Incorporation of CF3-pseudoprolines into peptides: A methodological study

Article abstract of DOI:10.1021/jo401494q

The peptide coupling reactions allowing the incorporation of trifluoromethyl substituted oxazolidine-type pseudoprolines (CF ₃-ΨPro) into peptide chains have been studied. While standard protocols can be used for the peptide coupling reaction at the C-terminal position of the CF₃-ΨPro, acid chloride activation has to be used for the peptide coupling reaction at the N-terminal position to overcome the decrease of nucleophilicity of the CF₃-ΨPro. We demonstrate that the N-amidification of a diastereomeric mixture of CF₃-ΨPro using Fmoc-protected amino acid chloride without base gave the corresponding dipeptides as a single diastereomer (6 examples). The ratio of the cis and trans amide bond conformers was determined by NMR study, highlighting the role of the Xaa side chains in the control of the peptide backbone conformation. Finally a tripeptide bearing a central CF₃-ΨPro has been successfully synthesized.

Full text of DOI:10.1021/jo401494q

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Article
Incorporation of CF3-Pseudoprolines into Peptides: a Methodological Study
Grégory Chaume, Julien Simon, Caroline Caupène, Nathalie Lensen, Emeric Miclet, and Thierry Brigaud
J. Org. Chem., Just Accepted Manuscript • DOI: 10.1021/jo401494q • Publication Date (Web): 13 Sep 2013
Downloaded from http://pubs.acs.org on September 15, 2013
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Incorporation of CF3-Pseudoprolines into Peptides: a Methodological Study
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Grégory Chaume,*,† Julien Simon,† Caroline Caupène,† Nathalie Lensen,† Emeric Miclet,‡
Thierry Brigaud*,†
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† Laboratoire SOSCO, Université de CergyꢀPontoise, EA 4505, 5 Mail GayꢀLussac, 95000
CergyꢀPontoise, France, ‡ Laboratoire des BioMolécules, UMR 7203, Ecole Normale Supérieure,
UPMC Paris 06, 4, Place Jussieu, 75005 Paris, France.
gregory.chaume@uꢀcergy.fr; thierry.brigaud@uꢀcergy.fr
R1
Cl
FmocHN
O
O
O
H
N
CO2tBu
* O
(1.1 equiv)
F3C
R1
F3C
CO2Me
N
N
F3C
CO2Me
O
N
no base
O
O
H
FmocHN
FmocHN
Single diastereomer
6 examples, 52-98% Yield
Abstract
The peptide coupling reactions allowing the incorporation of trifluoromethyl substituted
oxazolidineꢀtype pseudoprolines (CF3ꢀΨPro) into peptide chains have been studied. While
standard protocols can be used for the peptide coupling reaction at the Cꢀterminal position of the
CF3ꢀΨPro, acid chloride activation has to be used for the peptide coupling reaction at the Nꢀ
terminal position to overcome the decrease of nucleophilicity of the CF3ꢀΨPro. We demonstrate
that the Nꢀamidification of a diastereomeric mixture of CF3ꢀΨPro using Fmoc protected amino
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acid chloride without base gave the corresponding dipeptides as a single diastereomer (6
examples). The ratio of the cis and trans amide bond conformers was determined by NMR study,
highlighting the role of the Xaa side chains in the control of the peptide backbone conformation.
Finally a tripeptide bearing a central CF3ꢀΨPro has been successfully synthesized.
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Introduction
Because of its cyclic structure, proline residue is recognized to play a unique and important role
on the peptide backbone conformation. When included into a peptide, this feature prevents the
proline from acting as a hydrogen bond donor and restrains the φ diehedral angle to about –60°.1
The XaaꢀPro peptide bond is characterized by a small free energy difference (ꢀGtc) between the
trans and the cis amide bond conformers, combined with a high activation energy (ꢀG‡ ) for the
tc
cisꢀtrans isomerization (Figure 1).2ꢀ5 This isomerization is considered to play a key role for
regulating many important biological processes in proline containing peptides and proteins,
including the protein folding.6ꢀ9 Numerous proline surrogates have been used to design peptides
and peptidomimetics with defined conformation to improve their biological properties.10ꢀ13 In this
context, the use of pseudoprolines (ΨPro) appears as a very attractive approach.14 In their pioneer
works, Mutter et al. have shown that oxazolidines and thiazolidines derivatives could be obtained
by the cyclocondensation of serine, threonine, and cysteine amino acids with aldehydes, ketones,
and acetals.15,16 CδꢀSubstituted pseudoprolines have gained in popularity because they exhibit a
remarkable ability to induce cis amide bonds conformations in peptide backbone.17ꢀ20 While
unsubstituted pseudoprolines are similar to proline and favor the trans amide bond
conformation,21,22 disubstitution at Cδ leads to a high cis content and a decrease of the ꢀG‡
.
17,18
tꢀc
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FIGURE 1. Cis-Trans Conformer Equilibrium in Proline and Pseudoprolines (ΨR1,R2Pro)
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TS
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β
β
R2
R1
R2
R1
X
X
X = CH2, O, S
α
α
ꢀG‡
δ
δ
c-t
N
N
ꢀG‡
t-c
O
O
O
cis
ꢀGt-c
O
trans
cis conformer
trans conformer
Therefore, pseudoprolines proved to be a valuable tool to tailor the cisꢀtrans isomerization of the
XaaꢀΨPro amide bonds for various applications in peptide engineering. The incorporation of
pseudoprolines in small peptide chains is a powerful strategy to promote the headꢀtoꢀtail
cyclization.23ꢀ27 Pseudoprolines are also broadly used in solidꢀphase peptide synthesis (SPPS) as
temporary protecting groups to disrupt aggregates formation of the growing peptide chain and to
significantly enhance the yield of the peptide synthesis.28ꢀ34 Moreover, these proline surrogates
are very useful tools for investigating the peptide bioactive conformations.35ꢀ44 Despite these
interesting features, pseudoprolines exhibit varied chemical stability under acidic conditions
depending on the nature of the heteroatom and the degree of substitution at the Cδ carbon atom.29
This drawback limits the use of oxazolidineꢀtype pseudoprolines mostly as temporary protecting
group in SPPS while thiazolidineꢀtype pseudoprolines can also be exploited for the synthesis of
conformationally constrained peptides.
The introduction of fluorine atoms into biomolecules such as peptides is known to deeply
influence their chemical and biological properties. It can provide a better affinity for lipid
membranes, increased metabolic or thermal resistances, as well as improved autoassembly
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properties.45ꢀ51 Fluorinated peptides can also be used as efficient probes for 19F NMR studies.52ꢀ56
However, regioꢀ and stereoselective synthetic methods allowing the direct introduction of
fluorine atoms or fluorinated groups into biomolecules are still challenging. Therefore, the
development of syntheses of fluorinated biomolecules such as fluorinated amino acids has gained
a considerable interest in peptide and protein chemistry.57,58 Our group is focused on the
stereoselective synthesis of trifluoromethylated amino acids (CF3ꢀAAs)59ꢀ63 and their
incorporation into peptides.64 We have already reported the preparation of enantiomerically pure
trifluoromethylated pseudoprolines (CF3ꢀΨPro) and demonstrated that the stereoelectronic effects
imparted by the trifluoromethyl group strongly increased the chemical stability of the oxazolidine
core in acidic medium.65 Accordingly, CF3ꢀsubstituted pseudoprolines behave as hydrolytically
stable proline surrogates. We have also reported the electronic and geometric consequences due
to the CF3 group incorporation in CF3ꢀΨPro containing tripeptide models 1 and 2 by theoretical
calculations and NMR studies (Figure 2).66,67 In particular, we have demonstrated that the
incorporation of a Ser(ΨCF3,HPro) residue into a peptide chain can : lower the rotational barrier of
the cisꢀtrans peptide bond isomerization; enhance the cis population ratio; freeze the puckering of
the oxazolidine core and tune the peptide conformation depending of its absolute configuration
and the solvent polarity.
