Fluorinated Pseudopeptide Analogues of the Neuropeptide 26RFa: Synthesis, Biological, and Structural Studies

Article abstract of DOI:10.1002/cbic.201300325

A series of four fluorinated dipeptide analogues each containing a fluoro-olefin moiety as peptide bond surrogate has been designed and synthesized. These motifs have been successfully introduced into the bioactive C-terminal heptapeptide of the neuropeptide 26RFa by conventional SPPS. We then evaluated the ability of the generated pseudopeptides to increase [Ca²⁺]_i in GPR103-transfected cells. For these fluorinated analogues, greater stability in human serum was observed. Their conformations were also investigated, leading to the valuable identification of differences depending on the position of the fluoro-olefin moiety in the sequence.

Full text of DOI:10.1002/cbic.201300325

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DOI: 10.1002/cbic.201300325
Fluorinated Pseudopeptide Analogues of the
Neuropeptide 26RFa: Synthesis, Biological, and Structural
Studies
Camille Pierry,[a] Samuel Couve-Bonnaire,*[a] Laure Guilhaudis,[a] Cindy Neveu,[b]
Amꢀlie Marotte,[a] Benjamin Lefranc,[c] Dominique Cahard,[a] Isabelle Sꢀgalas-Milazzo,[a]
Jꢀrꢁme Leprince,[b, c] and Xavier Pannecoucke*[a]
A series of four fluorinated dipeptide analogues each contain-
ing a fluoro-olefin moiety as peptide bond surrogate has been
designed and synthesized. These motifs have been successfully
introduced into the bioactive C-terminal heptapeptide of the
neuropeptide 26RFa by conventional SPPS. We then evaluated
in GPR103-transfected cells. For these fluorinated analogues,
greater stability in human serum was observed. Their confor-
mations were also investigated, leading to the valuable iden-
tification of differences depending on the position of the
fluoro-olefin moiety in the sequence.
the ability of the generated pseudopeptides to increase [Ca2+
]
i
Introduction
26RFa is a neuropeptide of the RFamide family, originally iso-
lated from frog brain and subsequently cloned in human and
rat.[1] 26RFa is the endogenous ligand of the former orphan
receptor GPR103.[2] Analysis of the human 26RFa precursor in-
dicates that pre-pro26RFa might generate several additional
peptides including an N-terminal extended form (43RFa) and
a truncated form (26RFa(20–26) (GGFSFRF-NH2)) strictly conserved
throughout mammals.[3] In rodents, 26RFa and 43RFa induce
dose-dependent increases in food intake, stimulate secretion
of gonadotropins and aldosterone, and reduce glucose-in-
duced insulin release.[3,4] Moreover, GPR103-knockout mice
suffer from osteopenia and exhibit the characteristic kyphotic
humps of osteoporotic patients.[5] Altogether, these data indi-
cate that 26RFa is able to exert diverse biological activities in
vertebrates, such as the control of food intake, reproduction,
and osteogenesis.
[Nva23]26RFa(20–26), that was three times more potent than the
native heptapeptide.[7] More importantly, we have recently re-
ported the rational design of a potent, stable, and long-lasting
aza-b3-pseudopeptide (LV-2172) that paves the way to the de-
velopment of GPR103 drug candidates.[8] We have also report-
ed that the pseudopeptide LV-2045—[Gly20Y[CH2NH]Gly21]-
26RFa(20–26)—is as potent as 26RFa(20–26) in mobilizing intracellu-
lar calcium and that the replacement of the Gly–Gly motif by
a 4-(carboxymethyl)piperazine (Cmpi) unit leads to an analogue
(LV-2043) five times more potent than the heptapeptide.[8]
To find new potent GPR103 ligands and to increase the met-
abolic stabilities of the heptapeptides, we thus decided to
focus our attention on the two first peptide bonds of the N-
terminal region, Gly–Gly and Gly–Phe peptide bonds, which
are the first positions to undergo enzymatic degradation. To
this end, we decided in this work to turn our attention to fluo-
rinated molecules, as it nowadays well known that the intro-
duction of one or several fluorine atoms into a biomolecule
can change its physicochemical, physical, biochemical, and bio-
logical properties.[9] In this context, the fluoro-olefin moiety, de-
noted Y[CF=CH], can be regarded as a suitable peptide bond
mimetic because it exhibits isosteric and isoelectronic charac-
teristics similar to those of the native bond.[10] Moreover, in
terms of metabolic stability, the fluoro-olefin moiety increases
the resistance of a pseudopeptide to proteolysis.[11,12] To study
the importance of the configuration of the double bond, it
also seemed interesting to synthesize both Z (transoid) and E
(cisoid) analogues in order to obtain information about the
bioactive conformation of the heptapeptides.
It is important to note that the C-terminal heptapeptide,
26RFa(20–26), mimics the orexigenic and gonadotropic effects of
26RFa.[6] However, 26RFa(20–26) is about 75 times less potent
than 26RFa in increasing [Ca2+]i in cultured GPR103-transfected
CHO cells.[7] Structure–activity relationship studies showed that
replacement of Ser23 by a norvaline led to an analogue,
[a] Dr. C. Pierry, Dr. S. Couve-Bonnaire, Dr. L. Guilhaudis, A. Marotte,
Dr. D. Cahard, Prof. I. Sꢀgalas-Milazzo, Prof. X. Pannecoucke
UMR 6014 COBRA, INSA and University of Rouen
IRCOF, 1 rue Tesniꢁre, 76130 Mont-Saint-Aignan (France)
E-mail: samuel.couve-bonnaire@insa-rouen.fr
xavier.pannecoucke@insa-rouen.fr
[b] Dr. C. Neveu, Dr. J. Leprince
INSERM U982, IRIB, University of Rouen
place Emile Blondel, 76821 Mont-Saint-Aignan (France)
Although many pseudopeptides featuring a fluoro-olefin
have already been designed, only a few biological data[11] sup-
port the concept of a fluoro-olefin as a peptide bond replace-
ment because of the difficulties in synthesizing such halogen-
[c] B. Lefranc, Dr. J. Leprince
PRIMACEN, IRIB, University of Rouen
place Emile Blondel, 76821 Mont-Saint-Aignan (France)
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Results and Discussion
containing pseudopeptides. On the basis of our expertise in
the synthesis of fluoro-olefin-containing compounds,[11b,13] we
decided to develop fluorinated SPPS-convenient pseudodipep-
tides (Scheme 1) in order to incorporate them into the C-termi-
nal heptapeptide of 26RFa (Scheme 2), which can be used as
an ideal molecular scaffold for the design of GPR103 peptide
ligands of low molecular weight.[7,8]
In order to access the new fluorinated peptide targets, a series
of SPPS-amenable fluoroalkene-containing pseudodipeptides
was first synthesized. These were then incorporated into the C-
terminal heptapeptides of 26RFa by conventional SPPS, and
the functional activities of the fluorinated pseudoheptapepti-
des were evaluated by determining the calcium-mobilizing re-
sponses in GPR103-transfected cells. Their stabilities in human
serum were assessed in vitro, and their conformations were
studied by NMR spectroscopy.
Synthesis of fluorinated pseudodipeptides
We first undertook the synthesis of the simplest Fmoc-GlyY-
[CF=CH]Gly-OH pseudodipeptides (E)-1 and (Z)-1 as depicted
in Scheme 3.
Scheme 1. Target fluorinated pseudodipeptides 1 and 2.
Scheme 3. Synthesis of Fmoc-GlyY[CF=CH]Gly-OH isomers (E)-1 and (Z)-1.
a) TBDPS-Cl, nBuLi, THF, 84%; b) IBX, AcOEt, 98%; c) CBr2FCO2Et, ZnEt2, PPh3,
THF, 85% (Z/E 62:38); d) LiAlH4, THF; e) IBX, AcOEt, 84% for two steps;
f) NH2SOtBu, Ti(OEt)4, THF, reflux, 89% (separation of each diastereoisomer);
g) NaBH4, THF, 91% (Z) and 84% (E); h) HCl 4m in dioxane, MeOH, quantita-
tive; i) Fmoc-OSu, NaHCO3, dioxane/H2O, 72% (Z) and 70% (E); j) Jones re-
agent, acetone, 70% (Z) and 50% (E).
Monoprotection of one hydroxy group in commercially avail-
able propane-1,3-diol (3) was carried out, followed by oxida-
tion of the free hydroxy group into an aldehyde and a fluoro-
olefination reaction to afford the ethyl a-fluoroacrylate 4.[14]
The ester moiety was converted into an aldehyde through a
reduction/oxidation sequence. The sulfinylimine 5 was then
obtained, and at this stage the E and the Z diastereoisomers
were easily separated by column chromatography. The sulfiny-
limines 5 were reduced by sodium borohydride, followed by
simultaneous deprotection of amine and alcohol functions to
furnish compounds 6, which were subjected to Fmoc protec-
tion and Jones oxidation to give the two Fmoc-GlyY[CF=
CH]Gly-OH isomers (E)-1 and (Z)-1 in 17 and 24% overall yields,
respectively, for this ten-step synthesis.
We next turned our attention to the synthesis of the more
challenging analogues 2 Fmoc-GlyY[CF=CH]Phe-OH featuring
Scheme 2. 26RFa, C-terminal heptapeptide 26RFa(20–26), and fluorinated pseu-
doheptapeptide targets.
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(E)-2 derivative were unsuccessful, due to the degradation of
some intermediates.
Solid-phase peptide synthesis
We next incorporated these fluoropseudodipeptides in appro-
priate positions of 26RFa(20–26) by conventional solid-phase au-
tomated peptide synthesis. It should be noted that the cou-
pling of the fluorinated analogues was performed manually,
because of the small amounts of compounds available
(Scheme 5).
Scheme 4. Synthesis of (Z)-2 (Fmoc-GlyY[CF=CH]Phe-OH). a) (4R,5S)-(+)-4-
Methyl-5-phenyl-2-oxazolidinone, nBuLi, THF, 96%; b) BnOCH2Cl, DIPEA, TiCl4,
CH2Cl2, 88% (de >95%); c) LiAlH4, THF; d) PySO3, NEt3, DMSO, CH2Cl2, 55%
for two steps; e) CBr2FCO2Et, ZnEt2, PPh3, THF, 55% (Z) and 34% (E) (Z/E
64:36); f) LiAlH4, THF; g) PySO3, NEt3, DMSO, CH2Cl2, 70% for two steps;
h) NH2SOtBu, Ti(OEt)4, THF, reflux, 95%; i) NaBH4, THF, 95%; j) BCl3, CH2Cl2,
ꢀ788C, 58%; k) HCl 4m in dioxane, MeOH, quantitative; l) Fmoc-OSu,
NaHCO3, dioxane/H2O, 95%; m) Jones reagent, acetone, 80%.
Scheme 5. Solid-phase synthesis of fluorinated pseudoheptapeptide ana-
logues. a) SPPS cycles: i: 20% piperidine, NMP, ii: Fmoc-AA-OH (10 equiv),
HBTU (10 equiv), HOBt (10 equiv), DIEA (20 equiv), NMP. b) Manual coupling:
i: 20% piperidine, NMP, ii: Fmoc-GlyY[CF=CH]AA-OH (1.5 equiv), HATU
(1.5 equiv), HOAt (1.5 equiv), DIEA (3 equiv), NMP. c) i: 20% piperidine, NMP,
ii: TFA/TIS/H2O 99.5:0.25:0.25 (v/v/v).
a stereogenic center. For this purpose we developed an asym-
metric synthesis to control the stereogenic center at the C-ter-
minal side of the pseudodipeptide (Scheme 4).
An Evans oxazolidinone was condensed with the commer-
cially available acyl chloride 7, followed by a highly diastereo-
selective alkoxymethylation (de>95%) to set up the desired
configuration at the C-terminal side of the dipeptide ana-
logue.[15] The chiral auxiliary was removed under reductive con-
ditions, and the released hydroxy group was oxidized to give
aldehyde 10. An olefination reaction furnished Z and E diaste-
reoisomers 11 as a separable mixture. The completion of the
synthetic route was done only with the (Z)-11 stereoisomer.
Ester 11 was transformed into aldehyde 12, condensation of
which with tert-butanesulfinamide gave compound 13. Reduc-
tion of the imine and debenzylation yielded product 14. Fur-
ther removal of the tert-butanesulfinamide, Fmoc protection,
and Jones oxidation allowed us to obtain (Z)-2—Fmoc-GlyY-
[CF=CH]Phe-OH—in an overall yield of 6% in this 13-step syn-
thesis. It should be noted that several attempts to obtain the
Finally, the crude pseudoheptapeptides were analyzed by
analytical reversed-phase (RP) HPLC, purified by preparative
RP-HPLC, and characterized by MALDI-TOF mass spectrometry
with a-cyano-4-hydroxycinnamic acid as a matrix.
The two pseudoheptapeptides LV-2098 and LV-2094, derived
from (E)-1 and (Z)-1 GlyY[CF=CH]Gly, respectively, were each
obtained as a single RP-HPLC peak, as illustrated for LV-2094 in
Figure 1.
Surprisingly, the chromatogram of the pseudoheptapeptide
harboring the (Z)-2 GlyY[CF=CH]Phe moiety revealed the pres-
ence of two peaks of equal area and showing the same molec-
ular weight (Figure 2). We suspected epimerization of com-
pound (Z)-2 during the late stage of our pseudodipeptide syn-
thesis. Indeed, compound 14 was a single stereoisomer before
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Table 1. Effect of 26RFa and 26RFa(20–26) analogues on basal [Ca2+
hGPR103-CHO cells.
