Incorporation of trifluoromethylated proline and surrogates into peptides: Application to the synthesis of fluorinated analogues of the neuroprotective glycine-proline-glutamate (GPE) tripeptide

Article abstract of DOI:10.1021/acs.joc.6b00704

The incorporation into a peptide chain of highly hindered and weakly nucleophilic trifluoromethylated prolines, pseudoprolines and oxazolidines has been achieved. As an application, the synthesis of a new class of fluorinated analogues of the neuroprotective tripeptide glycine-proline-glutamate (GPE) is reported. These analogues have been elaborated from a panel of five-membered ring trifluoromethylated amino acids (Tfm-AAs) through the coupling reaction with a glutamate residue at the C-terminus and a glycine at the N-terminus. Although the peptide coupling reaction at the C-terminal position of the fluorinated amino acid was conveniently performed under standard conditions, the very challenging coupling reaction at the highly deactivated N-terminal position proved to be much more problematic. A methodological study was needed to identify suitable reaction conditions for this difficult peptide coupling.

Full text of DOI:10.1021/acs.joc.6b00704

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Article
Incorporation of Trifluoromethylated Proline and Surrogates into
Peptides: Application to the Synthesis of Fluorinated Analogues
of the Neuroprotective Glycine-Proline-Glutamate (GPE) Tripeptide
Julien Simon, Julien Pytkowicz, Nathalie Lensen, Grégory Chaume, and Thierry Brigaud
J. Org. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.joc.6b00704 • Publication Date (Web): 13 Jun 2016
Downloaded from http://pubs.acs.org on June 16, 2016
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The Journal of Organic Chemistry is published by the American Chemical Society.
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Incorporation of Trifluoromethylated Proline and Surrogates into Peptides: Application to the
Synthesis of Fluorinated Analogues of the Neuroprotective Glycine-Proline-Glutamate (GPE)
Tripeptide
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Julien Simon, Julien Pytkowicz, Nathalie Lensen,* Grégory Chaume,* Thierry Brigaud *
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Laboratoire de Chimie Biologique (LCB), Université de CergyꢀPontoise, EA 4505, 5 Mail Gayꢀ
Lussac, 95000 CergyꢀPontoise, France.
Table of Contents/Abstract Graphic:
CF
O
3 H
N
CF3
H
N
CF3
CO2H
CO2H
CO2H
CO2H
CO2H
CO2H
F3C
N
N
CO2H
CO2H
N
H
N
O
H
O
O
O
CF3-Prolines
NH2
NH2
X
X
F3C
F3C
CF
CO2R
CO2R
O
N
H
N
CF
3 H
3H
N
H
N
CO2H
N
N
CF3-pseudoprolines
X = O, S
O
O
O
O
O
CF3
CO2H
NH2
NH2
N
CO2R
CF3-oxazolidine
H
CF3-GPE analogues
ABSTRACT: The incorporation into a peptide chain of highly hindered and weakly nucleophilic
trifluoromethylated prolines, pseudoprolines and oxazolidines has been achieved. As an application,
the synthesis of a new class of fluorinated analogues of the neuroprotective tripeptide glycineꢀprolineꢀ
glutamate (GPE) is reported. These analogues have been elaborated from a panel of fiveꢀmembered
ring trifluoromethylated amino acids (TfmꢀAAs) through the coupling reaction with a glutamate
residue at the Cꢀterminus and a glycine at the Nꢀterminus. Although the peptide coupling reaction at
the Cꢀterminal position of the fluorinated amino acid was conveniently performed under standard
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conditions, the very challenging coupling reaction at the highly deactivated Nꢀterminal position proved
to be much more problematic. A methodological study was needed to identify suitable reaction
conditions for this difficult peptide coupling.
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INTRODUCTION
The use of peptides as therapeutic agents has been quite limited due to several major drawbacks such
as structural flexibility, rapid degradation by peptidases and low lipophilicity. However, because of
recent advances in peptides chemistry and biology, peptideꢀbased drug discovery constitutes now a
serious alternative for addressing new therapeutic challenges.1 The introduction of fluorine atoms into
biomolecules such as peptides is known to deeply influence their chemical and biological properties2ꢀ5
and consequently, represents an option to develop the therapeutic potential of peptides. The synthesis
of fluorinated amino acids has gained a considerable interest in peptide and protein chemistry6ꢀ8 as
they are known to increase the chemical stability, the metabolic resistances9,10 and to provide a better
affinity of peptides for lipid membranes. Moreover, their incorporation into peptides can induce
stabilization of particular conformations and better autoꢀassembly.11ꢀ15 Fluorinated peptides can also be
used as efficient probes for 19F NMR studies.16,17 Trifluoromethylated amino acids (TfmꢀAAs)
represent a special class of highly constrained nonꢀproteogenic amino acids. While the coupling
reactions of the TfmꢀAAs at their Cꢀtermini can be efficiently achieved using standard protocols, their
incorporation into peptides at the Nꢀterminal position still remains challenging due to the stereoꢀ
electronic effects imparted by the CF3 group which strongly decrease the nitrogen nucleophilicity.18
Our group has developed the stereoselective synthesis of a panel of acyclic and cyclic TfmꢀAAs in
enantiopure form19ꢀ26 and is now mainly focused on the development of efficient methodologies for
their incorporation into peptides.27ꢀ30 Although αꢀCF3ꢀalanine has been incorporated into peptides, the
peptide coupling at the Nꢀterminal position of αꢀCF3ꢀproline has not been reported so far. We report
herein our investigations about the incorporation of αꢀCF3ꢀproline and various cyclic surrogates into
short peptide sequences. The usefulness of this methodology for the synthesis of peptides of biological
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interest is illustrated by the synthesis of new CF3ꢀtripeptide analogues of the neuroprotective glycineꢀ
prolineꢀglutamate peptide (GPE) (Figure 1). It is assumed that the GPE is the result of the proteolytic
cleavage of the insulinꢀlike growth factor (IGFꢀ1).31 Even if the GPE neither binds to IGFꢀ1 receptors
nor has any neurotrophic effect, it displays remarkable CNS activities.32ꢀ33 In different animal models,
the GPE shows neuroprotective effects on neurodegenerative processes, such as Alzheimer’s,
Parkinson’s and Huntington’s diseases. There is also evidence that the GPE exhibits neuromodulatory
activities.34ꢀ36 Due to its structural simplicity, the GPE emerged as a lead for the development of potent
neuroprotective agents for the treatment of various CNS injuries.37 Numerous GPE analogues have
been reported in the literature but most of them suffer of a lack of chemical stability and short halfꢀ
time bioavailability.38ꢀ45 It has been shown that proline residue is crucial due to its unique
conformational constraint.46 The tripeptide NNZꢀ2566, containing a 2ꢀmethylproline residue, shows
higher activity compared to GPE and other analogues and is currently in phase II for several
neurological indications.47 Replacement of the proline residue by a CF3ꢀcontaining surrogate is
expected to enhance the chemical stability of the GPE tripeptide together with its bioavailability
profile.
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Figure 1. Chemical structure of GPE, NNZ-2566 and CF3-GPE analogues
Three distinctive trifluoromethylated cyclic surrogates were considered for replacing the proline
residue in the GPE sequence varying the position and the configuration of the CF3 group along the 5ꢀ
membered ring : αꢀCF3ꢀproline 1, CF3ꢀpseudoprolines 2 and 3 derived from serine and cysteine
respectively and CF3ꢀoxazolidines 4 and 5 derived from ethanolamine and (R)ꢀphenylglycinol (Figure
2). Except for the oxazolidine 4, the synthesis of enantiopure 1,20,22 2,23 323 and 520,21 have been
previously described by our group.
Figure 2. Chemical structure of Tfm-AAs as proline surrogates
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RESULTS AND DISCUSSION
CF3-proline containing GPE analogues. The synthesis of CF3ꢀGPE analogues was first investigated
using the (S)ꢀ and (R)ꢀαꢀCF3ꢀproline 1. Due to the very short GPE sequence, the synthesis was
achieved in solution phase using the standard peptide elongation methodology. The coupling reaction
between the glutamic acid residue (Glu) and the αꢀCF3ꢀproline 1 was performed following our
reported procedure in order to avoid the diketopiperazine formation.27 Because of the strong
deactivation of the αꢀCF3ꢀproline 1 amino group induced by the electronꢀwithdrawing effect of the
neighboring CF3 group,17 its Nꢀprotection was not required to perform the coupling reaction in contrast
with the nonꢀfluorinated series. The four enantiopure dipeptides 6 [(S,S)ꢀ6, (R,S)ꢀ6, (R,R)ꢀ6, (S,R)ꢀ6]
were obtained by coupling (R)ꢀ and (S)ꢀαꢀCF3ꢀprolines 1 with Lꢀ or Dꢀglutamic acids (Scheme 1).
Therefore, the addition of 1 equivalent of αꢀCF3ꢀproline 1 to 2 equivalent amount of glutamic acid
dibenzyl ester in the presence of coupling reagents afforded the corresponding dipeptides 6 without
any trace of diketopiperazine. The expected dipeptides were obtained in 42 to 89% yield depending on
the Dꢀ or Lꢀseries of the glutamic acid (Scheme 1).
