Synthesis of protected enantiopure (R) and (S)-α-trifluoromethylalanine containing dipeptide building blocks ready to use for solid phase peptide synthesis

Article abstract of DOI:10.1007/s00726-016-2200-9

Considering the increasing importance of fluorinated peptides, the development of efficient and reliable synthetic methods for the incorporation of unnatural fluorinated amino acids into peptides is a current matter of interest. In this study, we report t

Full text of DOI:10.1007/s00726-016-2200-9

Amino Acids
DOI 10.1007/s00726-016-2200-9
ORIGINAL ARTICLE
Synthesis of protected enantiopure (R)
and (S)‑α‑trifluoromethylalanine containing dipeptide building
blocks ready to use for solid phase peptide synthesis
Emmanuelle Devillers1 · Julien Pytkowicz1 · Evelyne Chelain1 · Thierry Brigaud1
Received: 13 November 2015 / Accepted: 11 February 2016
© Springer-Verlag Wien 2016
Abstract Considering the increasing importance of fluori-
nated peptides, the development of efficient and reliable syn-
thetic methods for the incorporation of unnatural fluorinated
amino acids into peptides is a current matter of interest. In
this study, we report the convenient Boc/benzyl and Cbz/tert-
butyl protection of both enantiomers of the quaternarized
amino acid α-trifluoromethylalanine [(R)- and (S)-α-Tfm-
Ala]. Because of the deactivation of the nitrogen atom of
this synthetic amino acid by the strong electron withdrawing
trifluoromethyl group, the peptide coupling on this position
is a challenge. In order to provide a robust synthetic meth-
odology for the incorporation of enantiopure (R)- and (S)-α-
trifluoromethylalanines into peptides, we report herein the
preparation of dipeptides ready to use for solid phase pep-
tide synthesis. The difficult peptide coupling on the nitrogen
atom of the α-trifluoromethylalanines was performed in solu-
tion phase by means of highly electrophilic amino acid chlo-
rides or mixed anhydrides. The synthetic effectiveness of this
fluorinated dipeptide building block strategy is illustrated by
the solid phase peptide synthesis (SPPS) of the Ac-Ala-Phe-
(R)-α-Tfm-Ala-Ala-NH2 tetrapeptide.
Keywords Fluorinated amino acids ·
α-Trifluoromethylalanine · Dipeptides · Mixed anhydrides
activation · Solid phase peptide synthesis · Fluorinated
peptides
Abbreviations
α-Tfm-AAs α-Trifluoromethylamino acids
Boc
Tert-butyloxycarbonyl
Cbz
Carboxybenzyl
Fmoc
DIPEA
DMAP
HPLC
HATU
Fluorenylmethoxy carbonyl
N,N-Diisopropylethylamine
4-Dimethylaminopyridine
High performance liquid chromatography
N-[(dimethylamino)-1H-1,2,3-triazolo[4,
5-b]pyridino-1-ylmethylene]-N-methylmeth-
anaminium hexafluorophosphate
Isobutyl chloroformate
IBCF
NMM
SPPS
TFA
N-Methylmorpholine
Solid phase peptide synthesis
Trifluoroacetic acid
TIS
Triisopropylsilane
Introduction
Communicated by J. Bode.
Electronic supplementary material The online version of this
article (doi:10.1007/s00726-016-2200-9) contains supplementary
material, which is available to authorized users.
In the last decades, the incorporation of non-natural fluori-
nated amino acids into peptides or proteins became a the-
matic of major concern since the fluorine atom allows to
beneficially modify the chemical and biological proper-
ties of these molecules of high biological and medicinal
interest (Salwiczek et al. 2012). Depending on the fluo-
rine substitution site and (or) the nature of the fluorine-
containing amino acid, different issues are observed. For
examples, fluorinated analogues of aromatic amino acids
have been used to stabilize protein–protein interactions
* Julien Pytkowicz
julien.pytkowicz@u-cergy.fr
* Thierry Brigaud
thierry.brigaud@u-cergy.fr
1
Laboratoire de Chimie Biologique (LCB), EA4505,
Université de Cergy-Pontoise, 5 Mail Gay-Lussac, Neuville
sur Oise, 95000 Cergy-Pontoise cedex, France
1 3

E. Devillers et al.
by electrostatic attraction in a model peptide (Zheng and
Gao 2010) and to stabilize the folded secondary struc-
ture of a nuclease by increasing the hydrogen bond donor
character of a tyrosine (Thorson et al. 1995). The substi-
tution of the methyl group by a trifluoromethyl group on
the side chain of an aliphatic amino acid (Leu, Val) has
been used to increase the thermal stability of “leucine zip-
per” type dimers by fluorine–fluorine interactions (Bilgicer
et al. 2001; Tang et al. 2001) or increase the stability of an
alpha helix (Gottler et al. 2008a) or a beta strand (Gerling
et al. 2014; Horng and Raleigh 2003; Chiu et al. 2009) by
increasing the hydrophobicity and the steric hindrance of
the residues. Moreover it has been demonstrated than the
replacement of leucines and isoleucines of the antimicro-
bial peptide pexiganan could increase its metabolic stability
in the presence of a lipid bilayer (Gottler et al. 2008b). The
authors hypothesized a strong peptide-lipids auto assem-
bly favored by the presence of the fluorinated residues. In
other respect it has been shown that the 14-helix stability
of a model beta peptide could be greatly reinforced by the
presence of trifluoromethyl groups in β3 position, increas-
ing the H-bond donor character of the amide functional
group (Cho et al. 2014). Among the panel of fluorinated
amino acids reported in the literature, the Cα trifluorome-
thyl substituted amino acids (α-Tfm-AAs) are of particular
interest. The trifluoroalanine is quite unstable and readily
epimerizes because of the increased acidity of the proton
in α-position of both the trifluoromethyl and the carboxylic
groups. This important drawback is solved by the quater-
narization of the α-carbon avoiding the amino acid race-
mization and the dehydrofluorination reaction. However,
because of the poor nucleophilicity of the vicinal Nα limit-
ing the use of classical coupling reactions, there are only
few examples of peptides where the trifluoromethyl group
is introduced directly on the peptide backbone. Neverthe-
less, the connection of the strong electron withdrawing
and sterically hindered trifluoromethyl group directly on
the backbone provides important modifications in terms
of hydrophobicity, preferred conformations and increased
hydrogen bonding donating effect. Firstly synthesized
in their racemic form (Osipov et al. 1986; Burger and
Gaa 1990; Burger and Sewald 1990; Kukhar et al. 1990),
α-Tfm-AAs can now be prepared in their enantiopure form
(Smits et al. 2008; Acena et al. 2012). However, α-Tfm-
AAs have mainly been incorporated into peptides in their
racemic form. The poor nucleophilicity of the Nα and the
steric hindrance brought by the trifluoromethyl group
require harsh coupling conditions or completely new cou-
pling strategies (Hollweck et al. 1997; Burger et al. 1998)
limiting their use by the peptide chemists community.
To our knowledge, the incorporation of an enantiopure
α-Tfm-AA at its C and N position in a peptide chain has
been very scarcely reported in the literature (Koksch et al.
2004; Chaume et al. 2009; Botz et al. 2015). Considered
as an aminoisobutyric acid (Aib) fluorinated analogue, the
α-trifluoromethylalanine provided an improved resistance
towards chymotrypsin digestion when incorporated into a
model peptide (Koksch et al. 1997). This fluorinated amino
acid has also been used as a 19F NMR probe to study the
structuration and the orientation of the antimicrobial pep-
tide alamethicin in membranes (Maisch et al. 2009). For
this study, the α-trifluoromethylalanine has been incorpo-
rated in racemic form in tripeptide building blocks. After
separation via chromatography on silica gel and identifica-
tion of their configuration, these tripeptides were engaged
in the SPPS of the antimicrobial peptide. Since several
years, we are involved in the synthesis of enantiopure
α-Tfm-AAs (Huguenot and Brigaud 2006; Caupene et al.
2009; Simon et al. 2011; Chaume et al. 2009) in order to
investigate the physicochemical and biological conse-
quences of their incorporation into peptides. In this context
we showed that the incorporation of a α-Tfm-proline at
the N-terminal position of a MIF-1 analogue increased its
biological activity (Jlalia et al. 2013; Bocheva et al. 2013).
