Design, synthesis and evaluation of β-lactam antigenic peptide hybrids; Unusual opening of the β-lactam ring in acidic media

Article abstract of DOI:10.1039/c003877f

β-Lactam peptides were envisioned as conformational constraints in antigenic peptides (APs). Three different β-lactam tripeptides of varying flexibility were prepared in solution and incorporated in place of the central part of the altered melanoma associ

Full text of DOI:10.1039/c003877f

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PAPER
www.rsc.org/obc | Organic & Biomolecular Chemistry
Design, synthesis and evaluation of b-lactam antigenic peptide hybrids;
unusual opening of the b-lactam ring in acidic media†
Marion Tarbe,a Itxaso Azcune,b Eva Balentova´,b John J. Miles,c Emily E. Edwards,c Kim M. Miles,c
Priscilla Do,d Brian M. Baker,d Andrew K. Sewell,c Jesus M. Aizpurua,b Ce´line Douat-Casassus*a and
Ste´phane Quideau*a
Received 5th March 2010, Accepted 2nd September 2010
DOI: 10.1039/c003877f
b-Lactam peptides were envisioned as conformational constraints in antigenic peptides (APs). Three
different b-lactam tripeptides of varying flexibility were prepared in solution and incorporated in place
of the central part of the altered melanoma associated antigenic peptide Leu27-Melan-A26–35 using solid
phase synthesis techniques. Upon TFA cleavage from the solid support, an unexpected opening of the
b-lactam ring occurred with conservation of the amide bond. After adaptation of the solid phase
synthesis strategy, b-lactam peptides were successfully obtained and both opened and closed forms
were evaluated for their capacity to bind to the antigen-presenting class-I MHC HLA-A2 protein
system. None of the closed b-lactam peptides bound to HLA-A2, but their opened variants were shown
to be moderate to good HLA-A2 ligands, one of them being even capable of stimulating a
Melan-A-specific T cell line.
Introduction
The incorporation of conformationally constrained b-turn mimet-
ics in therapeutically relevant peptide structures has long been
considered as a valuable approach in the design of peptidomimet-
ics with enhanced affinity and/or improved metabolic stability
compared to those of their peptide parents.1 In particular, since
their first utilization by Freidinger in 1980, dipeptide lactams are
among the most popular b-turn mimetics.2,3 In this context, our
current efforts towards the design of antigenic peptidomimetics
led us to envision the use of the b-lactam scaffolds I–III4,5
Fig. 1 b-lactam scaffolds envisioned as conformationally constrained
surrogates of antigenic peptide central segments.
(Fig. 1) as surrogates of the central part of a tumor-associated
antigenic peptide (AP) and to assess the consequences of such
antigen-presenting cells. Given the pivotal role played by APs in
structural modifications on the immunogenicity of the resulting
launching this cellular defense mechanism, it is not surprising that
peptidomimetics.
these small peptides have been considered by immunologists as
promising leads in the development of immunotherapeutic drugs,
Tumor-associated APs, usually 8 to 10 amino acids long,6 are
generated in cancer cells by proteolysis of self over-expressed
as well as synthetic vaccines. Unfortunately, the poor biostability
proteins in the proteasome and are then exposed to the cell
and globally unsatisfactory pharmacokinetic properties inherent
surface by class-I major histocompatibility (MHC-I) complexes
to their peptidic nature have precluded the general use of APs
to be screened by circulating CD8+ cytotoxic T lymphocytes.7
as therapeutic agents. Thus, designing bioresistant antigenic
Recognition of these peptide-MHC-I (pMHC-I) complexes by T
peptidomimetics capable of engaging both MHC-I and TCR
cell receptors (TCRs) is the causal event in the triggering of T
proteins in the formation of complexes inducing enhanced immune
cell-mediated immune responses that result in the destruction of
responses remains at the forefront of modern immunology.