Scheme 1. Synthesis of dipeptides 6
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The next step involved the coupling reaction of the glycine residue (Gly) at the Nꢀterminal position of
the dipeptides 6. Despite the small steric hindrance of the Gly residue, this reaction remained highly
challenging because of the very low nucleophilicity of the dipeptide amino group and the steric
bulkiness of the ꢀCF ꢀgroup. Only very few examples are found in the literature for the Nꢀterminal
α
3
coupling of acyclic ꢀCF ꢀamino acids.9,48ꢀ50 All these examples required specific activation methods,
α
3
such as mixed anhydrides or acyl halides, to promote the peptide coupling in good yield. To our
knowledge, the Nꢀterminal coupling reaction has never been reported so far for cyclic ꢀCF ꢀamino
α
3
acids such as ꢀCF ꢀprolines. Firstly, we checked the use of a mixed anhydride obtained from
α
3
isobutylchloroformate and Cbzꢀglycine. However no coupling product was obtained from the
dipeptide (S,S)ꢀ whatever the solvent used (ethyl acetate or DMF) and only starting material was
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recovered. The use of acyl halide activation was then investigated. Amino acid fluorides are less
moisture sensitive than amino acid chlorides and are recognized as excellent coupling reagents for
both solution and solidꢀphase peptide syntheses.51 They are particularly efficient for the coupling of
sterically hindered amino acids but require the treatment with a silylating agent such as N,Oꢀ
bis(trimethylsilyl)acetamide (BSA).52 A coupling test between the preꢀactivated dipeptide (S,S)ꢀ and
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the Fmocꢀglycine fluoride, prepared according to Olah’s method,53 was attempted without success and
only starting material was recovered. We postulated that the reactivity of the acyl fluoride was not
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enough to overcome the stereoelectronic deactivation of the fluorinated dipeptide and we decided to
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try the more reactive amino acid chlorides. Because of the hydrolysis risk, epimerization and other
side reactions, the acyl chlorides have been considered for a long time as overactivated species.54
Thanks to the significant contribution of Carpino and his group, the use of the Fmocꢀamino acid
chlorides have turned out to be one of the most efficient coupling strategies.55 However, particular
attention is required to ensure the coupling. The Fmocꢀglycyl chloride was prepared by treatment of
the Fmocꢀglycine with thionyl chloride in DCM under ultrasonication and obtained in pure form by
crystallization from pentane.56 Because of the release of HCl during the amide bond formation, the
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coupling reaction of the Fmocꢀglycyl chloride with the dipeptide (S,S)ꢀ was first performed in the
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presence of DIEA. As summarized in Table 1, no reaction occurred at room temperature and only the
starting material was recovered even after 48 h (Table 1, entry 1). The increase of the temperature at
60°C in a sealed tube led to the formation of the tripeptide (S,S)ꢀ7 in a low yield (Table 1, entry 2). We
postulated that the neutralization of the HCl released from the reaction would prevent the acidic
activation of the amino acid chloride. As the amino group of the fluorinated dipeptide is strongly
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deactivated, we anticipated that the HCl release would not be a problem and the reaction was
performed without base. Indeed, the coupling reaction of the Fmocꢀglycyl chloride with the dipeptide
(S,S)ꢀ at 60°C in the absence of DIEA gave the corresponding tripeptide (S,S)ꢀ in a very good yield
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(88%) without epimerization (Table 1, entry 3). The substitution of the conventional heating method
by microwave irradiation, reported to accelerate the peptide syntheses, allowed the coupling in about
30 min but the yield decreased to 54% (Table 1, entry 4).
Table 1. Optimization of the synthesis of the tripeptide (S,S)-7 using amino acid chloride
activation
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Tripeptide (S,S)ꢀ7
Entry
Base (equiv)
Temperature (°C)
Time (h)
Yield (%)
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4
DIEA (1)
r.t.
60b
48
24
24
0.5
0a
DIEA (2)
15c
88c
54c
ꢀ
ꢀ
60b
Microwave
aStarting material was recovered. bReaction performed in a sealed tube. cObtained as a single diastereomer.
The optimized coupling conditions (Table 1, entry 3) were then successfully applied to the dipeptides
(R,S)ꢀ , (S,R)ꢀ and (R,R)ꢀ to afford the corresponding tripeptides in 52ꢀ67% yield without
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epimerization (Scheme 2).
Scheme 2. Synthesis of tripeptides 7
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The access to the ꢀCF ꢀproline containing GPE analogues required the full removal of the protecting
α
3
groups. The sequential cleavages were first considered in classical manner as usually reported in
peptide chemistry when using orthogonal protecting groups. Unexpectedly the standard Fmoc
deprotection by treatment with piperidine in DMF led to degradation. The use of DBU in DCM gave
the same result. Since both Fmoc and Bn protecting groups are sensitive to hydrogenolysis, their
removal in a single step was envisioned. The hydrogenolysis of (S,S)ꢀ and (R,S)ꢀ tripeptides was
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carried out under hydrogen atmosphere (1 bar) in methanol in the presence of an excess amount of
Pd/C catalyst ( 140 %mol of Pd) to afford the CF ꢀGPE analogues (S,S)ꢀ and (R,S)ꢀ in 55% and 54%
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yield respectively (Scheme 3). Surprisingly the use of higher hydrogen pressure (3ꢀ5 bars) led only to
degradation.
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Scheme 3. Synthesis of CF3-proline containing GPE analogues (S,S)-8 and (R,S)-8
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CF3-pseudoproline containing GPE analogues. We then focused our attention on the CF3ꢀ
pseudoproline template. It is anticipated that this 5ꢀCF3ꢀproline surrogate should constitute a valuable
tool to control the cisꢀtrans isomerisation of the glycylꢀpseudoprolyl bond.23,28,30 The synthesis was
first attempted following the same peptide elongation sequence as used for the αꢀCF3ꢀproline. The
pseudoproline (S,S)ꢀ9 was prepared according to our reported procedure by condensation of serine
methyl ester with fluoral.23 After saponification of the ester function of the pseudoproline 9, its
coupling reaction with Lꢀglutamic acid dibenzyl ester was carried out at the Cꢀterminal position
according to the standard procedure using EDCI and HOBt (Scheme 4). The corresponding dipeptide
(S,S,S)ꢀ10 was obtained in 72% yield as a diastereomeric mixture.
Scheme 4. C-terminal coupling reaction of the pseudoproline (S,S)-9
At this stage, the absolute configuration of the minor diastereomer could not be unambiguously
assigned. This synthetic pathway being stereochemically irrelevant, we decided to adopt a reverse
strategy for the peptide elongation and to start with the Nꢀterminal coupling of pseudoprolines . We
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have recently reported the coupling reactions of a mixture of cis and trans pseudoprolines with
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various Fmoc protected amino acid chlorides in base free conditions.30 We demonstrated that the
reaction involved a dynamic kinetic resolution (DKR) process. Indeed only the corresponding
dipeptides bearing a cis oxazolidine were obtained regardless the configuration at the Cδ position of
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the starting pseudoproline . We postulated that the trans (S,S)ꢀ and the cis (R,S)ꢀ pseudoprolines
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interconvert rapidly throughout a ring opening equilibrium promoted by the acidic conditions (Scheme
5). Due to steric effects, the Nꢀacylation of the trans pseudoproline (S,S)ꢀ is strongly disfavored and
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only the cis (R,S)ꢀ pseudoproline reacts to afford exclusively the dipeptide bearing the cis (R,S)ꢀ
9
oxazolidine.57,58 It is important to note that no epimerization of the Cδ occurs once the pseudoprolines
are Nꢀacylated.
Scheme 5. Dynamic kinetic resolution process during N-coupling of pseudoprolines 9
Applied to Fmocꢀglycine chloride, the reaction of a diastereomeric mixture of pseudoprolines (S,S)ꢀ
9
and (R,S)ꢀ gave the dipeptide (R,S)ꢀ in 97% yield as a single diastereomer (Scheme 6). Compared
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to the conditions used with the CF3ꢀproline template (Table 1), the reaction was carried out in milder
conditions at room temperature using only a slight excess amount (1.1 equiv) of acyl chloride.
Scheme 6. N-terminal coupling reaction of pseudoprolines 9
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These conditions were applied to the thiazolidines
prepared by condensation of cysteine methyl
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ester with fluoral.23 Starting from a diastereomeric mixture of the trans (S,R)ꢀ and cis (R,R)ꢀ
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thiazolidines, the coupling reaction led to the expected dipeptide (R,R)ꢀ but with a low conversion.
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The monitoring of the reaction progress by TLC and 19F NMR analyses shown a complete
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disappearance of the cis thiazolidine (R,R)ꢀ without any reaction of the trans (S,R)ꢀ diastereomer.
12 12
The coupling reactions with FmocꢀGlyꢀCl were then performed separately on the trans (S,R)ꢀ and
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cis (R,R)ꢀ thiazolidines (Scheme 7). The thiazolidine (R,R)ꢀ afforded the expected dipeptide
12 12
(R,R)ꢀ
13
in 89% yield as a single diastereomer while no reaction occurred with the (S,R)ꢀ
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thiazolidine (Scheme 7).
Scheme 7. N-terminal coupling reaction of pseudoprolines 12
As observed with the oxazolidines 9, the Nꢀcoupling reaction is strongly dependent of steric effects
and only the cis thiazolidine (R,S)ꢀ12 reacts to afford the corresponding (R,S)ꢀ13 dipeptide. In
agreement with the literature in the nonꢀfluorinated series,59,60 we assumed that, unlike the
oxazolidines 9, the trifluoromethylated thiazolidines (R,R)ꢀ12 and (S,R)ꢀ12 are stable towards acidic
media and cannot interconvert throughout a ring opening equilibrium. In a previous work,23 we
reported that a Lewis acid treatment (BF3·OEt2) can mediate the ring opening and promote the
epimerization of the trans (S,S)ꢀ9 oxazolidine into the cis (R,S)ꢀ9 (Scheme 8). The higher
thermodynamic stability of the trans (S,S)ꢀ9 oxazolidine compared to the cis (R,S)ꢀ9 one induces a
slow equilibrium shift toward the trans (S,S)ꢀ9. In contrast, no epimerization occurred when the trans
(S,R)ꢀ12 thiazolidine was treated with BF3·OEt2, Ti(OiPr)4 or Ti(OiPr)3Cl as Lewis acids. The reaction
with TiCl4 or SnCl4 gave only degradation products.