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FIGURE 2. Structures of the CF3-ΨPro Containing Tripeptide Models66,67
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O
4
2
O
F3C
N
O
O
HN
O
4
2
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2
O
O
F3C
F3C
N
N
HN
HN
O
NH
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Ac-Ser(ΨH,CF3Pro)-NHMe
trans-(2S,4S)-1 cis-(2R,4S)-1
Ac-Ala-Ser(ΨH,CF3Pro)-NHMe
2
Herein, we report a methodological study for the development of efficient coupling reactions
allowing the incorporation of trifluoromethyl substituted oxazolidineꢀtype pseudoprolines into
peptide chains. We investigated the peptide coupling reaction at the Cꢀ and the Nꢀtermini of the
CF3ꢀΨPro residue. We also report the NMR study on five FmocꢀXaaꢀSer(ΨCF3,HPro)ꢀOMe
derivatives highlighting the role of the Xaa side chains in the control of the peptide backbone
conformation.
Results and Discussion
Coupling reaction at the C-Terminal Position: Synthesis of N-Terminal CF3-Pseudoproline
Containing Dipeptides
To demonstrate the incorporation of the Ser(ΨCF3,HPro) residue into peptide chains, we first
investigated the peptide coupling reaction at its Cꢀterminal position. Because of the decrease of
the nucleophilicity of the nitrogen atom due to the CF3 group electron withdrawing effect, no
protection of the pseudoproline amino group is required.64 The syntheses of the Nꢀterminal
Ser(ΨCF3,HPro) containing dipeptides 4 were achieved using a twoꢀsteps procedure involving the
saponification of the ester function followed by the amidification reaction with alanine benzyl
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ester using a standard coupling protocol. Starting from oxazolidines transꢀ(2S,4S)ꢀ3a and cisꢀ
(2R,4S)ꢀ3b, the dipeptides 4a and 4b were obtained in respectively 58% and 82% yields without
diketopiperazine side product formation and without epimerization at the Cꢀ2 or the Cꢀ4 center
(Scheme 1). As we already observed for similar amidification reactions,65 the yield difference
between 4a and 4b could be due to steric factors. These dipeptides, bearing a Ser(ΨCF3,HPro)
residue at the Nꢀterminal position, constitute new building blocks with an anticipated increased
lipophilicity since the presence of the vicinal CF3 group dramatically decreases the protonation
ability of the nitrogen atom.
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SCHEME 1. Synthesis of N-Terminal CF3-Pseudoproline Containing Dipeptides 4
O
2
O
1) LiOHaq, THF
2
F3C
H
4
F3C
CO2Bn
4
N
N
H
N
H
2) HCl.H2N-Ala-OBn,
NaHCO3,
CO2Me
O
HOBt, EDCI,DMF
(2S,4S)-3a
4a (58%)
O
2
O
2
same conditions
F3C
H
4
F3C
4
CO2Bn
N
N
H
N
CO2Me
H
O
(2R,4S)-3b
4b (82%)
Coupling at the N-Terminal Position: Synthesis of C-Terminal CF3-Pseudoproline
Containing Dipeptides
The synthesis of Cꢀterminal Ser(ΨCF3,HPro) containing dipeptides was then investigated. Because
of the lack of nucleophilicity of the amino group of the oxazolidines 3, the amide bond formation
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using amino acids activated as an ester, a symmetric anhydride68 or a mixed anhydride69 failed to
give the expected dipeptides. In contrast, we previously reported the peptide coupling reaction of
the cisꢀ(2R,4S)ꢀ3b oxazolidine using large amounts (7 equiv) of oꢀNbsꢀalanine chloride 5 in the
presence of collidine according to a reported procedure.70,71 The expected dipeptide 6 was
obtained in 91% yield as a 73:27 mixture of two diastereomers (Table 1, entry 1).65 The major
diastereomer of 6 was isolated by flash chromatography and fully characterized by 2D NMR
spectroscopy. The assignment of its absolute configuration was confirmed by Xꢀray
crystallographic analysis.67,72 At this stage, we were not able to unambiguously assign the
absolute configuration of the minor diastereomer of 6. When the reaction was performed starting
from the transꢀ(2S,4S)ꢀ3a oxazolidine, the same major dipeptide 6 was obtained with a similar
diastereomeric ratio indicating that an epimerization reaction occurred at the Cꢀ2 center of the
oxazolidine ring (Table 1, entry 2). As mentioned previously for the Nꢀbenzoylation reactions of
the CF3ꢀsubstituted oxazolidines,65 this result suggests an epimerization reaction of the transꢀ
(2S,4S)ꢀ3a oxazolidine into the cisꢀ(2R,4S)ꢀ3b oxazolidine under the reaction conditions. The Nꢀ
coupling reactions of the transꢀ(2S,4S)ꢀ3a and the cisꢀ(2R,4S)ꢀ3b oxazolidines occurred also
using a smaller amount of oꢀNbsꢀalanine chloride (2 equiv) without base to afford the dipeptide 6
in good yield (Table 1, entries 3 and 4). The presence or the absence of base for the coupling did
not affect the epimerization level since the diastereomeric ratio remained almost identical in all
cases. The coupling reactions of the transꢀ(2S,4S)ꢀ3a and the cisꢀ(2R,4S)ꢀ3b oxazolidines with oꢀ
Nbsꢀalanine uronium in the presence of collidine gave the dipeptide 6 in similar ranges of yield
and diastereomeric ratio (Table 1, entries 5 and 6).
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TABLE 1. Methodological study of the Peptide Coupling using o-Nbs-Alanine Chloride
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F3C
CO2Me
N
Conditions A, B or C
o-NbsHN
CO2H
O
O
o-NbsHN
2
4
9
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F3C
CO2Me
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N
H
(2S,4S)-3a or (2R,4S)-3b
(1 equiv)
Conditions A : 5 (7 equiv), 1-chloro-N,N-2-trimethyl-1-propenylamine (7 equiv), collidine (1 equiv), CH2Cl2
Conditions B : 5 (2 equiv), 1-chloro-N,N-2-trimethyl-1-propenylamine (2 equiv), CH2Cl2
Conditions C : 5 (2.2 equiv), SOCl2 (3.3 equiv), TMU (0.44 equiv), collidine (1 equiv), CH2Cl2
entry
conditions
CF3ꢀΨProꢀOMe 3
(2R,4S)ꢀ3b
(2S,4S)ꢀ3a
dipeptide 6a (dr)b
91% (73:27)c
79% (85:15)
86% (89:11)
79% (82:18)
78%, (87:13)
87%, (90:10)
1
2
3
4
5
6
A
A
B
B
C
C
(2S,4S)ꢀ3a
(2R,4S)ꢀ3b
(2S,4S)ꢀ3a
(2R,4S)ꢀ3b
a Isolated yield. b Measured by 19F NMR of the crude reaction mixture. The dr represents the ratio of the major
(2R,4S)-6 diastereomercompared to a minor diastereomer which configuration was not assigned. c see Ref. 65
The peptide coupling reaction of the transꢀ(2S,4S)ꢀ3a oxazolidine using the achiral protected
amino isobutyric acid (Aib) chloride derived from 7 afforded the peptide 8 in moderate yield
(66%) as a single diastereomer (Scheme 2). In accordance with the above results (see Table 1),
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the full NMR characterization of 8 revealed the relative (2,4)ꢀcis configuration of the oxazolidine
moiety indicating that the epimerization of the transꢀ(2S,4S)ꢀ3a oxazolidine into the cisꢀ(2R,4S)ꢀ
3b oxazolidine is total prior to the coupling reaction.
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SCHEME 2. Synthesis of C-Terminal CF3-Pseudoproline Containing Dipeptide 8
O
1) chloroenamine (5.7 equiv)
2
4
F3C
CH2Cl2
CO2Me
OH
N
o-NbsHN
7 (7 equiv)
O
O
2)
O
2
4
o-NbsHN
8 (66%, 100:0 dr)
F3C
CO2Me
N
H
(2S,4S)-3a (1 equiv)
collidine (1 equiv)
We then focused our attention on the Nꢀamidification reactions of oxazolidines 3 with Fmocꢀ
protected amino acid chloride prepared according to a reported procedure.73 This approach allows
the synthesis of Cꢀterminal Ser(ΨCF3,HPro) containing dipeptides with a suitable protecting group
for SPPS. We previously reported that the Fmocꢀalanine chloride was electrophilic enough for
the Nꢀcoupling of the (R)ꢀ(αꢀCF3)Alaꢀ
LꢀLeuꢀOBn dipeptide to afford the corresponding Fmocꢀ
Alaꢀ(R)ꢀ(αꢀCF3)Alaꢀ
L
ꢀLeuꢀOBn tripeptide in good yield.64 We also recently reported that the
amidification reaction of the cisꢀ(2R,4S)ꢀ3b oxazolidine with the Fmocꢀalanine chloride in the
presence of diisopropylethylamine (DIEA) gave the corresponding dipeptides 9a and 9b in 90%
yield as a 89:11 diastereomeric mixture (Table 2, entry 1).67 As observed with oꢀNbs protected
amino acids, the reaction led to the same diastereomeric mixture of dipeptides 9a and 9b in high
yield whatever was the Cꢀ2 absolute configuration of the starting oxazolidines 3 (Table 2, entries
2 and 3).