The structural data that have been gathered by biophysicists in
aUniversite´ de Bordeaux, Institut des Sciences Mole´culaires (UMR-CNRS
this field over the last decade have provided us with a precious
5255) and Institut Europe´en de Chimie et Biologie (IECB), 2 rue Robert
understanding of the mode of binding of APs to MHC-I proteins
and of their molecular recognition by TCRs.8 APs that efficiently
bind onto and in between the a-helical units of the MHC-I protein
cleft usually present anchoring amino acid residues at position
2 (P2) and at their C-terminus, whereas their central portion
(P4–P7) bulges out of the peptide binding cleft, sometimes even
zigzaging to interact with the TCR components (Fig. 2). Most
previous attempts in designing heteroclitic APs were essentially
aimed at developing MHC-I high affinity ligands so as to improve
immunogenicity.9 Based on the X-ray structure of the MHC-
I human leucocyte antigen (HLA)-A2 protein complex bearing
Escarpit, 33607 Pessac, France. E-mail: s.quideau@iecb.u-bordeaux.fr;
Fax: (+33) 5-4000-2215; Tel: (+33) 5-4000-3010
bDepartamento de Qu´ımica Orga´nica-I, Universidad del Pa´ıs Vasco, Joxe
Mari Korta R&D Center, Apdo. Tolosa-72, 20018 San Sebastian, Spain
cDepartment of Medical Biochemistry and Immunology, Henry Wellcome
Building, Cardiff University School of Medicine, Heath Park, Cardiff, CF14
4XN, UK
dDepartment of Chemistry and Biochemistry, 251 Nieuwland Science Hall,
University of Notre Dame, Notre Dame, IN 46556, USA
† Electronic supplementary information (ESI) available: NMR spectra of
b-lactam scaffolds and intermediates, and HPLC traces, mass spectra and
NMR TOCSY maps of b-lactam–peptide hybrids and opened variants.
See DOI: 10.1039/c003877f
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Fig. 2 (a) ELA peptide sequence showing HLA-A2 anchor and TCR-interacting residues, superimposed with its HLA-A2-bound X-ray structure.10,11
(b) Sequence of the envisioned b-lactam-containing peptidomimetics (1–3).
the altered melanoma-associated peptide epitope Leu27-Melan-
A26–35 (ELAGIGILTV, hereafter referred to as ELA),10 we recently
reported the successful replacement of the central segment GIGI
(i.e., P4–P7) of this AP by a nonpeptidic functionalizable scaffold
without compromising HLA-A2 binding.11 Some of the ELA pep-
tidomimetics thus designed even managed to efficiently stimulate
Melan-A-specific T cell clones.11 On the basis of these results, we
surmise that the replacement of the ELA central residues by a
b-lactam motif such as I, II or III (Fig. 1) thus incorporated in
ELA pseudopeptides 1–3 (Fig. 2) could advantageously serve the
purpose of mimicking their hydrophobic bulge.
Herein, we disclose the design and solid-phase synthesis of
these b-lactam-containing ELA mimetics, together with an un-
expected opening of the b-lactam ring during the release of the
peptidomimetics from the solid support. The three b-lactam-
containing peptidomimetics thus generated, as well as their opened
variants, were then evaluated for their binding affinity to the HLA-
A2 molecule and for their capacity to activate Melan-A-specific T
cells.