We wish now to develop convenient and efficient synthetic
routes for the incorporation of enantiopure α-Tfm-alanine
in peptides by solution or solid phase peptide synthesis
(SPPS). For this purpose we report herein the convenient
Boc/benzyl and Cbz/tert-butyl protection of both enan-
tiomers of α-Tfm-alanine and the synthesis of various
Fmoc-AA-α-Tfm-Ala-OH and Fmoc-AA-α-Tfm-Ala-OtBu
dipeptide building blocks ready to use for SPPS. Finally,
this strategy was illustrated by the SPPS incorporation of
(R)-α-Tfm-Ala into a model tetrapeptide.
Results and discussion
Orthogonal protection of (R)‑and (S)‑α‑Tfm‑alanine
Boc/benzyl protection
Multigram quantities of unprotected enantiopure (R)- and
(S)-α-Tfm-Ala (R)-1 and (S)-1 were conveniently prepared
following our previously described procedure involving a
Strecker-type reaction on (R)-phenylglycinol derived tri-
fluoromethyloxazolidines (Chaume et al. 2009). Surpris-
ingly, the direct esterification of the unprotected (R)-1 and
(S)-1 α-Tfm-Ala with benzyl alcohol in various standard
conditions only proceeded in extremely low yields. Thus
we chose to protect first the amino group with a Boc group
since the Boc/benzyl strategy is the most convenient for
liquid phase coupling reactions. The poor nucleophilic-
ity of the nitrogen atom did not allow the classical Boc
protection in the presence of Boc2O and an organic or an
inorganic base. After a short screening of solvents and
1 3

Synthesis of protected enantiopure (R) and (S)-α-trifluoromethylalanine containing dipeptide…
Scheme 1 Synthesis of Boc protected α-Tfm-Ala
Scheme 2 Synthesis of Cbz-protected α-Tfm-Ala
bases, we found out than the activation of Boc2O with a
submolar amount of DMAP was necessary to obtain the
Boc protected α-Tfm-Ala (R)-2 and (S)-2 in high yields
(Scheme 1). As the esterification of the Boc protected
α-Tfm-alanines with benzyl alcohol in various standard
conditions did not proceeded cleanly, we decided to take
advantage of the high acidity of the fluorinated carboxylic
acid to perform the alkylation of the carboxylate formed in
mild basic conditions. The benzylic esters (R)-3 and (S)-
3 were obtained in good yields by treatment of the Boc
protected α-Tfm-alanines with a slight excess of benzyl
bromide in the presence of potassium carbonate (Wipf
and Heimgartner 1987; Brasca et al. 2007; Li et al. 2011)
(Scheme 1).
Having in hand suitably protected α-Tfm-alanines
useful for peptide synthesis in solution, we directed our
efforts towards the solid phase peptide synthesis (SPPS).
Although we succeeded in the coupling of the unprotected
(R)-α-Tfm-Ala (R)-1 on the N-terminal position of a leu-
cine attached on a Wang resin, to date we failed in the
coupling of another Fmoc-amino acid on the deactivated
nitrogen atom of the fluorinated amino acid. To circum-
vent this lack of reactivity of the Tfm-alanine amino group
on resin, we designed to perform this difficult peptide cou-
pling in solution phase and to elaborate dipeptide build-
ing blocks suitably protected for solid phase peptide syn-
thesis. In order to synthesize these dipeptides we decided
first to prepare each orthogonally protected enantiomer of
Cbz-Tfm-Ala-OtBu.
Cbz/tert‑butyl protection
Unexpectedly, the tert-butylation reaction of the α-Tfm-
alanines 1 with isobutene or tert-butanol using standard
procedures of the literature failed to give the correspond-
ing esters in reasonable yield. Thus, the Cbz protection of
the nitrogen atom was achieved first (Li et al. 2013). The
α-Tfm-alanines (R)-1 and (S)-1 were N-protected in excel-
lent yields using benzyl chloroformate and potassium
carbonate in a water/dioxane mixture (Lai et al. 2005) to
give the Cbz-Tfm-Ala-OH (R)-4 and (S)-4 (Scheme 2).
As observed for the Boc protected α-Tfm-Ala, the stand-
ard protocols for the amino acids esterification reaction
using alcohols in acidic conditions were ineffective starting
from N-carboxybenzoyl protected Tfm-Ala (R)-4 or (S)-4.
However the alkylation of the amino acid carboxylate by
methyl iodide provided the Cbz-Tfm-Ala-OMe (R)-5 in a
good yield. Encouraged by this result we tried the substi-
tution on the sterically more hindered tert-butyl bromide.
Finally, this reaction took place in excellent yield using a
large excess of tert-butyl bromide and potassium carbonate
in the presence of one equivalent of triethylbenzylammo-
nium chloride (TEBAC) used to generate a looser and more
reactive ion pair (Ginisty et al. 2006) (Scheme 2).
1 3

E. Devillers et al.
Scheme 3 Synthesis of Fmoc-AA-(R)-α-Tfm-Ala-OH dipeptides
Synthesis of α‑Tfm‑Ala containing dipeptides designed
for solid phase peptide synthesis
chloride hydrolysis is difficult and requires tedious reverse
phase preparative HPLC purifications. For these reasons
we designed to perform the peptide coupling reactions at
the amino group on (R)- and (S)-α-trifluoromethylalanines
protected as their tert-butyl esters. The (R)-and (S)-α-Tfm-
Ala tert-butyl esters (R)-10 and (S)-10 were obtained in
nearly quantitative yields by hydrogenolysis of the Cbz-
protected α-Tfm-Ala (R)-6 and (S)-6. The highly volatile
tert-butyl esters were isolated as their hydrochlorides by a
short bubbling of gaseous hydrogen chloride in the crude
solution. Because of their low stability, the latter com-
pounds were directly engaged in the next coupling step.
The coupling reactions of various Fmoc-protected amino
acids was performed through mixed anhydride activa-
tion of the carboxylic acids with isobutylchloroformate in
the presence of N-methylmorpholine (Koksch et al. 1997;
Maisch et al. 2009). This activation mode is experimentally
more convenient to implement than the amino acid chloride
The coupling on the N-terminal position of the trifluoro-
methylalanine could be performed in high yield on the
unprotected fluorinated amino acid (Scheme 3). How-
ever this reaction required the prior preparation of highly
electrophilic Fmoc-protected amino acid chlorides (Patil
and Babu 2002; Chaume et al. 2013) and specific reaction
conditions (3 h at 100 °C in a sealed tube in acetonitrile).
After a reverse phase semi-preparative HPLC purification
the corresponding dipeptides (R)-7, (R)-8 and (R)-9 were
obtained in 85, 78 and 90 %, respectively (Scheme 3).
Although the dipeptides were obtained in good yields,
the main drawbacks of this methodology are: (1) it neces-
sitate a preliminary amino acid chloride preparation, (2) the
separation of the target free carboxylic dipeptide from the
remaining Fmoc-amino acid resulting from the amino acid
Table 1 Synthesis of of Fmoc-AA-α-Tfm-Ala-OtBu dipeptides
Entry
Fmoc-AA-OH
R1
R2
Product
Yield (%)
1
2
3
4
5
6
7
L-Ala
L-Ala
L-Phe
L-Pro
L-Pro
L-Leu
Aib
Me
CF3
Me
Me
CF3
Me
Me
CF3
Me
(R)-11
(S)-11
(R)-12
(R)-13
(S)-13
(R)-14
(R)-15
67
69
59
62
76
81
63
CF3
CF3
Me
CF3
CF3
1 3

Synthesis of protected enantiopure (R) and (S)-α-trifluoromethylalanine containing dipeptide…
Scheme 4 Orthogonal deprotection of the (R)‑12 dipeptide building block
Scheme 5 SPPS of the tetrapeptide 17
handling. The corresponding dipeptides were obtained in
quite good yields (Table 1). We can note that the yields are
similar regardless the enantiomer of the Tfm-Ala tert-butyl
ester (entries 1 and 2). Yields are also satisfactory in the
case of a cyclic amino acid such as proline (entries 4 and 5)
or bulky amino acid such as Aib (entry 7).
was carried out with the standard HATU activation in the
presence of DIPEA. A second Fmoc deprotection step
followed by the coupling of the dipeptide block (R)-16
allowed the efficient incorporation α-Tfm-Ala by SPPS.