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TABLE 2. Methodological Study of the Peptide Coupling Using Fmoc-Alanine Chloride in
Basic Conditions
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O
O
2
4
2
4
1) SOCl2, CH2Cl2, ))))
F3C
F3C
CO2Me
CO2Me
FmocHN
CO2H
+
N
N
O
2)
F3C
2
4
(1.1 equiv)
O
O
CO2Me
N
FmocHN
FmocHN
H
9a
9b
3 (1 equiv)
DIEA, CH2Cl2
DIEA (equiv)
1
entry
CF3ꢀΨProꢀOMe 3
(2R,4S)ꢀ3b
dipeptides 9a,ba (dr)b
90% (89:11)c
1
2
3
(2S,4S)ꢀ3a
94% (80:20)
1
1
3a/3b (1:1)
92% (82:18)
a Isolated yield. b Measured by 19F NMR of the crude reaction mixture. c Ref. 67.
It was anticipated that basic reaction conditions would be responsible of the Cꢀ4 epimerization
reaction. Indeed, when the reaction of a diastereomeric mixture of oxazolidines 3 was performed
without base, the dipeptide 9a was obtained as a single diastereomer in 66% yield (Table 3, entry
1). It should be stressed that these conditions constitute a great improvement since only 1.1
equivalents of Fmocꢀprotected amino acids is required and that the only side product of the
reaction is HCl.
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TABLE 3. Synthesis of C-Terminal Tfm-Pseudoproline Containing Dipeptides 9a-14
8
9
without Base
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O
1) SOCl2, CH2Cl2, ))))
2
4
F3C
CO2Me
Fmoc-AA-OH
(1.1 equiv)
N
2)
F3C
O
2
4
Fmoc-AA
O
CO2Me
AA : Gly, L-Ala, D-Ala,
Val, Pro, Aib
N
H
9a,10,11,12,13,14
3 (1 equiv)
single diastereomer
CH2Cl2
yield (%)a
CF3ꢀΨProꢀOMe 3
3a/3b (84:16)
3a/3b (84:16)
3a/3b (84:16)
3a/3b (84:16)
3a/3b (84:16)
3a/3b (100:0)
entry
FmocꢀAAꢀOH
dipeptide
1
2
3
4
5
6
L
ꢀAla
66
98
59
52
55b
79
9a
10
11
12
13
14
Gly
L
ꢀVal
ꢀPro
L
Aib
D
ꢀAla
a Isolated yield. b3 equiv of Fmoc-Aib-OH were used.
These optimized conditions were successfully applied to coupling reaction of various Fmocꢀ
protected amino acid chlorides with Tfmꢀpseudoprolines 3. The dipeptides 10, 11, 12, 13 and 14
derived respectively from glycine, valine, proline, aminoisobutyric acid (Aib) and Dꢀalanine were
obtained as a single diastereomer (Table 3, Entries 2–6). The yield of the reaction decreased with
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the steric hindrance of the Fmocꢀprotected amino acid chloride. Larger amount (3 equiv) of the
amino acid chloride was required in the case of Aib (Table 3, entry 4).
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10
11
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13
14
15
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Configuration assignments and epimerization explanation
The assignment of the relative configuration of the dipeptides 9aꢀ14 was then investigated by
NMR nOe analysis. When the coupling reactions were carried out without base, the exclusive
formation of the cisꢀ(2R,4S)ꢀoxazolidine containing dipeptides was observed. This result is
similar to those reported in the literature.74,75 While in the Nꢀunacetylated series the (2,4)ꢀtrans
oxazolidine is found to be more stable than the (2,4)ꢀcis isomer, the (2,4)-cis Nꢀacylated
oxazolidines are favoured compared to the corresponding (2,4)ꢀtrans Nꢀacylated isomers. This
preferred (2,4)ꢀcis relative configuration of Nꢀacylated oxazolidine moieties is due to the all trans
configuration minimizing the steric interactions (Scheme 3). The approach of the cisꢀoxazolidine
amino group by the acylating reagent is easy from the opposite side of the sterically hindered
face, while for the transꢀoxazolidine, both sides are hindered. Because of the complete
diastereoselectivity observed for the peptide coupling reactions without base, we can argue that
the acid catalyzed equilibrium between the (2,4)ꢀcis and the (2,4)ꢀtrans oxazolidines 3 before the
amide bond formation is critical for this outcome. The interconversion of the transꢀ(2S,4S)ꢀ3 to
the cisꢀ(2R,4S)ꢀ3 oxazolidine ring should proceed through an acidic promoted ring opening /ring
closing process involving a protonated hydroxyimine intermediate. As known for Nꢀacylated
oxazolidine compounds, there is no equilibrium between the cisꢀ(2R,4S)ꢀoxazolidine containing
dipeptides obtained and their transꢀ(2S,4S)ꢀ diastereomers.
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SCHEME 3. Mechanism of the N-Acylation Reaction without Base
5
MeO2C
MeO2C
6
7
8
9
4
4
H
CF3
H
H
H
O
O
H+
O
HN
2
O
2
HN
2
2
4
4
N
N
MeO2C
MeO2C
CF3
H
H
10
11
12
13
14
15
16
17
18
19
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22
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CF3
cis-(2R,4S)-3
CF3
trans-(2S,4S)-3
trans
(most stable)
cis
(less stable)
O
O
FmocAA
FmocAA
Cl
Cl
MeO2C
4
MeO2C
4
O
2
FmocAA
N
X
O
FmocAA
N
2
O
CF3
O
CF3
cis-(2R,4S)
trans-(2S,4S)
When the coupling reaction of the cisꢀ(2R,4S)ꢀ3b oxazolidine with the Fmocꢀalanine chloride
were carried out in the presence of base, the corresponding dipeptide was obtained as a mixture
of two diastereomers 9a and 9b (see Table 2). The use of basic reaction conditions would be
responsible of the epimerization reaction. The formation of the transꢀoxazolidine containing
dipeptide resulting from the Cꢀ2 epimerization of the oxazolidines ring was not considered
because of the great steric hindrance of this position. However, it was anticipated that the
epimerization reaction could occur either at the Cꢀ4 of the oxazolidine or at the Cα of the Ala
residue. To discriminate these hypotheses, the coupling reaction of the oxazolidines 3 was
performed using the FmocꢀDꢀalanine chloride applying our optimized conditions (Table 3, entry
6). The corresponding peptide 14 was obtained in 79% yield as a single diastereomer. Since the
1
optical rotation value of 14 was opposite to that of the minor diastereomer 9b and since their H
NMR spectra were indentical, 14 and 9b should be enantiomers and we assigned the (2S,4R)ꢀ
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9
configuration to the minor diastereomer 9b (Scheme 4). We concluded that the amidification of
Tfmꢀpseudoprolines in the presence of base led to the partial epimerization of the Cꢀ4 center. For
the reasons exposed above (Scheme 3), the epimerization of the Cꢀ4 center prompted the
epimerization of the Cꢀ2 center prior to the coupling and only the cisꢀ(2S,4R)ꢀoxazolidine 3b or
its cisꢀ(2R,4S)ꢀentꢀ3b enantiomer were Nꢀacylated (Scheme 4).