I–III. The methylene-composed b-alanine (in 1) and glycine (in
2) were chosen to maintain some degree of flexibility in the
peptidic backbone in order to allow the resulting constructs to best
orient themselves within the HLA-A2 binding cleft. Alternatively,
the inclusion of a constrained aminoisobutyric acid (Aib) unit,
known to be an efficient inducer of 310 helix,13 was also examined
(in 3). Moreover, our previous results11 prompted us to install
these b-lactam segments in place of the G29I30G31 tripeptide of
ELA so as to best mimic the zigzag conformation of its central
portion (Fig. 2),10 while maintaining adequate interactions of the
anchoring residue side chains at P2 and P10 within the HLA-
A2 binding cleft. Furthermore, our preliminary in silico docking
study of the three envisaged D-a-benzylserine-derived b-lactam–
peptide hybrids (Scheme 1)2,14 indicated that a substitution by the
D-enantiomer of isoleucine at P7 would be necessary to enable
the appropriate orientation of the N-terminal part of these b-
lactam pseudopeptides (data not shown). Finally, the N-terminal
glutamate residue (P1) was replaced by a b-alanine, as such
a substitution has previously been shown to be beneficial for
biostability without compromising neither the binding to the
HLA-A2 protein nor the recognition by Melan-A specific T-cell
clones.11,15
Results and discussion
Design of b-lactam scaffolds
Solid-phase synthesis of b-lactam pseudopeptides
Three different amino acids (Xaa) were selected to occupy the
b-turn (i+2) position in the central -(b-lactam)-Xaa- scaffolds
I–III of pseudopeptides 1–3 (Fig. 1 and 2). On the basis of
previous investigations stressing that TCR loops are sufficiently
flexible to accommodate rather bulky and hydrophobic central
side-chains on hapten-modified peptides12 and of results from our
preliminary in silico docking study (data not shown), a benzyl
group was selected in place of the sec-butyl central isoleucine
side-chain to equip the b-lactam motif of the three scaffolds
The b-lactam cores 5, 6 and 9 were prepared from methyl
D-a-benzylserinate 4 following previously described procedures
(Scheme 1).5 In the case of tripeptide scaffolds 7 and 8, allylic and
homoallylic amine moieties were respectively chosen as precursors
of the glycine and b-alanine residues. The oxidative cleavage of
their double bond by NaIO4 conveniently afforded the desired
carboxylic acid functions. All three N-Fmoc-protected tripeptide
scaffolds 7, 8 and 11 were installed on a D-Ile-Leu-Thr-Val peptide
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those expected for the desired b-lactam pseudopeptides 1–3. We
rapidly concluded that these observations were likely due to
a conventional hydrolysis of the b-lactam N1–C2 amide bond
during the final aqueous TFA cleavage step (Scheme 2a), which
would then furnish b-amino acid-containing peptides such as
15 from 1 (Scheme 3, path A). However, a careful examination
of the data we collected from standard 1D H and 2D H–1H
TOCSY NMR experiments performed on compound 12 clearly
indicated that 8 amidic protons were present in the molecule
(and not 7 as in 15), and that the “extra” amidic proton was
correlated with one of the methylene protons of the endo-b-alanine
residue (see ESI† for details). These data strongly suggest that
it is instead the N1–C4 bond of the b-lactam ring that suffered
hydrolytic cleavage (Scheme 3, path B), hence leading to the D-
a-benzylserine-containing peptides 12–14 shown in Scheme 2.
Cleavage of the N1–C4 bond has been previously observed or
forced in b-lactam systems bearing an electron-donating group
at C4.16–19 For example, the hydrogenolysis of 4-arylazetidin-2-
ones is a very well know methodology developed by Ojima for
the preparation of a-amino acids and peptidomimetics.17 More
recently, McMurray and co-workers described the N1–C4 bond
opening of 4-(4¢-hydroxyphenyl)-2-azetidinones under basic or
acidic conditions.18 In parallel, Alcaide and co-workers reported
novel N1–C4 bond breakages on b-lactams equipped with non-aryl
carbanionic moieties at C4.19 Moreover, according to the results
of detailed studies of acid-catalyzed hydrolyses,20 N-protonated
b-lactams intermediates undergo nucleophilic addition (AdN) of
a water molecule at the carbonyl sp2 carbon atom along a
classical Bu¨rgi–Dunitz trajectory,21 followed by rupture of the
N1–C2 bond by elimination (E). In the case of our b-lactams,
however, the Bu¨rgi–Dunitz trajectory of approach is hindered by
the a,a-substituents on both sides of the azetidinone ring, thus
energetically penalizing the AdN-E pathway (see path A in Scheme
3). Conversely, the unsubstituted b-methylene sp3 C4 atom hence
becomes more vulnerable to an SN2 attack by a water molecule that
1
1
Scheme 1 Synthesis of Fmoc-Ala-b-lactam-Xaa-OH tripeptide scaffolds
with C-terminal b-alanine (7), glycine (8) and Aib (11) residues. All yields
quoted correspond to overall transformations.
construct supported on a Wang resin by following standard Fmoc-
based solid-phase peptide synthesis (SPPS) protocols. Removal of
the Fmoc-protecting group, addition of the two remaining amino
acids (i.e., leucine and b-alanine) and release of the expected b-
lactam–peptide hybrids from the resin with concomitant removal
of all protecting groups completed the syntheses (Scheme 2a).
