic sulfate with NaN3, palladium-catalyzed selective hydro- plex of the chiral Schiff base of glycine which was introduced
genation, and oxidation. Zeng et al. described the synthesis of by Belokon et al. [23,24]. The chiral auxiliary (S)-N-(2-
the enantiomer (2R,3S)-Boc-CF3-Thr(Bzl) in four steps from benzoylphenyl)-1-benzylpyrrolidine-2-carboxamide (11) was
the (S)-Garner’s aldehyde [17,18]. The enantiomer (2S,3R)- obtained in good yield starting from the N-benzylation of
Boc-CF3-Thr(Bzl) was not described by Zeng et al. However, L-proline in the presence of KOH, then activation of the
we decided to follow this more straightforward methodology carboxylic acid functionality of 10 using SOCl2 at low tempera-
and we have adapted Zeng’s synthesis starting from the (R)- ture, followed by condensation with 2-aminobenzophenone
Garner’s aldehyde. (2S,3R)-Boc-CF3-Thr(Bzl) was obtained (Scheme 2). Complexation of 11 with nickel nitrate and glycine
with satisfactory yields (Scheme 1). In this synthetic pathway, under basic conditions gave the nickel Schiff base complex 12
Scheme 1: Synthesis of (2S,3R)-Boc-CF3-Thr(Bzl) 9.
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in 71% yield as red crystals. The nucleophilic glycine equiva- and 17d respectively, in satisfactory yields (61–87%). The
lent 12 went through the aldol reaction with trifluoroacetalde- pentapeptides 18a–c and 4a were obtained by deprotecting
hyde to give complex 13 in moderate yield (66%). Further tetrapeptides 17a–d with TFA and then performing the cou-
hydrolysis of complex 13 led to the recovery of the chiral auxil- pling reaction with Boc-L-Ala-OH in the presence of HBTU/
iary 11 and release of free (2S,3S)-CF3-threonine whose dia- HOBt/DIPEA or DMTMM(Cl−)/NMM. Catalytic hydrogena-
stereoselectivity was determined to be about 96% by 19F NMR. tion, using 10% Pd/C or Pd(OH)2, under H2 atmosphere, gave
Although in most of the reported cases, the free amino acid was pentapeptides 1a–3a in moderate to quantitative yield. After
released into the aqueous phase, and then purified by ion- acidic removal of the Boc group, the pentapeptide salts 1b–4b
exchange chromatography, we purified the free (2S,3S)-CF3- were obtained in quantitative yield.
threonine by another way. We first managed to remove the
Ni(II) by addition of 2.0 equivalents of NaSCN and 4.0 equiva- Conformational studies. The conformational properties of the
lents of pyridine to form the complex Ni(Py)4(SCN)2, which eight pentapeptides (1a–4a and 1b–4b) were examined by
precipitated from the aqueous phase. After filtration, we pro- NMR analyses in a protic solvent, which is more challenging
tected the free amino acid using Boc2O under basic conditions. than in aprotic organic solvents for maintaining intramolecular
The Boc-(2S,3S)-CF3-threonine 14 was then purified by silica hydrogen bond network. Methanol was used because of the
column chromatography and was obtained in 43% yield after limited solubility of these compounds in aqueous solutions. The
three steps from 13 (Scheme 2).
1H and 13C chemical shifts of these pentapeptides were
assigned using 1D 1H, 2D 1H,1H-TOCSY, 2D 1H,1H-ROESY,
Classical peptide synthesis in solution was used to prepare 2D 1H,13C-HSQC, and 2D 1H,13C-HMBC spectra. The 1H and
pentapeptides 1a–4a (Scheme 3). Boc-L-Val-OH was activated 13C chemical shift assignments of the 8 pentapeptides at 298 K
by isobutylchloroformate (IBCF) and then coupled with L-Leu- are given in Tables S1–S8 (Supporting Information File 1). A
OMe to afford dipeptide 15. Acidic hydrolysis of 15 using TFA, single set of chemical shifts was observed for all deprotected
and coupling with the third amino acid (Boc-L-Ser(Bzl)-OH, pentapeptides 1b–4b, whereas for the Boc-protected pentapep-
Boc-L-Thr(Bzl)-OH, (2S,3R)-Boc-CF3-Thr, (2S,3S)-Boc-CF3- tides 1a–4a, two chemical shift sets could be detected. This
Thr), using HBTU/HOBt in the presence of DIPEA in DMF, chemical shift heterogeneity involved in particular the t-Bu
afforded tripeptides 16a–d in good yields (48–83%). The tri- protons of the Boc group and the amide proton of the residue
peptides 16a–d were deprotected using TFA, and the salt of the Ala1. The chemical shift set of weaker intensity was assigned
free amine was coupled to Boc-L-Val-OH using HBTU/HOBt/ more easily by cooling down to 271 K because of significant
DIPEA or DMTMM(Cl−)/NMM to afford tetrapeptides 17a–c broadening near room temperature. Exchange peaks were ob-
Scheme 2: Synthesis of (2S,3S)-Boc-CF3-Thr 14.