After the coupling of the last amino acid, the acetylation
reaction with acetic anhydride and the cleavage from the
resin in TFA/TIS conditions gave the target tetrapeptide
Ac-Ala-Phe-(R)-Tfm-Ala-Ala-NH2 17 which was obtained
in 53 % yield after semi-preparative HPLC purification
(Scheme 5).
Solid phase peptide synthesis of a tetrapeptide
In order to demonstrate the practicability of the trifluo-
romethylalanine containing dipeptide building blocks, a
model tetrapeptide was prepared by SPPS. The dipeptide
Fmoc-Phe-(R)-Tfm-Ala-OtBu (R)-12 was treated with a
TFA/CH2Cl2 mixture (Scheme 4) to give quantitatively the
Fmoc-Phe-(R)-Tfm-AlaOH building block (R)-16 which
was engaged without further purification in the SPPS of the
tetrapeptide 17 (Scheme 5).
The Rink amide resin is commonly used in SPPS proto-
cols to prepare peptide amides from Fmoc-protected amino
acids. The peptide sequence is assembled under basic or
neutral conditions on Rink amide resin, and then the com-
pleted peptide is cleaved from the Rink resin under acidic
conditions. We have used these conditions for the synthesis
of the tetrapeptide Ac-Ala-Phe-(R)-Tfm-Ala-Ala-NH2 17.
After the Fmoc deprotection of the Fmoc-Rink-amide resin
using piperidine in DMF, the coupling of Fmoc-Ala-OH
Conclusion
We have reported the convenient Boc/benzyl and Cbz/tert-
butyl protection of both enantiomers of the fluorinated
quaternary amino acid α-trifluoromethylalanine (α-Tfm-
Ala). A strategy for the challenging SPPS incorpora-
tion of these hindered and deactivated amino acids into
peptides has been developed. Fmoc-protected dipeptides
incorporating the (R)- or (S)-α-trifluoromethylalanine
were efficiently prepared in solution phase and proved to
be highly valuable building blocks for the incorporation
of α-trifluoromethylalanines into peptides by SPPS. This
strategy was exemplified by the synthesis of a model tetra-
peptide. The use of the dipeptide block strategy constitutes
1 3

E. Devillers et al.
a nice alternative to circumvent the weak reactivity of the
amino group of α-Tfm-amino acids in peptide coupling
reactions and will be useful for the incorporation of various
α-Tfm-amino acids in more complex peptides.
(q, J = 284.7 Hz, CF3); 156.9 (CO tBu); 171.4 (COOH).
19F NMR (CD3OD, 376.2 MHz): δ −80.3 (s, 3F, CF3). IR
(neat/cm−1): 3325, 3249, 2974, 1730, 1699, 1652, 1396,
1382, 1370, 1155. MS (EI): m/z = 256.1 [M−H]−. HRMS
(EI) m/z calcd for C9H13F3NO4 [M−H]− 256.0797 found
256.0788.
Materials and methods
Boc‑(S)‑α‑Tfm‑Ala‑OH, (S)-2
Unless otherwise mentioned, all the reagents were pur-
chased from commercial source. All glassware was dried
in an oven at 100 °C prior to use. THF was distilled under
nitrogen from sodium/benzophenone prior to use. CH2Cl2
By means of the procedure described for (R)-2 starting
from (S)-α-Tfm-Ala-OH (S)-1 (800 mg, 5.1 mmol, 1.0
equiv), the compound (S)-2 (5.0 mmol) was obtained as a
white solid in 98 % yield and was then used without puri-
fication. Mp: 100–102 °C. [α]2D0 = +5.4 (c = 1.0, MeOH).
1H NMR (CD3OD, 400 MHz): δ 1.47 (s, 9H, tBu); 1.61 (s,
3H, Hβ). 13C NMR (CD3OD, 100.5 MHz): δ 20.4 (Cβ);
29.4 (CtBu); 63.4 (q, J = 27.8 Hz, Cα); 82.1 (CtBu); 126.9
(q, J = 284.7 Hz, CF3); 156.9 (CO tBu); 171.4 (COOH).
19F NMR (CD3OD, 376.2 MHz): δ −80.3 (s, 3F, CF3). IR
(neat/cm−1): 3325, 3249, 2974, 1730, 1699, 1652, 1396,
1382, 1370, 1155.
1
was distilled under nitrogen from CaH2 prior to use. H
NMR spectra, 13C NMR spectra and 19F NMR spectra were
1
recorded on a JEOL ECX-400 (400 MHz H, 100.5 MHz
13C and 376.2 MHz 19F). Chemical shift values (δ) were
reported in ppm downfield from Me4Si (δ 0.0 ppm), CDCl3
(δ 77.0 ppm) or C6F6 (δ −164.9 ppm) as internal standard.
Data are reported as follows: chemical shift (δ ppm), mul-
tiplicity (s = singlet, d = doublet, t = triplet, q = quartet,
p = pentet, m = multiplet), coupling constant (Hz), integra-
tion. Infrared (IR-FT) spectra were performed on a Brucker
Tensor 27. Specific rotations were measured on a JASCO
P1010 or an Anton Paar MCP 200 polarimeter. Microa-
nalysis and HRMS analyses were performed by the Service
Central d’Analyses of CNRS, or by IMAGIF/ICSN-CNRS.
Melting points (uncorrected) were measured in capillary
tubes on a Büchi apparatus. Column chromatography was
performed on 60A (40–63 μm) silica gel, employing mix-
ture of specified solvent as eluent. Silica TLC plates were
visualized under UV light, by a 10 % solution of phospho-
molybdic acid in ethanol followed by heating.
Boc‑(R)‑α‑Tfm‑Ala‑OBn, (R)-3
To a solution of Boc-(R)-α-Tfm-Ala-OH (R)-2 (770 mg,
3.0 mmol, 1.0 equiv) in DMF (8 mL) were successively
added K2CO3 (497 mg, 3.6 mmol, 1.2 equiv) and benzyl
bromide (427 μL, 1.2 equiv). The reaction mixture was
stirred at room temperature for 3 h and then diluted with
water (15 mL). The aqueous mixture was extracted with
AcOEt (3 × 30 mL) and the combined organic extracts
were dried over anhydrous MgSO4 and evaporated under
vacuum. The crude product was purified by silica gel chro-
matography (cyclohexane/AcOEt, 98/2–95/5) to afford
Boc-(R)-α-Tfm-Ala-OBn (R)-3 (854 mg, 2.46 mmol) as a
white solid in 82 % yield. Rf: 0.72 (cyclohexane/AcOEt,
Boc‑(R)‑α‑Tfm‑Ala‑OH, (R)-2
To a solution of (R)-α-Tfm-Ala-OH (R)-1 (700 mg,
4.5 mmol, 1.0 equiv) in THF (15 mL) was added DMAP
(163 mg, 1.3 mmol, 0.3 equiv). The resulting mixture
was stirred for 10 min at room temperature. Di-tert-
butyldicarbonate (1.24 mL, 5.4 mmol 1.2 equiv) was then
added slowly and the solution was stirred for 3 h at room
temperature. After completion of the reaction, the reac-
tion mixture was evaporated under vacuum and dissolved
in AcOEt (15 mL). The mixture was washed with a 1 M
HCl solution (10 mL) and the aqueous layer was then
extracted three times with AcOEt (3 × 15 mL). The com-
bined organic layers were dried over anhydrous MgSO4,
and evaporated under vacuum to give Boc-(R)-α-Tfm-
Ala-OH (R)-2 (1.102 g, 4.3 mmol) as a white solid in 96 %
yield. Mp: 107–108 °C. [α]2D0 = −5.7 (c = 0.7, MeOH).
1H NMR (CD3OD, 400 MHz): δ 1.47 (s, 9H, tBu); 1.62
(s, 3H, Hβ). 13C NMR (CD3OD, 100.5 MHz): δ 20.4 (Cβ);
29.4 (CtBu); 63.4 (q, J = 27.8 Hz, Cα); 82.1 (CtBu); 126.9
8/2). Mp: 94–95 °C. [α]2D0 = +3.7 (c = 1.0, CHCl3). H
1
NMR (CDCl3, 400 MHz): δ 1.41 (s, 9H, HBoc); 1.74 (s,
3 H, Hβ); 5.24 (s, 2H, CH2bzl); 7.35 (m, 5H, Haro). 13C
NMR (CDCl3, 100.5 MHz): δ 18.9 (Cβ); 28.4 (CHBoc);
62.5 (q, J = 28.8 Hz, Cα); 68.5 (Cbzl); 81.5 [C(CH3)3];
124.4 (q, J = 286.6 Hz, CF3); 128.6–128.9; 135.1 (Cq
bzl); 154.0 (CO bzl); 167.2 (CO tBu). 19F NMR (CDCl3,
376.2 MHz): δ −79.6 (s, 3F, CF3). IR (neat/cm−1): 3363,
3008, 1739, 1720, 1588. MS (EI): m/z = 370.1 [M+Na]+.