10
11
12
13
14
15
16
17
18
19
20
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27
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48
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60
SCHEME 4. Configuration Assignments of the Minor Diastereomer of Dipeptide 9b
O
O
2
4
Fmoc-Ala-Cl
F3C
CO2Me
F3C
CO2Me
N
N
H
3b
O
FmocHN
9a
O
2
4
F3C
CO2Me
N
H
3a
Base
C-4 epimerization
O
O
2
4
2
4
O
F3C
F3C
CO2Me
CO2Me
2
4
N
N
Fmoc-Ala-Cl
F3C
CO2Me
N
H
O
O
ent-3b
FmocHN
FmocHN
9b
14 (enantiomer of 9b)
[α]20D - 47.5 (c 1.00, CHCl3)
[α]20D + 42.8 (c 1.00, CHCl3)
O
2
4
F3C
CO2Me
N
H
ent-3a
Analysis of the Cis-Trans Amide Bond Conformer Ratio
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The ratio of the cis and trans amide bond conformers was determined for the dipeptides 9a, 10,
5
6
1
11, 12 and 13 at 274 K in CDCl3 by integration of both H and 19F NMR isolated conformer
7
8
9
resonances (Table 4). The XaaꢀΨPro amide bond conformation was assigned using interꢀresidue
10
11
12
13
14
15
16
17
18
19
20
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22
23
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45
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nOe correlations in the 2D NMR experiments. We observed a strong HαXaaꢀHδ Pro cross peak for
Ψ
the unique spin system of peptide 12 (Xaa = Pro) in agreement with a trans amide bond
conformation (Table 4, Entry 1). The dipeptides 9a, 10, 11 and 13 showed an additional HαXaaꢀ
Hα Pro correlation, indicating the presence of a stabilized cis conformation. The dipeptides 9a and
Ψ
11, bearing respectively the alanine and the valine residues, displayed a high proportion of the
trans population which seemed to be closely related with the steric hindrance of the amino acid
side chain (Table 4, Entries 2 and 3). The dipeptide 10, bearing the unsubstitued glycine residue,
exhibited a high proportion of cis conformer (61%, see Table 4, Entry 4). This level was quite
similar with the described disubstituted thiazolidine containing dipeptide CbzꢀGlyꢀ
Thr(ΨMe,MePro)ꢀOMe (75% cis).76 We suppose that the presence of the bulky Fmoc group could
be responsible for the destabilization of the cis conformer because the oꢀNbs Nꢀprotected peptide
6 showed a (55:45) isomeric ratio.67 The incorporation of the Aib residue increased significantly
the ratio of the cis conformer (85%, Table 4, Entry 5) indicating that the trans isomer is
destabilized by the presence of the methyl group substituting the Hα of the Ala residue.
TABLE 4. Cis-Trans Ratio for Dipeptides 9a-13
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7
8
9
β
β
O
δ
N
O
δ
N
F3C
H
F3C
CO2Me
H
H
CO2Me
H
α
α
H
H
R1
nOe
nOe
O
O
R1
NHFmoc
NHFmoc
10
11
12
13
14
15
16
17
18
19
20
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cis-amide bond conformer
trans-amide bond conformer
entry
AA
compound
cis/transa
0/100
7/93
1
2
3
4
5
L
ꢀPro
ꢀVal
ꢀAla
12
11
9a
10
13
L
L
12/88b
61/39
85/15
Gly
Aib
a Measured by 1H and 19F NMR at 274 K in CDCl3. b Ref 67
Synthesis of a CF3-Pseudoproline Containing Tripeptide
To demonstrate the potential incorporation of CF3ꢀpseudoprolines into peptide chains, a CF3ꢀ
pseudoproline containing tripeptide, bearing the Ser(ΨH,CF3Pro) residue at the central position,
was synthesized. To circumvent the risk of epimerization at both the Cꢀ2 and the Cꢀ4 centers of
the Ser(ΨH,CF3Pro) moiety, we adopted the unusual Nꢀterminal to Cꢀterminal peptide elongation
starting from the diastereomerically pure FmocꢀAlaꢀSer(ΨH,CF3Pro)ꢀOMe dipeptide 9a. Treatment
of 9a under smooth conditions with a 0.8 M NaOH solution in the presence of CaCl2 allowed the
ester hydrolysis without traces of Fmoc deprotection.77 Acidic treatment of the crude followed by
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the coupling of the Lꢀalanine tertꢀbutyl ester using BOPꢀCl afforded the tripeptide 15 in moderate
5
6
1
yield (Scheme 5). A 47:53 cis/trans population ratio was observed by H NMR contrasting with
7
8
9
the 12:88 ratio obtained for 9a. The very low field resonance of the Cꢀterminal alanine NH amide
proton (δΝΗ = 8.40 ppm) for the minor conformer suggested its involvement in a strong hydrogen
bond, which could account for the stabilization of the cis AlaꢀSer(ΨH,CF3Pro) peptide bond in
peptide 15. As already observed with the pseudoꢀtetrapeptide AcꢀAlaꢀSer(ΨCF3,HPro)ꢀNHMe, we
10
11
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14
15
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46
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53
54
55
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anticipated the existence of a type VI β
ꢀturn conformation for the minor conformer of 15.67
SCHEME 5. Synthesis of the Tripeptide 15
1) NaOH, (0.8 M) CaCl2
iPrOH/H2O (7:3)
2) H3O+
O
O
H
N
CO2tBu
F3C
CO2Me
F3C
N
N
O
3) HCl.H2N-Ala-OtBu
Et3N, BOP-Cl
DCM
O
O
FmocHN
9maj
FmocHN
15 (30% over 2 steps)
cis/trans (47:53)
Conclusion
Trifluoromethylꢀgroup containing pseudoprolines Ser(ΨH,CF3Pro) have been successfully
incorporated into a peptide chain at both Nꢀ and Cꢀtermini. The coupling reactions at the Cꢀ
terminus can be performed using standard protocols. The more challenging Nꢀamidification
reaction required specific coupling conditions to circumvent the lack of nucleophilicity of the
Ser(ΨCF3,HPro) nitrogen atom and to prevent the epimerization of the Ser(ΨH,CF3Pro) residue at
the Cꢀ4 center. The use of Fmocꢀprotected amino acid chlorides without base allowed efficient
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coupling to afford several FmocꢀXaaꢀSer(ΨCF3,HPro)ꢀOMe dipeptides as single diastereomers in
good yield. Conformational studies by NMR spectroscopy revealed that the geometry of the
amide bond was depending on both the nature of the side chain of the preceding amino acids.
Finally, we synthesized a tripeptide bearing the Ser(ΨH,CF3Pro) residue at the central position
suitably protected and ready to be used in solid phase peptide synthesis.
3
4
5
6
7
8
9
10
11
12
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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 were 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
1
13
obtained by Fourier transformation and wave numbers are given in cm–1. H NMR, C 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 spectrometer operating
at a 1H frequency of 400 MHz. 1H NMR (500.00 MHz), 13C NMR (125.75 MHz) were measured
on a spectrometer operating at a 1H frequency of 500 MHz and equipped with a triple resonance,
zꢀaxis pulsedꢀ5 fieldꢀgradient cryogenic probehead, optimized for 1H detection. Complete proton
assignments were obtained from the analysis of 2D total correlation spectroscopy (TOCSY)
experiments using 80 ms DIPSIꢀ2 mixtime, and 2D nuclear Overhauser effect spectroscopy
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(NOESY) experiments (typically 500 ms mixing time). Homonuclear experiments were typically
collected as 512 (t1) and 4096 (t2) timeꢀdomain matrices over a spectral width of 10 ppm, with 8
7
8
9
1
scans per t1 increment. Carbon assignment was deduced from heteronuclear 2D Hꢀ13C HSQC
1
and 2D Hꢀ13C CH2ꢀTROSY16 experiments, using 256 (t1) x 1024 (t2) timedomain matrices,
10
11
12
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1
with 32 scans per t1 increment. 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
19
parts per million downfield from CDCl3 as internal standard (δ = 77.0). Chemical shifts of F
NMR are expressed in parts per million downfield from C6F6 as an internal standard (δ = –164.9).
Coupling constants are reported in Hertz. Melting points were uncorrected. Highꢀresolution mass
spectra were obtained using electrospray ionization in positive ion mode and a TOF mass
analyzer. Mass spectra were recorded using electrospray ionization in positive ion mode and a
quadrupole mass analyzer.
Synthesis of N-Terminal CF3-Pseudoproline Containing Dipeptides.