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Beilstein J. Org. Chem. 2017, 13, 2842–2853.
Scheme 3: Synthesis of pentapeptides 1a–4a and 1b–4b.
served on ROESY spectra at 271 K (300 ms mixing time), for residues Val2 and Val4 in all of the eight pentapeptides
proving that the two forms interconvert in a slow exchange (Table 1 and Table 2) supports the predominance of extended
regime on the 1H NMR time scale. This equilibrium was conformations, as shown by downfield shifted Hα protons (pos-
ascribed to the existence of the syn- and anti-rotamers of the itive CSD values between 0.09 and 0.22 ppm, Table 1) and
carbamate group. The more stable forms (about 85% popula- upfield shifted Cα carbons (negative CSD values in the range of
tion at 271 K) were assigned to anti-rotamers based on litera- −2.5 to −1.6 ppm, Table 2).
ture results [25].
The high propensity for exploring extended backbone confor-
Different NMR parameters were examined to analyze back- mations was further confirmed for these pentapeptides by the
bone conformational propensities, namely 1Hα and 13Cα chemi- analysis of Hα–HN ROE correlations, showing that sequential
cal shift deviations (CSD), vicinal 3JHN-Hα coupling constants, Hαi–HNi+1 ROEs have much higher intensities than intra-
Hα-HN ROE correlations and temperature coefficient (ΔδHN/ residual Hαi–HNi ROEs. Few sequential HN–HN ROEs with
ΔT) of the amide protons. The 1Hα and 13Cα chemical shift de- weak intensities could be observed, indicating that turn or
viations (CSD) from random coil values provide information on helical conformers are sparsely populated.
backbone conformational space for each amino acid [26-31].
The terminal Ala1 and Leu5 residues were excluded from this Because of its Karplus dependence upon main chain φ dihedral
CSD analysis because of the absence of a neighboring residue, angle, the vicinal 3JHN-Hα coupling constant is also a valuable
as well as fluorinated Thr residues because of the absence of descriptor of peptide backbone conformations [32]. The cou-
known random coil values. The analysis of 1Hα and 13Cα CSDs pling constants in all pentapeptides (Table 3) exhibit large
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Table 1: 1Hα chemical shift deviations (CSD) of residues in pentapeptides 1a–4a and 1b–4b in CD3OH (298 K).
Peptide
Boc-protected (1a–4a)
Non-protected (1b–4b)
Ala1
Val2
X3
Val4
Leu5
Ala1
Val2
X3
Val4
Leu5
R-Ala-Val-Ser-Val-Leu-OMe (1)
R-Ala-Val-Thr-Val-Leu-OMe (2)
−0.23
−0.20
0.09
0.13
0.01
0.04
0.15
0.13
0.09
0.11
−0.35
−0.41
0.14
0.14
0.04
0.05
0.15
0.12
0.08
0.07
R-Ala-Val-(2S,3R)-CF3-Thr-Val-Leu
-OMe (3)
−0.22
−0.19
0.09
0.13
–
–
0.17
0.15
0.07
0.09
−0.33
−0.35
0.14
0.22
–
–
0.18
0.17
0.07
0.08
R-Ala-Val-(2S,3S)-CF3-Thr-Val-Leu
-OMe (4)
Table 2: 13Cα chemical shift deviations (CSD) of residues in pentapeptides 1a–4a and 1b–4b in CD3OH (298 K).