Scheme 8. Lewis acid-mediated epimerization of pseudoprolines 9 and 12
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In order to synthesize oxazolidineꢀtype CF3ꢀpseudoproline containing GPE analogues, the
saponification of the methyl ester dipeptide (R,S)ꢀ was carried out under mild conditions to avoid
11
early Fmoc deprotection.61 The treatment using NaOH in iPrOH/H2O (7:3) solution of CaCl2 (0.84 M)
gave the corresponding acid which was directly engaged in the coupling reaction with respectively Lꢀ
and Dꢀglutamic acid dibenzyl ester (Scheme 9). Surprisingly, the reaction under the standard
conditions (EDCI/HOBt) which were effective for the CF3ꢀproline template (Scheme 1) failed and the
starting material was recovered. The use of BOPꢀCl allowed the synthesis of the expected tripeptide
(R,S,S)ꢀ in 68% yield without epimerization (Scheme 9). The removal of all the protecting groups
14
was performed following the same procedure used for the CF3ꢀproline template. In a single step, the
hydrogenolysis of the tripeptide (R,S,S)ꢀ afforded the corresponding CF ꢀGPE analogues (R,S,S)ꢀ
14
15
3
in good yield (72%).
Scheme 9. Synthesis of CF3-pseudoproline containing GPE analogues (R,S,S)-14 and (R,S,S)-15
These optimized conditions were applied for the coupling of the thiazolidine containing dipeptide
(R,R)ꢀ13 with Lꢀglutamic acid dibenzyl ester. Unfortunately, the reaction led only to the recovery of
the starting material. Other attempts using EDCI/HOBt coupling reagents or the mixed anhydride
activation by treatment of (R,R)ꢀ13 with isobutylchloroformate in the presence of NMM failed.
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2-CF3-oxazolidine containing GPE analogues. The last investigated GPE analogue series ambitioned
to incorporate the trifluoropyruvate oxazolidine template in place of the proline. According to our
previously reported procedure,21 the CF3ꢀoxazolidine 16 was obtained in a high yield (95%) as a 75:25
diastereomeric mixture by the condensation of the ethyl trifluoropyruvate with NꢀBocꢀ(R)ꢀ
phenylglycinol in the presence of PPTS (Scheme 10). We already reported that this oxazolidine
proved to be a convenient building block for the synthesis of various enantiopure αꢀCF3ꢀamino acids20ꢀ
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22,24
and amino alcohols.62 In other respect, we consider herein this kind of oxazolidines as original
stable proline surrogates. Indeed, the conjugated electron withdrawing effect of both the
trifluoromethyl and the carboxylic group prevents the oxazolidine ring opening. Likewise, the
unsubstituted racemic oxazolidine 17 was obtained in 44% yield through the condensation reaction of
ethyl trifluoropyruvate with NꢀBocꢀethanolamine (Scheme 10). As both diastereomers of 16 and the
racemic oxazolidine 17 can be considered as 2ꢀCF3ꢀproline surrogates (2ꢀCF3ꢀΨPro), we decided to
investigate their incorporation into the GPE peptide sequence. Following the synthetic strategy
successfully employed in the CF3ꢀpseudoproline series based on the Nꢀ to Cꢀtermini peptide elongation
(vide supra), the coupling reaction between a diastereomeric mixture of oxazolidine 16 and the glycine
residue was attempted (Scheme 10). However, the expected dipeptide was not obtained whatever was
the glycine activation (acyl chloride or acyl bromide) or the protocol used (room temperature,
conventional heating or microwave activation, zinc activation, stoichiometry and solvent nature). In
order to decrease the steric impact of the oxazolidine, we decided to perform the coupling reaction
starting from the unsubstituted racemic oxazolidine 17 (Scheme 10). Therefore 17 was coupled with
Fmocꢀglycine chloride to afford the racemic dipeptide 18 in a good yield (75%).
Scheme 10. N-terminal coupling reaction of oxazolidines 16 and 17
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The rest of the synthesis to get the GPE analogues was achieved following the protocol described for
the CF3ꢀpseudoproline template. A saponification reaction followed by the coupling reaction of the
dipeptide
with the dibenzylated glutamic acid gave the tripeptide
as a 57:43 diastereomeric
19
18
mixture in 52% yield (Scheme 11). A pure fraction of each diastereomer was obtained by flash
chromatography separation. However, we were unable to assign the absolute configuration of the
oxazolidine core of each diastereomer. Finally, the isolated diastereomer of the tripeptide
was
19min
submitted to hydrogenolysis to give the CF ꢀGPE analogue in 49% yield.
20
3
Scheme 11. Synthesis of the 2-CF3-oxazolidine containing GPE analogue 20
CONCLUSIONS
We have developed convenient methods for the incorporation of various CF ꢀproline surrogates ( ꢀ
α
3
CF3ꢀprolines, CF3ꢀpseudoprolines and 2ꢀCF3ꢀoxazolidines) within peptide sequences. As a result of
this study, we demonstrated that the choice of the peptide elongation route (Cꢀ to Nꢀterminus or Nꢀ to
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Cꢀterminus) was crucial for the success of the peptide synthesis and also to guarantee its configuration
integrity. For the CF3ꢀproline template, the standard peptide elongation route (Cꢀ to Nꢀterminus) was
applied while the reverse strategy was required for the CF3ꢀpseudoproline and CF3ꢀoxazolidine
templates. Noteworthy, the Fmocꢀbenzyl strategy allowed the final full removal of the protecting
groups in single step by hydrogenolysis. The methods described in this paper have been successfully
applied to the solution phase synthesis of several novel trifluoromethylated analogues of the
neuroprotective GPE peptide. Evaluation of their neuroprotective effects on different types of neurons
from diverse induced injuries activity are in progress and will be reported in due time. Moreover, for
future developments, the fluorinated Fmocꢀprotected peptide intermediates elaborated in this work will
constitute suitable peptides bocks for the solid phase synthesis of longer peptides incorporating CF3ꢀ
proline or its surrogates.
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EXPERIMENTAL SECTION
General Methods. Unless otherwise mentioned, all the reagents were purchased from commercial
source. All glassware was dried in an oven at 150 °C prior to use. All solvents were purified and dried
by standard techniques and distilled prior to use. Dichloromethane was distilled over calcium hydride
under argon. THF was distilled over sodium benzophenone ketyl under argon. All organic extracts
were dried over MgSO4, unless otherwise noted. Silica gel (230−400 mesh) was used for flash column
chromatography, eluting (unless otherwise stated) with cyclohexane/ethyl acetate. Silica TLC plates
were visualized under UV light, by a 10% solution of phosphomolybdic acid in ethanol followed by
heating. Infrared spectra (IR) were obtained by Fourier transformation, and wave numbers are given in
cm−1. 1H NMR, 13C NMR, and 19F NMR spectra were recorded in CDCl3 (unless otherwise stated). 1H
NMR (400.00 MHz), 13C NMR (100.50 MHz) and 19F NMR (376.20 MHz) were measured on a
1
1
spectrometer operating at a H frequency of 400 MHz. Chemical shifts of H NMR are expressed in
parts per million downfield from tetramethylsilane (δ = 0) in CDCl3. Chemical shifts of 13C NMR are
expressed in parts per million downfield from CDCl3 as internal standard (δ = 77.0). Chemical shifts
of 19F NMR are expressed in parts per million downfield from C6F6 as an internal standard (δ =
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−164.9). Coupling constants are reported in Hertz. Assignments were obtained from the analysis of 2D
1H–13C heteronuclear correlation (HETCOR) spectroscopy. Correlation spectroscopy (COSY) was
used to correlate chemical shifts of protons coupled to one another. Melting points were uncorrected.
Highꢀresolution mass spectra were obtained using electrospray ionization (ESI) in positive ion mode
and a TOF mass analyzer or using direct inlet probe (DIꢀHRMS).
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CF3-proline containing GPE analogues.
General procedure for the synthesis of dipeptides (S,S)-6, (S,R)-6, (R,S)-6, (R,R)-6. Triethylamine
(4.1 equiv), HOBt (1.5 equiv), EDCI (1.5 equiv), and finally αꢀTfmꢀproline 1 (1 equiv) were
successively added at 0 °C to a stirred solution of glutamic dibenzylester tosylate salt (2 equiv) in
DMF (0.25 M/αꢀTfm amino acid). DMF was added and the resulting mixture was stirred at 0 °C for 20
min and then at room temperature for 24 h. The mixture was diluted with DCM and water. The
aqueous layer was extracted with DCM (3 ×) and the combined organic layers were washed with
water, dried with MgSO4, filtered, and concentrated under reduced pressure. The crude residue was
purified by flash chromatography to afford the corresponding dipeptides in 42–89% yields.
H-(S)-α-Tfm-Pro-L-Glu(OBn)-OBn (S,S)-6. The dipeptide (S,S)ꢀ6 was prepared by the General
Procedure, with Lꢀglutamic dibenzylester tosylate salt (961 mg, 1.92 mmol, 2 equiv), triethylamine
(540 ꢁL, 3.94 mmol, 4.1 equiv), HOBt (195 mg, 1.44 mmol, 1.5 equiv), EDCI (275 mg, 1.44 mmol,
1.5 equiv), and (S)ꢀαꢀTfm proline 1 (176 mg, 0.96 mmol) in DMF (4 mL). Purification on silica gel
(cyclohexane/AcOEt, 90:10) gave pure (S,S)ꢀ6 (388 mg, 82%) as a colorless oil; [α]25 –19.6 (c 0.9,
D
CHCl3); IR (neat) : 3021, 1740, 1685 cm–1; H NMR (400 MHz, CDCl3)
δ 1.70 (m, 1 H, Hγ ProꢀHa),
1
1.85 (m, 1 H, Hγ ProꢀHb), 2.07 (m, 1 H, Hβ GluꢀHa), 2.15–2.46 (m, 6 H, NH Pro, Hβ GluꢀHb, Hγ Glu
and Hβ Pro), 3.01–3.11 (m, 2 H, Hδ ProꢀHa), 4.62 (m, 1 H, Hα Glu), 5.09 (s, 2 H, Bzl CH2), 5.14 (d, J
= 12.4 Hz, 1 H, Bn CH2ꢀHa), 5.18 (d, J = 12.4 Hz, 1 H, Bn CH2ꢀHb), 7.30–7.38 (m, 10 H, Bn arom.),
8.11 (d, J = 6.9 Hz, 1 H, NH Glu); 13C NMR (100.5 MHz, CDCl3)
δ 25.4 (CH2, Cγ Pro), 26.9 (CH2, Cβ
Glu), 30.1 (CH2, Cγ Glu), 32.3 (CH2, Cβ Pro), 47.5 (CH2, Cδ Pro), 51.9 (CH, Cα Glu), 66.6 (CH2, Bn
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CH2), 67.4 (CH2, Bn CH2), 70.7 (q, J = 26.8 Hz, CH, Cα Pro), 125.9 (q, J = 284.7 Hz, CF3), 128.2,
4
5
6
7
128.3, 128.5, 128.6, 128.6, 135.0, 135.6, 169.5, 171.1, 172.3; 19F NMR (376.2 MHz, CDCl3)
(s, CF3); DIꢀHRMS calcd. for C25H27F3N2O5 [M]+: 492.1872; found 492.1873.