HRMS (EI) m/z calcd for C16H20F3NO4Na [M+Na]+
370.1242 found 370.1233.
Boc‑(S)‑α‑Tfm‑Ala‑OBn, (S)-3
By means of the procedure described for (R)-3 start-
ing from (S)-α-Tfm-Ala-OH (S)-2 (775 mg, 3.0 mmol,
1.0 equiv), the compound (S)-3 (900 mg, 2.6 mmol) was
1 3

Synthesis of protected enantiopure (R) and (S)-α-trifluoromethylalanine containing dipeptide…
obtained as a white solid in 86 % yield. Rf: 0.72 (Cyclohex-
ane/AcOEt, 8/2). Mp: 94–95 °C. [α]2D0 = –3.8 (c = 1.0,
CF3); 127.9; 128.8; 129.4; 137.7; 156.8 (CO Bzl); 170.1
(COOH). 19F NMR (CD3OD, 376.2 MHz): δ −80.1 (s, 3F,
CF3). IR (neat/cm−1): 3324, 3036, 2952, 1720, 1627, 1247.
MS (EI): m/z = 290.1 [M−H]−. HRMS (EI) m/z calcd for
C12H11F3NO4 [M−H]− 290.0640 found 290.0653.
1
CHCl3). H NMR (CDCl3, 400 MHz): δ 1.36 (s, 9 H,
HBoc); 1.74 (s, 3 H, Hβ); 5.24 (s, 2 H, CH2bzl); 7.35 (m,
5 H, Haro). 13C NMR (CDCl3, 100.5 MHz): δ 18.7 (Cβ);
28.4 (CHBoc); 62.5 (q, J = 28.8 Hz, Cα); 68.5 (Cbzl); 81.5
[C(CH3)3]; 124.4 (q, J = 286.6 Hz, CF3); 128.6–128.9;
135.1 (Cq bzl);154.0 (CO bzl); 167.2 (CO tBu). 19F NMR
(CDCl3, 376.2 MHz): δ −79.6 (s, 3 F, CF3). IR (neat/cm−1):
3363, 3008, 1739, 1720, 1588. MS (EI): m/z = 370.1
[M+Na]+. HRMS (EI): m/z calcd for C16H20F3NO4Na
[M+Na]+ 370.1242 found 370.1259.
Cbz‑(R)‑α‑Tfm‑Ala‑OMe (R)-5
To a solution of N-Cbz-(R)-α-Tfm-Ala-OH (R)-4 (1.5 g,
5.1 mmol, 1.0 equiv) in DMF (13 mL) was added K2CO3
(1.07 g, 6.1 mmol, 1.2 equiv). The mixture was stirred for
10 min at 0 °C and MeI (6.6 mmol, 1.3 equiv) was then
added slowly. The reaction mixture was stirred for 30 min
at 0 °C and 1 h at room temperature and was then diluted
with water (20 mL). The diluted mixture was extracted
with AcOEt (3 × 20 mL) and the combined organic lay-
ers were dried over anhydrous MgSO4 and evaporated
under vacuum. The crude product was purified by silica
gel chromatography (cyclohexane/AcOEt: 95/5–80/20)
to afford desired Cbz-(R)-α-Tfm-Ala-OMe (R)-5 (1.43 g,
4.7 mmol, 92 %) as a yellow oil. Rf: 0.68 (cyclohexane/
Cbz‑(R)‑α‑Tfm‑Ala‑OH, (R)-4
To a solution of enantiopure (R)-α-Tfm-Ala-OH (R)-
1 (500 mg, 3.2 mmol, 1.0 equiv) and K2CO3 (1.76 g,
12.7 mmol, 4.0 equiv) in water (9 mL) at 0 °C was added
dropwise CbzCl (1.14 mL, 8.0 mmol, 2.5 equiv, 1.4 M in
dioxane). The solution was stirred for 10 min at 0 °C and
3 h at room temperature. Another 2.5 equivalent (1.4 M
in dioxane) of CbzCl were added following the same pro-
cedure. After completion of the reaction, the solution was
washed with dichloromethane (3 × 3 mL). The aqueous
layer was acidified with 10 % HCl solution to pH 1–2 and
extracted with ethyl acetate (3 × 40 mL). The organic
layers were combined, dried over MgSO4, filtered and
evaporated under reduced pressure. The crude product
was purified by silica gel chromatography (DCM/MeOH
98/2) to afford Cbz-(R)-α-Tfm-Ala-OH (R)-4 (850 mg,
2.94 mmol) as a white solid in 92 % yield. Mp = 70–72 °C.
[α]2D0 = +67.2 (c = 1.3, MeOH). 1H NMR (CD3OD,
400 MHz): δ 1.68 (s, 3H, Hβ); 5.09 (m, 2H, CH2bzl); 7.28–
7.41 (m, 5H, Haro). 13C NMR (CD3OD, 100.5 MHz): δ
19.5 (Cβ); 62.7 (q, J = 30.7 Hz, Cα); 67.8 (Cbzl); 126.0 (q,
J = 284.7 Hz, CF3); 127.9; 128.8; 129.4; 137.7; 156.8 (CO
Bzl); 170.1 (COOH). 19F NMR (CD3OD, 376.2 MHz): δ
−80.1 (s, 3F, CF3). IR (neat/cm−1): 3324, 3036, 2952,
1720, 1627, 1247. MS (EI): m/z = 292.1 [M+H]+. HRMS
(EI) m/z calcd for C12H13F3NO4 [M+H]+ 292.0797 found
292.0801. Anal. calcd: %C: 49.49; %H: 4.15; %N: 4.81
found: %C: 49.27; %H: 4.04; %N: 4.71.
AcOEt, 7/3). [α]D20 = +2.4 (c = 1.0, CHCl3). H NMR
1
(CDCl3, 400 MHz): δ 1.78 (s, 3H, Hβ); 3.80 (s, 3H, OMe);
5.12 (s, 2H, CH2bzl); 6.00 (s, 2H, NH); 7.37–7.39 (m, 5H,
Har). 13C NMR (CDCl3, 100.5 MHz): δ 18.2 (Cβ); 53.2
(CO2CH3); 61.9 (q, J = 28.8 Hz, Cα); 67.2 (Cbzl); 123.9
(q, J = 285.6 Hz, CF3); 128.0; 128.2; 128.3; 135.5 (Car);
154.3 (CO Bzl); 167.3 (CO2CH3). 19F NMR (CDCl3,
376.2 MHz): δ −79.8 (s, 3F, CF3). IR (neat/cm−1): 3450–
3250, 1710, 1730, 1524, 1450. MS (EI): m/z = 306.1
[M+H]+. HRMS (EI): m/z calcd for C13H15F3NO4
[M+H]+ 306.0953 found 306.0960. Anal. calcd: %C:
51.15; %H: 4.62; %N: 4.59 found: %C: 51.17; %H: 4.63;
%N: 4.74.