Representative Procedure for the Peptide Coupling Reaction at the C-terminal position. To
a solution of oxazolidine 3a or 3b (1 equiv) in THF at 0 °C was added a 1 M aqueous solution of
LiOH (1.1 equiv). The reaction mixture was vigorously stirred for 4 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.65 The lithium carboxylate was
diluted in DMF, and Lꢀalanine benzylester hydrochloride (1.1 equiv), HOBt (1.0 equiv), NaHCO3
(3.0 equiv) and EDCI (1.1 equiv) were successively added at room temperature. The reaction
mixture was stirred overnight at room temperature, and then diluted with AcOEt and water. The
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layers were separated, and the aqueous layer was extracted with AcOEt (3 ×). The combined
organic layers were dried over MgSO4, filtered, and concentrated under reduced pressure.
Purification by flash chromatography gave dipeptides 4a and 4b in 58% and 82% yield
respectively.
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13
14
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(2S,4S)-H-Ser(ΨCF3,HPro)-Ala-OBn (4a). The dipeptide 4a was prepared according to the
representative procedure. The lithium carboxylate was prepared starting from oxazolidine
(2S,4S)-3a (796 mg, 4.0 mmol) in THF (22 mL), 1 M aqueous solution of LiOH (4.4 mL, 4.4
mmol, 1.1 equiv) as a yellow oil. The dipeptide 4a was obtained starting from the crude lithium
carboxylate in DMF (10.5 mL), Lꢀalanine benzylester hydrochloride (951 mg, 1.1 equiv, 4.4
mmol), HOBt (541 mg, 1.0 equiv, 4.0 mmol), NaHCO3 (1.00 g, 3.0 equiv, 12.0 mmol) and EDCI
(844 mg, 1.1 equiv, 4.4 mmol). Purification by flash chromatography (70:30 cyclohexane/ethyl
acetate) gave the dipeptide 4a (800 mg, 58%) as a white solid: mp 98–100 °C; Rf = 0.35 (70:30
cyclohexane/ethyl acetate); [α]26D –37.1 (c 1.8, CHCl3); IR (neat) 3308, 2977, 1730, 1654, 1549,
1455, 1143, 692 cm–1; 1H NMR (400 MHz, CDCl3)
δ 1.42 (d, J = 7.3 Hz, 3 H, Hβ AlaꢀH), 3.28 (t,
J = 8.2 Hz, 1 H, NH ΨPro), 3.83 (dd, J = 7.8, 6.9 Hz, 1 H, Hβ ΨProꢀHa), 3.98 (ddd, J = 8.2, 7.8,
6.9 Hz, 1 H, Hα ΨProꢀH), 4.19 (t, J = 7.8 Hz, 1 H, Hβ ΨProꢀHb), 4.63 (quint, J = 7.3 Hz, 1 H, Hα
AlaꢀH), 4.99 (dq, J = 8.2, 5.5 Hz, 1 H, Hδ ΨProꢀH), 5.14 (d, J = 12.4 Hz, 1 H, Bn CH2ꢀHa), 5.19
(d, J = 12.4 Hz, 1 H, Bn CH2ꢀHb), 6.95 (d, J = 7.3 Hz, 1 H, NH Ala), 7.28–7.42 (m, 5 H, Bn
13
arom.); C NMR (100.5 MHz, CDCl3)
δ 18.2 (CH3, Cβ Ala), 48.3 (CH, Cα Ala), 59.3 (CH, Cα
ΨPro), 67.4 (CH2, Bn CH2), 70.2 (CH2, Cβ ΨPro), 88.0 (q, J = 33.6 Hz, CH, Cδ ΨPro), 123.0 (q,
J = 282.7 Hz, CF3), 128.2 (2 × CH, Bn arom.), 128.5 (CH, Bn arom.), 128.6 (2 × CH, Bn arom.),
135.0 (C, Bn arom.), 169.4 (C, C=O), 172.3 (C, C=O); 19F NMR (376.2 MHz, CDCl3)
δ –84.6 (d,
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J = 5.5 Hz, CF3); EIMS m/z: M+ 346, 277, 211, 164, 140, 112, 91 (100); HRMS (ESIꢀTOF) calcd
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for C15H17F3N2O4 346.1140, found 346.1134.
7
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9
(2R,4S)-H-Ser(ΨCF3,HPro)-Ala-OBn (4b). The dipeptide 4b was prepared according to the
representative procedure. The lithium carboxylate was prepared starting from oxazolidine
(2R,4S)-3b (792 mg, 4.0 mmol) in THF (22 mL), 1 M aqueous solution of LiOH (4.4 mL, 4.4
mmol, 1.1 equiv) as a yellow oil. The dipeptide 4b was obtained starting from the crude lithium
carboxylate in DMF (10.5 mL), Lꢀalanine benzylester hydrochloride (970 mg, 1.1 equiv, 4.4
mmol), HOBt (554 mg, 1.0 equiv, 4.0 mmol), NaHCO3 (1.00 g, 3.0 equiv, 12.0 mmol) and EDCI
(876 mg, 1.1 equiv, 4.4 mmol). Purification by flash chromatography (70:30 cyclohexane/ethyl
acetate) gave the dipeptide 4b (1.18 g, 82%) as a colorless solid: mp 77–81 °C; Rf = 0.15 (60:40
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cyclohexane/ethyl acetate); [α]27 –14.6 (c 2.25, CHCl3); IR (neat) 3309, 2908, 1745, 1654,
D
1526, 1450, 1148, 1131, 804, 749 cm–1; 1H NMR (400 MHz, CDCl3)
δ 1.41 (d, J = 7.3 Hz, 3 H,
Hβ AlaꢀH), 3.25 (m, 1 H, NH ΨPro), 4.14 (dd, J = 8.0, 5.7 Hz, 1 H, Hβ ΨProꢀHa), 4.22–4.31 (m,
2 H, Hα ΨProꢀH, Hβ ΨProꢀHb), 4.64 (dq, J = 8.0, 7.3 Hz, 1 H, Hα AlaꢀH), 5.04 (q, J = 5.5 Hz, 1
H, Hδ ΨProꢀH), 5.17 (d, J = 12.4 Hz, 1 H, Bn CH2ꢀHa), 5.20 (d, J = 12.4 Hz, 1 H, Bn CH2ꢀHb),
7.32–7.41 (m, 5 H, Bn arom.), 7.63 (d, J = 8.0 Hz, 1 H, NH Ala); 13C NMR (100.5 MHz, CDCl3)
δ
18.1 (CH3, Cβ Ala), 47.7 (CH, Cα Ala), 59.3 (CH, Cα ΨPro), 67.1 (CH2, Bn CH2), 70.3 (CH2,
Cβ ΨPro), 87.6 (q, J = 34.5 Hz, CH, Cδ ΨPro), 123.2 (q, J = 283.7 Hz, CF3), 128.1 (2 × CH, Bn
arom.), 128.4 (CH, Bn arom.), 128.6 (2 × CH, Bn arom.), 135.3 (C, Bn arom.), 170.4 (C, C=O),
172.5 (C, C=O); 19F NMR (376.2 MHz, CDCl3)
δ
–83.8 (d, J = 5.5 Hz, CF3); EIMS m/z: M+ 346,
277, 211, 164, 140, 112, 91 (100); HRMS (ESIꢀTOF) calcd for C15H17F3N2O4 346.1140, found:
346.1154.
Synthesis of C-Terminal CF3-Pseudoproline Containing Dipeptides.
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Synthesis of o-Nbs Protected C-Terminal Dipeptides (6 and 8).
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7
Representative Procedure for the Peptide Coupling Reaction using o-Nbs-Amino Acid
8
9
Chloride.
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Method A: To a solution of the oꢀNbsꢀamino acid 5 or 7 (7 equiv) suspended in dichloromethane
under argon was added 1ꢀchloroꢀN,Nꢀ2ꢀtrimethylꢀ1ꢀpropenylamine (7 equiv) at 0 °C. The
resulting solution was stirred at 0 °C until the disappearance of the precipitate (usually 20 min).
The total conversion of the acid to chloride was checked by TLC after quenching with methanol.
The resulting oꢀNbsꢀamino acid chloride solution was added via cannula to a neat mixture of
pseudoproline 3a or 3b (1.0 equiv) and collidine (1.0 equiv) at 0 °C. The temperature was
allowed to warm to room temperature and the solution was concentrated twice using a stream of
argon. After 24 h, the resulting mixture was diluted with dichloromethane, and quenched with a
saturated aqueous solution of NaHCO3. The layers were separated, and aqueous layer was
extracted with dichloromethane. The combined organic layers were washed with water, dried
over MgSO4, filtered, and evaporated under reduced pressure. Purification by flash
chromatography gave dipeptides 665 and 8 in 79% and 66% yield respectively.