Much to our surprise, a structural analysis of the resulting
compounds (i.e., 12–14) by electrospray ionization mass spec-
trometry (ESIMS) indicated molecular masses exceeding by 18 Da
Scheme 2 Solid-phase synthesis of b-lactam–peptide hybrids 1–3 (b) and their opened variants 12–14 (a).
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The high hydrophobicity of the resulting crude pseudopeptides
caused us some difficulties during their purification by HPLC,
notably because of their tendency to aggregate. Consequently, the
three targeted pseudopeptides 1–3 were obtained in pure forms
in relatively poor yields (Scheme 2b), but nevertheless in largely
sufficient quantities to handle their immunological evaluation.
HLA-A2 binding
Having thus in hand both the b-lactam-containing ELA mimetics
1–3 and their opened variants 12–14, we investigated their relative
capacity to bind to the HLA-A2 protein via the T2 surface
binding assay (see Experimental section). Two peptides were used
as references: ELA as a positive control and a nonapeptide of
sequence GAGAGAGAG (termed GAG) containing no HLA-A2
anchor residues as a negative control. Hence, any peptide with a
binding affinity equal to or lower than GAG can be considered
as a very weak ligand or non-binding entity. The T2 binding
experiments revealed that the opened pseudopeptides 12–14 bind
relatively well to HLA-A2, whereas no binding was surprisingly
observed with the b-lactam-bearing peptidomimetics 1–3 (Fig. 3).
Importantly, compound 14 emerged as an extremely strong HLA-
A2 ligand, showing an apparent binding strength superior to that
of ELA.
Scheme 3 Proposed mechanism (path B) for the unusual N1–C4 b-lactam
ring opening observed during aqueous TFA treatment.
thus opens up the b-lactam ring with conservation of the amide
bond (see path B in Scheme 3). To the best of our knowledge, the
ring opening reactions we thus observed leading to 12–14 are the
first examples of nucleophilic N1–C4 bond cleavage occurring at
an unsubstituted C4 center of b-lactam rings.22
To gather additional information about this unusual N1–
C4 bond breakage, we also exposed the non-immobilized Aib-
containing tripeptide scaffold 11 and the methyl ester of its
nosylated precursor 95 to the same aqueous acidic conditions
(see ESI† for details). Interestingly, whereas the C3-amidated
b-lactam 11 was also rapidly converted into its N1–C4-opened
1
variant in quantitative yield, as confirmed by H NMR analysis
Fig. 3 Normalized HLA-A2 binding affinities for b-lactam-containing
ELA mimetics 1–3 and their opened variants 12–14 relative to that
of ELA (100) expressed as relative fluorescence intensities (RFI) from
HLA-A2-binding surface antibodies.
showing notably a characteristic downfield shift for the methylene
protons at C4,23 its C3-nosylated analogue 9 remained intact. These
observations raise the question of whether the presence of an
amide substitutent at C3 is compulsory for mediating the N1–C4
bond breakage. The significance of such a “peptide-like” factor
in favoring this unusual b-lactam ring opening will be assessed
in future studies aimed at delineating further the scope of the
reaction.