Peptide
Boc-Protected (1a–4a)
Non-Protected (1b–4b)
Ala1
Val2
X3
Val4
Leu5
Ala1
Val2
X3
Val4
Leu5
R-Ala-Val-Ser-Val-Leu-OMe (1)
R-Ala-Val-Thr-Val-Leu-OMe (2)
−0.6
−0.9
-2.0
−1.6
−1.8
−2.1
−2.2
−2.8
−2.9
−2.1
−2.1
−1.7
−1.6
−1.8
−1.9
−2.2
−2.3
−2.8
−2.9
−2.0
R-Ala-Val-(2S,3R)-CF3-Thr-Val-Leu
-OMe (3)
−0.8
−0.8
−2.5
−1.6
−
−
−2.4
−1.9
−2.7
−2.9
−2.2
−2.1
−2.0
−2.0
−
−
−2.4
−2.4
−2.7
−2.8
R-Ala-Val-(2S,3S)-CF3-Thr-Val-Leu
-OMe (4)
Table 3: Coupling constants 3JHN–Hα (Hz) of residues in pentapeptides 1a–4a and 1b–4b in CD3OH (271 K for most residues, * indicates values
measured at 298 K).
Peptide
Boc-protected (1a–4a)
Non-protected (1b–4b)
Ala1
Val2
X3
Val4
Leu5
Ala1
Val2
8.3
X3
Val4
Leu5
R-Ala-Val-Ser-Val-Leu-OMe (1)
R-Ala-Val-Thr-Val-Leu-OMe (2)
6.8
8.2
7.5
8.9
7.8
–
–
7.6*
8.4
8.6*
8.7
7.6*
7.7
broad
peak
6.9*
8.3*
8.4*
8.8*
7.8*
R-Ala-Val-(2S,3R)-CF3-Thr-Val-Leu
-OMe (3)
7.1
6.8
8.9
7.7
9.1
8.6
9.2
9.0
7.6
7.7
–
–
8.7
8.9
9.0
9.0
8.2
8.9
7.4
7.6
R-Ala-Val-(2S,3S)-CF3-Thr-Val-Leu
-OMe (4)
values (6.8–9.2 Hz range), that are systematically higher than We next examined the values of vicinal 3JHα-Ηβ coupling con-
average values found in the coil library (6.1, 7.0, and 7.5 Hz for stants which yield information on side-chain χ1 dihedral angle
Ala, Leu and Val, respectively) [33]. This clearly reflects a space (Table 4) [34]. Most residues exhibit average values that
preference of all backbone dihedral angles φ for values within indicate conformational equilibria between different side-chain
the range of −160° to −110°, as expected for extended confor- rotamers. Notably, the (2S,3S)-CF3-Thr residue in peptides 4a
mations. The three central residues presented higher 3JHN-Hα and 4b has a small coupling constant, indicating a gauche rela-
coupling constants than terminal Ala1 and Leu5 residues, thus tionship between Hα and Hβ protons. The analysis of
demonstrating stronger extended conformational propensities. intraresidual and sequential Hβ-HN ROEs led to the identifica-
Interestingly, the conformation of the central residue gets more tion of the χ1 gauche+ (+60°) conformation as the major side-
extended upon substitution of Ser (3JHN-Hα of 7.5 Hz) by the chain rotamer. As the local conformational space appears to be
β-branched Thr residue (3JHN-Hα 8.4 Hz) and trifluoromethyla- more restricted for both backbone and side chain of (2S,3S)-
tion of Thr further stabilizes extended conformations (3JHN-Hα CF3-Thr residue, we further characterized its conformation by
between 8.6 and 9.1 Hz).
recording 1H,19F heteronuclear NOEs in 1D 1H{19F} and 2D
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Table 4: 3JHα–Hβ coupling constants (Hz) for peptides 1a–4a and 1b–4b in CD3OH. Coupling constants were extracted from 1D 1H spectra on multi-
plets of Hβ protons for Ala or Hα protons for other residues.