] in
i
Peptides/pseudopeptides
EC50 [nm]
1
2
3
4
5
6
26RFa
26RFa(20–26)
LV-2094: [Z,Y(CF=CH)20,21]26RFa(20–26)
LV-2098: [E,Y(CF=CH)20,21]26RFa(20–26)
LV-2095: [Za,Y(CF=CH)21,22]26RFa(20–26)
LV-2096: [Zb,Y(CF=CH)21,22]26RFa(20–26)
10.4ꢁ1.5
739ꢁ149
618ꢁ104
538ꢁ13
6752
1720ꢁ1010
Figure 1. RP-HPLC chromatogram of crude LV-2094 [containing the (Z)-1
GlyY[CF=CH]Gly motif]. The dashed line shows the concentration of aceto-
nitrile in the eluting solvent.
Data are the meansꢁSEMs of at least three distinct experiments per-
formed in triplicate.
In addition, the presence of a local constraint, introduced by
replacement of the Gly–Gly motif by a Cmpi moiety, which can
be regarded as an inducer of a concomitant cis/trans peptide
bond increases the potency of the heptapeptide.[8]
Conversely, the pseudopeptides [Z,Y(CF=CH)21,22]26RFa(20–26)
(LV-2095 and LV-2096) were less potent than 26RFa(20–26); this
suggests a critical role of the Gly–Phe peptide bond in the bio-
active conformation of the peptide (Table 1). In support of this
hypothesis, we have recently reported that [azab3-Phe22]-
26RFa(20–26) (LV-2154), in which the Gly–Phe peptide bond is re-
placed by a Y[CONHNRCH2] pseudopeptide bond, is signifi-
cantly less active than the heptapeptide.[8] We may also point
out that the less potent designed fluorinated analogues LV-
2095 gave results similar to those for [d-Phe22]26RFa(20–26); this
suggests that LV-2095 harbors the (Z)-2 diastereoisomer with
a configuration similar to that of a d-phenylalanine residue.[7]
Figure 2. RP-HPLC chromatogram of crude pseudoheptapeptide containing
the (Z)-2 GlyY[CF=CH]Phe motif. The dashed line shows the concentration
of acetonitrile in the eluting solvent.
it was subjected to the last sequence (Scheme 3, steps k–m),
although these steps are commonly used in the laboratory
without any epimerization problem.[16] Compound (Z)-2 was
derivatized with (ꢀ)-menthol as chiral auxiliary, and NMR analy-
sis of the corresponding chiral esters revealed the existence of
two diastereoisomers—(Z)-2a and (Z)-2b—of the same intensi-
ty; this confirms the racemization of the molecule. Neverthe-
less, it appeared interesting to evaluate both diastereoisomers
of the pseudopeptides. For this, we purified both diastereoiso-
meric pseudopeptides containing (Z)-2 [LV-2095 (first eluted)
and LV-2096 (second eluted)], one containing an [l-Phe22]
amino acid analogue and the other a [d-Phe22] unit, and sub-
jected them to individually biological and structural studies.
Susceptibility to enzymatic degradation
The incorporation of a pseudopeptide bond usually enhances
the resistance of the pseudopeptide to enzymatic degrada-
tion.[16] We thus evaluated the breakdown of fluorinated pseu-
doheptapeptides in human serum by combining RP-HPLC and
MALDI-TOF MS characterization (Figure 3). The half-lives of LV-
2094 (52.9 min) and LV-2098 (52.5 min) in human serum were
about five times longer than that of 26RFa(20–26) (11.17 min).
Likewise, the half-lives of LV-2095 (28.11 min) and LV-2098
(19.07 min) were better than for 26RFa(20–26) although the
values were lower relative to LV-2094 and LV-2098. These re-
sults confirm that the fluoro-olefin moiety induced better sta-
bility of the biomolecules to enzymatic degradation and can
be used as an efficient tool to replace any peptide bond easily
prone to metabolic degradation as long as the biological activ-
ity is not impaired.
Biological activity
The functional activities of fluorinated pseudopeptide ana-
logues LV-2094, LV-2095, LV-2096, and LV-2098 were evaluated
by assessing the highly sensitive calcium-mobilizing response
in GPR103-transfected cells as previously reported.[7,8] The
pseudopeptides [Z,Y(CF=CH)20,21]26RFa(20–26) (LV-2094) and
[E,Y(CF=CH)20,21]26RFa(20–26) (LV-2098) were both slightly more
potent than 26RFa(20–26) in increasing [Ca2+]i; this suggests on
one hand the efficiency of the fluoro-olefin mimetic and on
the other, the lack of structuration of the Gly–Gly region, with
the cisoid and transoid orientations of the Gly–Gly peptide
bond having no impact on the biological activity of the pseu-
doheptapeptides (Table 1).
The better stability of LV-2095 than of LV-2096 seems to con-
firm the hypothesis relating to the presence of a d-Phe residue
in the pseudopeptide. In fact, it is well established that d-
amino acid substitution prevents degradation of peptide
bonds by peptidases.[17] We also speculate that the differences
in stability between GlyY[CF=CH]Gly (LV-2094 and LV-2098)
and GlyY[CF=CH]Phe (LV-2095 and LV-2096) bonds could be
explained by the involvement of the native Gly-Phe bond in
intramolecular stabilized structures, resulting in weaker effects
of its replacement than in the unstructured case (Gly–Gly).
In support of this statement, the Gly–Gly peptide bond can
be reduced (LV-2045) without impairing the agonist activity.[8]
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a detailed analysis of sequential and medium-range inter-resi-
due NOEs.
The Ha secondary chemical shifts calculated for 26RFa(20–26)
suggested the presence of a helical conformation between res-
idues 22 and 26 (Figure 4). These data are in good agreement
Figure 4. Ha secondary shifts in DPC/water versus 26RFa(20–26) peptide se-
1
1
quence. Ha secondary shifts were calculated with the aid of Ha random
coil values in water from Wishart et al.[19] (n.d.: not determined).
with the different number of characteristic NOE crosspeaks
present in the NOESY spectra. Indeed, the observation of
medium-to-weak N,N (i,i+1), a,N (i,i+2), and a,N (i,i+3) correla-
tions between residues 22 and 26 reinforces the hypothesis of
a helical conformation in this region of the molecule (Figure 5).
The absence of a,b (i,i+3) and a,N (i,i+4) correlations indicates
that the secondary structure of 26RFa(20–26) is composed of
a series of turns rather than of a canonical helix, in agreement
with our previous CD results.[8]
Figure 3. Stabilities of the fluoro-olefin pseudoheptapeptides. A)–D) Degra-
) as well as of A) [Z,Y(CF=CH)20,21]26RFa(20–26)
~
dation kinetics of 26RFa(20–26)
(
(LV-2094), B) [E,Y(CF=CH)20,21]26RFa(20–26) (LV-2098), C) [Za,Y(CF=CH)21,22]-
26RFa(20–26) (LV-2095), and D) [Zb,Y(CF<C=<CH)21, 22]26RFa(20–26) (LV-2096; ),
evaluated by RP-HPLC after incubation of the compounds in human serum.
Each point is the meanꢁSEM of two independent experiments.
&
Figure 5. Summary of the sequential and medium-range NOEs used for sec-
ondary structure evaluation of 26RFa(20–26). The NOEs are classified into three
categories (strong <2.5 ꢄ, medium ꢂ2.5 ꢄ and ꢃ3.5 ꢄ, weak >3.5 ꢄ) by
crosspeak volume. The intensities are indicated by the thicknesses of the
bars.
Solution conformation analysis by NMR
To evaluate the influence of the fluoro-olefin moiety on the
conformations of the pseudoheptapeptides, we performed
structural studies on 26RFa(20–26) and on the four fluorinated
analogues by NMR.
The studies were conducted in a dodecylphosphocholine
(DPC)/water mixture in order to mimic the membrane environ-
ment of GPR103, the target of 26RFa. Proton resonance assign-
ment for all molecules was carried out by the strategy pro-
posed by Wꢃthrich and co-workers.[18] Spin systems were iden-
tified with a combination of 2D TOCSY and COSY spectra, and
neighboring residues were connected through 2D NOESY
experiments.
The influence of the fluoro-olefin moiety on the pseudohep-
tapeptide conformation was first evaluated by comparing the
Ha backbone chemical shifts of each analogue with those of
26RFa(20–26) (Figure 6). For pseudoheptapeptide analogues LV-
2094 [containing (Z)-1 dipeptide] and LV-2098 [containing (E)-
1 dipeptide], with the exception of residue 21, the upfield shift
of which was consistent with the shielding effect of the intro-
duced fluoro-olefin moiety relative to the amide group, very
small Ha chemical shift differences (DHa<0.03 ppm) were de-
tected; this indicates that the fluoro-olefin insertion between
residues 20 and 21 was structurally conservative.
A first assessment of the secondary structure of 26RFa(20–26)
was obtained by analyzing Ha chemical shifts, because their
deviations from the random coil values reflect peptide/protein
secondary structures.[19] A NOESY spectrum with a mixing time
of 150 ms was also recorded to improve this estimate with
For pseudoheptapeptide analogues LV-2095 and LV-2096
[containing (Z)-2a and (Z)-2b dipeptides, respectively], in addi-
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ration for residue 22. These data confirmed our previous hy-
pothesis based on the biological activity and stability studies.
To characterize the influence of the fluoro-olefin moiety on
the secondary structures of the pseudoheptapeptides in detail,
an analysis of sequential and medium-range inter-residue
proton distances was performed (Figure 8).
Figure 6. 1Ha chemical shift difference between each analogue (LV-2094, LV-
2095, LV-2096, or LV-2098) and 26RFa(20–26) (n.d.: not determined).
tion to the upfield shift of residue 22, more significant chemi-
cal shift differences (jDHaj <0.08 ppm) were observed for res-
idues 21, 23, 24, and 25; this suggests a greater structural
impact of the fluoro-olefin insertion between residues 21 and
22.
Because the incorporation of a pseudopeptide bond might
also induce modifications in the side chain conformations, we
then compared the Hb chemical shifts of each analogue with
those of 26RFa(20–26) (Figure 7).
Figure 8. Summary of the sequential and medium-range NOEs used for sec-
ondary structure evaluation of analogues: A) LV-2094, B) LV-2098, C) LV-2095,
and D) LV-2096. The NOEs are classified into three categories (strong <2.5 ꢄ,
medium ꢂ2.5 ꢄ and ꢃ3.5 ꢄ, weak >3.5 ꢄ) based on the crosspeak volumes.
The intensities are indicated by the thicknesses of the bars. Ambiguous
NOEs are represented by gray lines. In each diagrams the CH part of the
fluoro-olefin moiety is regarded as an NH moiety in the modified residue.
Figure 7. 1Hb chemical shift difference between each analogue (LV-2094, LV-
2095, LV-2096, or LV-2098) and 26RFa(20–26). The reported value for each resi-
due corresponds to the mean of the differences observed for each of the
two b protons (n.a.: not applicable).
For analogues LV-2094 and LV-2098, very few differences
(jDHbmeanj<0.03 ppm) were observed; this confirms the small
influence of Gly–Gly peptide bond modification on the hepta-
peptide conformation.
Consistently with the Ha and Hb chemical shift comparisons,
we observed very few modifications in the NOE crosspeak pat-
terns of analogues LV-2098 and LV-2094 relative to 26RFa(20–26)
.
The absence of the N,N (i,i+1) correlation between residues 24
and 25 or the shifts to the ambiguous category were due to
chemical shift overlaps in the spectra of the two analogues.
Only three correlations were not detected: for LV-2094 the
weak a,N (i,i+2) correlation between Ser23 and Arg25 and the
weak N,N (i,i+2) correlation involving Phe24 and Phe26, and
for analogue LV-2098 the weak a,N (i,i+3) correlation between
Ser23 and Phe26. These findings indicate that the insertion of
the fluoro-olefin between residues 20 and 21, either in a Z or
in an E configuration, induces very small modifications of the
For analogues LV-2095 and LV-2096, in addition to the ex-
pected variation observed for residue 22, significant chemical
shift differences were detected for Ser23, Phe24, and Arg25;
this confirms that the conformation of the molecule is modi-
fied by the incorporation of the fluoro-olefin moiety in posi-
tion 21. Interestingly, for analogue LV-2095 an upfield shift
(DHbmean =ꢀ0.16 ppm) was observed for the b protons of resi-
due 23. This suggests that this diastereoisomer [containing (Z)-
2a dipeptide] could be the one harboring a modified configu-
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heptapeptide secondary structure and confirms the lack of
structuration of this N-terminal part of the heptapeptides.
Conversely, the NOE crosspeak patterns of the two pseudo-
peptides [Z,Y(CF=CH)21,22]26RFa(20–26) were quite different from
that of the native heptapeptide. For analogue LV-2095, addi-
tional NOE crosspeaks characteristic of turns [i.e., a,N (i,i+2),
N,N (i,i+1), and N,N (i,i+2)] were observed between Gly20 and
Ser23; this suggests that the secondary structure extends to
the N-terminal part of the peptide. In addition, the two a,N
(i,i+3) correlations are each moved by one residue; this indi-
cates that the C-terminal conformation is also altered.
dation. Nevertheless, higher stability did not necessarily imply
better biological activity, and careful position modification has
to be done to produce a concomitant effect. Fluoro-olefin-con-
taining pseudopeptides can be used as interesting tools to
design bioactive compounds as well as for conformation analy-
sis of peptides.