δ –77.8
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H-(R)-α-Tfm-Pro-D-Glu(OBn)-OBn (R,R)-6. The dipeptide (R,R)ꢀ6 was prepared by the General
Procedure, from Dꢀglutamic dibenzylester tosylate salt (332 mg, 0.67 mmol, 2 equiv), triethylamine
(181 ꢁL, 1.36 mmol, 4.1 equiv), HOBt (68 mg, 0.50 mmol, 1.5 equiv), EDCI (96 mg, 0.50 mmol, 1.5
equiv), and (R)ꢀαꢀTfm proline 1 (61 mg, 0.33 mmol) in DMF (1.5 mL). Purification on silica gel
(cyclohexane/AcOEt, 90:10) gave pure (R,R)ꢀ6 (110 mg, 67%) as a colorless oil; [α]25 +22.9 (c 1.0,
D
CHCl3); The spectral data of (R,R)ꢀ6 were identical to those of (S,S)ꢀ6; DIꢀHRMS calcd. for
C25H27F3N2O5 [M]+: 492.1872; found 492.1873.
H-(R)-α-Tfm-Pro-L-Glu(OBn)-OBn (R,S)-6. The dipeptide (R,S)ꢀ6 was prepared by the General
Procedure, with Lꢀglutamic dibenzylester tosylate salt (764 mg, 1.53 mmol, 2 equiv), triethylamine
(420 ꢁL, 3.14 mmol, 4.1 equiv), HOBt (155 mg, 1.15 mmol, 1.5 equiv), EDCI (219 mg, 1.15 mmol,
1.5 equiv), and (R)ꢀαꢀTfm proline 1 (140 mg, 0.76 mmol) in DMF (3 mL). Purification on silica gel
(cyclohexane/AcOEt, 90:10) gave pure (R,S)ꢀ6 (334 mg, 89%) as a colorless oil; [α]25 +18.1 (c 0.9,
D
1
CHCl3); IR (neat): 3332, 3019, 2925, 1736, 1685 cm–1; H NMR (400 MHz, CDCl3)
δ 1.69 (m, 1 H,
Hγ ProꢀHa), 1.79 (m, 1 H, Hγ ProꢀHb), 2.04 (m, 1 H, Hβ GluꢀHa), 2.11–2.48 (m, 6 H, NH Pro, Hβ Gluꢀ
Hb, Hγ Glu and Hβ Pro), 3.00–3.12 (m, 2 H, Hδ Pro), 4.60 (m, 1 H, Hα Glu), 5.09 (s, 2 H, Bn CH2),
5.12 (d, J = 12.1 Hz, 1 H, Bn CH2ꢀHa), 5.18 (d, J = 12.1 Hz, 1 H, Bn CH2ꢀHb), 7.28–7.37 (m, 10 H,
Bn arom.), 8.29 (d, J = 8.5 Hz, 1 H, NH Glu); 13C NMR (100.5 MHz, CDCl3)
δ 25.1 (CH2, Cγ Pro),
26.9 (CH2, Cβ Glu), 30.2 (CH2, Cγ Glu), 32.0 (CH2, Cβ Pro), 47.5 (CH2, Cδ Pro), 51.8 (CH, Cα Glu),
66.5 (CH2, Bn CH2), 67.4 (CH2, Bn CH2), 71.0 (q, J = 26.8 Hz, CH, Cα Pro), 125.8 (q, J = 283.7 Hz,
CF3), 127.9, 127.9, 128.0, 128.2, 128.3, 135.1, 135.7, 169.4, 171.1, 172.4; 19F NMR (376.2 MHz,
CDCl3)
δ
–77.8 (s, CF3); DIꢀHRMS calcd. for C25H27F3N2O5 [M]+: 492.1872; found 492.1874.
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H-(S)-α-Tfm-Pro-
Procedure, with
D
-Glu(OBn)-OBn (S,R)-6. The dipeptide (S,R)ꢀ6 was prepared by the General
D
ꢀglutamic dibenzylester tosylate salt (605 mg, 1.12 mmol, 2 equiv), triethylamine
6
7
8
(326 ꢁL, 2.46 mmol, 4.1 equiv), HOBt (122 mg, 0.90 mmol, 1.5 equiv), EDCI (173 mg, 0.90 mmol,
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1.5 equiv), and (S)ꢀαꢀTfm proline 1 (111 mg, 0.60 mmol) in DMF (3 mL). Purification on silica gel
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(cyclohexane/AcOEt, 90:10) gave pure (S,R)ꢀ6 (124 mg, 42%) as a colorless oil; [α]25 –16.6 (c 1.0,
D
CHCl3); The spectral data of (S,R)ꢀ6 were identical to those of (R,S)ꢀ6; DIꢀHRMS calcd. for
C25H27F3N2O5 [M]+: 492.1872; found 492.1874.
General procedure for the synthesis of dipeptides (S,S)-7, (S,R)-7, (R,S)-7, (R,R)-7. To a solution
of dipeptide 6 (1 equiv) in DCM under argon was added FmocꢀGlyꢀCl (2 equiv) prepared according to
our reported procedure.30 The mixture was refluxed for 24 h and then quenched by saturated NaHCO3
solution. The aqueous phase was extracted by DCM (3 ×) and the combined organic extracts were
dried over MgSO4 and concentrated under reduced pressure. The crude residue was purified by flash
chromatography to afford the corresponding tripeptides in 52–88% yield.
Fmoc-Gly-(S)-α-Tfm-Pro-L-Glu(OBn)-OBn (S,S)-7. The tripeptide (S,S)ꢀ7 was prepared following
the General Procedure, using dipeptide (S,S)ꢀ6 (552 mg, 1.12 mmol, 1 equiv) and FmocꢀGlyꢀCl (708
mg, 2.24 mmol, 2 equiv) in DCM (4 mL). Purification by flash chromatography (70:30
cyclohexane/ethyl acetate) gave pure (S,S)ꢀ7 (757 mg, 88 %) as a white solid. Mp 110–112°C; [α]25
–
D
53.7 (c 0.23, CHCl3); IR (neat): 3300, 1728, 1681 cm–1; 1H NMR (400 MHz, CDCl3)
δ 1.95–2.15 (m,
3 H, Hγ Pro and Hβ GluꢀHa), 2.29 (m, 1 H, Hβ GluꢀHb), 2.30–2.59 (m, 4 H, Hγ Glu and Hβ Pro), 3.60
(q, J = 8.0 Hz 1 H, Hδ ProꢀHa), 3.71 (q, J = 8.0 Hz, 1 H, Hδ ProꢀHb), 4.03–4.09 (m, 2 H, Hα Gly), 4.20
(t, J = 7.2 Hz, 1 H, Fmoc CH), 4.30–4.40 (m, 2 H, Fmoc CH2), 4.66 (m, 1 H, Hα Glu), 5.09 (s, 2 H, Bn
CH2), 5.14 (d, J = 12.1 Hz, 1 H, Bn CH2ꢀHa), 5.16 (d, J = 12.1 Hz, 1 H, Bn CH2ꢀHb), 5.73 (s, 1 H, NH
Gly), 7.21 (d, J = 6.9 Hz, 1 H, NH Glu), 7.27–7.35 (m, 12 H, Fmoc arom. and Bn arom.) 7.41 (t, J =
7.3 Hz, 2 H, Fmoc arom.), 7.60 (d, J = 7.3 Hz, 2 H, Fmoc arom.), 7.76 (d, J = 7.3 Hz, 2 H, Fmoc
arom.); 13C NMR (100.5 MHz, CDCl3)
δ 23.3 (CH2, Cγ Pro), 26.4 (CH2, Cβ Glu), 29.9 (CH2, Cγ Glu),
34.1 (CH2, Cβ Pro), 44.1 (CH2, Cα Gly), 47.0 (CH, Fmoc CH), 48.5 (CH2, Cδ Pro), 52.7 (CH, Cα Glu),
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66.5 (CH2, Bn CH2), 67.1 (CH2, Fmoc CH2), 67.4 (CH2, Bn CH2), 72.0 (q, J = 28.8 Hz, CH, Cα Pro),
119.9, 124.9 (q, J = 286.6 Hz, CF3), 125.1, 127.0, 127.6, 128.1, 128.2, 128.3, 128.5, 128.6, 135.0,
6
7
8
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135.6, 141.2, 143.8, 156.1, 165.9, 167.9, 170.9, 172.9; 19F NMR (376.2 MHz, CDCl3)
CF3); HRMS (ESIꢀTOF) calcd. for C42H41F3N3O8 [M+H]+: 772.2846; found 772.2881.