Peptide
Boc-protected (1a–4a)
Non-protected (1b–4b)
Ala1
Val2
X3
Val4
Leu5
Ala1
Val2
X3
Val4
Leu5
R-Ala-Val-Ser-Val-Leu-OMe (1)
R-Ala-Val-Thr-Val-Leu-OMe (2)
7.1
7.2
6.7
7.3
6/6
4.9
6.6
6.9
5.1/10.6
5.0/10.6
7.2
7.2
6.6
7.1
7.5/7.5
4.6
6.6
7.1
5.3/10.3
5.6/10.0
R-Ala-Val-(2S,3R)-CF3-Thr-Val-
Leu-OMe (3)
7.3
7.0
7.1
7.0
6.4
2.5
6.5
7.1
6/10
7.3
7.2
7.3
8.2
6.7
2.0
7.0
7.5
5.3/10.3
5.4/10.5
R-Ala-Val-(2S,3S)-CF3-Thr-Val-
Leu-OMe (4)
5.0/10.5
1H,19F-HOESY experiments (Figures S19 and S20, Supporting no long-range HN/HN or Hα/Hα ROEs could be detected in the
Information File 1). Heteronuclear NOEs involving the CF3 8 pentapeptides, indicating that transient intermolecular associa-
group confirmed the previous assignment of χ1 rotamer and tion, if any, is too fast to be detected by ROE magnetization
revealed an i/i+2 interaction with the Ala1 methyl group, as ex- transfer.
pected in peptides exploring major β-strand like conformations
(Figure S21, Supporting Information File 1).
In summary, the NMR analysis shows that the pentapeptides
with the sequence RNH-Ala-Val-X-Val-Leu-OMe (X = Ser,
The chemical shift of amide protons generally displays a tem- Thr, (2S,3R)-CF3-Thr and (2S,3S)-CF3-Thr) explore predomi-
perature dependence [35,36] which can be used to get informa- nantly extended backbone conformations in CD3OH. No major
tion on the presence and the stability of hydrogen bonds [37]. In difference could be observed between the Boc protected
aqueous and alcoholic solvents, small negative temperature pentapeptides 1a–4a and their respective deprotected amine an-
coefficients (ΔδHN/ΔT > –4.5 ppb K−1) usually characterize alogues 1b–4b. This β-propensity can be ascribed to the pres-
amide protons that are engaged in intramolecular hydrogen ence of two Val residues, as β-branched residues are known to
bonds, while more negative values (ΔδHN/ΔT < –6 ppb K−1) explore more extended conformations [38]. Such an effect is
rather indicate that they are exposed to solvent. The analysis of also observed for the central residue upon replacement of Ser by
the temperature coefficient of the amide bond NH protons the β-branched Thr residue and the incorporation of a trifluoro-
(ΔδHN/ΔT) reveals negative values in the range of −9.0 ppb/K methyl group in Thr or allo-Thr further increases the β-propen-
to −5.0 ppb/K for most protons, which indicates that they are sity of these residues. The presence of self-association involv-
not engaged in stable intra- (or inter-)molecular hydrogen bonds ing intermolecular β-sheet formation was not detectable. Never-
with carbonyl groups (Table 5). Interestingly, residue Val4 theless, the unique i/i+2 periodicity of amide proton tempera-
displays the smallest temperature coefficient (around ture coefficients in peptides 4a–4b incorporating the (2S,3S)-
–4.5 ppb K−1) in pentapeptides 4a and 4b while residue Val2 CF3-threonine residue might be explained by transient intermo-
shows intermediate values of −5.5 and −5.9 ppb K−1. This i/i+2 lecular β-strand contacts.
periodicity may reflect transient intermolecular β-strand
contacts involving hydrogen bonding through Val2 and Val4 In order to gain a more detailed insight into the structural be-
residues (Figure S16, Supporting Information File 1). However, havior of the pentapeptides according to their central fluori-
Table 5: Temperature coefficients Δδ/ΔT (ppb K−1) for HN protons in pentapeptides 1a–4a and 1b–4b in CD3OH (298 K).
Peptide
Boc-protected (1a–4a)
Non-protected (1b–4b)
Ala1
Val2
X3
Val4
Leu5
Ala1
Val2
X3
Val4
Leu5
R-Ala-Val-Ser-Val-Leu-OMe (1)
R-Ala-Val-Thr-Val-Leu-OMe (2)
−7.8
−7.4
−6.4
−5.8
−7.7
−7.5
−8.0
−7.6
−7.4
−7.9
−
−
−6.1
−5.5
−7.8
−8.1
−7.8
−8.0
−7.0
−7.7
R-Ala-Val-(2S,3R)-CF3-Thr-Val-Leu
-OMe (3)
−6.5
−7.4
−5.3
−5.9
−7.4
−8.9
−7.4
−4.5
−5.0
−9.0
−
−
−6.6
−5.5
−8.5
−8.3
−9.0
−4.6
−6.0
−7.9
R-Ala-Val-(2S,3S)-CF3-Thr-Val-Leu
-OMe (4)
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Beilstein J. Org. Chem. 2017, 13, 2842–2853.