At this stage and for the sake of the continuation of this
study, other conditions of release of our pseudopeptide targets
from the solid support had thus to be found to avoid this
unprecedented b-lactam ring opening. A solution was found by
replacing the Wang resin by an amino PEGA resin bearing a
base-labile hydroxymethylbenzoic acid (HMBA) linker (Scheme
2b).24 The tert-butyl protection of the threonine (Thr) side-chain
was removed by a TFA treatment, and immediately followed
by an acetylation step. The rest of the synthesis, including the
installation of the b-lactam scaffolds 7, 8 and 11, was carried out
by standard SPPS techniques. After removal of the N-terminal
b-Ala Fmoc protecting group, pseudopeptides were released by
treating the resin for 3 h with a 0.1 M aqueous solution of NaOH,
which also enabled cleavage of the Thr acetate protecting group.
One possible explanation for the lack of binding of the b-lactam-
bearing constructs 1–3 is that their built-in conformational con-
straints in fact altered the orientation of the anchor residues within
the HLA-A2 binding pockets. In contrast, the opened b-lactam–
peptide hybrids 12–14, which possess a D-a-benzylserinate at their
core, appeared to fold well inside the HLA-A2 groove. These HLA-
A2 binding data also reveal that the substitution by a D-isoleucine
residue at P7 does not affect the binding efficiency of these opened
pseudopeptides, since all three compounds expressed affinity in
the same range as that of the ELA control. Moreover, the fact that
the best HLA-A2 ligand 14 possesses an Aib residue suggests that
the rotational restriction this residue likely imposes contributes to
a proper positioning of the pseudopeptide to best match the HLA-
A2 binding requirements. The structure–affinity relationship trend
observed with the three pseudopeptides 12–14 is in agreement
with this hypothesis, since their binding affinity decreases with an
increase of their flexibility. Thus, compound 12, which possesses
a more flexible sequence-embedded b-alanine residue in place of
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the more constrained Aib next to the D-a-benzylserine, exerts a
two-fold weaker binding affinity compared to that of 14.
Given the remarkably good binding affinity 14 for HLA-A2, we
then decided to evaluate the thermal stability of the resulting com-
plex, since it is now well established that a highly stable pMHC-I
complex is more likely to productively interact with circulating
T cells.25 Measurements of thermal stability based upon circular
dichroism-monitored protein denaturation have previously been
performed for a number of pMHC-I complexes,26 and in general,
a higher melting temperature (Tm) correlates with tighter peptide
binding. As shown in Fig. 4, 14 forms a complex with HLA-A2 that
is more stable at physiological temperatures than that formed with
the native Melan-A26–35 decapeptide (EAAGIGILTV), but some-
what less stable than that formed with ELA. The thermal stabilities
thus measured have globally a good concordance with the binding
affinities measured by the T2 surface binding assay. Notwithstand-
ing the disappointing behavior of our initially designed b-lactam-
ELA mimetics vis-a`-vis the HLA-A2 protein, the good binding
results obtained with pseudopeptides 12–14 then prompted us to
investigate their capacity to specifically activate T cells.
Fig. 5 Flow cytometry detection of secretion of the stimulatory cytokine
INF-g and the LAMP CD107a after stimulation of Melan-A-specific T
cells with EAA (native peptide used as positive control) or peptides 12–14
at 500 mM.
a CD107a response in tetramer gated T cells, albeit in weaker
amounts in comparison to the response elicited by the native
peptide EAA. Furthermore, 14 elicited an IFNg response in the
same range as that of EAA. These data clearly indicate that a
peptidomimetic structure such as 14 can specifically stimulate T
cells in a MHC-I-restricted context.
Conclusion
None of the designed b-lactam-ELA peptidomimetics displayed
HLA-A2 binding affinity, but the unprecedented acid-catalyzed
opening of their b-lactam ring led to the serendipitous discovery
of the HLA-A2 binding and T cell stimulation capacities of
their opened variants. Taken together, the results described herein
provide us with highly informative insights into the possibilities
offered to medicinal chemists to modify the structures of MHC-
I-restricted antigenic peptides with the aim of better stimulating
T cells. The simple fact that the T cell stimulating pseudopeptide
14 exhibits binding properties similar to those of ELA suggests
that the same type of molecular interactions upon binding to
the HLA-A2 protein are at play. Future work will address these
issues through X-ray crystallographic studies of the 14–HLA-A2
complex.