Experimental Section
Pseudodipeptide synthesis: All organometallic reagents were
commercially available. Reactions with organometallics were car-
ried out under argon. THF was distilled prior to use from sodium
benzophenone ketyl under nitrogen, and dichloromethane from
CaH2. Analytical thin layer chromatography was performed on silica
gel aluminum plates with F-254 indicator and visualized by UV
fluorescence and/or by staining with KMnO4 or phosphomolybdic
acid. Flash column chromatography purifications were carried out
In analogue LV-2096, the modification of the Gly–Phe pep-
tide bond induces an opposite effect. Indeed, we observed the
absence of several NOE crosspeaks between residues 21 and
25; this indicates a loss of secondary structure.
Both the fluoro-olefin insertion and the chirality[20] of Phe22
thus induced marked modifications of the heptapeptide secon-
dary structure. It is more than likely that these structural modi-
fications are responsible for the failure of compounds LV-2095
and LV-2096 to activate GPR103.
1
with silica gel (70–230 mesh). H NMR, 13C NMR, and 19F NMR (CFCl3
as internal reference) were recorded at 300.13, 75.47, and
282.40 MHz, respectively, with a Bruker DXP 300. IR spectra were
recorded with a PerkinElmer Spectrum 100. Absorption bands are
reported in cmꢀ1. ESI-MS experiments were performed with a
Bruker–Esquire mass spectrometer. Electronic impact (EI, 70 eV),
chemical ionization (CI, 200 eV), or high-resolution MS experiments
were recorded with a JEOL AX 500 mass spectrometer and use of
a mass resolution of 5000. Elemental analyses were performed
with a CE Instruments EA 110 CHNS-O instrument.
As mentioned above, 26RFa(20–26) contains interlaced turns
between residues 22 and 26, and so the carbonyl group of
Phe22 is probably involved in hydrogen bonding. We have
shown that the substitution of the native Gly20–Gly21 peptide
bond by a fluoro-olefin did not impair the molecule conforma-
tion. On the contrary, the modification of the native Gly21–
Phe22 peptide bond is associated with marked alterations of
the secondary structure. These results suggest that the fluoro-
olefin moiety can substitute the peptide bond without induc-
ing significant structural modifications if the substitution takes
place in a flexible region of the peptide.
3-{[tert-Butyl(diphenyl)silyl]oxy}propan-1-ol (Scheme 3, step a):
nBuLi (2.62 mL of a 2.5m solution in hexane, 6.57 mmol, 1 equiv)
and tert-butyldiphenylchlorosilane (1.7 mL, 6.57 mmol, 1 equiv)
were added at ꢀ788C to a solution of propane-1,3-diol (3, 0.47 mL,
6.57 mmol, 1 equiv) in dry THF (15 mL). The reaction mixture was
stirred for 15 min at ꢀ788C and for 30 min at room temperature
and was finally heated at reflux for 3 h 30 min. It was quenched
with a saturated aqueous solution of NH4Cl. The mixture was
extracted with Et2O (2ꢅ), and the combined organic layers were
dried over MgSO4, filtered, and then concentrated under reduced
pressure. The crude mixture was purified by column chromatogra-
phy on silica gel (PE/Et2O 80:20 to 60:40) to afford a white crystal-
line solid (1.75 g, 84%). M.p. 398C; Rf =0.25 (hexane/EtOAc 75:25);
1H NMR (300 MHz, CDCl3): d=1.05 (s, 9H), 1.81 (m, 2H), 3.85 (t,
3JH,H =5.7 Hz, 2ꢅ2H), 7.37–7.47 (m, 6H), 7.66–7.70 ppm (m, 4H);
13C NMR (75.4 MHz, CDCl3): d=19.1, 26.8, 34.4, 61.7, 63.1, 127.8,
129.8, 133.3, 135.6 ppm; IR (neat): n˜ =3349, 3071, 2931, 2858, 1472,
1428, 1112, 823, 737, 702, 688, 614, 505 cmꢀ1; MS: m/z: 257.00
[MꢀtBu]+; elemental analysis calcd (%) for C19H26O2Si: C 72.56, H
8.33; found: C 72.50, H 8.27.
Conclusions
We have reported the syntheses of four fluorinated pseudodi-
peptides, of Fmoc-GlyY[CF=CH]Gly-OH and Fmoc-Gly-Y[CF=
CH]Phe-OH type, convenient for Fmoc-SPPS. These dipeptides
have been used in peptide synthesis to design new fluorinated
analogues of the 26RFa(20–26) heptapeptide. These fluorinated
pseudopeptides have been subjected to biological evaluation,
enzymatic degradation, and conformation analysis. The results
suggested that the fluoro-olefin moiety can be employed as
an effective mimic of the peptide bond with great enhance-
ment of the peptide stability. Moreover, the conformation anal-
ysis showed that the fluoro-olefin moiety induces only slight
modification of the secondary structure as long as the replaced
peptide bond is not involved in hydrogen bonding. Indeed,
very few impacts on the conformation were observed with the
Gly–Gly dipeptide analogues, whereas with the Gly–Phe ana-
logues alteration of the structure was observed, this region
being involved in the secondary structure of the heptapeptide
26RFa(20–26). The biological activity, measured by the calcium-
mobilizing response in GPR103-transfected cells, is fairly inter-
esting for the two fluorinated pseudopeptides LV-2094 and LV-
2098, which exhibit slightly better activities than the native
peptide. To conclude, a fluoro-olefin unit can be used as a pep-
tide bond mimic to stabilize peptides against enzymatic degra-
3-{[tert-Butyl(diphenyl)silyl]oxy}propanal (Scheme 3, step b): IBX
(5.5 g, 19.67 mmol, 3 equiv) was added to a solution of the protect-
ed alcohol (2.06 g, 6.55 mmol, 1 equiv) in EtOAc (50 mL). The reac-
tion mixture was heated to reflux for 5 h, filtered through a plug
of celite, and then concentrated under reduced pressure to afford
a colorless oil (2.01 g, 98%). M.p. 418C; Rf =0.25 (PE/EtOAc 95:5);
1H NMR (300 MHz, CDCl3): d=1.04 (s, 9H), 2.61 (dt, 3JH,H =6.0 Hz,
3JH,H =2.2 Hz, 2H), 4.02 (t, 3JH,H =6.0 Hz, 2H), 7.37–7.44 (m, 6H),
7.64–7.68 (m, 4H), 9.82 ppm (t, 3JH,H =2.2 Hz, 1H); 13C NMR
(75.4 MHz, CDCl3): d=19.2, 26.8, 46.5, 58.4, 127.9, 129.9, 133.3,
135.6, 202.0 ppm; IR (neat): n˜ =3437, 3071, 3050, 2959, 2932, 2858,
1728, 1428, 1112, 703, 506 cmꢀ1; MS: m/z: 256.00 [MꢀtBu]+; ele-
mental analysis calcd (%) for C19H26O2Si: C 73.03, H 7.74; found: C
72.98, H 7.64.
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Fluorinated acrylates (Z)-4 and (E)-4 (Scheme 3, step c): Diethyl-
zinc (137 mL of a 1m solution in hexane, 0.137 mol, 4 equiv) was
added rapidly to a solution of triphenylphosphine (36 g, 0.137 mol,
4 equiv) and ethyl dibromofluoroacetate (9.63 mL, 0.068 mol,
2 equiv) in dry THF (350 mL). The reaction mixture was stirred for
10 min (until the internal temperature had returned to ambient
temperature), and the aldehyde (2.04 g, 6.49 mmol, 1 equiv) dis-
solved in THF (20 mL) was added rapidly. After 45 min, the mixture
was quenched with ethanol, stirred for 15 min, and then concen-
trated under reduced pressure. The crude mixture was purified by
column chromatography on silica gel (EtOAc in PE, 1%) and the
two diastereoisomers were separated to afford (Z)-4 and (E)-4 as
two colorless oils (2.09 g, 80%).
2H), 5.23 (dt, 3JH,H =8.3 Hz, 3JH,F =20.7 Hz, 1H), 7.36–7.43 (m, 6H),
7.65–7.66 ppm (m, 4H); 13C NMR (75.4 MHz, CDCl3): d=19.2, 26.9,
3
2
4
25.5 (d, JC,F =8.7 Hz), 57.6 (d, JC,F =31.8 Hz), 63.3 (d, JC,F =3.3 Hz),
105.6 (d, 2JC,F =21.4 Hz), 127.8, 129.9, 133.4, 135.7, 159.4 ppm (d,
1JC,F =249.0 Hz); 19F NMR (282.5 MHz, CDCl3): d=111.5 ppm (q); IR
(neat): n˜ =3356, 3072, 2931, 2858, 1714, 1589, 1471, 1427, 1390,
1111, 1020 cmꢀ1; MS: m/z: 359.19 [M+H]+; elemental analysis calcd
(%) for C21H27FO2Si: C 70.35, H 7.59; found: C 70.21, H 7.43.
(E/Z)-5-{[tert-Butyl(diphenyl)silyl]oxy}-2-fluoropent-2-enal
(Scheme 3, step e): IBX (7.23 g, 25.83 mmol, 3 equiv) was added to
a solution of the above mixture of alcohols (3.08 g, 8.61 mmol,
1 equiv) in EtOAc (60 mL). The reaction mixture was heated to
reflux for 6 h, filtered through a plug of celite, and then concen-
trated under reduced pressure to afford the desire aldehyde
(2.95 g, 97%) as a yellow oil. Rf(E/Z) =0.20 (cyclohexane/EtOAc 90:10)
Isomer (Z)-4: Ethyl (Z)-5-{[tert-butyl(diphenyl)silyl]oxy}-2-fluoropent-
2-enoate. Rf(Z) =0.27 (cyclohexane/EtOAc 95:5); 1H NMR (300 MHz,
3
CDCl3): d=1.06 (s, 9H), 1.33 (t, JH,H =7.0 Hz, 3H), 2.49 (m, 2H), 3.74
1
Z isomer: H NMR (300 MHz, CDCl3): d=1.06 (s, 9H), 2.59 (m, 2H),
3
(t, 3JH,H =6.4 Hz, 2H), 4.29 (q, 3JH,H =7.0 Hz, 2H), 6.23 (dt, JH,H
=
3
3
3
3.82 (t, JH,H =6.2 Hz, 2H), 6.02 (dt, JH,H =7.6 Hz, JH,F =32.5 Hz, 1H),
7.5 Hz, 3JH,F =33.5 Hz, 1H), 7.32–7.44 (m, 6H), 7.65–7.68 ppm (m,
7.37–7.47 (m, 6H), 7.63–7.67 (m, 4H), 9.18 ppm (d, 3JH,F =18.4 Hz,
3
4H); 13C NMR (75.4 MHz, CDCl3): d=14.2, 19.3, 26.9, 27.9 (d, JC,F
=
1H); 13C NMR (75.4 MHz, CDCl3): d=18.2, 25.8, 27.2 (d, JC,F
=
3
2.2 Hz), 61.6, 62.1 (d, 4JC,F =2.2 Hz), 117.6 (d, 2JC,F =11.0 Hz), 127.8,
2.3 Hz), 60.7, 105.6 (d, 2JC,F =21.2 Hz), 126.7, 128.8, 132.8, 134.5,
2
129.8, 133.6, 135.6, 148.8 (d, 1JC,F =256.1 Hz), 160.8 ppm (d, JC,F
=
=
155.9 (d, 1JC,F =262.0 Hz), 182.5 ppm (d, 2JC,F =25.2 Hz); 19F NMR
3
35.6 Hz); 19F NMR (282.5 MHz, CDCl3): d=129.8 ppm (d, JF,H
(282.5 MHz, CDCl3): d=132.9 ppm (dd, 3JF,H =33.0 Hz, JF,H
=
3
33.5 Hz); IR (neat): n˜ =3071, 2960, 2931, 2858, 1730, 1669, 1428,
1375, 1325, 1216, 1119, 938, 823, 702, 613, 505 cmꢀ1; MS: m/z:
423.18 [M+Na]+; elemental analysis calcd (%) for C23H29FO3Si: C
68.97, H 7.30; found: C 68.80, H 7.29.
18.4 Hz); IR (neat): n˜ =2957, 2930, 2857, 1703, 1472, 1428, 1361,
1112, 938, 702, 613, 506 cmꢀ1; MS: m/z: 299.1 [MꢀtBu]+; elemental
analysis calcd (%) for C21H25FO2Si: C 70.75, H 7.07; found: C 70.59,
H 7.16.