Method B: To a solution of the oꢀNbsꢀalanine 5 (2 equiv) suspended in dichloromethane under
argon, was added 1ꢀchloroꢀN,Nꢀ2ꢀtrimethylꢀ1ꢀpropenylamine (2 equiv) at 0 °C. The resulting
solution was stirred at 0 °C until the disappearance of the precipitate (usually 20 min). The total
conversion of the acid to chloride was checked by TLC after quenching with methanol. The
resulting oꢀNbsꢀalanine chloride solution was added via cannula to neat pseudoproline 3a or 3b
(1.0 equiv) at 0 °C. The temperature was allowed to warm to room temperature, and the solution
was concentrated twice using a stream of argon. After 24 h, the resulting mixture was diluted
with dichloromethane and quenched with a saturated aqueous solution of NaHCO3. The layers
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were separated and aqueous layer was extracted with dichloromethane. The combined organic
layers were washed with water, dried over MgSO4, filtered, and evaporated under reduced
pressure. Purification by flash chromatography gave dipeptide 665 in 79ꢀ86% yield.
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Method C: To a solution of oꢀNbsꢀalanine 5 (2.2 equiv) in THF at 0 °C under argon was added
dropwise freshly distilled SOCl2 (3.3 equiv) and TMU (0.44 equiv). The resulting mixture was
stirred for 2 h at 0 °C, then temperature was allowed to warm to room temperature, and solvent
was removed under vacuum. The corresponding oꢀNbsꢀalanine chloride was directly used in the
peptidic coupling reaction without further purification. A solution of pseudoproline 3a or 3b (1.0
equiv) and collidine (1.0 equiv) in dichloromethane was added at 0°C via cannula to the freshly
prepared oꢀNbsꢀalanine chloride (neat). The resulting mixture was stirred overnight at room
temperature, diluted with dichloromethane, and quenched with a saturated aqueous solution of
NaHCO3. The layers were separated and aqueous layer was extracted with dichloromethane. The
combined organic layers were washed with brine, dried over MgSO4, filtered, and evaporated
under reduced pressure. Purification by flash chromatography gave dipeptide 665 in 78ꢀ87%
yield.
o-Nbs-Aib-Ser(ΨCF3,Hpro)-OMe (8). The dipeptide 8 was prepared according to the method A
starting from oꢀNbsꢀ2ꢀmethylalanine 7 (2.02 g, 7.0 mmol, 7.0 equiv) in dichloromethane (7 mL),
1ꢀchloroꢀN,Nꢀ2ꢀtrimethylꢀ1ꢀpropenylamine (750 ꢁL, 5.67 mmol, 5.7 equiv), pseudoproline
(2S,4S)ꢀ3a (260 mg, 1.0 mmol, 1.0 equiv) and collidine (133 ꢁL, 1.0 mmol, 1.0 equiv). The crude
was purified by flash chromatography (70:30 cyclohexane/ethyl acetate) to give the dipeptide 8
(310 mg, 66%) as 100% cis rotational isomer in CDCl3 at 273 K: pale yellow solid; mp 62–63
°C; Rf = 0.68 (60:40 petroleum ether/ethyl acetate); [α]22D +77.4 (c 1.02, CHCl3); IR (neat) 3338,
2957, 1743, 1677, 1539, 1175, 1145 cm–1; 1H NMR (400 MHz, CDCl3, 298 K) (single rotational
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isomer):
δ
1.45 (s, 3 H, Hβ AibꢀH), 1.60 (s, 3 H, Hβ AibꢀH), 3.81(s, 3 H, OMe), 4.41 (t, J = 7.8
5
6
7
8
Hz, 1 H, Hβ ΨProꢀHa), 4.63 (dd, J = 7.8, 2.8 Hz, 1 H, Hβ ΨProꢀHb), 5.84 (dd, 1 H, J = 7.8, 2.8
Hz, Hα ΨProꢀH), 6.00 (s, 1 H, NH Aib), 6.05 (q, J = 4.6 Hz, 1 H, Hδ ΨProꢀH), 7.79–7.84 (m, 2
H, oꢀNbs arom.), 7.96 (m, 1 H, oꢀNbs arom.), 8.18 (m, 1 H, oꢀNbs arom.); 13C NMR (100.5
9
10
11
12
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MHz, CDCl3, 298 K) (single rotational isomer):
δ 26.6 (CH3, Cβ Aib), 27.4 (CH3, Cβ Aib), 52.9
(CH3, OMe), 58.8 (CH, Cα ΨPro), 61.4 (C, Cα Aib), 70.3 (CH2, Cβ ΨPro), 86.1 (q, J = 36.4 Hz,
CH, Cδ ΨPro), 122.4 (q, J = 285.6 Hz, CF3), 125.8 (CH, oꢀNbs arom.), 131.1 (CH, oꢀNbs arom.),
133.6 (CH, oꢀNbs arom.), 134.2 (CH, oꢀNbs arom.), 135.4 (C, oꢀNbs arom.), 147.6 (C, oꢀNbs
arom.), 169.6 (C, C=O), 171.6 (C, C=O); 19F NMR (376.2 MHz, CDCl3, 298 K) (single rotational
isomer):
δ
–80.8 (d, J = 4.6 Hz, CF3); ESIMS m/z: 492.17 [M + Na]+, 470.19 [M + H]+; HRMS
(ESIꢀTOF) calcd for C16H18F3N3O8SNa 492.0664, found: 492.0659.
Synthesis of Fmoc protected C-Terminal Dipeptides (9-14).
Representative procedure for the preparation of Fmoc-aminoacid chloride assisted by
ultrasonication:73 To a 0.2 M solution of the Fmocꢀaminoacid (1.0 equiv) suspended in
dichloromethane under argon, was added freshly distilled SOCl2 (13.8 equiv). The mixture was
sonicated at room temperature until the complete disappearence of the precipitate (from 30 min to
1 h), then solvent and excess of SOCl2 were removed in vacuo to give the Fmocꢀaminoacid
chloride as a white solid directly used without further purification.
DIEA representative procedure for peptide coupling reaction: To a solution of
pseudoprolines 3 (1.0 equiv) in dichloromethane was added DIEA (1.0 equiv). The resulting
mixture was added via cannula to the freshly prepared Fmocꢀamino acid chloride solid (1.1
equiv). The reaction mixture was stirred for 24 h at room temperature, diluted with
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dichloromethane, and washed with 1 M aqueous solution of HCl. The organic layer was dried
over MgSO4, filtered, and evaporated under reduced pressure. Purification by flash
chromatography gave a diastereomeric mixture of dipeptides 9a and 9b 67 in 90ꢀ94% yield.
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Base free representative procedure for the peptide coupling reaction: To a solution of
pseudoprolines 3 (1.0 equiv) in dichloromethane was added Fmocꢀamino acid chloride (1.1
equiv). The reaction mixture was stirred for 18 h at room temperature under inert atmosphere,
then the solvent was evaporated under reduced pressure. Purification by flash chromatography
gave pure dipeptides 9a-14 in 52ꢀ98% yield.