nated or non-fluorinated residue, all-atom molecular dynamics
(MD) simulations were performed using the GROMACS 4.5
package, with the OPLS-AA force field in combination with the
SPC/E water model (for a complete description of the method,
see Supporting Information File 1).
The conformational ensembles generated for each of the eight
pentapeptides in water, were first characterized by the average
coupling constants 3JHN-Hα of their five residues and then com-
pared to available NMR measurements at 298 K (Figure S23,
Supporting Information File 1). Water solvent was chosen in
order to better anticipate the peptide conformations in a solvent
closer to physiological conditions. Nevertheless, we verified for
compounds 2b and 4b that the simulations conducted in MeOH
and in water were very similar (Figure S23, Supporting Infor-
mation File 1). Overall, the theoretical 3JHN-Hα coupling values
are in fair agreement with the experimental ones, indicating that
the peptide conformational ensembles were sampled quite faith-
fully by the MD trajectories. Excepting the first residue Ala1, all
the theoretical coupling constants have high values above 7 Hz,
confirming that the pentapeptides have locally extended back-
bone conformations. It could be noted that the 3JHN-Hα experi-
mental value of the central residue in compounds 3a, 3b, and 4b
are significantly higher than in the simulations. This discrep-
ancy between the NMR and MD 3JHN-Hα coupling values for
the fluorinated central residues indicates that their conforma-
tions are less frequently extended in the simulations than in ex-
periments. However, the 3JHN-Hα coupling constants alone
cannot unambiguously discriminate between α- or β-structures
for each residues and, above all, cannot determine the peptide
global structure. In that context, MD simulations can provide
useful complementary structural information.
Figure 2: Probability distribution of the peptide conformations as a
function of end-to-end distance (defined as the distance between the
nitrogen of residue Ala1 and the carbon of the C-terminal carbonyl).
tribution functions centered around −30° or +140°, respectively.
The probability of the three central residues to be in β-confor-
In particular, MD trajectories revealed significant differences mation is reported in Table 6 for all studied peptides. It can be
between the conformations of the fluorinated and non-fluori- seen that, except the (2S,3R)-CF3-Thr residue in the Boc-pro-
nated peptides. Indeed, when their end-to-end distances are tected peptide 3a, all central residues predominantly adopt local
analyzed (Figure 2), it can be noted that both the Boc-protected β-conformations, with probabilities ranging from 50 to 92%, in
and non-protected peptides 4a and 4b have significantly larger agreement with the NMR CSDs and 3JHN-Hα coupling constant
populations of extended conformations than the other three se- values. The probability of each peptide to have all its three
quences whose distributions are broader and shifted toward central residues in β-conformation (which is equal to the prod-
lower values.
uct of the three central residue probabilities) is a good indica-
tion of its propensity to adopt a global extended structure. Ac-
This global structural characteristic is reflected at a local level cording to this criterion, almost 50% of the 4a and 4b confor-
when the distributions of the backbone ψ dihedral angle values mations are globally extended, whereas less than 30% of the
are examined (Figure 3). In contrast with other peptides, all the other sequence conformations are in that case (Table 6).
three central ψ dihedral angles of peptides 4a and 4b clearly
have a higher propensity to populate the β basin (90° to 180°) The most prevalent conformations of each peptide were deter-
than the α region (−70° to +40°), endowing it with the afore- mined by clustering their conformational ensembles, using the
mentioned extended conformations. More specifically, the prob- “gromos” method implemented in GROMACS with a RMSD
ability of each residue to be in α- or β-conformation can be threshold of 0.2 nm. Visual inspections of the representative
quantified by calculating the area under the peaks of the ψ dis- structure of the most populated clusters (Figures S24 and S25,
2849

Beilstein J. Org. Chem. 2017, 13, 2842–2853.