Isomer (E)-4: Ethyl (E)-5-{[tert-butyl(diphenyl)silyl]oxy}-2-fluoropent-
2-enoate. Rf(E) =0.30 (cyclohexane/EtOAc 95:5). 1H NMR (300 MHz,
1
E isomer: H NMR (300 MHz, CDCl3): d=1.05 (s, 9H), 2.68 (m, 2H),
3
3
3
3.78 (t, JH,H =6.0 Hz, 2H), 6.24 (q, JH,H =8.8 Hz, JH,F =17.9 Hz, 1H),
3
CDCl3): d=1.05 (s, 9H), 1.33 (t, JH,H =7.1 Hz, 3H), 2.49 (m, 2H), 3.75
7.37–7.47 (m, 6H), 7.63–7.67 (m, 4H), 9.68 ppm (d, 3JH,F =16.6 Hz,
3
(t, 3JH,H =6.2 Hz, 2H), 4.28 (q, 3JH,H =7.1 Hz, 2H), 6.16 (dt, JH,H
=
1H); 13C NMR (75.4 MHz, CDCl3): d=18.2, 25.8, 27.2 (d, JC,F
=
3
7.7 Hz, 3JH,F =21.5 Hz, 1H), 7.33–7.44 (m, 6H), 7.64–7.67 ppm (m,
2.3 Hz), 60.7, 105.6 (d, 2JC,F =21.2 Hz), 126.7, 128.8, 132.8, 134.5,
155.9 (d, 1JC,F =262.0 Hz), 181.0 ppm (d, 2JC,F =25.2 Hz); 19F NMR
(282.5 MHz, CDCl3): d=126.6 ppm (t); IR (neat): n˜ =2957, 2930,
2857, 1703, 1472, 1428, 1361, 1112, 938, 702, 613, 506 cmꢀ1; MS:
m/z: 299.1 [MꢀtBu]+; elemental analysis calcd (%) for C21H25FO2Si:
C 70.75, H 7.07; found: C 70.56, H 7.13.
3
4H); 13C NMR (75.4 MHz, CDCl3): d=14.2, 19.3, 26.9, 29.1 (d, JC,F
=
4.9 Hz), 61.4, 62.8, 120.6 (d, 2JC,F =19.2 Hz), 127.8, 129.8, 133.6,
135.6, 147.8 (d, 1JC,F =251.7 Hz), 161.0 ppm (d, 2JC,F =36.2 Hz);
19F NMR (282.5 MHz, CDCl3): d=121.5 ppm (d, 3JF,H =21.5 Hz); IR
(neat): n˜ =3072, 2931, 2858, 1732, 1427, 1375, 1325, 1217,
1111 cmꢀ1; MS: m/z: 423.18 [M+Na]+; elemental analysis calcd (%)
for C23H29FO3Si: C 68.97, H 7.30; found: C 68.83, H 7.31.
N-(5-{[tert-Butyl(diphenyl)silyl]oxy}-2-fluoropent-2-enylidene)-2-
methyl-2-propanesulfinamide—fluorinated sulfinylimines (Z)-5
and (E)-5 (Scheme 3, step f): A solution of Ti(OEt)4 (2.14 mL,
10.2 mmol, 2.5 equiv) and the Z/E mixture of aldehydes (1.45 g,
4.08 mmol, 1 equiv) in dry THF (60 mL) was prepared under argon.
tert-Butylsulfinylamine (1.23 g, 10.2 mmol, 2.5 equiv) was then
added, and the mixture was heated to reflux for 1 h 15 min. Once
cooled, the mixture was poured into an equal volume of brine
with fast stirring. The resulting suspension was filtered through
a plug of celite, and the filter cake was washed with EtOAc. The
brine layer was extracted once with EtOAc. The combined organic
layers were dried over MgSO4, filtered, and concentrated under
reduced pressure. The crude mixture was purified by chromatogra-
phy on silica gel (PE/EtOAc 95:5!62:38) to afford both isomers of
5 as a yellow oil (1.67 g, 89%).
(E/Z)-5-{[tert-Butyl(diphenyl)silyl]oxy}-2-fluoropent-2-en-1-ol
(Scheme 3, step d): LiAlH4 (705 mg, 18.57 mmol, 1.1 equiv) was
added at 08C to a mixture of diastereoisomers (Z/E)-4 (6.76 g,
16.88 mmol, 1 equiv) in dry THF (150 mL). The reaction mixture
was stirred for 35 min and then slowly quenched with H2SO4 (5%)
and concentrated. It was then extracted with CH2Cl2 (3ꢅ), and the
combined organic layers were washed with a saturated aqueous
solution of NaCl, dried over MgSO4, filtered, and then concentrated
under reduced pressure to afford the alcohols as a colorless oil
(5.26 g, 87%). Rf(E/Z) =0.20 (PE/EtOAc 90:10).
1
Z isomer: H NMR (300 MHz, CDCl3): d=1.06 (s, 9H), 2.37 (m, 2H),
3.69 (t, 3JH,H =6.4 Hz, 2H), 4.08 (dd, 3JH,OH =6.4 Hz, 3JH,F =15.6 Hz,
2H), 4.90 (dt, 3JH,H =7.3 Hz, 3JH,F =37.1 Hz, 1H), 7.36–7.43 (m, 6H),
7.65–7.66 ppm (m, 4H); 13C NMR (75.4 MHz, CDCl3): d=19.3, 26.9,
1
Z isomer: Rf(Z) =0.21 (cyclohexane/EtOAc 90:10); H NMR (300 MHz,
3
2
4
27.1 (d, JC,F =3.8 Hz), 61.3 (d, JC,F =32.3 Hz), 63.1 (d, JC,F =1.6 Hz),
104.6 (d, 2JC,F =13.7 Hz), 127.7, 129.7, 133.9, 135.7, 158.5 ppm (d,
1JC,F =254.4 Hz); 19F NMR (282.5 MHz, CDCl3): d=120.1 ppm (dt,
3
CDCl3): d=1.05 (s, 9H), 1.23 (s, 9H), 2.57 (m, 2H), 3.78 (t, JH,H
=
3
3
5.6 Hz, 2H), 5.73 (dt, JH,H =7.5 Hz, JH,F =33.2 Hz, 1H), 7.35–7.46 (m,
6H), 7.63–7.66 (m, 4H), 7.95 ppm (d, 3JH,F =19.6 Hz, 1H); 13C NMR
(75.4 MHz, CDCl3): d=19.3, 22.5, 26.8, 28.4 (d, 3JC,F =2.2 Hz), 58.0,
62.1 (d, 4JC,F =1.6 Hz), 123.3 (d, 2JC,F =13.2 Hz), 127.8, 129.9, 133.5,
135.6, 155.1 (d, 2JC,F =21.4 Hz), 155.7 ppm (d, 1JC,F =254.4 Hz);
19F NMR (282.5 MHz, CDCl3): d=126.6 ppm (dd, 3JF,H =19.6 Hz,
3JF,H =33.2 Hz); IR (neat): n˜ =2959, 2858, 1664, 1592, 1473, 1428,
1363, 1186, 1111, 1088, 823, 702, 613, 504 cmꢀ1; MS: m/z: 460.33
3
3JF,H =15.6 Hz, JF,H =37.1 Hz); IR (neat): n˜ =3356, 3072, 2931, 2858,
1714, 1589, 1471, 1427, 1390, 1111, 1020 cmꢀ1; MS: m/z: 359.19
[M+H]+; elemental analysis calcd (%) for C21H27FO2Si: C 70.35, H
7.59; found: C 70.06, H 7.39.
1
E isomer: H NMR (300 MHz, CDCl3): d=1.06 (s, 9H), 2.27 (m, 2H),
3.64 (t, 3JH,H =6.4 Hz, 2H), 4.18 (dd, 3JH,OH =6.4 Hz, 3JH,F =19.9 Hz,
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1
[M+H]+; elemental analysis calcd (%) for C25H34FNO2SSi: C 65.32, H
2JC,F =20.8 Hz), 127.7, 129.7, 133.5, 135.5, 157.0 ppm (d, JC,F
=
7.45, N 3.05, S 6.98; found: C 65.36, H 7.47, N 3.03, S 6.94.
248.4 Hz); 19F NMR (282.5 MHz, CDCl3): d=109.5 ppm (q); IR (neat):
n˜ =3201, 3071, 2957, 2930, 2858, 1702, 1473, 1427, 1390, 1363,
1155, 1117, 823, 738, 702, 614, 505 cmꢀ1; MS: m/z: 461.93 [M]+; ele-
mental analysis calcd (%) for C25H36FNO2SSi: C 65.03, H 7.86, N 3.03,
S 6.94; found: C 65.29, H 7.38, N 3.04, S 6.92.
1
E isomer: Rf(E) =0.23 (cyclohexane/EtOAc 90:10). H NMR (300 MHz,
3
CDCl3): d=1.04 (s, 9H), 1.24 (s, 9H), 2.60 (m, 2H), 3.74 (t, JH,H
=
3
3
6.4 Hz, 2H), 6.01 (dt, JH,H =8.5 Hz, JH,F =18.5 Hz, 1H), 7.36–7.44 (m,
6H), 7.63–7.65 (m, 4H), 8.37 ppm (d, 3JH,F =19.9 Hz, 1H); 13C NMR
(75.4 MHz, CDCl3): d=19.2, 22.5, 26.8, 28.6 (d, 3JC,F =6.6 Hz), 58.0,
62.4 (d, 4JC,F =2.7 Hz), 120.7 (d, 2JC,F =20.3 Hz), 127.8, 129.8, 133.2,
135.5, 151.7 (d, 2JC,F =21.9 Hz), 154.6 ppm (d, 1JC,F =246.8 Hz);
19F NMR (282.5 MHz, CDCl3): d=120.1 ppm (t); IR (neat): n˜ =2932,
2959, 2858, 1664, 1592, 1473, 1428, 1363, 1186, 1111.3, 1088, 823,
702, 613, 504 cmꢀ1; MS: m/z: 460.33 [M+H]; elemental analysis
calcd (%) for C25H34FNO2SSi: C 65.32, H 7.45, N 3.05, S 6.98; found:
C 65.35, H 7.44, N 3.02, 6.92.
(E)-2-Fluoro-5-hydroxypent-2-en-1-aminium
chloride
[(E)-6,
Scheme 3, step h]: HCl in dioxane (4m, 750 mL, 3.00 mmol, 2 equiv)
was added to a solution of the above (E)-fluoro tert-butylsulfina-
mide (693 mg, 1.50 mmol, 1 equiv) in dry MeOH (7 mL). The mix-
ture was stirred at room temperature for 1 h and then concentrat-
ed under reduced pressure to near dryness. The crude mixture was
used in the next step without further purification.
9H-Fluoren-9-ylmethyl (Z)-2-fluoro-5-hydroxypent-2-enylcarba-
mate (Scheme 3, step i): NaHCO3 (944 mg, 11.24 mmol, 3 equiv)
was added at 08C to a solution of amine hydrochloride derivative
(Z)-6 (583 mg, 3.74 mmol, 1 equiv) in dioxane (4 mLmmolꢀ1 of
amine hydrochloride) and water (4 mLmmolꢀ1 of amine hydrochlo-
ride), followed by Fmoc-OSu (1.51 g, 4.49 mmol, 1.2 equiv). The re-
action mixture was stirred at 08C for 2 h 30 min and was then
poured into ice-cooled HCl (1n, 8 mLmmolꢀ1 of amine hydrochlo-
ride) and extracted with AcOEt (3ꢅ). The combined organic layers
were washed with a saturated aqueous solution of NaCl, dried
over Na2SO4, filtered, and concentrated under reduced pressure.
The crude mixture was purified by column chromatography (PE/
EtOAc 70:30!45:55), to afford a white solid (920 mg, 72%). Rf =
N-((Z)-5-{[tert-Butyl(diphenyl)silyl]oxy}-2-fluoropent-2-enyl)-2-
methyl-2-propanesulfinamide
(Scheme 3,
step g):
NaBH4
(286.4 mg, 7.57 mmol, 1.5 equiv) was added at 08C to a solution of
imine (Z)-5 (2.32 g, 5.05 mmol, 1 equiv) in dry THF (60 mL). The
reaction mixture was stirred for 2 h 30 min and then slowly
quenched with a saturated aqueous solution of NH4Cl. It was ex-
tracted with EtOAc (3ꢅ), and the combined organic layers were
washed with a saturated aqueous solution of NaCl, dried over
Na2SO4, filtered, and then concentrated under reduced pressure.
The crude mixture was purified by chromatography on silica gel
(PE/EtOAc 60:40) to afford (Z)-6 as a colorless oil (2.12 g, 91%). Rf =
0.25 (PE/AcOEt 70:30); 1H NMR (300 MHz, CDCl3): d=1.05 (s, 9H),
1
3
3
0.22 (PE/AcOEt 50:50); m.p. 1128C; H NMR (300 MHz, CDCl3): d=
1.21 (s, 9H), 2.36 (m, 2H), 3.46 (t, JNH,H =6.4 Hz, 1H), 3.68 (t, JH,H
=
2.35 (m, 2H), 3.64 (t, 3JH,H =6.2 Hz, 2H), 3.88 (dd, 3JH,F =14.3 Hz,
3
3
6.4 Hz, 2H), 3.72–3.88 (m, 2H), 4.90 (dt, JH,H =7.3 Hz, JH,F =36.5 Hz,
3
3
3JH,NH =5.8 Hz, 2H), 4.21 (t, JH,H =6.8 Hz, 2H), 4.43 (d, JH,H =7.0 Hz,
1H), 7.34–7.45 (m, 6H), 7.62–7.65 ppm (m, 4H); 13C NMR (75.4 MHz,
3
3
CDCl3): d=19.2, 22.5, 26.8, 27.1 (d, 3JC,F =3.8 Hz), 46.2 (d, JC,F
=
2
2H), 4.89 (dt, JH,H =7.5 Hz, JH,F =36.5 Hz, 1H), 5.09 (brt, 1H), 7.29–
7.34 (m, 2H), 7.38–7.43 (m, 2H), 7.67 (d, 3JH,H =7.3 Hz, 2H),
7.76 ppm (d, 3JH,H =7.3 Hz, 2H); 13C NMR (75.4 MHz, CDCl3): d=
4
2
31.2 Hz), 56.1, 62.9 (d, JC,F =1.6 Hz), 105.2 (d, JC,F =13.2 Hz), 127.7,
129.7, 133.8, 135.5, 156.5 ppm (d, 1JC,F =255.0 Hz); 19F NMR
3
2
4
3
3
27.11 (d, JC,F =3.8 Hz), 41.7 (d, JC,F =32.9 Hz), 47.1, 61.5 (d, JC,F
=
(282.5 MHz, CDCl3): d=116.3 ppm (dt, JF,H =14.4 Hz, JF,H =36.5 Hz);
IR (neat): n˜ =3201, 2958, 2930, 2858, 1712, 1473, 1428, 1390, 1363,
1117, 1058, 823, 738, 702, 613, 505 cmꢀ1; MS: m/z: 462.13 [M+H]+;
elemental analysis calcd (%) for C25H36FNO2SSi: C 65.03, H 7.86, N
3.03, S 6.94; found: C 65.30, H 7.41, N 3.02, 6.91.