Fig. 4 Thermal stability measurements of complexes of HLA-A2 with
EAA, ELA, and 14.
ELA-specific T cell stimulation
Experimental
The capacity of T cells to functionally recognize pseudopeptides
12–14 in a HLA-A2-restricted context was determined by per-
forming an intracellular cytokine stain (ICS) to measure the level
of cytokines secreted by activated T cells via flow cytometry. Thus,
briefly, a Melan-A-specific T cell line was generated by stimulating
HLA-A2+ peripheral blood mononuclear cells (PBMC) with
10 mM of the native Melan-A26–35 epitope (i.e., EAA). The PBMC
were cultured for 10 days in T cell media, which resulted in
approximately 15% of the total T cell population staining with
a Melan-A-HLA-A2 tetramer (data not shown). The T cell
line was then divided into 1 ¥ 105 cell samples, pulsed with
500 mM of either EAA used as a positive control or 12–14,
washed and incubated overnight in an ICS assay. The T cell
line was then examined by flow cytometry for expression of the
lysosomal-associated membrane protein (LAMP) CD107a27 and
the stimulatory cytokine interferon-g (INFg); both produced by
the tetramer gated T cells (Fig. 5). Once again, 14 emerged as
the most interesting compound, as it was the only one to elicit
General
All reagents were purchased from either Aldrich, Acros, or Fluka.
Amino acids, Wang resin, amino-PEGA resin, and HBTU were
purchased from Novabiochem (Switzerland). Tetrahydrofuran
(THF) and diethyl ether (Et2O) were dried through PS-MD-2
columns. Extra pure dichloromethane (CH2Cl2), hexane (Hex) and
ethyl acetate (EtOAc) were bought from Sharlau. All other organic
solvents were of analytical quality and Milli-Q (Millipore) water
was used for reverse phase (RP) HPLC analyses and purifications.
All solution-phase reactions were carried out under nitrogen
atmosphere in oven- or flame-dried glassware with magnetic
stirring and dry solvents were used. Solid-phase peptide syntheses
were performed on a Chemspeed ASW100 automated synthesizer,
in double-jacket glass reactors.
RP-HPLC analyses were performed on a Thermo system using
a Chromolith performance RP-18e column (4.6 ¥ 100 mm, 5 mm)
with P1000 XR pumps. The mobile phase was composed of 0.1%
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(v/v) TFA-H2O (Solvent A) and 0.1% TFA-CH3CN (Solvent B),
unless otherwise noted. A gradient elution (0–10 min: 100% to
50% A) was applied at a flow rate of 3 mL min-1. Column effluent
was monitored by UV detection at 214 and 254 nm using a Thermo
UV 6000 LP diode array detector. Semi-preparative purifications
of peptides were performed on a Varian PrepStar system with SD-
residue, which was purified by flash chromatography, eluting with
EtOAc–Hex (2 : 1), to furnish 5 (220 mg, 85%) as a yellow oil. [a]D25
-100.6 (c 0.6 in CH2Cl2); nmax(KBr)/cm-1 3421 (NH), 1750 (C O),
1556 (NO2); dH(500 MHz, CDCl3) 2.08–2.12 (2 H, m, NCH2CH2),
3.12 (2 H, s, CH2Ph), 3.12–3.21 (2 H, m, NCH2CH2), 3.44 (1 H,
d, J 5.5, lactam CH2), 3.72 (1 H, d, J 5.5, lactam CH2), 5.00–5.05
(2 H, m, CH CH2), 5.59–5.67 (1 H, m, CH CH2), 5.89 (1 H,
s, NH), 7.23–7.29 (5 H, m, Ph), 7.70–7.78 (2 H, m, Ns), 7.88 (1
H, d, J 8.0, Ns), 8.17 (1 H, d, J 7.5, Ns); dC(125 MHz, CDCl3)
166.0, 147.4, 135.4, 134.6, 133.5, 132.9, 131.0, 130.0, 128.8, 127.7,
125.3, 117.1, 70.0, 52.8, 40.8, 40.7, 31.5; HRMS (ESI) calcd. for
C20H22N3O5S [M+H]+ 416.1280, found 416.1296.