Inhibition of Aβ1-42 fibrillization. In the frame of our interest
in modulators of protein–protein interactions involving β–sheet
structures, in particular in the field of Aβ1-42 peptide aggrega-
tion involved in Alzheimer’s disease [15,39-42], we evaluated
the activity of the pentapeptides on this process. The objective
of this preliminary study was to analyze the influence of the tri-
fluoromethyl group and of the propensity of the pentapeptides
to adopt an extended structure, on their ability to modulate
Aβ1-42 peptide aggregation. For that purpose, the classical
fibrillization assay was performed using thioflavin-T (ThT)
fluorescence spectroscopy [14,15,39-42]. The fluorescence
curve of the control peptide (Aβ1-42 10 µM, purple curve,
Figure S26, Supporting Information File 1) displayed a typical
sigmoid pattern with a lag phase corresponding to the nucle-
ation process, an elongation phase and a final plateau linked to
the morphology and the amount of fibrils formed at the end of
the aggregation process. Compounds 1a–4a and 1b–4b were
tested at compound/Aβ1-42 ratios of 10:1 and 1:1. None of the
Boc-N-protected pentapeptides 1a–4a displayed inhibitory ac-
tivity even at a 10:1 compound/Aβ1-42 ratio (data not shown)
while some N-deprotected compounds displayed inhibitory ac-
tivity at this ratio, by decreasing the fluorescence plateau at
40 hours (see Supporting Information File 1, Table S9). This
result is in accordance with our previous demonstration that a
free amine is crucial to establish ionic interactions with acidic
residues of Aβ1-42 [15,39-41]. No activity was observed at a 1:1
ratio for the fluorinated compounds 3b and 4b, while an
increase of the fluorescence plateau was observed in the pres-
ence of the Ser and Thr containing compounds 1b and 2b. At a
10:1 compound/Aβ1-42 ratio the less extended Ser containing
Figure 3: Probability distribution of the peptide dihedral angles ψ for
the three central residues Val2 (black), X3 (red) and Val4 (green).
Supporting Information File 1SI) confirm that the peptides 4a pentapeptide 1b was found to be inactive (Figure 4 and Table
and 4b visit extended β-strand-like structures more frequently S9, Supporting Information File 1). The Thr containing
than the other three which have higher propensities to form pentapeptide 2b reduced the fluorescence plateau intensity by
compact α-helix-like conformations.
22%, suggesting a slight reduction of the amount of fibrils
formed after 40 hours (Figure 4 and Table S9, Supporting Infor-
All together, the theoretical study shows that the replacement of mation File 1). The reduction of the fluorescence intensity after
the methyl group of the threonine side chain in the RNH-Ala- 40 hours was much more pronounced for the two CF3-Thr de-
Val-Thr-Val-Leu-OMe pentapeptide by a trifluoromethyl in- rivatives 3b and 4b, reaching 60% (Figure 4 and Table S9, Sup-
duces an increase of the population of global extended confor- porting Information File 1), indicating that the presence of
mations.
fluorine atoms probably increased the interaction of pentapep-
Table 6: Probability (%) of the three central residues of the eight studied peptides to be in β-conformation. The column P indicates the probability for
each peptide to have all its three central residues in β-conformation.
peptide
Boc-protected (1a–4a)
Non-protected (1b−4b)
Val2
X3
Val4
P
Val2
X3
Val4
P
RNH-Ala-Val-Ser-Val-Leu-OMe (1)
59.9
53.5
59.7
67.1
51.4
91.1
31.7
82.7
55.8
61.0
69.9
75.6
17.2
29.7
13.2
42.0
76.5
64.8
63.3
77.9
50.3
73.5
59.4
85.6
64.9
59.1
74.7
74.8
25.0
28.2
28.1
49.9
RNH-Ala-Val-Thr-Val-Leu-OMe (2)
RNH-Ala-Val-(3R)-CF3-Thr-Val-Leu-OMe (3)
RNH-Ala-Val-(3S)-CF3-Thr-Val-Leu-OMe (4)
2850

Beilstein J. Org. Chem. 2017, 13, 2842–2853.
tides with Aβ1-42 and their inhibitory effect on Aβ1-42 aggrega- interaction of the trifluoromethyl group with the Ala1 methyl
tion.
group side chain, as observed in 1H,19F heteronuclear NOEs in
1D 1H{19F} and 2D 1H,19F HOESY experiments. Thus, both
conformational studies demonstrated the trifluoromethyl effect
on peptide conformations that promotes an extended conforma-
tion in order to mimic a β-strand structure. Interestingly in the
MD results, we found that the deprotected pentapeptides 1b, 3b
and 4b showed increased propensities to adopt extended confor-
mations compared to the Boc-protected counterparts 1a, 3a and
4a (a similar propensity to be in β-conformation was observed
for 2a and 2b).