Cbz‑(R)‑α‑Tfm‑Ala‑OtBu (R)-6
To a solution of enantiopure Cbz-(R)-α-Tfm-Ala-OH (R)-4
(650 mg, 2.2 mmol, 1.0 equiv) in acetonitrile (20 mL) were
successively added benzyl triethylammonium chloride
(505 mg, 2.2 mmol, 1.0 equiv), K2CO3 (6.17 g, 44.7 mmol,
20 equiv) and tert-butyl bromide (8.8 mL, 78.2 mmol, 35
equiv) at room temperature. The reaction mixture was then
heated to 48 °C and stirred for 20 h at 48 °C. The mixture
was cooled to room temperature and evaporated under vac-
uum. The crude product was diluted with water (20 mL)
and extracted with AcOEt (3 × 20 mL). The combined
organic layers were dried over anhydrous MgSO4, and con-
centrated under vacuum. The crude product was purified
by silica gel chromatography (cyclohexane/AcOEt, 90/10)
to afford enantiopure Cbz-(R)-α-Tfm-Ala-OtBu (R)-6
(729 mg, 2.1 mmol) as a white solid in 94 % yield. Rf: 0.78
(cyclohexane/AcOEt, 8/2). Mp: 103–104 °C. [α]2D0 = +5.2
Cbz (S)‑α‑Tfm‑Ala‑OH, (S)-4
By means of the procedure described for (R)-4 starting from
(S)-α-Tfm-Ala-OH (S)-1 (470 mg, 3.0 mmol, 1.0 equiv),
the compound (S)-4 (835 mg, 2.9 mmol) was obtained as
a white solid in 96 % yield. Mp: 71–73 °C. [α]2D0 = –65.4
1
(c = 1.2, MeOH). H NMR (CD3OD, 400 MHz): δ 1.68
(s, 3H, Hβ); 5.09 (m, 2H, CH2bzl); 7.28–7.41 (m, 5H,
Haro). 13C NMR (CD3OD, 100.5 MHz): δ 19.5 (Cβ); 62.7
(q, J = 30.7 Hz, Cα); 67.8 (Cbzl); 126.0 (q, J = 284.7 Hz,
1 3

E. Devillers et al.
1
(c = 1.0, CHCl3). H NMR (CDCl3, 400 MHz): δ 1.46
3J = 7 Hz, 1 H, CHFmoc); 4,23 (q, 3J = 7 Hz, 1 H, Hα1);
3
3
(s, 9H, HtBu); 1.79 (s, 3H, Hβ); 5.10 (s, 2H, CH2bzl);
5.67 (s, 1H, NH); 7.33–7.35 (m, 5H, Haro). 13C NMR
(CDCl3, 100.5 MHz): δ 18.0 (Cβ); 27.8 (CHtBu); 63.0
(q, J = 28.8 Hz, Cα); 67.4 (Cbzl); 84.6 [C(CH3)3]; 124.5
(q, J = 286.6 Hz, CF3); 128.5; 128.6; 128.8; 136.2; 154.6
(CObzl); 166.0 (COtBu). 19F NMR (CDCl3, 376.2 MHz):
δ −79.4 (s, 3F, CF3). IR (neat/cm−1): 3278, 1745, 1700,
1543. MS (EI): m/z = 370.1 [M+Na]+. HRMS (EI) calcd
for C16H20F3NO4Na [M+Na]+ 370.1242 found 370.1232.
Anal. calcd: %C: 55.33; %H: 5.80; %N: 4.03 found: %C:
55.27; %H: 5.81; %N: 3.89.
4,33 (t, J = 7 Hz, 2 H, CH2Fmoc); 7,29 (t, J = 7 Hz,
3
2 H, HFmoc); 7,36 (t, J = 7 Hz, 2 H, HFmoc); 7,64 (t,
3J = 8 Hz, 2 H, HFmoc); 7,76 (d, 3J = 8 Hz, 2 H, HFmoc);
8,74 (s, 1 H, NH) ppm. 13C NMR (100.5 MHz, CD3OD): δ
19.1 (Cβ1), 19.7 (Cβ2); 47.1 (CHFmoc); 50,2 (Cα1), 61.2
(Cα2, J = 28,8 Hz); 68.8 (CH2Fmoc); 121.7 (Car Fmoc);
124.6 (CF3, J = 284.7 Hz); 127.1 (Car Fmoc); 141.5 (Car
Fmoc); 143.8 (Car Fmoc); 144.0 (Car Fmoc); 156.9 (CO);
168.2 (CO)173.6 (CO2H) ppm. 19F NMR (376.2 MHz,
D2O): δ −80.3 (s, 3 F, CF3) ppm. IR (neat/cm−1): 3246,
2840, 2250, 2238, 1500. MS (EI): m/z (%) = 450.1 [M]+.
Cbz‑(S)‑α‑Tfm‑Ala‑OtBu, (S)-6
Fmoc‑Leu‑(R)‑α‑Tfm‑Ala‑OH, (R)-8
By means of the procedure described for (R)‑6 start-
ing from (S)‑4 (579 mg, 2.0 mmol), the compound (S)-
6 (635 mg, 1.8 mmol) was obtained as a white solid in
92 % yield. Rf: 0.78 (cyclohexane/AcOEt, 8/2). Mp: 102–
To a suspension of (R)-α-Tfm-Ala-OH (R)-1 (80 mg,
0.51 mmol, 1.0 equiv) in THF (4 mL), were added Fmoc-
Leu-Cl (216 mg, 0.61 mmol, 1.2 equiv) and K2CO3
(141 mg, 1.02 mmol, 2.0 equiv). The reaction mixture was
heated at 100 °C in a sealed tube for 1 h. The crude mixture
was then cooled to room temperature, and the K2CO3 was
filtered. The filtrated product was evaporated under vacuum
and purified by semi-preparative HPLC (water/acetonitrile
90/10–70/30) to afford the desired dipeptide Fmoc-Leu-
(R)-α-Tfm-Ala-OH (R)-8 (221 mg, 0.45 mmol, 88 %) as
a white hygroscopic solid. [α]2D0 = +6.3 (c = 0.9, MeOH).
103 °C. [α]2D0 = −6.7 (c = 1.0, CHCl3). H NMR (CDCl3,
1
400 MHz): δ 1.46 (s, 9H, HtBu); 1.80 (s, 3H, Hβ); 5.10 (s,
2H, CH2bzl); 5.60 (s, 1H, NH); 7.26–7.37 (m, 5H, Haro).
13C NMR (CDCl3, 100.5 MHz): δ 18.0 (Cβ); 27.9 (CHtBu);
63.1 (q, J = 28.8 Hz, Cα); 67.5 (Cbzl); 84.7 [C(CH3)3];
124.5 (q, J = 286.6 Hz, CF3); 128.5; 128.6; 128.9; 136.3;
154.6 (CO bzl); 166.0 (COtBu). 19F NMR (CDCl3,
376.2 MHz): δ −79.5 (s, 3F, CF3). IR (neat/cm−1): 3281,
1744, 1701, 1542. MS (EI): m/z (%) = 370.1 [M+Na]+.
HRMS (EI) m/z calcd for C16H20F3NO4Na [M+Na]+
370.1242 found 370.1239. Anal. calcd: %C: 55.33; %H:
5.80; %N: 4.03 found: %C: 55.53; %H: 5.88; %N: 3.91.
3
1H NMR (400 MHz, CD3OD): δ 0.88 (d, J = 5,5 Hz, 3
3
H, Hδ1); 0,91 (d, J = 5,5 Hz, 3 H, Hδ1′); 1,60 (m, 3 H,
3
Hβ1 et Hγ1); 1,69 (s, 3 H, Hβ2); 4,17 (t, J = 6.9 Hz, 1
H, HFmoc); 4,36 (m, 3 H, Hα1 et CH2Fmoc); 6,02 (s,
3
1 H, NH); 7,27 (t, J = 7.3 Hz, 2 H, HFmoc); 7,37 (t,
3
3J = 7.3 Hz, 2 H, HFmoc); 7,54 (t, J = 8.2 Hz, 2 H,
3
Syntheses of Fmoc‑AA‑(R)‑α‑Tfm‑Ala‑OH dipeptides
HFmoc); 7,72 (d, J = 7.3 Hz, 2 H, HFmoc) ppm. 13C
NMR (100.5 MHz, CD3OD): δ 18.6 (Cβ2), 22,1 (Cδ1);
22,9 (Cδ1′); 24,8 (Cγ1); 41,0 (Cβ1); 47,2 (CH Fmoc); 53,5
(Cα1); 62.1 (q, J = 29.7 Hz, Cα2), 68,1 (CH2Fmoc); 120,4
(Car Fmoc); 124,1 (q, J = 282.8 Hz, CF3); 125,4 (Car
Fmoc); 127.5 (Car Fmoc); 128.2 (Car Fmoc); 141.6 (Cq
ar Fmoc); 143.8 (Cq ar Fmoc); 157.4 (CO); 168.6 (CO);
173.7 (CO); 168.2 (CO2H) ppm. 19F NMR (376.2 MHz,
CD3OD): δ −79.0 (s, CF3) ppm. IR (neat/cm−1): 3402,
2937, 1645, 1611, 1545.
The Fmoc-amino acid chlorides were prepared according
to previously reported procedures (Patil and Babu 2002;
Chaume et al. 2013).