δ –71.3 (s,
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Fmoc-Gly-(S)-α-Tfm-Pro-D-Glu(OBn)-OBn (S,R)-7. To a solution of dipeptide (S,R)ꢀ6 (67 mg,
1.12 mmol, 1 equiv) in DCM (1 mL) under argon was added FmocꢀGlyꢀCl (86 mg, 2.24 mmol, 2
equiv). Purification by flash chromatography (70:30 cyclohexane/ethyl acetate) gave pure (S,R)ꢀ7 (55
mg, 52%) as a white solid. Mp 110–112°C; [α]25D –21.4 (c 0.44, CHCl3); IR (neat): 1728, 1681, 1519,
1
1450 cm–1; H NMR (400 MHz, CDCl3)
δ 1.92–2.15 (m, 3 H, Hγ Pro and Hβ GluꢀHa), 2.20 (m, 1 H,
Hβ GluꢀHb), 2.34–2.52 (m, 4 H, Hγ Glu and Hβ Pro), 3.55 (q, J = 8.0 Hz 1 H, Hδ ProꢀHa), 3.67 (q, J =
8.0 Hz, 1 H, Hδ ProꢀHb), 3.97 (dd, J = 17.4, 4.0 Hz, 1 H, Hα GlyꢀHa), 4.11–4.21 (m, 2 H, Hα GlyꢀHb
and Fmoc CH), 4.28 (dd, J = 10.3, 6.0 Hz, 1 H, Fmoc CH2ꢀHa), 4.37 (dd, J = 10.3, 7.3 Hz, 1 H, Fmoc
CH2ꢀHb), 4.63 (m, 1 H, Hα Glu), 5.01 (d, J = 12.1 Hz, 1 H, Bn CH2ꢀHa), 5.09 (d, J = 12.1 Hz, 1 H, Bn
CH2ꢀHb), 5.10 (d, J = 12.1 Hz, 1 H, Bn CH2ꢀHa), 5.19 (d, J = 12.1 Hz, 1 H, Bn CH2ꢀHb), 5.71 (m, 1
H, NHꢀGly), 7.27–7.35 (m, 13 H, NH Glu, Fmoc arom. and Bn arom.), 7.40 (t, J = 7.6 Hz, 2 H, Fmoc
arom.), 7.57 (d, J = 7.6 Hz, 2 H, Fmoc arom.), 7.76 (d, J = 7.6 Hz, 2 H, Fmoc arom.); 13C NMR (100.5
MHz, CDCl3)
δ 22.9 (CH2, Cγ Pro), 25.7 (CH2, Cβ Glu), 30.0 (CH2, Cγ Glu), 34.4 (CH2, Cβ Pro), 44.2
(CH2, Cα Gly), 46.9 (CH, Fmoc CH), 48.5 (CH2, Cδ Pro), 52.3 (CH, Cα Glu), 66.8 (CH2, Bn CH2),
67.1 (CH2, Fmoc CH2), 67.3 (CH2, Bn CH2), 72.0 (q, J = 27.8 Hz, CH, Cα Pro), 119.9, 124.5 (q, J =
286.6 Hz, CF3), 125.1, 127.0, 127.7, 127.9, 128.3, 128.4, 128.5, 128.6, 135.0, 135.3, 141.2, 143.8,
156.1, 166.2, 167.9, 171.0, 174.2; 19F NMR (376.2 MHz, CDCl3)
calcd. for C42H41F3N3O8 [M+H]+: 772.2846; found 772.2864.
δ –71.1 (s, CF3); HRMS (ESIꢀTOF)
Fmoc-Gly-(R)-α-Tfm-Pro-L-Glu(OBn)-OBn (R,S)-7. The tripeptide (R,S)ꢀ7 was prepared following
the General Procedure, using dipeptide (R,S)ꢀ6 (190 mg, 0.39 mmol, 1 equiv) and FmocꢀGlyꢀCl (243
mg, 0.77 mmol, 2 equiv) in DCM (2 mL). Purification by flash chromatography (70:30
cyclohexane/ethyl acetate) gave pure (R,S)ꢀ7 (159 mg, 53%) as a white solid; Mp 110ꢀ112°C; [α]25
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+23.1 (c 0.45, CHCl3); The spectral data of (R,S)ꢀ7 were identical to those of (S,R)ꢀ7; HRMS (ESIꢀ
4
5
TOF) calcd. for C42H41F3N3O8 [M+H]+: 772.2846; found 772.2864.
6
7
8
Fmoc-Gly-(R)-α-Tfm-Pro-D-Glu(OBn)-OBn (R,R)-7. The tripeptide (R,R)ꢀ7 was prepared following
9
the General Procedure, using dipeptide (R,R)ꢀ6 (62 mg, 0.13 mmol, 1 equiv) in DCM (1 mL) under
argon was added FmocꢀGlyꢀCl (80 mg, 0.26 mmol, 2 equiv. Purification by flash chromatography
(70:30 cyclohexane/ethyl acetate) gave pure (R,R)ꢀ7 (70 mg, 72%) as a white solid; Mp 110–112°C;
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[α]25 +52.2 (c 0.25, CHCl3); The spectral data of (R,R)ꢀ7 were identical to those of (S,S)ꢀ7; HRMS
D
(ESIꢀTOF) calcd. for C42H41F3N3O8 [M+H]+: 772.2846; found 772.2864.
H-Gly-(S)-α-Tfm-Pro-L-Glu(OH)-OH (S,S)-8. A solution of tripeptide (S,S)ꢀ7 (440 mg, 0.57 mmol,
1 equiv) in MeOH (2.5 mL) was hydrogenated over 20% Pd/C (440 mg) at room temperature for 24 h
under hydrogen atmosphere (1 bar). The reaction mixture was filtered and evaporated. The crude was
precipitated in pentane and filtrated to afford pure (S,S)ꢀ8 (116 mg, 55%) as a white solid; [α]25D –55.5
1
(c 1.3, H2O); IR (neat): 3600ꢀ2750, 1667, 1538 cm–1; H NMR (400 MHz, D2O)
δ 1.90 (m, 1 H, Hβ
GluꢀHa), 1.98–2.18 (m, 3 H, Hγ ProꢀH and Hβ GluꢀHb), 2.28–2.38 (m, 3 H, Hβ ProꢀHa and Hγ GluꢀH),
2.55 (m, 1 H, Hβ ProꢀHb), 3.65 (q, J = 8.0 Hz 1 H, Hδ ProꢀHa), 3.79–3.88 (m, 1 H, Hδ ProꢀHb), 3.98
(d, J = 16.7 Hz,1 H, Hα GlyꢀHa), 4.09 (d, J = 16.7 Hz,1 H, Hα GlyꢀHb), 4.21 (m, 1 H, Hα GluꢀH); 13
NMR (100.5 MHz, D2O) 22.7 (CH2, Cγ Pro), 27.0 (CH2, Cβ Glu), 30.9 (CH2, Cγ Glu), 34.5 (CH2, Cβ
Pro), 41.3 (CH2, Cα Gly), 48.7 (CH2, Cδ Pro), 54.4 (CH, Cα Glu), 71.8 (q, J = 28.8 Hz, CH, Cα Pro),
C
δ
124.5 (q, J = 285.6 Hz, CF3), 166.0, 167.8, 176.8, 178.6; 19F NMR (376.2 MHz, D2O)
δ –71.7 (s, CF3);
HRMS (ESIꢀTOF) calcd. for C13H19F3N3O6 [M+H]+: 370.1226; found 370.1221.
H-Gly-(R)-α-Tfm-Pro-L-Glu(OH)-OH (R,S)-8. A solution of tripeptide (R,S)ꢀ7 (159 mg, 0.21 mmol,
1 equiv) in MeOH (1.0 mL) was hydrogenated over 20% Pd/C (159 mg) at room temperature for 24 h
under hydrogen atmosphere (1 bar). The reaction mixture was filtered and evaporated. The crude was
precipitated in pentane and filtrated to afford pure (R,S)ꢀ8 (41 mg, 54%) as a white solid; [α]25D –49.3
1
(c 0.85, H2O); IR (neat): 3600ꢀ2750, 1667, 1538 cm–1; H NMR (400 MHz, D2O)
δ 1.90 (m, 1 H, Hβ
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GluꢀHa), 2.00–2.18 (m, 3 H, Hγ ProꢀH and Hβ GluꢀHb), 2.28–2.48 (m, 3 H, Hβ ProꢀHa and Hγ GluꢀH),
2.58 (m, 1 H, Hβ ProꢀHb), 3.69 (m, 1 H, Hδ ProꢀHa), 3.87 (m, 1 H, Hδ ProꢀHb), 4.01 (d, J = 16.5 Hz, 1
H, Hα GlyꢀHa), 4.15 (d, J = 16.5 Hz,1 H, Hα GlyꢀHb), 4.20 (m, 1 H, Hα GluꢀH); 13C NMR (100.5
9
MHz, D2O)
δ 22.7 (CH2, Cγ Pro), 26.1 (CH2, Cβ Glu), 31.7 (CH2, Cγ Glu), 34.5 (CH2, Cβ Pro), 41.5
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(CH2, Cα Gly), 48.9 (CH2, Cδ Pro), 54.9 (CH, Cα Glu), 71.8 (q, J = 27.8 Hz, CH, Cα Pro), 124.5 (q, J =
285.6 Hz, CF3), 166.3, 168.0, 177.0, 179.6; 19F NMR (376.2 MHz, D2O)
(ESIꢀTOF) calcd. for C13H19F3N3O6 [M+H]+: 370.1226; found 370.1222.
δ –71.5 (s, CF3); HRMS
CF3-pseudoproline containing GPE analogues.