2.2 Hz), 66.8, 104.0 (d, 2JC,F =13.7 Hz), 120.0, 125.0, 127.1, 127.7,
141.3, 143.8, 156.5, 156.6 ppm (d, 1JC,F =255.0 Hz); 19F NMR
3
3
(282.5 MHz, CDCl3): d=116.5 ppm (dt, JF,H =14.5 Hz, JF,H =36.1 Hz);
IR (neat): n˜ =3339, 2952, 1702, 1676, 1542, 1450, 1307, 1273, 1044,
983, 757, 734, 642, 620 cmꢀ1; MS: m/z: 359.13 [M+H2O]; elemental
analysis calcd (%) for C20H20FNO3: C 70.37, H 5.91, N 4.10; found: C
70.49, H 6.01, N 4.12.
(Z)-2-Fluoro-5-hydroxypent-2-en-1-aminium
chloride
[(Z)-6,
Scheme 3, step h]: HCl in dioxane (4m, 1.9 mL, 7.49 mmol 2 equiv)
was added to a solution of the above (Z)-fluoro tert-butylsulfina-
mide (1.73 g, 3.74 mmol, 1 equiv) in dry MeOH (18 mL). The mix-
ture was stirred at room temperature for 50 min and then concen-
trated under reduced pressure to near dryness. The crude mixture
was used in the next step without further purification.
Dipeptide analogue Fmoc-GlyY[CF=CH]Gly-OH, (Z)-5-{[(9H-fluo-
ren-9-ylmethoxy)carbonyl]amino}-4-fluoropent-3-enoic acid [(Z)-
1, Scheme 3, step j]: Jones’ reagent (2.74n, 3 equiv) was added at
08C to a solution of the above (Z)-N-protected amino alcohol
(497.1 mg, 1.45 mmol, 1 equiv) in acetone (10 mLmmolꢀ1 of alco-
hol). The reaction mixture was stirred at 08C for 1 h, after which
isopropyl alcohol (10 equiv) and water (13 mLmmolꢀ1 of alcohol)
were added. The mixture was extracted with AcOEt (3ꢅ), and the
combined organic layers were washed with a saturated aqueous
solution of NaCl, dried over Na2SO4, filtered, and concentrated
under reduced pressure. The crude mixture was purified by
column chromatography (PE/EtOAc 60:40!30:70 with 0.1% of
acetic acid) to afford (Z)-1 as a white solid (360.7 mg, 70%). M.p.
N-((E)-5-{[tert-Butyl(diphenyl)silyl]oxy}-2-fluoropent-2-enyl)-2-
methyl-2-propanesulfinamide
(Scheme 3,
step g):
NaBH4
(118.9 mg, 3.14 mmol, 1.5 equiv) was added at 08C to a solution of
imine (E)-5 (963.8 mg, 2.096 mmol, 1 equiv) in dry THF (62 mL). The
reaction mixture was stirred for 2 h and then slowly quenched
with a saturated aqueous solution of NH4Cl. It was then extracted
with EtOAc (3ꢅ), and the combined organic layers were washed
with a saturated aqueous solution of NaCl, dried over Na2SO4, fil-
tered, and then concentrated under reduced pressure. The crude
mixture was purified by chromatography on silica gel (PE/EtOAc
65:35) to afford the title compound as a colorless oil (811.6 mg,
84%). Rf =0.32 (PE/AcOEt 70:30); 1H NMR (300 MHz, CDCl3): d=
1
3
1448C; H NMR (300 MHz, (CD3)2CO): d=3.13 (d, JH,H =7.0 Hz, 2H),
3.92 (dd, 3JH,F =12.4 Hz, 3JH,NH =6.0 Hz, 2H), 4.23 (t, 3JH,H =7.0 Hz,
3
3
3
1H), 4.36 (d, JH,H =7.0 Hz, 2H), 5.07 (dt, JH,H =7.1 Hz, JH,F =36.5 Hz,
1H), 5.09 (brt, 1H), 7.30–7.35 (m, 2H), 7.39–7.44 (m, 2H), 7.71 (d,
3JH,H =7.3 Hz, 2H), 7.66 ppm (d, 3JH,H =7.3 Hz, 2H); 13C NMR
3
1.04 (s, 9H), 1.19 (s, 9H), 2.25 (m, 2H), 3.33 (t, JNH,H =5.7 Hz, 1H),
3
3
2
3.64 (t, JH,H =6.4 Hz, 2H), 3.70–3.91 (m, 2H), 5.22 (dt, JH,H =8.3 Hz,
(75.4 MHz, (CD3)2CO): d=29.2 (d, 3JC,F =5.5 Hz), 41.7 (d, JC,F
=
3JH,F =20.3 Hz, 1H), 7.35–7.43 (m, 6H), 7.63–7.66 ppm (m, 4H);
13C NMR (75.4 MHz, CDCl3): d=19.2, 22.5, 26.8, 28.8 (d, JC,F
32.9 Hz), 47.9, 67.1, 100.3 (d, 2JC,F =12.1 Hz), 120.7, 126.0, 127.9,
128.5, 142.1, 145.0, 157.1, 158.5 (d, JC,F =256.6 Hz), 172.1 ppm (d,
4JC,F =1.6 Hz); 19F NMR (282.5 MHz, (CD3)2CO): d=114.6 ppm (dt,
3
1
=
2
4
8.2 Hz), 42.0 (d, JC,F =29.1 Hz), 56.1, 63.2 (d, JC,F =2.7 Hz), 106.1 (d,
ꢂ 2013 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
ChemBioChem 2013, 14, 1620 – 1633 1628

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3
3JF,H =12.4 Hz, JF,H =37.1 Hz); IR (neat): n˜ =3330, 3018, 1692, 1542,
was stirred at this temperature for 2 h, then allowed to warm to
room temperature, and stirred for 1 h 30 min. The reaction mixture
was quenched with a saturated aqueous solution of NaHCO3, and
the resulting mixture was concentrated under reduced pressure.
The residue was extracted with Et2O (2ꢅ), and the combined or-
ganic layers were dried over MgSO4 and then concentrated under
reduced pressure to afford 8 as white needles (4.19 g, 96%). Rf =
0.18 (cyclohexane/EtOAc 90:10); m.p. 958C; ½aꢄ2D0 =+29.8 (c=0.7,
CHCl3); 1H NMR (300 MHz, CDCl3): d=0.88 (d, 3JH,H =6.6 Hz, 3H),
1451, 1318, 1265, 1224, 1142, 1048, 963, 758, 735 cmꢀ1; MS: m/z:
378.13 [M+H2O]; elemental analysis calcd (%) for C20H18FNO4: C
67.60, H 5.11, N 3.94; found: C 67.50, H 5.02, N 3.91.
9H-Fluoren-9-ylmethyl (E)-2-fluoro-5-hydroxypent-2-enylcarba-
mate (Scheme 3, step i): NaHCO3 (378.4 mg, 3.0 mmol, 3 equiv)
was added at 08C to a solution of amine hydrochloride derivative
(E)-6 (233.6 mg, 1.50 mmol, 1 equiv) in dioxane (4 mLmmolꢀ1 of
amine hydrochloride) and water (4 mLmmolꢀ1 of amine hydrochlo-
ride), followed by Fmoc-OSu (607.8 mg, 1.80 mmol, 1.2 equiv). The
reaction mixture was stirred at 08C for 2 h 30 min and was then
poured into ice-cooled HCl (1n, 8 mLmmolꢀ1 of amine hydrochlo-
ride) and extracted with AcOEt (3ꢅ). The combined organic layers
were washed with saturated aqueous NaCl, dried over Na2SO4, fil-
tered, and concentrated under reduced pressure. The crude mix-
ture was purified by column chromatography (PE/EtOAc 75:25!
55:45), affording the product as a white solid (359.5 mg, 70%). Rf =
0.31 (PE/AcOEt 50:50); m.p. 928C; 1H NMR (300 MHz, CDCl3): d=
2.30 (m, 2H), 3.62 (t, 3JH,H =6.2 Hz, 2H), 3.92 (dd, 3JH,F =20.7 Hz,
3
3.02 (m, 2H), 3.30 (m, 2H), 4.75 (m, 1H), 5.63 (d, JH,H =7.3 Hz, 1H),
7.18–7.45 ppm (m, 10H); 13C NMR (75.4 MHz, CDCl3): d=14.6, 30.3,
37.3, 54.8, 79.0, 125.6, 126.3, 128.5, 128.6, 128.7, 128.8, 133.3, 140.5,
153.1, 172.2 ppm; IR (neat): n˜ =2924, 1776, 1698, 1452, 1402, 1373,
1359, 1300, 1122, 988, 960, 767, 753, 700, 521 cmꢀ1; MS: m/z:
310.19 [M+H]+; elemental analysis calcd (%) for C19H19NO3: C
73.77, H 6.19, N 4.53; found: C 73.97, H 6.14, N 4.48.
(4R,5S)-3-[(2S)-2-Benzyl-3-(benzyloxy)propanoyl]-4-methyl-5-
phenyl-1,3-oxazolidin-2-one (9, Scheme 4, step b): TiCl4 (1.56 mL,
14.22 mmol, 1.05 equiv) was added at 08C to a solution of 8
(4.19 g, 13.54 mmol, 1 equiv) in dry CH2Cl2 (40 mL). After the reac-
tion mixture had been stirred for 5 min, diisopropylethylamine
(2.23 mL, 13.54 mmol, 1 equiv) was added, and the mixture was
stirred for 1 h. Benzyl chloromethyl ether (3.76 mL, 27.08 mmol,
2 equiv) was added, and the reaction mixture was stirred at 08C
for a further 5 h and then quenched with a saturated aqueous so-
lution of NH4Cl. The mixture was extracted with CH2Cl2 (3ꢅ), and
the combined organic layers were dried over MgSO4 and then con-
centrated under reduced pressure. The yellow oil was purified by
column chromatography on silica gel (PE/EtOAc 90:10!88:12) to
afford 9 as a colorless oil (4.56 g, 88%). Rf =0.19 (cyclohexane/
EtOAc 90:10); ½aꢄ2D0 =ꢀ17.6 (c=0.81, CHCl3); 1H NMR (300 MHz,
3JH,NH =6.0 Hz, 2H), 4.19 (t, JH,H =6.8 Hz, 2H), 4.43 (d, JH,H =7.0 Hz,
3
3
3
3
2H), 5.20 (dt, JH,H =8.3 Hz, JH,F =20.5 Hz, 1H), 5.57 (brt, 1H), 7.27–
7.33 (m, 2H), 7.37–7.42 (m, 2H), 7.57 (d, 3JH,H =7.3 Hz, 2H),
7.75 ppm (d, 3JH,H =7.5 Hz, 2H); 13C NMR (75.4 MHz, CDCl3): d=
3
2
4
28.75 (d, JC,F =8.2 Hz), 38.0 (d, JC,F =29.1 Hz), 47.1, 61.5 (d, JC,F
=
2.7 Hz), 67.1, 106.0 (d, 2JC,F =20.8 Hz), 120.0, 125.1, 127.1, 127.8,
141.3, 143.8, 156.7, 156.9 ppm (d, 1JC,F =248.4 Hz); 19F NMR
(282.5 MHz, CDCl3): d=109.5 ppm (q); IR (neat): n˜ =3338, 2951,
1697, 1675, 1536, 1451, 1282, 1255, 1142, 1047, 985, 757, 732,
620 cmꢀ1; MS: m/z: 359.00 [M+H2O]; elemental analysis calcd (%)
for C20H20FNO3: C 70.37, H 5.91, N 4.10; found: C 70.47, H 5.99, N
4.12.