Fmoc‑Ala‑(R)‑α‑Tfm‑Ala‑OH, (R)-7
To a suspension of (R)-α-Tfm-Ala-OH (R)-1 (100 mg,
0.64 mmol, 1.0 equiv) in THF (4 mL), were added Fmoc-
Ala-Cl (250 mg, 0.76 mmol, 1.2 equiv) and K2CO3
(176 mg, 1.27 mmol, 2.0 equiv). The reaction mixture was
heated at 100 °C in a sealed tube for 1 h. The crude mixture
was then cooled to room temperature, and the K2CO3 was
filtered. The filtrated product was evaporated under vacuum
and purified by semi-preparative HPLC (water/acetonitrile
90/10–70/30) to afford the desired dipeptide Fmoc-Ala-
(R)-α-Tfm-Ala-OH (R)-7 (262 mg, 0.59 mmol, 92 %) as
a white hygroscopic solid. Mp: 148 °C. [α]2D0 = +17.9
Fmoc‑Pro‑(R)‑α‑Tfm‑Ala‑OH, (R)-9
To a suspension of (R)-α-Tfm-Ala-OH (R)-1 (99 mg,
0.64 mmol, 1.0 equiv) in THF (4 mL), were added Fmoc-
Pro-Cl (259 mg, 0.76 mmol, 1.2 equiv) and K2CO3
(176 mg, 1.27 mmol, 2.0 equiv). The reaction mixture was
heated at 100 °C in a sealed tube for 1 h. The crude mix-
ture was then cooled to room temperature, and the K2CO3
was filtered. The filtrated product was evaporated under
vacuum and purified by semi-preparative HPLC (water/
1
(c = 0.9, HCl). H NMR (400 MHz, CD3OD): δ 1.36
3
(d, J = 7 Hz, 3 H, Hβ1); 1,72 (s, 3 H, Hβ2); 4,18 (t,
1 3

Synthesis of protected enantiopure (R) and (S)-α-trifluoromethylalanine containing dipeptide…
acetonitrile 90/10–70/30) to afford the desired dipeptide
Fmoc-Pro-(R)-α-Tfm-Ala-OH (R)-9 (270 mg, 0.57 mmol,
90 %) as a white hygroscopic solid. [α]2D0 = +23.4
equiv) in THF (10 mL) was added to the reaction mix-
ture and the resulting mixture was stirred 1 h at −20 °C
and 2 h at room temperature. The reaction was evapo-
rated under vacuum and diluted with water (15 mL). The
aqueous solution was extracted with AcOEt (3 × 15 mL)
and the combined organic extracts were dried with anhy-
drous MgSO4, and evaporated under vacuum. The crude
product was purified by silica gel chromatography
(cyclohexane/AcOEt 90/10–70/30) to afford the dipeptide
Fmoc-AA-α-Tfm-Ala-OtBu.
1
(c = 0.9, MeOH). H NMR (400 MHz, CD3OD): δ 1.71
(s, 3 H, Hβ2); 1.92 (m, 2 H, Hγ1 and Hγ1′); 2.19 (m, 1
H, Hβ1); 2.39 (m, 1 H, Hβ1′); 3.44 (m, 1 H, Hδ1); 3.25
3
(m, 1 H, Hδ1′); 4.32 (t, J = 6.9 Hz, 1 H, HFmoc); 4.36–
4.46 (m, 3 H, Hα1 et CH2 Fmoc); 6.70 (s, 1 H, NH); 7.32
3
3
(t, J = 7.3 Hz, 2 H, HFmoc); 7.41 (t, J = 7.3 Hz, 2 H,
3
HFmoc); 7.60 (d, J = 7.3 Hz, 2 H, Hfmoc); 7.65 (s, 1
H, NH); 7.77 (d, J = 7.3 Hz, 2 H, HFmoc). 13C NMR
3
(100.5 MHz, CD3OD): δ 18.3 (Cβ2); 26.0 (Cγ1); 29.2
(Cβ1)′; 47.4 (CHFmoc); 47.9 (δ1); 60.7 (Cα1); 62.6 (q,
J = 28.8 Hz, Cα2); 68.2 (CH2 Fmoc); 121.1 (Car Fmoc);
123.9 (q, J = 285.6 Hz, CF3); 125.2 (Car Fmoc); 127.2
(Car Fmoc); 128.1 (Car Fmoc); 141.6 (Cqar Fmoc); 144.0
(Cqar Fmoc); 156.8 (CO); 165.6 (CO); 170.8 (CO). 19F
NMR (376.2 MHz, CD3OD): δ −79.1 (s, CF3) ppm. IR
(neat/cm−1): 3506, 3257, 2959, 2592, 1650, 1607, 1532,
1156.
Fmoc‑Ala‑(R)‑α‑Tfm‑Ala‑OtBu, (R)-11
According to the general procedure described above start-
ing from (R)-10 (182 mg, 0.9 mmol), the compound (R)-
11 (190 mg, 0.6 mmol) was obtained as a white solid in
67 % yield. Rf: 0.35 (cyclohexane/AcOEt, 7/3). Mp:
53–55 °C. [α]2D0 = +12.7 (c = 0.9, MeOH). H NMR
1
3
(CDCl3, 400 MHz): δ 1.41 (d, J = 6.9 Hz, 3H, Hβ1,);
1.47 (s, 9H, HtBu); 1.73 (s, 3H, Hβ2); 4.20 (t, 3J = 6.9 Hz,
1H, Hfmoc); 4.38–4.39 (m, 3H, CH2 Fmoc et Hα1);
3
General procedure for the synthesis of the α‑Tfm‑Ala
containing dipeptides (11–15)
5.61 (d, J = 7.3 Hz, 1H, NH); 7.22 (s, 1H, NH); 7.31 (t,
3J = 7.8 Hz, 2H, Fmoc); 7.40 (t, 3J = 7.3 Hz, 2H, HFmoc);
7.58 (d, 3J = 7.3 Hz, 2H HFmoc); 7.76 (d, 3J = 7.3 Hz, 2H,
Fmoc). 13C NMR (CDCl3, 100.5 MHz): δ 18.1 (Cβ2); 19.0
(Cβ1); 27.9 (CtBu); 47.3 (CH Fmoc); 50.6 (Cα1); 62.9 (q,
J = 28.8 Hz, Cα2); 67.5 (CH2 Fmoc); 84.2 (CtBu); 120.3;
124.5 (q, J = 285.6 Hz,CF3); 125.4; 127.4; 128.0; 141.6;
143.9; 156.4 (CO); 165.4 (CO); 172.0 (CO). 19F NMR
(CDCl3, 376.2 MHz): δ −79.1 (s, 3F, CF3). IR (neat/cm−1):
3400, 1747, 1673, 1538. MS (EI): m/z = 507.2 [M+H]+.
HRMS (EI) m/z calcd for C26H30F3N2O5 [M+H]+:
507.2107 found 507.2111. Anal. calcd: %C: 61.65; %H:
5.77; %N: 5.53 found: %C: 61.97; %H: 6.00; %N: 5.11.
HCl.(R)‑α‑Tfm‑Ala‑OtBu, (R)‑10
Pd/C (250 mg, 0.23 mmol, 0.2 equiv) was added to a solu-
tion of enantiopure N-Cbz-(R)-α-Tfm-Ala-OtBu (R)-6
(345 mg, 1.0 mmol, 1.0 equiv) in AcOEt (15 mL) under
a nitrogen atmosphere. The reaction mixture was stirred
under a hydrogen atmosphere (three bars) for 2 h. The cata-
lyst was separated by filtration and HCl gas was bubbled
through the solution for 10 min. The resulting solution was
evaporated under vacuum to afford enantiopure HCl.(R)-
α-Tfm-Ala-OtBu (R)-10 (210 mg, 0.99 mmol) as a white
solid in 99 % yield which was directly used in the next step
Fmoc‑Ala‑(S)‑α‑Tfm‑Ala‑OtBu, (S)-11
1
1
without further purification after H NMR checking. H
NMR (CD3OD, 400 MHz): δ 1.60 (s, 9H, tBu); 1.82 (s, 3H,
Hβ). 19F NMR (CD3OD, 376.2 MHz): δ −80.0 (s, 3F, CF3).