Fmoc-Gly-L-Ser(ΨCF3,Hpro)-OMe (10). The reaction was performed following the base free
representative procedure starting from pseudoproline (2S,4S)-3a (256 mg, 1.29 mmol, 1.0 equiv)
in dichloromethane (4 mL) and FmocꢀGlyꢀCl (446 mg, 1.42 mmol, 1.1 equiv). Purification by
flash chromatography (60:40 cyclohexane/ethyl acetate) gave the pure dipeptide 10 (604 mg,
98%) as a 61/39 inseparable mixture of cis/trans rotational isomers in CDCl3 at 274 K : white
solid; mp 88–92 °C; Rf = 0.24 (60:40 cyclohexane/ethyl acetate); [α]23D –36.0 (c 1.06, CHCl3); IR
1
(neat) 3338, 2955, 1686, 1518, 1150, 727 cm–1; H NMR (500 MHz, CDCl3, 274 K) : (trans
rotamer)
δ 3.79 (s, 3 H, OMe), 4.00 (m, 1 H, Hα GlyꢀHa), 4.20 (m, 1 H, Hα GlyꢀHb), 4.23 (t, J =
7.2 Hz, 1 H, Fmoc CH), 4.39 (d, J = 7.2 Hz, 2 H, Fmoc CH2), 4.48ꢀ4.54 (m, 2 H, Hβ ΨproꢀH),
5.05 (t, J = 7.9 Hz, 1 H, Hα ΨproꢀH), 5.67 (q, J = 4.7 Hz, 1 H, Hδ ΨproꢀH), 5.70ꢀ5.75 (m, 1 H,
NH Gly), 7.32 (t, J = 7.6 Hz, 2 H, Fmoc arom.), 7.42 (t, J = 7.4 Hz, 2 H, Fmoc arom.), 7.60 (d, J
= 7.4 Hz, 2 H, Fmoc arom.), 7.78 (d, J = 7.6 Hz, 2 H, Fmoc arom.); (cis rotamer)
δ 3.82 (s, 3 H,
OMe), 4.05 (m, 1 H, Hα GlyꢀHa), 4.22 (m, 1 H, Hα GlyꢀHb), 4.23 (t, J = 7.2 Hz, 1 H, Fmoc CH),
4.39 (d, J = 7.2 Hz, 2 H, Fmoc CH2), 4.54 (d, J = 6.8 Hz, 2 H, Hβ ΨproꢀH), 4.80 (t, J = 6.8 Hz, 1
H, Hα ΨproꢀH), 5.70ꢀ5.75 (m, 1 H, NH Gly), 5.97 (q, J = 5.0 Hz, 1 H, Hδ ΨproꢀH), 7.32 (t, J =
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7.6 Hz, 2 H, Fmoc arom.), 7.42 (t, J = 7.4 Hz, 2 H, Fmoc arom.), 7.60 (d, J = 7.4 Hz, 2 H, Fmoc
5
6
13
arom.), 7.78 (d, J = 7.6 Hz, 2 H, Fmoc arom.); C NMR (100.5 MHz, CDCl3, 298 K) : (trans
7
8
9
rotamer)
δ 42.8 (CH2, Cα Gly), 46.2 (CH, Fmoc CH), 52.4 (CH3, OMe), 56.3 (CH, Cα Ψpro),
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66.8 (CH2, Fmoc CH2), 68.2 (CH2, Cβ Ψpro), 83.6 (q, J = 36.4 Hz, CH, Cδ Ψpro), 119.4 (2 × CH,
Fmoc arom.), 121.8 (q, J = 286.6 Hz, CF3), 124.9 (2 × CH, Fmoc arom.), 126.9 (2 × CH, Fmoc
arom.), 127.6 (2 × CH, Fmoc arom.), 140.6 (2 × C, Fmoc arom.), 143.0 (2 × C, Fmoc arom.),
155.7 (C, C=O), 167.2 (C, C=O), 167.9 (C, C=O); (cis rotamer)
δ 42.5 (CH2, Cα Gly), 46.3 (CH,
Fmoc CH), 53.0 (CH3, OMe), 56.1 (CH, Cα Ψpro), 66.7 (CH2, Fmoc CH2), 69.9 (CH2, Cβ Ψpro),
83.7 (q, J = 36.4 Hz, CH, Cδ Ψpro), 119.4 (2 × CH, Fmoc arom.), 121.8 (q, J = 286.6 Hz, CF3),
124.5 (2 × CH, Fmoc arom.), 126.4 (2 × CH, Fmoc arom.), 127.1 (2 × CH, Fmoc arom.), 140.6 (2
× C, Fmoc arom.), 143.0 (2× CH, Fmoc arom.), 155.6 (C, C=O), 167.8 (C, C=O), 167.9 (C,
C=O); 19F NMR (376.2 MHz, CDCl3, 298 K) : (trans rotamer)
δ –81.8 (s, CF3); (cis rotamer) δ –
82.2 (s, CF3); ESIMS m/z: 501.18 [M + Na]+, 479.20 [M + H]+; HRMS (ESIꢀTOF) calcd for
C23H21F3N2O6 478.1352, found: 478.1408.
Fmoc-L-Val-L-Ser(ΨCF3,Hpro)-OMe (11). The reaction was performed starting from a 84:16
diastereomeric mixture of (2S,4S)-3a and (2R,4S)-3b pseudoprolines (2.00 g, 10.0 mmol, 1.0
equiv) in dichloromethane (30 mL) and FmocꢀLꢀValꢀCl (3.95 g, 11.0 mmol, 1.1 equiv).
Purification by flash chromatography (80:20 cyclohexane/ethyl acetate) gave the pure dipeptide
11 (3.09 g, 59%) as a 7/93 inseparable mixture of cis/trans rotational isomers in CDCl3 at 300 K:
white solid; mp 112 °C; Rf = 0.42 (70:30 cyclohexane/ethyl acetate); [α]25D –74.5 (c 1.0, CHCl3);
1
IR (neat) 3312, 2928, 1764, 1716 cm–1; H NMR (500 MHz, CDCl3, 300 K) : (trans rotamer)
δ
1.03 (d, J = 7.0 Hz, 3 H, Hγ ValꢀHa), 1.05 (d, J = 7.3 Hz, 3 H, Hγ ValꢀHb), 2.06ꢀ2.15 (m, 1 H,
Hβ ValꢀH), 3.75 (s, 3 H, OMe), 3.97 (t, J = 9.0 Hz, 1 H, Hα ValꢀH), 4.19 (t, J = 6.9 Hz, 1 H,
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Fmoc CH), 4.31 (dd, J = 9.8, 6.9 Hz, 1 H, Hβ ΨproꢀHa), 4.33 (d, J = 7.8 Hz, 1 H, Fmoc CH2ꢀHa),
4.46 (d, J = 7.8 Hz, 1 H, Fmoc CH2ꢀHb), 4.47 (dd, J = 9.8, 6.4 Hz, 1 H, Hβ ΨproꢀHb), 5.10 (t, J =
7
8
9
8.2 Hz, 1 H, Hα ΨproꢀH), 5.26 (d, J = 8.7 Hz, 1 H, NH Val), 6.40 (q, J = 4.6 Hz, 1 H, Hδ Ψproꢀ
H), 7.31 (t, J = 7.3 Hz, 2 H, Fmoc arom.), 7.40 (t, J = 7.3 Hz, 2 H, Fmoc arom.), 7.56 (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, 298
10
11
12
13
14
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19
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K) : (trans rotamer)
δ 18.5 (CH3, Cγ Val), 18.6 (CH3, Cγ Val), 31. 6 (CH, Cβ Val), 47.0 (CH,
Fmoc CH), 52.7 (CH3, OMe), 56.5(CH, Cα Ψpro), 58.6 (CH, Cα Val), 67.3 (CH2, Fmoc CH2),
68.7 (CH2, Cβ Ψpro), 85.0 (q, J = 37.4 Hz, CH, Cδ Ψpro), 120.0 (2 × CH, Fmoc arom.), 122.7 (q,
J = 287.5 Hz, CF3), 124.9 (CH, Fmoc arom.), 125.0 (CH, Fmoc arom.), 127.0 (2 × CH, Fmoc
arom.), 127.8 (2 × CH, Fmoc arom.), 141.3 (2 × C, Fmoc arom.), 143.5 (C, Fmoc arom.), 143.6
(C, Fmoc arom.), 156.5 (C, C=O), 168.6 (C, C=O), 172.5 (C, C=O); 19F NMR (376.2 MHz,
CDCl3, 298 K) : (trans rotamer)
δ –81.0 (d, J = 4.6 Hz); HRMS (ESIꢀTOF) calcd for
C26H27F3N2O6 520.1821, found: 520.1906.