3
CDCl3): d=0.81 (d, JH8,H9 =6.6 Hz, 3H, H8), 2.94 (m, 2H), 3.73 (dd,
Dipeptide analogue Fmoc-GlyY[CF=CH]Gly-OH, (E)-5-{[(9H-fluo-
ren-9-ylmethoxy)carbonyl]amino}-4-fluoropent-3-enoic acid [(E)-
1, Scheme 3, step j]: Jones’ reagent (2.74n, 3 equiv) was added at
08C to a solution of the (E)-N-protected amino alcohol (240.2 mg,
0.703 mmol, 1 equiv) in acetone (10 mLmmolꢀ1 of alcohol). The
reaction mixture was stirred at 08C for 2 h 30 min and was then
quenched with isopropyl alcohol (10 equiv) and water
(13 mLmmolꢀ1 of alcohol). The mixture was extracted with AcOEt
(3ꢅ), and the combined organic layers were washed with saturated
aqueous NaCl, dried over Na2SO4, filtered, and concentrated under
reduced pressure. The crude mixture was purified by column chro-
matography (PE/EtOAc 70:30!50:50 with 0.1% of acetic acid) to
3
2JH,H’ =4.1 Hz, 3JH,H =7.3 Hz, 1H), 3.73 (dd, 2JH,H =4.1 Hz, JH,H
=
7.3 Hz, 1H’), 4.52 (s, 2H), 4.59 (m, 2H), 5.25 (d, 3JH,H =7.1 Hz, 1H),
7.19–7.42 ppm (m, 15H); 13C NMR (75.4 MHz, CDCl3): d=14.5, 35.4,
45.2, 54.9, 70.6, 73.1, 78.7, 125.6, 126.6, 127.6, 127.6, 128.3, 128.5,
128.6, 128.7, 129.2, 133.3, 138.2, 138.6, 152.7, 173.9 ppm; IR (neat):
n˜ =3027, 2965, 2880, 1763, 1712, 1490, 1454, 1353, 1248, 1186,
1099, 959, 751, 700, 670, 512 cmꢀ1; MS: m/z: 429.87 [M+H]+; ele-
mental analysis calcd (%) for C27H27NO4: C 69.65, H 7.67, N 2.54;
found: C 69.47, H 7.85, N 2.52.
(2R)-2-Benzyl-3-(benzyloxy)propan-1-ol (Scheme 4, step c): LiAlH4
(402 mg, 10.61 mmol, 1 equiv) was added at 08C to a solution of 9
(4.56 g, 10.61 mmol, 1 equiv) in dry THF (45 mL), and the reaction
mixture was stirred for 2 h. The reaction mixture was then
quenched with H2SO4 (5%) and concentrated. The reaction mixture
was extracted with CH2Cl2 (3ꢅ), and the combined organic layers
were washed with a saturated aqueous solution of NaCl, dried
over MgSO4, and then concentrated under reduced pressure. The
crude mixture was purified by column chromatography on silica
gel (PE/EtOAc 86:14!80:20) to afford the title alcohol as a colorless
oil (1.90 g, 70%). Rf =0.28 (PE/EtOAc 80:20); ½aꢄ2D0 =+25.1 (c=0.69,
1
afford (E)-1 as a white solid (124.9 mg, 50%). M.p. 1248C; H NMR
(300 MHz, (CD3)2CO): d=3.08 (m, 2H), 3.83 (m, 2H), 4.02–4.10 (m,
1H), 4.36 (m, 2H), 4.83–4.95 (m, 1H), 5.23 (brt, 1H), 7.20–7.29 (m,
4H), 7.46–7.47 (m, 2H), 7.64–7.66 ppm (m, 2H); 13C NMR (75.4 MHz,
(CD3)2CO): d=29.3 (d, 3JC,F =5.5 Hz), 38.1 (d, 2JC,F =29.1 Hz), 47.1,
67.2, 101.5 (d, 2JC,F =25.8 Hz), 120.1, 125.1, 127.2, 127.8, 141.3,
143.7, 156.6, 158.7 (d, JC,F =254.2 Hz), 176.1 ppm (d, JC,F =1.6 Hz);
19F NMR (282.5 MHz, (CD3)2CO): d=107.4 ppm (q); IR (neat): n˜ =
3330, 3018, 1692, 1542, 1451, 1318, 1265, 1224, 1142, 1048, 963,
758, 735 cmꢀ1; MS: m/z: 378.13 [M+H2O]; elemental analysis calcd
(%) for C20H20FNO3: C 67.60, H 5.11, N 3.94; found: C 67.35, H 4.78,
N 3.90.
1
4
1
CHCl3); H NMR (300 MHz, CDCl3): d=2.06 (m, 1H), 2.49 (brs, 1H,
OH), 2.58 (m, 2H), 3.45 (dd, 2JH,H’ =4.1 Hz, 3JH,H =9.6 Hz, 1H), 3.55
2
(dd, 2JH,H =4.1 Hz, 3JH,H =9.6 Hz, 1H), 3.63 (m, 2H), 4.40 (d, JH,H
=
4.1 Hz, 2H), 7.07–7.30 ppm (m, 10H); 13C NMR (75.4 MHz, CDCl3):
d=34.6, 42.7, 65.5, 73.0, 73.6, 126.2, 127.8, 127.9, 128.5, 128.6,
129.2, 138.1, 140.1 ppm; IR (neat): n˜ =3412, 3027, 2863, 1603, 1495,
1453, 1364, 1206, 1088, 1029, 741, 699 cmꢀ1; MS: m/z: 257.00
[M+H]+; elemental analysis calcd (%) for C17H20O2: C 78.65, H 7.86;
found: C 78.81, H 7.92.
(4R,5S)-4-Methyl-5-phenyl-3-(3-phenylpropanoyl)-1,3-oxazolidin-
2-one (8, Scheme 4, step a): A solution of nBuLi (9.2 mL of a 1.6m
solution in hexane, 14.81 mmol, 1.05 equiv) was added dropwise
to
a solution of (4R,5S)-(+)-4-methyl-5-phenyloxazolidin-2-one
(2.5 g, 14.10 mmol, 1 equiv) in dry THF (23 mL). After 45 min,
hydrocinnamoyl chloride 7 (2.30 mL, 15.51 mmol, 1.1 equiv) in dry
THF (3 mL) was added dropwise at ꢀ788C. The reaction mixture
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(2S)-2-Benzyl-3-(benzyloxy)propanal (10, Scheme 4, step d): Et3N
(3.03 mL, 21.86 mmol, 3 equiv), DMSO (10.8 mL, 152.6 mmol,
21 equiv), and PySO3 (3.46 g, 21.80 mmol, 3 equiv) were added at
08C to a solution of the above alcohol (1.86 g, 7.26 mmol, 1 equiv)
in dry CH2Cl2 (40 mL). The reaction mixture was stirred at this tem-
perature for 1 h 15 min and was then quenched with a saturated
aqueous solution of NH4Cl. The mixture was extracted with CH2Cl2
(3ꢅ), and the combined organic layers were washed with a saturat-
ed aqueous solution of NaCl, dried over MgSO4, and then concen-
trated under reduced pressure. The crude mixture was purified by
column chromatography on silica gel (PE/EtOAc 96:4!90:10) to
afford 10 as a colorless oil (1.46 g, 79%). Rf =0.37 (PE/EtOAc
90:10); ½aꢄ2D0 =+6.7 (c=0.5, CHCl3); 1H NMR (300 MHz, CDCl3): d=
2.77–2.88 (m, 2H), 3.09 (m, 1H), 3.58–3.72 (m, 2H), 4.49 (s, 2H),
7.14–7.39 (m, 10H), 9.80 ppm (d, 3JH,H =1.1 Hz, 1H); 13C NMR
(75.4 MHz, CDCl3): d=31.7, 53.4, 67.5, 73.4, 126.5, 127.8, 127.9,
128.5, 128.7, 129.2, 138.0, 138.8, 203.2 ppm; IR (neat): n˜ =3029,
2926, 2827, 1706, 1495, 1454, 1363, 1207, 1113, 1028, 740,
698 cmꢀ1; MS: m/z: 272.07 [M+H2O]; elemental analysis calcd (%)
for C17H18O2: C 80.28, H 7.13; found: C 80.42, H 7.29.
(2Z,4R)-4-Benzyl-5-(benzyloxy)-2-fluoropent-2-en-1-ol (Scheme 4,
step f): LiAlH4 (209 mg, 5.52 mmol, 1.5 equiv) was added at 08C to
a solution of (Z)-11 (1.26 g, 3.68 mmol, 1 equiv) in dry THF (40 mL),
and the reaction mixture was stirred for 35 min. It was then
quenched with H2SO4 (5%), concentrated, and extracted with
CH2Cl2 (3ꢅ), and the combined organic layers were washed with
a saturated aqueous solution of NaCl, dried over MgSO4, and then
concentrated under reduced pressure to afford the alcohol as a col-
orless oil (965 mg, 87%). Rf =0.16 (PE/EtOAc 85:15); ½aꢄ2D0 =ꢀ11.4
1
(c=0.52, CHCl3); H NMR (300 MHz, CDCl3): d=2.23 (brs, 1H, OH),
2.59 (dd, 2JH,H =7.5 Hz, 3JH,H =13.6 Hz, 1H), 2.79 (dd, 2JH,H =7.0 Hz,
3
3JH,H =13.6 Hz, 1H), 3.08 (m, 1H), 3.30 (d, JH,H =5.6 Hz, 2H), 3.90 (d,
3
3JH,F =15.1 Hz, 1H), 4.42 (d, 2JH,H =2.8 Hz, 2H), 4.72 (dd, JH,H
=
9.6 Hz, 3JH,F =37.1 Hz, 1H), 7.27–7.48 ppm (m, 10H); 13C NMR
(75.4 MHz, CDCl3): d=36.4 (d, 4JC,F =2.7 Hz), 37.8 (d, 3JC,F =1.6 Hz),
2
4
2
61.2 (d, JC,F =32.9 Hz), 72.0 (d, JC,F =1.6 Hz), 73.0, 108.7 (d, JC,F
=
13.1 Hz), 126.1, 127.7, 127.8, 128.3, 128.5, 129.3, 138.4, 139.6,
158.4 ppm (d, 1JC,F =256.1 Hz); 19F NMR (282.5 MHz, CDCl3): d=
ꢀ118.6 ppm (dt, 3JF,H =15.0 Hz, 3JF,H =37.1 Hz); IR (neat): n˜ =3390,
3028, 2925, 2858, 1714, 1602, 1459, 1454, 1363, 1263, 1207, 1101,
1075, 1028, 843, 747, 699 cmꢀ1; MS: m/z: 323.2 [M+Na]+; elemen-
tal analysis calcd (%) for C19H21FO2: C 75.97, H 7.05; found: C 76.07,
H 7.13.
Compounds (E)- and (Z)-11 (Scheme 4, step e): Diethylzinc
(28.5 mL of a 1m solution in hexane, 28.5 mmol, 4 equiv) was
added rapidly to
a solution of triphenylphosphine (7.48 g,
28.47 mmol, 4 equiv) and ethyl dibromofluoroacetate (1.98 mL,
14.23 mmol, 2 equiv) in dry THF (70 mL). The reaction mixture was
stirred for 10 min (until the internal temperature had returned to
ambient temperature), and aldehyde 10, dissolved in THF (20 mL),
was added rapidly. After 45 min, the mixture was quenched with
ethanol, stirred for 15 min, and then concentrated under reduced
pressure. The crude mixture was purified by column chromatogra-
phy on silica gel (PE/EtOAc 90:10) to afford (E)-11 (826.2 mg, 34%)
and (Z)-11 (1.34 g, 55%), both as colorless oils.
(2Z,4R)-4-Benzyl-5-(benzyloxy)-2-fluoropent-2-enal
(12,
Scheme 4, step g): Et3N (1.34 mL, 9.64 mmol, 3 equiv), DMSO
(4.79 mL, 67.5 mmol, 21 equiv), and PySO3 (1.53 g, 9.64 mmol,
3 equiv) were added at 08C to a solution of the above alcohol
(965 mg, 3.21 mmol, 1 equiv) in dry CH2Cl2 (15 mL). The reaction
mixture was stirred at this temperature for 1 h and then quenched
with a saturated aqueous solution of NH4Cl. The mixture was ex-
tracted with CH2Cl2 (3ꢅ), and the combined organic layers were
washed with a saturated aqueous solution of NaCl, dried over
MgSO4, and then concentrated under reduced pressure. The crude
mixture was purified by column chromatography on silica gel (PE/
Ethyl (2E,4R)-4-benzyl-5-(benzyloxy)-2-fluoropent-2-enoate [(E)-11]:
Rf =0.20 (PE/EtOAc 96:4); ½aꢄ2D0 =ꢀ15.2 (c=0.5, CHCl3); 1H NMR
EtOAc 90:10) to afford 12 as a colorless oil (781 mg, 81%). Rf =0.18
2
(300 MHz, CDCl3): d=1.28 (t, 3JH,H =7.2 Hz, 3H), 2.59 (dd, JH,H
=
(PE/EtOAc 90:10); 1H NMR (300 MHz, CDCl3): d=2.69 (dd, JH,H
=
2
7.2 Hz, 3JH,H =13.4 Hz, 1H), 2.75 (dd, 2JH,H =7.2 Hz, 3JH,H =13.4 Hz,
7.3 Hz, 3JH,H =13.6 Hz, 1H), 2.88 (dd, 2JH,H =7.5 Hz, 3JH,H =13.6 Hz,
3
2
1H), 3.29 (m, 2H), 3.63 (m, 1H), 4.12 (q, JH,H =7.2 Hz, 2H), 4.38 (d,
1H), 3.24 (m, 1H), 3.40 (m, 2H), 4.44 (d, JH,H =2.5 Hz, 2H), 5.91 (dd,
2JH,H =2.2 Hz, 2H), 5.82 (dd, JH,H =10.5 Hz, JH,F =21.7 Hz, 1H), 7.24–
3
3
3
3JH,H =9.8 Hz, JH,F =32.8 Hz, 1H), 7.16–7.41 (m, 10H), 9.09 ppm (d,
7.46 ppm (m, 10H); 13C NMR (75.4 MHz, CDCl3): d=14.1, 37.8 (d,
2JH,F =18.3 Hz, 1H); 13C NMR (75.4 MHz, CDCl3): d=37.0 (d, JC,F
=
4
4JC,F =2.2 Hz), 38.0 (d, JC,F =4.9 Hz), 61.4, 71.5 (d, JC,F =1.6 Hz), 73.0,
124.4 (d, 2JC,F =18.1 Hz), 126.2, 127.6, 128.3, 128.4, 129.2, 138.3,
139.0, 147.4 (d, 1JC,F =254.4 Hz), 160.8 ppm (d, 2JC,F =35.6 Hz);
19F NMR (282.5 MHz, CDCl3): d=121.0 ppm (d, 3JF,H =21.6 Hz); IR
(neat): n˜ =3063, 3028, 2859, 1727, 1665, 1496, 1454, 1375, 1325,
1221, 1104, 1027, 747, 699 cmꢀ1; MS: m/z: 343.07 [M+H]+; elemen-
tal analysis calcd (%) for C21H23FO3: C 73.66, H 6.77; found: C 73.70,
H 6.82.