By means of the general procedure described above start-
ing from (S)-10 (145 mg, 0.7 mmol), the compound (S)-
11 (238 mg, 0.5 mmol) was obtained as a white solid in
69 % yield. Rf: 0.45 (cyclohexane/AcOEt, 7/3). Mp:
HCl.(S)‑α‑Tfm‑alanine‑OtBu, (S)‑10
51–54 °C. [α]2D0 = −23.5 (c = 0.5, CHCl3). H NMR
1
3
By means of the procedure described for (R)-10 start-
ing from (S)-6 (345 mg, 1.0 mmol), the compound (S)-10
(206 mg, 0.97 mmol) was obtained as a white solid in 97 %
(CDCl3, 400 MHz): δ 1.40 (d, J = 6.9 Hz, 3H, Hβ1,);
1.47 (s, 9H, HtBu); 1.73 (s, 3H, Hβ2); 4.20 (t, 3J = 6.9 Hz,
1H, HFmoc); 4.33–4.43 (m, 3H, CH2 Fmoc et Hα1);
1
3
yield. H NMR (CD3OD, 400 MHz): δ 1.60 (s, 9H, tBu);
5.60 (d, J = 6.9 Hz, 1H, NH); 7.18 (s, 1H, NH); 7.30 (t,
1.82 (s, 3H, Hβ). 19F NMR (CD3OD, 376.2 MHz): δ −80.0
3J = 6.9 Hz, 2H, Fmoc); 7.40 (t, J = 7.3 Hz, 2H, Fmoc);
3
3
3
(s, 3F, CF3).
7.57 (d, J = 7.3 Hz, 2H, HFmoc); 7.76 (d, J = 7.3 Hz,
2H, HFmoc). 13C NMR (CDCl3, 100.5 MHz): δ 18.3
(Cβ2); 18.7 (Cβ1); 27.9 (CtBu); 47.3 (CH Fmoc); 50.7
(Cα1); 62.7 (q, J = 28.8 Hz, Cα2); 67.6 (CH2 Fmoc); 84.2
(CtBu); 120.3; 124.5 (q, J = 285.6 Hz,CF3); 125.3; 127.4;
128.1; 141.6; 143.9; 156.5 (CO); 165.5 (CO); 172.0 (CO).
To a solution of Fmoc-AA-OH (0.9 mmol, 1.0 equiv) in
THF (15 mL) at −20 °C was added N-methylmorpholine
(1.1 equiv) and isobutylchloroformiate (1.1 equiv). After
10 min at −20 °C, a solution of enantiopure HCl.α-Tfm-
Ala-OtBu 10 (0.9 equiv) and N-methylmorpholine (1.1
1 3

E. Devillers et al.
19F NMR (CDCl3, 376.2 MHz): δ −78.9 (s, 3F, CF3). IR
δ 1.46 (s, 9H, HtBu); 1.76 (s, 3H, Hβ2); 1.96 (m, 2H, Hγ1
and Hγ1′); 2.19 (m, 1H, Hβ1); 2.42 (m, 1H, Hβ1′); 3.44
(neat/cm−1): 3301, 2981, 1745, 1680, 1508.
3
(m, 1H, Hδ1); 3.55 (m, 1H, Hδ1′); 4.27 (t, J = 6.9 Hz,
Fmoc‑Phe‑(R)‑α‑Tfm‑Ala‑OtBu, (R)-12
1H, HFmoc); 4.36–4.46 (m, 3H, Hα1 et CH2 Fmoc); 6.70
3
(s, 1H, NH); 7.32 (t, J = 7.3 Hz, 2H, HFmoc); 7.41 (t,
By means of the general procedure described above start-
ing from (R)-10 (215 mg, 1.0 mmol), the compound (R)-
12 (342 mg, 0.59 mmol) was obtained as a white solid in
3J = 7.3 Hz, 2H, HFmoc); 7.60 (d, 3J = 7.3 Hz, 2H, fmoc);
3
7.65 (s, 1H, NH); 7.77 (d, J = 7.3 Hz, 2H, Fmoc). 13C
NMR (CDCl3, 100.5 MHz): δ 17.7 (Cβ2); 24.6 (Cγ1); 27.4
(CtBu and Cβ1)′; 47.0 (CHFmoc); 47.9 (δ1); 60.1 (Cα1);
62.5 (q, J = 28.8 Hz, Cα2); 67.9 (CH2 Fmoc); 83.2 (CtBu);
119.9; 124.1 (q, J = 285.6 Hz, CF3); 124.9; 127.0; 127.7;
141.2; 143.5; 143.7; 156.4 (CO); 165.1 (CO); 170.5 (CO).
19F NMR (CDCl3, 376.2 MHz): δ −79.4 (s, 3F, CF3). IR
(neat/cm−1): 3350, 2977, 1738, 1710, 1690.
1
59 % yield. Mp: 170 °C. H NMR (CDCl3, 400 MHz): δ
1.48 (s, 9H, HtBu); 1.69 (s, 3H, Hβ2); 3.08 (s, 2H, Hβ1);
4.18 (t, 3J = 7.3 Hz, 1H, HFmoc); 4.28 (t, 3J = 6.9 Hz, 1H,
3
CH2Fmoc); 4.37 (t, J = 8.2 Hz, 1H, CH2Fmoc); 4.57 (m,
3
1H, Hα1); 5.66 (d, J = 7.3 Hz, 1H, NH); 7.14–7.27 (m,
3
5H, HarPhe); 7.31 (t, J = 7.3 Hz, 2H, HFmoc); 7.41 (t,
3J = 7.3 Hz, 2H, HFmoc); 7.54 (m, 2H, HFmoc); 7.77 (d,
3J = 7.3 Hz, 2H, HFmoc). 13C NMR (CDCl3, 100.5 MHz):
δ 17.9 (Cβ2); 27.9 (CtBu); 38.7 (Cβ1); 47.2 (CH Fmoc);
56.4 (Cα1); 62.7 (q, J = 28.8 Hz, Cα2); 67.5 (CH2 Fmoc);
84.2 (CtBu); 120.3; 124.3 (q, J = 285.6 Hz, CF3); 125.4;
127.4; 128.1; 141.6; 144.0; 156.4 (CO); 165.4 (CO); 170.8
(CO). 19F NMR (CDCl3, 376.2 MHz): δ −78.7 (s, 3F, CF3).
IR (neat/cm−1): 3428, 3303, 3290, 1750, 1746, 1495.
Fmoc‑Leu‑(R)‑α‑Tfm‑Ala‑OtBu, (R)-14
By means of the general procedure described above start-
ing from (R)-10 (170 mg, 0.8 mmol), the compound (R)-14
(355 mg, 0.65 mmol) was obtained as a white solid in 81 %
yield. Mp: 147–148 °C. [α]2D0 = –25.0 (c = 0.6, CHCl3).
1H NMR (CDCl3, 400 MHz): δ 0.94 (s, 6H, Hδ1 et Hδ1′);
1.45 (s, 9H, HtBu); 1.53 (m, 1H, Hγ1); 1.73 (m, 2H, Hβ1);
1.74 (s, 3H, Hβ2); 4.20–4.28 (m, 2H, Hα1 et HFmoc); 4.41
(d, 3J = 6.9 Hz, 2H, CH2 Fmoc); 5.25 (d, 3J = 7.8 Hz, 1H,
NH); 6.96 (s, 1H, NH); 7.31 (t, 3J = 7.3 Hz, 2H, HFmoc);
Fmoc‑Pro‑(R)‑α‑Tfm‑Ala‑OtBu, (R)-13
By means of the general procedure described above start-
ing from (R)-10 (150 mg, 0.7 mmol), the compound (R)-
13 (232 mg, 0.44 mmol) was obtained as a white solid
in 62 % yield. Mp: 162–163 °C. [α]2D0 = −78.8 (c = 1.2,
CHCl3). 1H NMR (CDCl3, 400 MHz): δ 1.44 (s, 9H,
HtBu); 1.71 (s, 3 H, Hβ2); 1.95 (m, 2H, Hγ1 and Hγ1′);
2.19 (m, 1H, Hβ1); 2.39 (m, 1H, Hβ1′); 3.44 (m, 1H, Hδ1);
3
3
7.40 (t, J = 7.8 Hz, 2H, HFmoc); 7.58 (d, J = 7.3 Hz,
2H, HFmoc); 7.77 (d, J = 7.3 Hz, 2H, HFmoc). 13C
3
NMR (CDCl3, 100.5 MHz): δ 18.1 (Cβ2); 22.2 (Cδ1); 23.2
(Cδ1′); 24.9 (Cγ1); 27.9 (CtBu); 41.4 (Cβ1); 47.4 (CH
Fmoc); 53.8 (Cα1); 62.9 (q, J = 28.8 Hz, Cα2); 67.5 (CH2
Fmoc); 84.3 (CtBu); 120.4; 124.5 (q, J = 285.6 Hz, CF3);
125.4; 127.4; 128.1; 128.7; 141.6; 144.0; 156.7 (CO); 165.4
(CO); 171.8 (CO). 19F NMR (CDCl3, 376.2 MHz): δ −79.1
(s, 3F, CF3). IR (neat/cm−1): 3432, 3297, 1750, 1748, 1497.