H-(S)-Ser(ΨCF3,HPro)-L-Glu(OBn)-OBn (S,S,S)-10. To a solution of oxazolidine (S,S)ꢀ9 (2 g, 10.0
mmol) in THF (40 mL) was slowly added at 0°C 1 M aqueous solution of LiOH (11 mL, 11 mmol, 1.1
equiv). The solution mixture was vigorously stirred at 0°C until the disappearance of the starting
material (usually 1 h). Subsequently, Et2O was added, and the reaction mixture was extracted with
water (3×). The aqueous layers were combined, and water was removed under reduced pressure to
give the corresponding lithium carboxylate, which was directly used without further purification. A
fraction of the crude lithium carboxylate (210 mg, 1.1 mmol) was diluted in DMF (6 mL), and Lꢀ
glutamic acid dibenzylester tosylate salt (1.1 g, 2.2 mmol, 2.0 equiv), NEt3 (630 ꢀL, 4.5 mmol, 4.1
equiv), HOBt (223 mg, 1.65 mmol, 1.5 equiv),and EDCI (316 mg, 1.65 mmol, 1.5 equiv) were
successively added at room temperature. The reaction mixture was stirred overnight at room
temperature and then diluted with DCM and water. The layers were separated, and the aqueous layer
was extracted with DCM (3×). The combined organic layers were dried over MgSO4, filtered, and
concentrated under reduced pressure. Purification by flash chromatography (70:30 cyclohexane/ethyl
acetate) gave the dipeptide (S,S,S)ꢀ10 (392 mg, 72%) as an inseparable 75:25 diastereomeric mixture.
1H NMR (400 MHz, CDCl3) (major)
δ 2.08–2.12 (m, 1 H, Hβ GluꢀHa), 2.20ꢀ2.31 (m, 1 H, Hβ Gluꢀ
Hb), 2.32–2.50 (m, 2 H, Hγ Glu), 2.85 (m, 1 H, NH ΨPro), 3.81 (t, J = 7.0 Hz, 1 H, Hβ ΨProꢀHa), 3.93
(t, J = 7.0 Hz, 1 H, Hα ΨPro), 4.17 (t, J = 7.8 Hz, 1 H, Hβ ΨProꢀHb), 4.62–4.69 (m, 1 H, Hα Glu), 5.01
(q, J = 5.5 Hz, 1 H, Hδ ΨPro), 5.10 (s, 2 H, Bn CH2ꢀH), 5.14 (d, J = 12.1 Hz, 1 H, Bn CH2ꢀHa), 5.18
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(d, J = 12.1 Hz, 1 H, Bn CH2ꢀHb), 7.10 (d, J = 7.1 Hz, 1 H, NHꢀGlu), 7.26–7.40 (m, 10 H, Bn arom.);
4
(minor) δ 2.08–2.12 (m, 1 H, Hβ GluꢀHa), 2.20ꢀ2.31 (m, 1 H, Hβ GluꢀHb), 2.32–2.50 (m, 2 H, Hγ Glu),
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2.85 (m, 1 H, NH ΨPro), 3.86ꢀ3.93 (m, 2 H, Hβ ΨProꢀHa and Hα ΨPro), 4.11 (t, J = 7.1 Hz, 1 H, Hβ
ΨProꢀHb), 4.62–4.69 (m, 1 H, Hα Glu), 4.90 (q, J = 5.1 Hz, 1 H, Hδ ΨPro), 5.10 (s, 2 H, Bn CH2ꢀH),
5.14 (d, J = 12.1 Hz, 1 H, Bn CH2ꢀHa), 5.18 (d, J = 12.1 Hz, 1 H, Bn CH2ꢀHb), 7.10 (d, J = 7.1 Hz, 1
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H, NHꢀGlu), 7.26–7.40 (m, 10 H, Bn arom.); 13C NMR (100.5 MHz, CDCl3) (major)
δ 26.7 (CH2, Cβ
Glu), 30.2 (CH2, Cγ Glu), 52.0 (CH, Cα Glu), 59.3 (CH, Cα ΨPro), 66.7 (CH2, Bn CH2), 67.6 (CH2, Bn
CH2), 70.2 (CH2, Cβ ΨPro), 87.9 (q, J = 33.6 Hz, CH, Cδ ΨPro), 123.1 (q, J = 283.7 Hz, CF3), 128.2,
128.3, 128.4, 128.5, 128.6, 128.7, 134.9, 135.5, 170.0, 171.1, 172.7; (minor)
δ 26.8 (CH2, Cβ Glu),
30.2 (CH2, Cγ Glu), 51.8 (CH, Cα Glu), 59.3 (CH, Cα ΨPro), 66.6 (CH2, Bn CH2), 67.6 (CH2, Bn CH2),
70.0 (CH2, Cβ ΨPro), 87.9 (q, J = 33.6 Hz, CH, Cδ ΨPro), 123.0 (q, J = 282.8 Hz, CF3), 128.3, 128.3,
128.4, 128.5, 128.6, 128.7, 134.9, 135.6, 170.2, 171.0, 172.5; 19F NMR (376.2 MHz, CDCl3) (major)
δ
–84.6 (d, J = 5.5 Hz, CF3); (minor) δ –84.4 (d, J = 5.1 Hz, CF3); HRMS (ESIꢀTOF) calcd. for
C24H26F3N2O6 [M+H]+: 495.1743; found 495.1732.
The FmocꢀGlyꢀ(R)ꢀSer(ΨCF3,HPro)ꢀOMe (R,S)ꢀ11 was prepared according to our previously reported
procedure.30
Fmoc-Gly-(R)-Cys(ΨCF3,HPro)-OMe (R,R)-13. To a solution of (R,R)ꢀ12 (3.0 g, 13.9 mmol, 1 equiv)
in DCM (150 mL) was added FmocꢀGlyꢀCl (4.820 g, 15.3 mmol, 1.1 equiv).The mixture is stirred at
room temperature for 24 h then quenched with saturated NaHCO3 solution (150 mL). The aqueous
solution was extracted with DCM (3 x 100 mL). The combined organic extracts were washed with
brine (150 mL) and were dried over MgSO4, filtered and concentrated under reduced pressure. The
crude mixture is purified by flash chromatography (80:20 cyclohexane/ethyl acetate) to afford pure
dipeptide (R,R)ꢀ13 (6.08 g, 89%) as a 57/43 inseparable mixture of rotational isomers in CDCl3 at 298
K: yellow solid. Mp 72–74°C; [α]25 –95.9 (c 1.07, CHCl3); IR (neat): 3342, 1716, 1682, 1519, 1118
D
1
cm–1; H NMR (400 MHz, CDCl3, 323 K)
δ
3.33ꢀ3.65 (m, 2 H, Hβ ΨPro), 3.79 (s, 3 H, OMe), 4.12
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(m, 1 H, Hα GlyꢀHa), 4.22 (t, J = 6.9 Hz, 1 H, Fmoc CH), 4.35ꢀ4.47 (m, 3 H, Hα GlyꢀHb and Fmoc
CH2), 5.03 (m, 1 H, Hα ΨPro), 5.69 (m, 1 H, NH, Gly), 6.02 (m, 1 H, Hδ ΨPro), 7.31 (t, J = 7.3 Hz, 2
H, Fmoc arom.), 7.40 (t, J = 7.3 Hz, 2 H, Fmoc arom.), 7.59 (d, J = 7.3 Hz, 2 H, Fmoc arom.), 7.76 (d,
J = 7.3 Hz, 2 H, Fmoc arom.); 13C NMR (100.5 MHz, CDCl3, 323 K)
δ 31.9 (CH2, Cβ ΨPro), 43.2
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(CH2, Cα Gly), 47.2 (CH, Fmoc CH), 53.0 (CH3, OMe), 62.2 (q, J = 30.7 Hz, Cδ ΨPro), 62.8 (CH, Cα
ΨPro), 67.4 (CH2, Fmoc CH2), 119.9, 124.2 (q, J = 281.8 Hz, CF3), 125.0, 127.1, 127.7, 141.3, 143.8,
156.3, 168.4, 168.9; 19F NMR (376.2 MHz, CDCl3, 298 K)
δ (majo rotamer) δ –77.1 (s, CF3), (mino
rotamer) δ –77.3 (s, CF3); HRMS (ESIꢀTOF) calcd. for C23H21F3N2O5SNa [M+Na]+: 517.1021; found:
517.1028.
Fmoc-Gly-(R)-Ser(ΨCF3,HPro)-
L-Glu(OBn)-OBn (R,S,S)-14. The saponification of the dipeptide
methyl ester (R,S)ꢀ11 (15.0 g, 31.4 mmol) was performed following a described method by addition of
NaOH (1.5 g, 37.6 mmol, 1.2 equiv) to a 0.8 M CaC12 solution in iPrOHꢀH2O 7:3 (715 mL).61 The
reaction mixture was stirred for 12 h at room temperature, quenched with 1 M HCl, concentrated
under reduced pressure and diluted with H2O (200 mL). The aqueous solution was extracted with ethyl
acetate (3 ×). The combined organic layers were washed with H2O, dried over MgSO4, filtered and
concentrated under reduced pressure. The crude acid was used in the next step without further
purification. To a solution of the crude acid (6.0 g, 12.9 mmol, 1 equiv) in DCM (690 mL) were added
L
ꢀglutamic dibenzylester tosylate salt (8.06 g, 16.1 mmol, 1.25 equiv) and Et3N (5.5 mL, 39.7 mmol,
3.2 equiv). After 20 min at room temperature BOPꢀCl (4.26 g, 16.8 mmol, 1.3 equiv) was added and
the mixture was stirred at room temperature for 24 h then quenched with water. The aqueous solution
was extracted with DCM (3 x 200 mL). The organic layer was washed with brine (100 mL). The
combined organic extracts were dried over MgSO4, filtered and concentrated under reduced pressure.