3
4
1.6 Hz), 38.0 (d, 3JC,F =1.6 Hz), 70.8 (d, 4JC,F =1.6 Hz), 73.3, 126.7,
127.8, 12.9, 128.5, 128.6, 129.1, 131.9 (d, 2JC,F =10.4 Hz), 138.0,
138.4, 156.4 (d, 1JC,F =262.7 Hz), 183.8 ppm (d, 2JC,F =24.5 Hz);
19F NMR (282.5 MHz, CDCl3): d=ꢀ132.2 ppm (dd, 3JF,H =18.3 Hz,
3JF,H =32.8 Hz); IR (neat): n˜ =3029, 2859, 1701, 1465, 1454, 1273,
1100, 1028, 742, 699 cmꢀ1; MS: m/z: 299.53 [M+H]+; elemental
analysis calcd (%) for C19H19FO2: C 76.49, H 6.42; found: C 76.52, H
6.55.
Ethyl (2Z,4R)-4-benzyl-5-(benzyloxy)-2-fluoropent-2-enoate [(Z)-11]:
N-[(2Z,4R)-4-Benzyl-5-(benzyloxy)-2-fluoropent-2-enylidene]-2-
methyl-2-propanesulfinamide (13, Scheme 4, step h): Ti(OEt)4
(1.22 mL, 5.82 mmol, 2.5 equiv) and (S)-tert-butylsulfinylamine
(705.9 mg, 5.82 mmol, 2.5 equiv) were added to a solution of alde-
hyde (694.6 mg, 2.33 mmol, 1 equiv) in dry THF (25 mL). The reac-
tion mixture was heated to reflux for 1 h and, once cooled, poured
into an equal volume of brine with fast stirring. The resulting sus-
pension was filtered through a plug of celite, and the filter cake
was washed with EtOAc. The organic layer was washed with brine,
the brine layer was extracted (3ꢅ) with EtOAc, and the combined
organic layers were dried over MgSO4, filtered, and concentrated
under reduced pressure. The crude mixture was purified by
column chromatography on silica gel (PE/EtOAc 85:15), affording
13 as a colorless oil (884 mg, 95%). Rf =0.20 (PE/EtOAc 85:15);
Rf =0.17 (PE/EtOAc 96:4); ½aꢄ2D0 =ꢀ17.5 (c=0.5, CHCl3); 1H NMR
2
(300 MHz, CDCl3): d=1.29 (t, 3JH,H =7.1 Hz, 3H), 2.71 (dd, JH,H
=
7.3 Hz, 3JH,H =13.6 Hz, 1H), 2.85 (dd, 2JH,H =7.3 Hz, 3JH,H =13.6 Hz,
3
3
1H), 3.18 (m, 1H), 3.38 (d, JH,H =5.7 Hz, 2H), 4.20 (q, JH,H =7.1 Hz,
3
2H), 4.45 (d, 2JH,H =3.8 Hz, 2H), 6.10 (dd, 3JH,H =9.9 Hz, JH,F
=
33.5 Hz, 1H), 7.22–7.47 ppm (m, 10H); 13C NMR (75.4 MHz, CDCl3):
d=14.2, 37.2 (d, 4JC,F =1.6 Hz), 37.6, 61.7, 71.1 (d, 4JC,F =2.2 Hz),
73.2, 121.3 (d, 2JC,F =11.0 Hz), 126.4, 127.8, 128.4, 128.5, 129.2,
1
2
138.2, 138.9, 148.3 (d, JC,F =257.2 Hz), 160.7 ppm (d, JC,F =35.6 Hz);
19F NMR (282.5 MHz, CDCl3): d=ꢀ128.8 ppm (d, 3JF,H =33.5 Hz); IR
(neat): n˜ =3028, 2932, 2859, 1735, 1677, 1454, 1371, 1314, 1232,
1098, 738, 699 cmꢀ1; MS: m/z: 343.07 [M+H]+; elemental analysis
calcd (%) for C21H23FO3: C 73.66, H 6.77; found: C 73.72, H 6.80.
ꢂ 2013 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
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½aꢄ2D0 =+63.7 (c=0.62, CHCl3); H NMR (300 MHz, CDCl3): d=1.18 (s,
(2Z,4R)-4-Benzyl-2-fluoro-5-hydroxypent-2-en-1-aminium chlo-
ride (15, Scheme 4, step k): HCl in dioxane (4m, 269 mL,
1.07 mmol, 2 equiv) was added to a solution of 14 (168.1 mg,
0.54 mmol, 1 equiv) in dry MeOH (2 mL). The mixture was stirred at
room temperature for 1 h and then concentrated under reduced
pressure to near dryness. The crude mixture 15 was used in the
1
9H), 2.68 (dd, 2JH,H =7.5 Hz, 3JH,H =13.6 Hz, 1H), 2.87 (dd, JH,H
7.5 Hz, JH,H =13.6 Hz, 1H), 3.22 (m, 1H), 3.38 (d, JH,H =5.3 Hz, 2H),
4.43 (s, 2H), 5.61 (dd, 3JH,H =9.8 Hz, 3JH,F =33.0 Hz, 1H), 7.15–7.41
(m, 10H), 7.84 ppm (d, 2JH,F =19.4 Hz, 1H); 13C NMR (75.4 MHz,
=
2
3
2
4
CDCl3): d=22.5, 37.3, 38.2, 58.0, 71.0 (d, JC,F =1.1 Hz), 73.2, 126.4,
126.9 (d, 2JC,F =12.6 Hz), 127.7, 127.8, 128.4, 128.5, 129.2, 138.1,
138.7, 155.1 (d, 2JC,F =21.4 Hz), 155.1 ppm (d, 1JC,F =255.5 Hz);
19F NMR (282.5 MHz, CDCl3): d=ꢀ125.7 ppm (dd, 3JF,H =19.4 Hz,
3JF,H =33.0 Hz); IR (neat): n˜ =3028, 2958, 2862, 1661, 1592, 1454,
1363, 1177, 1086, 1029, 772, 746, 699 cmꢀ1; MS: m/z: 402.07
[M+H]+; elemental analysis calcd (%) for C19H19FO2: C 68.80, H
7.03, N 3.49, S 7.99; found: C 68.85, H 7.10, N 3.53, S 8.04.
next step without further purification. H NMR (300 MHz, D2O): d=
2.52 (dd, 2JH,H =9.6 Hz, 3JH,H =13.6 Hz, 1H), 2.84 (dd, 2JH,H =9.6 Hz,
1
3
3JH,H =13.6 Hz, 1H), 3.08 (m, 1H), 3.46–3.74 (m, 4H), 5.04 (dd, JH,H
=
10.0 Hz, 3JH,F =36.7 Hz, 1H), 7.25–7.37 ppm (m, 5H); 13C NMR
4
2
(75.4 MHz, D2O): d=36.4, 38.8 (d, JC,F =1.1 Hz), 39.7, 46.5 (d, JC,F
=
4
2
30.7 Hz), 64.2 (d, JC,F =1.1 Hz), 113.6 (d, JC,F =12.6 Hz), 126.3, 128.4,
129.1, 139.8, 152.4 ppm (d, 1JC,F =251.7 Hz); 19F NMR (282.5 MHz,
D2O): d=ꢀ117.0 ppm (q).
N-[(2Z,4R)-4-Benzyl-5-(benzyloxy)-2-fluoropent-2-enyl]-2-methyl-
2-propanesulfinamide (Scheme 4, step i): NaBH4 (31.6 mg,
0.83 mmol, 1.1 equiv) was added at 08C to a solution of 13
(305.1 mg, 0.76 mmol, 1 equiv) in dry THF (18 mL). The reaction
mixture was stirred at 08C for 2 h 30 min and then quenched with
a saturated aqueous solution of NH4Cl. The mixture was extracted
with EtOAc (3ꢅ), and the combined organic layers were washed
with a saturated aqueous solution of NaCl, dried over Na2SO4, fil-
tered, and then concentrated under reduced pressure. The crude
mixture was purified by column chromatography on silica gel (PE/
EtOAc 60:40!35:65), affording the amine as a colorless oil
(297.6 mg, 97%). Rf =0.30 (PE/EtOAc 50:50); ½aꢄ2D0 =+35.5 (c=0.63,
9H-Fluoren-9-ylmethyl (2Z,4R)-4-Benzyl-2-fluoro-5-hydroxypent-
2-enyl carbamate (Scheme 4, step l): NaHCO3 (92 mg, 1.1 mmol,
3 equiv) was added at 08C to a solution of 15 (89.5 mg, 0.36 mmol,
1 equiv) in dioxane (4 mLmmolꢀ1 of amine hydrochloride) and
water (4 mLmmolꢀ1 of amine hydrochloride), followed by Fmoc-
OSu (122.7 mg, 0.36 mmol, 1 equiv). The reaction mixture was
stirred at 08C for 1 h 30 min and was then poured into ice-cold
HCl (1n, 8 mLmmolꢀ1 of amine hydrochloride) and extracted with
AcOEt (3ꢅ). The combined organic layers were dried over Na2SO4,
filtered, and concentrated under reduced pressure. The crude mix-
ture was purified by column chromatography on silica gel (PE/
EtOAc 70:30!50:50), affording the product as a colorless oil
(149.8 mg, 95%). Rf =0.19 (PE/EtOAc 70:30); ½aꢄ2D0 =ꢀ13.15 (c=
1
2
CHCl3); H NMR (300 MHz, CDCl3): d=1.13 (s, 9H), 2.53 (dd, JH,H
=
7.9 Hz, 3JH,H =13.4 Hz, 1H), 2.80 (dd, 2JH,H =7.9 Hz, 3JH,H =13.4 Hz,
3
2
1
1H), 3.08 (m, 1H), 3.17 (t, JNH,H =6.8 Hz, 1H), 3.30 (d, JH,H =5.6 Hz,
0.95, CHCl3); H NMR (300 MHz, CDCl3): d=2.53–2.80 (m, 2H+OH),
3
3
3
2H), 3.62–3.84 (m, 2H), 4.42 (s, 2H), 4.71 (dd, JH,H =9.8 Hz, JH,F
=
3.01 (m, 1H), 3.42–3.61 (m, 2H), 3.79 (dd, 3JH,F =14.3 Hz, JH,NH
=
36.3 Hz, 1H), 7.14–7.40 ppm (m, 10H); 13C NMR (75.4 MHz, CDCl3):
d=22.6, 37.3, 38.1 (d, 3JC,F =1.6 Hz), 46.3 (d, 2JC,F =31.3 Hz), 58.0,
71.0 (d, 4JC,F =1.1 Hz), 73.2, 109.6 (d, 2JC,F =13.2 Hz), 126.1, 127.7,
128.2, 128.5, 129.3, 138.1, 139.6, 156.4 ppm (d, 1JC,F =256.6 Hz);
19F NMR (282.5 MHz, CDCl3): d=ꢀ115.1 ppm (dt, 3JF,H =18.6 Hz,
3JF,H =36.3 Hz); IR (neat): n˜ =3205, 2925, 2861, 1702, 1495, 1454,
1364, 1099, 1059, 746, 699 cmꢀ1; MS: m/z: 404.20 [M+H]+; elemen-
tal analysis calcd (%) for C23H30FNO2S: C 68.45, H 7.49, N 3.47, S
7.95; found: C 68.89, H 6.57, N 3.42, S 7.99.
5.8 Hz, 2H), 4.19 (t, 3JH,H =6.8 Hz, 1H), 4.41 (d, 3JH,H =6.8 Hz, 2H),
4.68 (dd, 3JH,H =9.8 Hz, 3JH,F =36.7 Hz, 1H), 5.37 (t, 3JNH,H =5.6 Hz,
1H, NH), 7.13–7.18 (m, 3H), 7.22–7.33 (m, 4H), 7.38–7.43 (m, 2H),
7.58 (d, JH,H =7.3 Hz, 2H), 7.76 ppm (d, JH,H =7.3 Hz, 2H); 13C NMR
3
3
2
(75.4 MHz, CDCl3): d=37.5, 39.0, 41.2 (d, JC,F =32.3 Hz), 47.1, 64.9,
66.8, 108.6 (d, 2JC,F =12.6 Hz), 120.0, 125.0, 126.1, 127.1, 127.7,
1
128.2, 129.1, 139.4, 141.3, 143.8, 156.4, 156.5 ppm (d, JC,F
=
=
256.1 Hz); 19F NMR (282.5 MHz, CDCl3): d=ꢀ114.7 ppm (dt, JF,H
3
3
14.5 Hz, JF,H =37.1 Hz); IR (neat): n˜ =3410, 3326, 2926, 1704, 1520,
1450, 1257, 1031, 753, 741, 621 cmꢀ1; MS: m/z: 454.33 [M+Na]+; el-
emental analysis calcd (%) for C27H26FNO3: C 75.15, H 6.07, N 3.25;
found: C 75.28, H 6.15, N 3.29.