MS (EI): m/z = 549.3 [M+H]+. HRMS (EI) m/z calcd for
C29H36F3N2O5 [M+H]+ 549.2576 found 549.2579.
3
3.55 (m, 1H, Hδ1′); 4.28 (t, J = 6.9 Hz, 1H, HFmoc);
4.36–4.46 (m, 3H, Hα1 et CH2 Fmoc); 6.70 (s, 1H, NH);
3
3
7.32 (t, J = 7.3 Hz, 2H, HFmoc); 7.41 (t, J = 7.3 Hz,
2H, HFmoc); 7.60 (d, 3J = 7.3 Hz, 2H, fmoc); 7.65 (s, 1H,
NH); 7.77 (d, J = 7.3 Hz, 2H, Fmoc). 13C NMR (CDCl3,
3
100.5 MHz): δ 17.9 (Cβ2); 24.7 (Cγ1); 27.6 (CtBu and
Cβ1)′; 47.1 (CHFmoc); 47.5 (δ1); 60.3 (Cα1); 62.2 (q,
J = 28.8 Hz, Cα2); 67.9 (CH2 Fmoc); 83.4 (CtBu); 120.0;
124.1 (q, J = 285.6 Hz, CF3); 125.0; 127.0; 127.8; 141.2;
143.7; 156.4 (CO); 165.2 (CO); 170.4 (CO). 19F NMR
(CDCl3, 376.2 MHz): δ –79.1 (s, 3F, CF3). IR (neat/cm−1):
3354, 2975, 1735, 1709, 1693. MS (EI): m/z = 533.2
[M+H]+. HRMS (EI) m/z calcd for C28H32F3N2O5
[M+H]+ 533.2263 found 533.2266.
Fmoc‑Aib‑(R)‑α‑Tfm‑Ala‑OtBu (R)-15
By means of the general procedure described above
starting from (R)-10 (0.8 mmol), the compound (R)-15
(0.5 mmol) was obtained as a white solid in 63 % yield.
1
Mp: 146–149 °C. H NMR (CDCl3, 400 MHz): δ 1.47 (s,
9H, HtBu); 1.53 (s, 6H, Hβ1 et Hβ1′); 1,78 (s, 3H, Hβ2);
3
3
4,22 (t, J = 6.4 Hz, 1H, HFmoc); 4,44 (d, J = 5.0 Hz,
3
2H, CH2Fmoc); 5,29 (s, 1H, NH); 7.33 (t, J = 7.3 Hz,
3
Fmoc‑Pro‑(S)‑α‑Tfm‑Ala‑OtBu, (S)-13
2H, HFmoc); 7.42 (t, J = 7.3 Hz, 2H, HFmoc); 7.60
3
(m, 2H, HFmoc); 7.77 (d, J = 7.3 Hz, 2H, HFmoc). 13C
By means of the general procedure described above start-
ing from (S)-10 (157 mg, 0.63 mmol), the compound (S)-
13 (255 mg, 0.48 mmol) was obtained as a white solid in
76 % yield. Mp: 158–159 °C. 1H NMR (CDCl3, 400 MHz):
NMR (CDCl3, 100.5 MHz): δ 17.3 (Cβ2); 25.2 (Cβ1 et
Cβ1′); 27,5 (CtBu); 47.0 (CH Fmoc); 57,2 (Cα1); 62.5 (q,
J = 28.8 Hz, Cα2); 66.7 (CH2 Fmoc); 84.9 (CtBu); 119.9;
120.0; 124.2 (q, J = 285.6 Hz, CF3); 124.9; 127.0; 127.7;
1 3

Synthesis of protected enantiopure (R) and (S)-α-trifluoromethylalanine containing dipeptide…
141.3; 143.6; 143.7; 155.3 (CO); 165.5 (CO); 173.4 (CO).
19F NMR (CDCl3, 376.2 MHz): δ −79.2 (s, 3F, CF3). IR
(neat/cm−1): 3420, 3293, 1747, 1752, 1490.
(CH3CO); 38.6 (Cβ2); 51.3 (Cα1 or Cα4); 51.7 (Cα1
or Cα4); 57.3 (Cα2); 64.4 (q, J = 25.9 Hz, Cα3); 126.7
(q, J = 283.7 Hz, CF3); 128.8; 130.4; 131.2;138.8; 168.9
(CO); 174.2 (CO); 174.6 (CO);176.5 (CO); 178.2 (CO).
19F RMN (CD3OD, 376.2 MHz): δ −79.1 (s, 3F, CF3). IR
(neat/cm−1): 3402, 3350, 3250, 1740, 1628, 1550. MS (EI):
m/z = 488.2 [M+H] +. HRMS (EI) m/z for C21H29F3N5O5
[M+H]+: 488.2121 found 488.2122.
Ac‑Ala‑Phe‑(R)‑α‑Tfm‑Ala‑Ala‑NH2, 17
A
sample of N-Fmoc-Rink-amide resin (loading
0.64 mmol/g, 0.13 mmol, 1.0 equiv) was placed in a dried
15 mL glass reaction vessel, stirred in a mixture of dry
DCM (2 mL) and DMF (2 mL) for 30 min, and drained. The
Fmoc group was removed by treatment with 10 ml of 20 %
piperidine in DMF (3 × 10 min) and the resin was washed
with DMF (3 × 1 min) and DCM (3 × 1 min). To the resin
were added a solution of N-Fmoc-Ala-OH (0.65 mmol,
5.0 equiv), DIPEA (0.65 mmol, 5.0 equiv) and HATU
(0.65 mmol, 5.0 equiv) in DMF (4 mL). The coupling reac-
tion was shaken for 2 h at room temperature, after which the
resin was washed with 5 mL of DMF (3 × 2 min) and 5 mL
of DCM (3 × 2 min). The Fmoc group was removed with
10 ml of 20 % piperidine in DMF (3 × 10 min) and the resin
was washed with DMF (3 × 1 min) and DCM (3 × 1 min).
Independently, the dipeptide (R)-12 (0.26 mmol) was
treated with a solution of trifluoroacetic acid in CH2Cl2
(4 M, 3 mL) for 3 h at room temperature and evaporated
to dryness to give the N-Fmoc-Phe-(R)-α-Tfm-Ala-OH
dipeptide (R)-16 which was used in the next step without
further purification. To the resin were then added the dipep-
tide (R)-16 (0.26 mmol, 2.0 equiv), DIPEA (0.26 mmol, 2.0
equiv) and HATU (0.26 mmol, 2.0 equiv) in DMF (2 mL).
The coupling reaction was shaken for 3 h at room tempera-
ture, after which the resin was washed with 5 mL of DMF
(3 × 2 min) and 5 mL of DCM (3 × 2 min). The same pro-
cedure was repeated for the coupling of the Fmoc-Ala-OH
(0.65 mmol, 5.0 equiv). A 10 ml solution of acetic anhy-
dride/pyridine/DMF (1:2:3) was transferred to the peptidyl
resin and was reacted at room temperature (2 × 60 min).
The resin was drained, washed with DMF (3 × 1 min)
and DCM (3 × 1 min). A total of 6 ml of TFA/TIS/water/
EDT (94:1:2.5:2.5) was added to the acetylated peptidyl
resin (0.26 mmol) and shaked at room temperature for 3 h.
The resin was then removed by filtration and washed twice
with 100 % TFA. The filtrate was evaporated under vacuum
and purified by semi-preparative HPLC (water/acetonitrile
90/10–70/30) to afford the desired tetrapeptide Ac-Ala-Phe-
(R)-α-Tfm-Ala-Ala-NH2 17 (341 mg, 0.07 mmol, 53 %) as
a white hygroscopic solid. [α]2D0 = −1.6 (c = 0.5, MeOH).
Compliance with ethical standards
Conflict of interest The authors declare that they have no conflict
of interest.
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1 3