The structural information obtained in this study provides valu-
able insights to explore novel β-strand mimics containing tri-
fluoromethylated analogues of threonine as inhibitors of pro-
tein–protein interactions involving β-sheet structures. As a
proof of concept, we demonstrated that the incorporation of the
CF3-Thr residues in hydrophobic pentapeptides allowed their
interaction with the amyloid protein Aβ1-42, in order to reduce
its aggregation process. The inhibitory effect seems more pro-
nounced by combining both the use of extended pentapeptides
and the introduction of fluorine atoms. This positive effect of
the trifluoromethylation can be due to the increased polarity of
the hydroxy group in the CF3-Thr residue, acting as a β-sheet
Figure 4: Effects of compounds 1–4 on Aβ1-42 fibrillization assessed
by ThT-fluorescence spectroscopy at 10:1 compound/Aβ ratios (the
concentration of Aβ1-42 is 10 μM) and compared to the values ob-
tained for Aβ1-42 alone. (See Supporting Information File 1 for the
calculation of the change of fluorescence intensity at the plateau).
Conclusion
We synthesized eight pentapeptides 1a–4a and 1b–4b having breaker element and thus preventing the interactions between
the sequence RHN-Ala-Val-X-Val-Leu-OMe, where the central Aβ species [15].
residue X is L-serine, L-threonine, (2S,3R)-L-CF3-threonine
and (2S,3S)-L-CF3-threonine, respectively. The fluorinated The introduction of such fluorinated peptides in larger struc-
amino acid (2S,3R)-Boc-CF3-Thr(Bzl) was prepared through a tures, such as glycopeptide or β-hairpin compounds can be
nucleophilic trifluoromethylation of Ruppert’s reagent on the envisaged. Indeed we have previously demonstrated that small
(R) Garner’s aldehyde, while (2S, 3S)-Boc-CF3-Thr (mimic of peptides/peptidomimetics that displayed inhibitory activity at
the natural threonine) was obtained through the aldol reaction of high ratios show greater aggregation inhibitory activity at 1:1
trifluoroacetaldehyde with the Ni(II) complex of the chiral ratio or even less, when they are incorporated in such designed
Schiff base of glycine.
structures [15,39-41].
The conformational analysis of these pentapeptides was con-
ducted by the combined use of NMR spectroscopy and molecu-
lar dynamics simulations. NMR conformational studies showed
that the eight pentapeptides (1a–4a and 1b–4b) adopt mainly
extended backbone conformations in a polar solvent (CD3OH).
The MD simulated conformations were in fair agreement with
the NMR results. Overall we conclude that the CF3-Thr-con-
taining pentapeptides were experimentally found more extend-
ed than the L-Ser-, L-Thr derivatives, with the (2S,3S)-CF3-Thr-
residue more prone to induce extended conformations than the
(2S,3R)-CF3-Thr, as suggested by MD simulations. The temper-
ature coefficients observed in both Boc-protected and depro-
tected (2S,3S)-CF3-Thr pentapeptides (4a and 4b) suggest that
these pentapeptides could transiently form intermolecular
β-strand contacts. This higher propensity of 4a and 4b to adopt
extended structures can be explained by a strong hydrophobic
Supporting Information
Supporting Information File 1
Description of synthetic procedures and characterization of
compounds. Additional NMR data, computational methods
and additional figures and tables. Experimental procedure
for fluorescence-detected ThT binding assay and
representative curves of ThT fluorescence assays.
[http://www.beilstein-journals.org/bjoc/content/
supplementary/1860-5397-13-276-S1.pdf]
Acknowledgements
Central Glass Co. Ltd. is gratefully acknowledged for kindly
providing fluoral. The China Scholarship Council is thanked for
financial support for Yaochun Xu. The Laboratory BioCIS is a
2851

Beilstein J. Org. Chem. 2017, 13, 2842–2853.
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