Fmoc-L-Pro-L-Ser(ΨCF3,Hpro)-OMe (12). The reaction was performed following the base free
representative procedure starting from a 84:16 diastereomeric mixture of (2S,4S)-3a and (2R,4S)-
3b pseudoprolines (1.02 g, 5.13 mmol, 1.0 equiv) in dichloromethane (7.5 mL) and FmocꢀLꢀProꢀ
Cl (2.00 g, 5.62 mmol, 1.1 equiv). Purification by flash chromatography (80:20
cyclohexane/ethyl acetate) gave the pure dipeptide 12 (1.39 g, 52%) as a trans rotational isomer
in CDCl3 at 300 K: white solid; mp 115 °C; Rf = 0.26 (70:30 cyclohexane/ethyl acetate); [α]21
–
D
1
56.1 (c 1.0, CHCl3); IR (neat) 2956, 1763, 1689 cm–1; H NMR (500 MHz, CDCl3, 300 K) :
(trans rotamer)
δ 1.94ꢀ2.05 (m, 1 H, Hγ ProꢀHa), 2.10ꢀ2.26 (m, 2 H, Hβ ProꢀH), 2.27ꢀ2.39 (m, 1
H, Hγ ProꢀHb), 3.52ꢀ3.60 (m, 1 H, Hδ ProꢀHa), 3.67ꢀ3.74 (m, 1 H, Hδ ProꢀHb), 3.77 (s, 3 H,
OMe), 4.23 (t, J = 7.1 Hz, 1 H, Fmoc CH), 4.32 (dd, J = 8.5, 6.9 Hz, 1 H, Hβ ΨproꢀHa), 4.34 (d,
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J = 7.1 Hz, 2 H, Fmoc CH2ꢀH), 4.36ꢀ4.40 (m, 1 H, 1 H, Hα ProꢀH), 4.50 (t, J = 8.5 Hz, 1 H, Hβ
5
6
7
8
ΨproꢀHb), 5.06 (t, J = 7.8 Hz, 1 H, Hα ΨproꢀH), 6.23 (q, J = 4.8 Hz, 1 H, Hδ ΨproꢀH), 7.31 (t, J
= 7.3 Hz, 2 H, Fmoc arom.), 7.40 (t, J = 7.3 Hz, 2 H, Fmoc arom.), 7.56 (dd, J = 11.0, 7.6 Hz, 2
H, Fmoc arom.), 7.77 (d, J = 7.3 Hz, 2 H, Fmoc arom.); 13C NMR (100.5 MHz, CDCl3, 298 K) :
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
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(trans rotamer)
δ 24.8 (CH2, Cγ Pro), 30.9 (CH2, Cβ Pro), 47.1 (CH2, Cδ Pro), 47.2 (CH, Fmoc
CH), 52.8 (CH3, OMe), 56.9 (CH, Cα Ψpro), 58.3 (CH, Cα Pro), 67.7 (CH2, Fmoc CH2), 68.9
(CH2, Cβ Ψpro), 85.1 (q, J = 30.7 Hz, CH, Cδ Ψpro), 120.1 (2 × CH, Fmoc arom.), 122.9 (q, J =
286.6 Hz, CF3), 125.2 (2 × CH, Fmoc arom.), 127.1 (2 × CH, Fmoc arom.), 127.8 (2 × CH, Fmoc
arom.), 141.4 (2 × C, Fmoc arom.), 143.8 (C, Fmoc arom.), 143.9 (C, Fmoc arom.), 155.4 (C,
19
C=O), 168.8 (C, C=O), 173.0 (C, C=O); F NMR (376.2 MHz, CDCl3, 298 K) : (trans rotamer)
δ
–82.0 (d, J = 4.8 Hz); ESIMS m/z: 519.28 [M + H]+; Anal. Calcd for C26H25F3N2O6 (518.17):
C, 60.23; H, 4.86; N, 5.40, found: C, 60.22; H, 4.84; N, 5.51.
Fmoc-Aib-Ser(ΨCF3,Hpro)-OMe (13). The reaction was performed following the base free
representative procedure starting from a 84:16 diastereomeric mixture of (2S,4S)-3a and (2R,4S)-
3b pseudoprolines (500 mg, 2.51 mmol, 1.0 equiv) in dichloromethane (7.5 mL) and FmocꢀAibꢀ
Cl (2.59 g, 7.74 mmol, 3.0 equiv). Purification by flash chromatography (70:30
cyclohexane/ethyl acetate) gave the pure dipeptide 13 (700 mg, 55%) as a 85/15 inseparable
mixture of cis/trans rotational isomers in CDCl3 at 274 K: white solid; mp 107ꢀ112 °C; Rf = 0.24
(70:30 cyclohexane/ethyl acetate); [α]25.0 –28.4 (c 0.95, CHCl3); IR (neat) 3321, 2952, 1709,
D
1677, 1513, 1144, 739 cm–1; H NMR (500 MHz, CDCl3, 274 K)
δ 1.54 (br s, 6 H, Hβ AibꢀH),
1
3.69 (s, 3 H, OMe), 4.16 (m, 1 H, Hβ ΨproꢀHa), 4.18 (m, 1 H, Fmoc CH), 4.39 (m, 1 H, Hβ Ψproꢀ
Hb), 4.50ꢀ4.52 (m, 2 H, Fmoc CH2ꢀH), 5.10 (s, 1 H, NH Aib), 5.14 (dd, J = 7.5, 4.1 Hz, 1 H, Hα
ΨproꢀH), 6.04 (q, J = 4.9 Hz, 1 H, Hδ ΨproꢀH), 7.34 (t, J = 7.4 Hz, 2 H, Fmoc arom.), 7.43 (t, J =
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The Journal of Organic Chemistry
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7.4 Hz, 2 H, Fmoc arom.), 7.57 (d, J = 7.4 Hz, 2 H, Fmoc arom.), 7.76 (d, J = 7.4 Hz, 2 H, Fmoc
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7
13
arom.); C NMR (100.5 MHz, CDCl3, 298 K)
δ 25.7 (2 × CH3, Cβ Aib), 46.5 (CH, Fmoc CH),
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52.4 (CH3, OMe), 57.6 (CH, Cα Ψpro), 57.9 (C, Cα Aib), 65.9 (CH2, Fmoc CH2), 69.5 (CH, Cβ
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Ψpro), 84.9 (q, J = 35.5 Hz, CH, Cδ Ψpro), 119.4 (2 × CH, Fmoc arom.), 121.9 (q, J = 285.6 Hz,
CF3), 124.1 (2 × CH, Fmoc arom.), 126.5 (2 × CH, Fmoc arom.), 127.3 (2 × CH, Fmoc arom.),
140.7 (2 × C, Fmoc arom.), 142.7 (2 × C, Fmoc arom.), 154.7 (C, C=O), 169.0 (C, C=O), 172.6
(C, C=O); 19F NMR (376.2 MHz, CDCl3, 298 K)
+ H]+; HRMS (ESIꢀTOF) calcd for C25H25F3N2O6Na 529.1562, found: 529.1554.
δ –80.9 (d, J = 5.2 Hz); ESIMS m/z: 507.29 [M
Fmoc-D-Ala-L
-Ser(ΨCF3,Hpro)-OMe (14). The reaction was performed following the base free
representative procedure starting from pseudoproline (2S,4S)ꢀ3a (300 mg, 1.51 mmol, 1.0 equiv)
in dichloromethane (5 mL) and FmocꢀDꢀAlaꢀCl (547 mg, 1.66 mmol, 1.1 equiv). Purification by
flash chromatography (70:30 cyclohexane/ethyl acetate) gave the pure dipeptide 14 (545 mg,
79%) as a 92/8 inseparable mixture of cis/trans rotational isomers in CDCl3 at 300 K: white
solid; mp 64–66 °C; Rf = 0.40 (70:30 cyclohexane/ethyl acetate); [α]23 –47.5 (c 1.0, CHCl3);
D
HRMS (ESIꢀTOF) calcd for C24H23F3N2O6Na 515.1406, found: 515.1400; spectral data of 14 are
similar to those of 9b67.
Synthesis of the Tripeptide. Fmoc-
L
-Ala-
L
-Ser(ΨCF3,Hpro)-
L-Ala-OtBu (15). Saponification
of the dipeptide methyl ester 9a (1.41 g, 2.86 mmol) was performed following a described
method by addition of NaOH (137 mg, 2.44 mmol, 1.2 equiv) to a 0.8 M CaC12 solution in
iPrOHꢀH20 7:3 (68 mL).75 The reaction mixture was stirred for 4 h at room temperature,
quenched with 1 M HCl, concentrated under reduced pressure and diluted with H2O (20 mL). The
aqueous solution was extracted with ethyl acetate (3 × 50 mL). The combined organic layers
were washed with H2O (20 mL), dried over MgSO4, filtered and concentrated under reduced
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