N-[(2Z,4R)-4-Benzyl-2-fluoro-5-hydroxypent-2-enyl]-2-methyl-2-
propanesulfinamide (14, Scheme 4, step j): BCl3 (4.2 mL of a 1m
solution in DCM, 28.5 mmol, 5 equiv) was slowly added at ꢀ788C
to a solution of the above sulfinamide (336.5 mg, 0.83 mmol,
1 equiv) in dry DCM (10 mL). The reaction mixture was stirred at
ꢀ788C for 10 min and then quenched with a saturated aqueous
solution of NaHCO3. The mixture was extracted with DCM (3ꢅ),
and the combined organic layers were washed with a saturated
aqueous solution of NaHCO3 and with H2O and were then dried
over Na2SO4, filtered, and concentrated under reduced pressure.
The crude mixture was purified by column chromatography on
silica gel (DCM/MeOH 98.5:1.5!80:20), affording 14 as a colorless
Fmoc-GlyY[CF=CH]Phe-OH, (2R,3Z)-2-benzyl-5-{[(9H-fluoren-9-yl-
methoxy)carbonyl]amino}-4-fluoropent-3-enoic acid, dipeptide
analogue (Z)-2 (Scheme 4, step m): Jones’ reagent (2.74n,
3 equiv) was added at 08C to a solution of the above (Z)-N-protect-
ed amino alcohol (240.3 mg, 0.55 mmol, 1 equiv) in acetone
(10 mLmmolꢀ1 of alcohol). The reaction mixture was stirred at 08C
for 1 h and then quenched with isopropyl alcohol (10 equiv) and
water (13 mLmmolꢀ1 of alcohol). The mixture was extracted with
AcOEt (3ꢅ), and the combined organic layers were washed with
a saturated aqueous solution of NaCl, dried over Na2SO4, filtered,
and concentrated under reduced pressure. The crude mixture was
purified by column chromatography on silica gel (PE/EtOAc
80:20!70:30, then 50:50, with 0.1% of acetic acid), affording the
dipeptide analogue as a white solid (192.2 mg, 80%). 1H NMR
(300 MHz, CDCl3): d=2.85–3.22 (m, 2H), 3.73–3.89 (m, 3H, H2), 4.25
1
oil (156.3 mg, 60%). Rf =0.18 (DCM/MeOH 98:2); H NMR (300 MHz,
2
3
CDCl3): d=1.21 (s, 9H), 2.61 (dd, JH,H =8.3 Hz, JH,H =13.7 Hz, 1H),
2
3
2.84 (dd, JH,H =8.3 Hz, JH,H =13.7 Hz, 1H), 3.03 (m, 1H), 3.48–3.75
3
3
(m, 5H), 4.78 (dd, JH,H =9.8 Hz, JH,F =37.1 Hz, 1H), 7.10–7.31 ppm
4
(m, 5H); 13C NMR (75.4 MHz, CDCl3): d=22.6, 37.5 (d, JC,F =1.6 Hz),
4
39.3 (d, 3JC,F =1.1 Hz), 46.5 (d, 2JC,F =31.8 Hz), 56.4, 65.1 (d, JC,F
=
1.6 Hz), 109.4 (d, 2JC,F =12.6 Hz), 126.2, 128.3, 129.2, 139.6,
157.3 ppm (d, 1JC,F =257.2 Hz). 19F NMR (282.5 MHz, CDCl3): d=
ꢀ113.3 ppm (dt, 3JF,H =14.5 Hz, 3JF,H =37.1 Hz); elemental analysis
calcd (%) for C16H24FNO2S: C 61.31, H 7.72, N 4.47, S 10.23; found:
C 61.45, H 7.85, N 4.52, S 10.25.
(t, JH,H =6.4 Hz, 1H), 4.48 (d, JH,H =6.2 Hz, 2H), 4.99 (3JH,H =9.6 Hz,
3JH,F =35. 2 Hz, 1H), 5.11 (brs, 1H, NH), 7.23–7.48 (m, 9H), 7.63 (d,
3JH,H =7.1 Hz, 2H), 7.82 ppm (d, 3JH,H =7.6 Hz, 2H); 13C NMR
3
3
2
(75.4 MHz, CDCl3): d=38.4, 41.2 (d, JC,F =31.8 Hz), 42.5, 47.1, 67.0,
104.7 (d, 2JC,F =12.1 Hz), 120.1, 125.0, 126.7, 127.1, 127.8, 128.4,
129.1, 137.9, 141.3, 143.8, 156.3, 156.8 (d, 1JC,F =259.4 Hz),
ꢂ 2013 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
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3
178.0 ppm; 19F NMR (282.5 MHz, CDCl3): d=ꢀ113.5 ppm (dt, JF,H
=
lamp was used as excitation source. The wavelengths of excitation
(485 nm) and emission (525 nm) of Fluo-4AM were selected by use
of two monochromators included in the device, equipped with
a bottom reading probe.
3
13.4 Hz, JF,H =35.1 Hz); IR (neat): n˜ =3320, 3066, 3028, 2941, 1708,
1522, 1450, 1251, 1162, 760, 739, 699 cmꢀ1; MS: m/z: 891.00 [2M<
M+ >H]+; elemental analysis calcd (%) for C27H26FNO4: C 72.80, H
5.43, N 3.14; found: C 72.92, H 5.52, N 3.17.
Susceptibility of pseudopeptides to enzymatic degradation: The
stabilities of the 26RFa analogues LV-2094, LV-2095, LV-2096, and
LV-2098 were tested in vitro as previously described.[8] Pseudopep-
tides (250 mgmLꢀ1) were incubated at 378C with pooled human
serum (healthy donors). The reaction was stopped after different
times by adding TFA (10% of the final volume), and the samples
were diluted five times in PBS (pH 7.4). After centrifugation, the
supernatants were collected and analyzed by RP-HPLC on a Vydac
218MS54 C18 column (0.46ꢅ25 cm) with a linear gradient (10–60%
over 50 min) of acetonitrile/TFA (99.9:0.1, v/v) at a flow rate of
1 mLminꢀ1. HPLC peak areas were used to calculate the percentag-
es of intact compounds remaining at various time points during
the incubation. Half-life times (t1/2) were calculated with the Prism
software (Graphpad Software, San Diego, CA) from exponential
decay curves. Mass spectra of the intact peptide and its metabo-
lites were performed by MALDI-TOF MS with a Voyager DE-PRO
(Applera-France) in the reflector mode with a-cyano-4-hydroxycin-
namic acid as a matrix.
Peptide and pseudopeptide solid-phase synthesis: Peptides
26RFa and 26RFa(20–26) were synthesized by solid-phase methodolo-
gy as previously described.[7] The pseudopeptides [Z,CF=CH20,21]-
26RFa(20–26) (LV-2094), [E,CF=CH20,21]26RFa(20–26) (LV-2098), and [Z,CF=
CH21,22]26RFa(20–26) (LV-2095 and LV-2096) were synthesized
(0.1 mmol scale) by the solid-phase methodology on a Rink amide
4-methylbenzhydrylamine resin (VWR, Fontenay-Sous-Bois, France)
with a 433A Applied Biosystems peptide synthesizer (Applera-
France, Courtaboeuf, France) and the standard manufacturer’s
Fmoc procedure. All Fmoc-amino acids (1 mmol, 10 equiv, Christof
Senn Laboratories, Dielsdorf, Switzerland) were coupled by in situ
activation with HBTU/HOBt (1.25 mmol:1.25 mmol, 12.5 equiv) and
DIEA (2.5 mmol, 25 equiv) in NMP. The Fmoc-GlyY[CF=CH]AA-OH
pseudodipeptides (1.5 equiv, AA=Gly, Phe) were manually coupled
with the aid of HATU (1.5 equiv), HOAt (1.5 equiv), and DIEA
(3 equiv) in NMP for 2 h. Reactions were monitored by use of the
Kaiser test. Pseudopeptides were deprotected and cleaved from
the resin by adding a TFA/TIS/H2O (99.5:0.25:0.25, v/v/v, 10 mL)
mixture (120 min at room temperature). After filtration, crude pep-
tides were precipitated by addition of tert-butyl methyl ether
(TBME), centrifuged (4500 rpm), washed twice with TBME, and
freeze-dried. The synthetic pseudopeptides were purified by re-
versed-phase HPLC with a 2.2ꢅ25 cm Vydac 218TP1022 C18 column
(Grace, Epernon, France) and use of a linear gradient (10–50% over
NMR spectroscopy for conformation studies: All NMR experi-
ments were performed with a Bruker Avance III 600 MHz NMR
spectrometer (Wissembourg, France), fitted with a triple resonance
cryoprobe including shielded z-gradients. All peptides were dis-
solved at concentrations of about 1 mm in D2O/H2O (10%) in the
presence of [D38]DPC (150 mm, C/D/N isotopes, Pointe-Claire,
1
Canada). DSS was added as an internal H chemical shift reference.
45 min) of CH3CN/TFA (99.9:0.1, v/v) at a flow rate of 10 mLminꢀ1
.
Conventional COSY, TOCSY, and NOESY two-dimensional experi-
ments were carried out at 298 K. TOCSY experiments were per-
formed with an 80 ms DIPSI2 spin lock mixing pulse. NOESY spec-
tra were collected at mixing times of 120 and 150 ms. Water sup-
pression was achieved by use of the excitation sculpting sequence
except in the case of COSY experiments, in which a low power pre-
saturation was applied during the relaxation delay. TOCSY and
NOESY experiments were performed in the phase-sensitive mode,
with use of proportional phase incrementation method for quadra-
ture detection (States-TPPI). Proton chemical shifts are reported rel-
ative to DSS taken as an internal reference. Distance restraints for
NOE diagrams were derived from crosspeaks in the NOESY spectra
recorded with mixing times of 150 ms. NOE crosspeaks were inte-
grated into distances by volume integration with FelixNMR (San
Diego, USA). The NOE volumes were calibrated from well-resolved
geminal Hb crosspeaks.
Analytical HPLC, performed with a 0.46ꢅ25 cm Vydac 218TP54 C18
column (Grace), showed that the purities of the pseudopeptides
were >99.5% (Table 2). The purified pseudopeptides were charac-
terized by MALDI-TOF mass spectrometry (Table 2) with a Voyager
DE PRO (Applera-France) in the reflector mode with a-cyano-4-
hydroxycinnamic acid as a matrix.
Table 2. Chemical data for pseudopeptides.
Code
HPLC
MS
tR [min][a]
Purity [%]
Calcd[b]
Found[c]
LV-2094
LV-2098
LV-2095
LV-2096
26.2
26.3
25.4
27.4
100
100
100
100
816.41
816.41
816.41
816.41
817.28
817.44
817.47
817.56
[a] Retention times determined by RP-HPLC. [b] Theoretical monoisotopic
molecular weights. [c] m/z values assessed by MALDI-TOF MS.
Acknowledgements
Calcium mobilization assays: Changes in [Ca2+
] induced by
i
This work was promoted by the inter-regional Norman chemistry
network (CRUNCh); the “Rꢀgion Haute-Normandie” is gratefully
thanked for its financial support. This study was also supported
by INSERM (U982), PNR-RE, and FEDER (ERDF). The authors also
thank the Centre de Ressources Informatiques de Haute-Norman-
die (France) for NMR facilities. C.P. and C.N. were the recipients of
fellowships from the CRUNCh and the LARC-Neuroscience net-
work, respectively. A.M. is funded by the IS:CE-Chem project,
which was selected under the European cross-border cooperation
programme Interreg IV A France-(Channel)-England, cofunded by
the ERDF.
26RFa and 26RFa(20–26) analogues in hGPR103-transfected CHO cells
were measured with a benchtop scanning fluorometer Flexstatio-
n III (Molecular Devices, Sunnyvale, CA) as previously described.[7,8]
Briefly, 96-well assay black plates with clear bottom (Corning Inter-
national, Avon, France) were seeded at a density of 40000 cells per
well 24 h prior to assay. Cells were loaded with 2 mm Fluo-4AM (In-
vitrogen) for 1 h, washed three times, and incubated for 30 min
with standard Hank’s balanced salt solution (HBSS) containing pro-
benecid (2.5 mm) and HEPES (5 mm). Compounds to be tested
were added at final concentrations ranging from 10ꢀ12 to 10ꢀ5 m,
and the fluorescence intensity was measured over 2 min. A Xenon
ꢂ 2013 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
ChemBioChem 2013, 14, 1620 – 1633 1632

CHEMBIOCHEM
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Keywords: biological evaluation · conformation analysis ·
fluoro-olefins · peptides · pseudopeptides
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Received: May 21, 2013
Published online on August 12, 2013
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ChemBioChem 2013, 14, 1620 – 1633 1633