The crude mixture is purified by flash chromatography (80:20 cyclohexane/ethyl acetate) to give pure
(R,S,S)ꢀ14 (6.82 g, 68%) as a 57/43 inseparable mixture of rotational isomers in CDCl3 at 298 K:
1
yellow oil; [α]25 –27.2 (c 0.9, CHCl3); IR (neat): 3310, 2928, 1731, 1677, 1529, 1150, 737 cm–1; H
D
NMR (400 MHz, CDCl3)
δ
(mixture of rotational isomers) 2.00ꢀ2.12 (m, 1 H, Hβ GluꢀHa), 2.21ꢀ2.32
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(m, 1 H, Hβ GluꢀHb), 2.34ꢀ2.54 (m, 2 H, Hγ Glu), 3.82ꢀ4.02 (m, 2 H, Hα Gly), 4.17ꢀ4.26 (m, 2 H,
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Fmoc CH and Hβ ΨProꢀHa), 4.34ꢀ4.44 (m, 3 H, Fmoc CH2 and Hβ ΨProꢀHb), 4.67ꢀ4.77 (m, 1 H, Hα
Glu), 5.02ꢀ5.20 (m, 5 H, Hα ΨPro and Bn CH2), 5.69ꢀ6.09 (m, 2 H, Hδ Pro and NHꢀGly), 7.12ꢀ7.21
(m, 1 H, NH Glu), 7.27ꢀ7.37 (m, 12 H, Bn arom. and Fmoc arom.), 7.41 (t, J = 7.3 Hz, 2 H, Fmoc
arom.), 7.62 (d, J = 7.3 Hz, 2 H, Fmoc arom.), 7.77 (d, J = 7.3 Hz, 2 H, Fmoc arom.); 13C NMR (100.5
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MHz, CDCl3)
δ (mixture of rotational isomers) 26.8 (CH2, Cβ Glu), 30.0 (CH2, Cγ Glu), 43.3 and 44.0
(CH2, Cα Gly), 46.8 (CH, Fmoc CH), 51.6 and 51.9 (CH, Cα Glu), 58.4 (CH, Cα ΨPro), 66.4 (CH2, Bn
CH2), 67.0 (CH2, Fmoc CH2), 67.2 (CH2, Bn CH2), 67.3 (Fmoc CH2), 67.4 (CH2, Cβ ΨPro), 84.7 (CH,
Cδ Pro), 119.8, 122.5 (q, J = 287.5 Hz, CF3), 124.9, 126.9, 127.6, 128.0, 128.1, 128.3, 128.4, 134.9,
135.4, 141.0, 143.5, 143.6, 156.5, 168.0, 169.2, 170.9, 171.3, 172.4, 172.5; 19F NMR (376.2 MHz,
CDCl3)
δ (mixture of rotational isomers) –81.8 (s, CF3) and ꢀ81.3 (s, CF3); HRMS (ESIꢀTOF) calcd.
for C41H39F3N3O9 [M+H]+: 774.2638; found 774.2653.
H-Gly-(R)-Ser(ΨCF3,HPro)-
L-Glu(OH)-OH-(R,S,S)-15. A solution of tripeptide (R,S,S)ꢀ14 (4.0 g,
5.16 mmoles, 1 equiv.) in MeOH (40 mL) was hydrogenated over 20% Pd/C (4.0 g) at room
temperature for 24 h under 1 bar pressure of hydrogen. The reaction mixture was filtered and
concentrated under reduced pressure. Water and Et2O were added, aqueous phase was washed with
Et2O (3 x 50 mL). The aqueous phase was concentrated under reduced pressure to give pure
deprotected peptide (R,S,S)ꢀ15 (1.75 g, 72 %) as a single rotational isomer in D2O at 353 K: yellow
solid; [α]25 –31.1 (c 1.2, H2O); IR (neat): 3600ꢀ2750, 1667, 1538 cm–1; H NMR (400 MHz, D2O,
1
D
353K)
δ 2.38ꢀ2.52 (m, 1 H, Hβ GluꢀHa), 2.56ꢀ2.70 (m, 1 H, Hβ GluꢀHb), 2.82ꢀ2.91 (m, 2 H, Hγ Glu),
4.57ꢀ4.62 (m, 2 H, Hα Gly), 4.80ꢀ4.92 (m, 2 H, Hα Glu, Hβ ΨProꢀHa), 5.10ꢀ5.20 (m, 1 H, Hβ ΨProꢀ
Hb), 5.40ꢀ5.50 (m, 1 H, Hα ΨPro), 6.45ꢀ6.52 (m, 1 H, Hδ ΨPro); 13C NMR (100.5 MHz, D2O, 353K)
δ
27.7 (CH2, Cβ Glu), 31.7 (CH2, Cγ Glu), 41.6 (CH2, Cα Gly), 55.3 (CH, Cα Glu), 59.4 (CH, Cα ΨPro),
71.1 (CH2, Cβ ΨPro), 85.5 (q, J = 34.5 Hz, Cδ ΨPro), 123.0 (q, J = 285.6 Hz, CF3), 168.3, 169.1,
177.2, 178.8; 19F NMR (376.2 MHz, D2O, 353K)
δ –80.4 (s, CF3); HRMS (ESIꢀTOF) calcd. for
C12H17F3N3O7 [M+H]+: 372.1019; found 372.1019.
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2-CF3-oxazolidine containing GPE analogues
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The compound 16 was prepared as a mixture of diastereomer (75:25) according to our previously
7
8
reported procedure.20
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2-CF3-ΨPro-OEt-17. To a solution of NꢀBocꢀethanolamine (5.200 g, 33 mmol, 1 equiv) in toluene
(100 mL) was added PPTS (829 mg, 3.3 mmol, 0.1 equiv) and ethyl trifluoropyruvate (5.610 g, 102
mmol, 1.2 equiv). After stirring the mixture for 1 h at room temperature, the reaction was heated to
reflux with a Dean Stark apparatus for 24 h then cooled to 0°C, filtrated and concentrated under
reduced pressure. Purification by flash chromatography (90:10 cyclohexane/ethyl acetate) gave a
racemic mixture of 17 (5.320 g, 44 %) as a yellow oil. IR (neat): 3342, 2987, 2904, 1742, 1448, 1372,
1322, 1279, 1232, 1177, 1016, 987, 943, 858, 817, 747, 681 cm–1; 1H NMR (400 MHz, CDCl3)
δ 1.35
(t, 3H, J = 7.1 Hz, HEt), 3.16 (q, 1H, J = 9.9 Hz, HδꢀHa), 3.32 (bs, 1H, NH), 3.38ꢀ3.45 (m, 1H, HδꢀHb),
3.77 (q, 1H, J = 7.9 Hz, Hγ−Ha), 4.15 (ddd, 1H, J = 7.9 Hz, 6.9 Hz, 2.9 Hz, HγꢀHb), 4.27ꢀ4.42 (m, 2H,
HEt); 13C NMR (100.5 MHz, CDCl3)
δ 13.6 (CEt), 46.6 (Cδ), 63.6 (CEt), 68.5 (Cγ), 93.3 (q, J= 31.6 Hz,
Cα), 122.7 (q, J = 286.6 Hz, CF3), 166.2 (CO); 19F NMR (376.2 MHz, CDCl3)
δ –83.1 (s, CF3).
HRMS (ESIꢀTOF) calcd. C7H11F3NO3 [M+H]+: 214.0691, found 214.0695.
Fmoc-Gly-2-CF3-ΨPro-OEt (18). To a solution of 17 (220 mg, 1.03 mmol, 1 equiv) in DCM (3 mL)
was added FmocꢀGlyꢀCl (490 mg, 1.55 mmol, 1.5 equiv). The mixture is stirred at room temperature
for 24 h then quenched with saturated NaHCO3 solution (5 mL). The aqueous solution was extracted
with DCM (3 x 10 mL). The organic layer was washed with a saturated NaCl solution (10 mL). The
combined organic extracts were dried over MgSO4, filtered and concentrated under reduced pressure.
The crude mixture is purified by flash chromatography (80:20 cyclohexane/ethyl acetate) to give the
racemic dipeptide 18 (378 mg, 75 %) as a white solid. Mp 54ꢀ56°C; IR (neat): 3330, 2922, 1758,
1
1715, 1682, 1203, 1170, 1033, 759, 738 cm–1; H NMR (400 MHz, CDCl3)
δ 1.30 (t, J = 7.3 Hz, 3H,
Et), 3.7 (dd, J = 8.2, 7.8 Hz,1H, Hδ ΨProꢀHa), 3.92 (m, 1H, Hδ ΨProꢀHb), 4.02 (dd, J = 17.4, 4.6 Hz,
1H, Hα GlyꢀHa), 4.11 (dd, J = 17.4, 6.9 Hz, 1H, Hα GlyꢀHb), 4.18 (dd, J = 7.3, 7.3 Hz, 1H, CHFmoc),
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4.29 (q, J = 7.3 Hz, 2H, HEt), 4.35ꢀ4.39 (m, J = 7.3 Hz, 4H, Hγ ΨPro and CH2Fmoc), 5.72 (bs, 1H,
NH), 7.28 (t, J = 7.3, 2H, HArFmoc), 7.38 (t, J = 7.3 Hz, 2H, HArFmoc), 7.57 (d, J = 7.3 Hz, 2H,
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HArFmoc), 7.74 (d, J = 7.3 Hz, 2H, HArFmoc); 13C NMR (100.5 MHz, CDCl3)
δ 13.9 (CEt), 44.1
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(Cα Gly), 45.2 (Cδ ΨPro), 47.1 (CHFmoc), 63.1 (CEt), 67.4(Cγ ΨPro), 69.0 (CH2Fmoc), 89.5 (q, J =
32.6 Hz, Cα ΨPro), 120.1 (CFmoc), 122.4 (q, J = 288.5 Hz, CF3), 125.2, 127.2, 127.9, 141.4, 143.8
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(CFmoc), 156.3, 163.5, 167.1 (CO); 19F NMR (376.2 MHz, CDCl3)
for C24H23F3N2O6 [M]+: 492.1508, Found 492.1491.
δ –79.7 (s, CF3); DIꢀHRMS calcd.
Fmoc-Gly-2-CF3-ΨPro-L-Glu(OBn)-OBn (19). To a solution of calcium chloride (0.84 M) in
isopropanol/water (7:3) (11 mL) were added the compound 18 (237 mg, 0.48 mmol, 1 equiv) and
NaOH (23 mg, 0.58 mmol, 1.2 equiv).61 The mixture was stirred at room temperature for 24 h then
quenched by HCl 1 M. The aqueous solution was extracted with ethyl acetate (3 x 10 mL). The
organic layer was washed with a saturated NaCl solution (10 mL). The combined organic extracts
were dried over MgSO4, filtered and concentrated under reduced pressure to give the intermediate acid
which was used directly in the next step without further purification. To a solution of the crude
carboxylic acid intermediate (173 mg, 0.37 mmol, 1 equiv) in DCM (20 mL) were added LꢀGlu(OBn)ꢀ
OBn (240 mg, 0.48 mmol, 1.30 equiv) and Et3N (120 mg, 1.19 mmol, 3.2 equiv). After 20 min at
room temperature BOPꢀCl (122 mg, 254.57 mmol, 1.3 equiv) was added and the mixture was stirred at
room temperature for 24 h then quenched with water. The aqueous solution was extracted with DCM
(3 x 10 mL). The organic layer was washed with a saturated NaCl solution (10 mL). The combined
organic extracts were dried over MgSO4, filtered and concentrated under reduced pressure. The crude
mixture was purified by flash chromatography (50:50 cyclohexane/ethyl acetate) to give access to the
separate tripeptide diastereomers 19maj (85 mg, 30 %) and 19min (64 mg, 22 %).
The diastereomer 19maj was obtained as colorless foam. [α]20 +15.0 (c 0.2, CHCl3); IR (neat): 3375,
D
1
2924, 1694, 1524, 1451, 1164, 1125, 1003, 740, 696 cm–1; H NMR (400 MHz, CDCl3)
δ 2.01ꢀ2.10
(m, 1H, Hβ GluꢀHa), 2.19ꢀ2.29 (m, 1H, Hβ GluꢀHb), 2.36ꢀ2.43 (m, 2H, Hγ Glu), 3.71 (dd, J = 8.2, 7.3
Hz, 1H, Hδ ΨProꢀHa), 3.86 (dd, J = 8.0, 6.2 Hz, 1H, Hδ ΨProꢀHb), 4.02ꢀ4.07 (m, 2H, Hα Gly), 4.18
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(dd, J = 7.3, 6.9 Hz, 1H, CHFmoc), 4.29ꢀ4.40 (m, 4H, CH2Fmoc and Hγ ΨPro), 4.63ꢀ470 (m, 1H, Hα
Glu), 5.03 (d, J = 14.6 Hz,1H, HBn), 5.06 (d, J = 14.6 Hz, 1H, HBn), 5.12 (d, J = 12.4 Hz, 1H, HBn),
5.14 (s, 1H, NH), 5.19 (d, J = 12.4 Hz, 1H, HBn), 5.65 (s, 1H, NH), 7.28ꢀ7.33 (m, 12 H, HArFmoc and
HAr), 7.40 (t, J = 7.3 Hz, 2 H, HArFmoc), 7.51 (d, J = 7.3 Hz, 1H, NH), 7.59 (d, J = 7.8 Hz, 2 H,
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HArFmoc), 7.75 (d, J = 7.8 Hz, 2 H, HArFmoc); 13C NMR (100.5 MHz, CDCl3)
δ 26.6 (Cβ Glu), 30.0
(Cγ Glu), 44.3 (Cα Gly), 45.5 (Cδ ΨPro), 47.1 (CHFmoc), 52.4 (Cα Glu), 66.8, 67.4, 67.7, 67.9 (CBn
CH2Fmoc and Cγ ΨPro), 90.9 (q, J = 30.7 Hz, Cα ΨPro), 120.1 (CFmoc), 122.6 (q, J = 289.5 Hz, CF3),
125.2, 127.2, 127.8, 128.2, 128.4, 128.7, 128.7 (CFmoc), 135.5, 141.2, 143.7 (CFmoc), 156.2, 162.6,
167.0, 170.7, 173.2 (CO); 19F NMR (376.2 MHz, CDCl3)
for C41H39F3N3O9 [M+H]+: 774.2638, found 774.2643.
δ –77.5 (s, CF3). HRMS (ESIꢀTOF) calcd.
The diastereomer 19min is obtained as colorless foam. [α]20 −15.5 (c 0.5, CHCl3); IR (neat): 3333,
D
2916, 1694, 1246, 1207, 1164, 1125, 1103, 1081, 1002, 758, 740, 697 cm–1; H NMR (400 MHz,
1
CDCl3)
δ 2.00ꢀ2.17 (m, 1H, Hβ GluꢀHa), 2.20ꢀ2.36 (m, 1H, Hβ GluꢀHb), 2.39ꢀ2.53 (m, 2H, Hγ Glu),
3.73 (dd, J = 15.1, 7.3 Hz, 1H, Hδ ΨProꢀHa), 3.94 (dd, J = 15.1, 6.9 Hz, 1H, Hδ ΨProꢀHb), 4.05ꢀ4.09
(m, 2H, Hα Gly), 4.21 (dd, J = 7.8, 6.9 Hz, 1H, CHFmoc), 4.30ꢀ4.40 (m, 4H, CH2Fmoc and Hγ ΨPro),
4.60ꢀ4.70 (m, 1H, Hα Glu), 5.08 (AB, 2H, HBn), 5.14 (AB, 2H, HBn), 5.75 (s, 1H, NH), 7.25ꢀ7.43 (m,
15 H, HArFmoc, HAr, and NH), 7.60 (d, J = 7.3 Hz, 2 H, HArFmoc), 7.76 (d, J = 7.3 Hz, 2H, HArFmoc );
13C NMR (100.5 MHz, CDCl3)
δ 26.4 (Cβ Glu), 29.9 (Cγ Glu), 44.1 (Cα Gly), 45.5 (Cδ ΨPro), 47.0
(CHFmoc), 52.3 (Cα Glu), 66.5, 67.2, 67.4, 67.8 (CBzl CH2Fmoc and Cγ ΨPro), 90.7 (q, J = 32.6 Hz,
Cα ΨPro), 119.9 (CFmoc), 122.4 (q, J = 291.7 Hz, CF3), 125.0, 127.0, 127.6, 128.1, 128.2, 128.4,
128.5 (CFmoc), 134.9, 135.6, 141.1, 143.7 (CFmoc), 156.2, 162.7, 167.2, 170.8, 172.8 (CO); 19F
NMR (376.2 MHz, CDCl3)
774.2638, Found 774.2660.
δ
–77.5 (s, CF3). HRMS (ESIꢀTOF) calcd. for C41H39F3N3O9 [M+H]+:
H-Gly-2CF3-ΨPro-L-Glu(OH)-OH (20). The diastereomer 19min (86 mg, 0.11 mmole, 1 equiv) in
MeOH (2 mL) was hydrogenated over 10% Pd/C (86 mg) at room temperature for 24 h under 1 bar
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pressure of hydrogen. The reaction mixture was filtered, concentrated under reduced pressure and
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purified by reverse phase semiꢀpreparative HPLC to give pure deprotected tripeptide 23 as a mixture
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of two conformers (59/41 determined by 19F NMR) (20 mg, 49 %). White solid. H NMR (400 MHz,
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D2O, 353K)
δ 1.99 (ddd, J = 14.2, 7.1, 6.9 Hz, 1H, Hβ GluꢀHa), 2.22 (ddd, J = 14.2, 8.2, 6.9 Hz, 1H,
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Hβ GluꢀHb), 2.42 (t, J = 6.9 Hz, 2H, Hγ Glu), 3.91 (dt, J = 8.2, 7.1 Hz, 1H, Hα GlyꢀHa), 4.02ꢀ4.10 (m,
3 H, Hα GlyꢀHb, Hδ ΨPro), 4.42ꢀ4.52 (m, 3 H, Ha Glu, Hγ ΨPro); 13C NMR (100.5 MHz, D2O, 353K)
δ
27.3 (CH2, Cβ Glu minor), 27.8 (CH2, Cβ Glu major), 31.1 (CH2, Cγ Glu minor), 31.5 (CH2, Cγ Glu
major), 42.4 (CH2, Cα Gly major), 45.1 (CH2, Cα Gly minor), 46.6 (CH2, Cδ ΨPro major), 46.9 (CH2,
Cδ ΨPro minor), 54.4 (CH, Cα Glu), 67.8 (CH2, Cγ ΨPro minor), 69.5 (CH2, Cγ ΨPro major), 91.8
(Cα ΨPro), 127.3 (q, J = 302.9 Hz, CF3), 162.5 (CO Gly minor), 164.9 (CO Gly major), 165.9 (CO
ΨPro), 175.9 (Cδ Glu), 177.0 (CO Glu); 19F NMR (376.2 MHz, D2O, 353K)
δ –79.9 (s, CF3 major), –
78.8 (s, CF3, minor); HRMS (ESIꢀTOF) calcd. for C12H17F3N3O7 [M+H]+: 372.1019, Found 372.1023.
AUTHOR INFORMATION
Corresponding Authors
*Eꢀmail: nathalie.lensen@uꢀcergy.fr
*Eꢀmail: gregory.chaume@uꢀcergy.fr
*Eꢀmail: thierry.brigaud@uꢀcergy.fr
Notes
The authors declare no competing financial interest.
ACKNOWLEDGEMENTS
We thank the Agence Nationale de la Recherche for financial support (No. ANRꢀ09ꢀJCJCꢀ0060). We
also thank the French Fluorine Network. This work has benefited from the facilities and expertise of
the Small Molecule Mass Spectrometry platform of IMAGIF (Centre de Recherche de Gif ꢀ
www.imagif.cnrs.fr).
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ASSOCIATED CONTENT
Supporting Information
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NMR spectra for all new compounds. This material is available free of charge via the Internet at
http://pubs.acs.org.
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