R
1 Dynamaxꢀ pumps, using a Microsorb C18 column (21.4 mm ¥
˚
250 mm, 100 A pore size, 5 mm). The mobile phase was similar
as for the analytic system, unless otherwise notified. A gradient
elution (0–40 min: 90% to 50% A) was applied at a flow rate of
20 mL min-1. Column effluent was monitored by UV detection at
214 and 254 nm using a Varian UV-Vis Prostar 325 diode array
detector.
Purification of reaction products was carried out by flash
chromatography using silica gel 60 (230–400 mesh) and the
indicated solvents. Optical rotation were obtained using a Perkin-
N-Fmoc-Ala-(b-lactam)-b-Ala-OH (7). To a solution of 5
(0.38 mmol, 157 mg) in CH3CN (20 mL) were added K2CO3
(0.38 mmol, 52 mg) and thiophenol (PhSH, 0.75 mmol, 0.077 mL).
The mixture was allowed to stir for 3 h at room temperature. The
suspension was filtered through a celite pad and the mixture was
concentrated to dryness. The residue was used without further
purification. It was dissolved in CH2Cl2 (20 mL) at -10 ◦C. N-
Fmoc-Ala-OH (0.44 mmol, 138 mg) and N-ethoxycarbonyl-2-
ethoxy-1,2-dihydroquinoline (EEDQ, 0.67 mmol, 164 mg) were
added successively and the suspension was allowed to warm
up to room temperature overnight. The reaction mixture was
then diluted with CH2Cl2, washed with 1 M HCl, dried over
MgSO4, and evaporated to give a residue, which was purified by
flash chromatography, eluting with EtOAc–Hex (1 : 2), to furnish
the expected N-Fmoc-Ala-(b-lactam)-but-3-ene (172 mg, 87%)
1
Elmer 243B polarimeter. H NMR and 13C NMR spectra were
recorded using Bruker Avance spectrometers DPX-400 and DPX-
500. Chemical shifts (d) are given in ppm and J values are given
in Hz. Mass spectra were obtained either under electron impact
(EI, 70 eV) or electrospray ionization (ESI) conditions after direct
injection (HRMS) or using GC-MS coupling (column: fused
silica gel, 15 m, 0.25 mm, 0.25 nm phase SPB-5). Peptides were
characterized by electrospray ionization low- and high-resolution
(ESI, HRMS) obtained from the Mass Spectrometry Laboratory
at the European Institute of Chemistry and Biology (IECB),
Pessac, France.
◦
(R)-3-Benzyl-1-(but-3-enyl)-3-(2-nitrophenylsulfonamido)-azeti-
din-2-one (5). To a solution of methyl (R)-2-benzylserinate
45 (1.05 mmol, 219 mg) in CH3CN (40 mL) were added 2-
nitrobenzenesulfonyl chloride (NsCl, 2.31 mmol, 511 mg) and
KHCO3 (5.25 mmol, 525 mg). The reaction mixture was stirred
overnight under reflux. The temperature was decreased to 50 ◦C,
then homoallylic amine hydrochloride (1.57 mmol, 169 mg) and
KHCO3 (1.57 mmol, 157 mg) were added and the solution
was allowed to stir at 50 ◦C until full consumption of the N-
nosyl aziridine. The reaction mixture was concentrated and the
residue was purified by flash chromatography, eluting with EtOAc–
Hex (3 : 1), to furnish the expected methyl (R)-2-benzyl-3-(but-
3-enylamino)-2-(2-nitrophenylsulfonamido)-propanoate (340 mg,
as a white solid, mp 65–67 C. [a]2D5 -20.4 (c 1.18 in CH2Cl2);
n
max(KBr)/cm-1 3298 (NH), 1745, 1666 (C O); dH(500 MHz,
CDCl3) 1.35 (3 H, br s, CH3), 2.04 (2 H, s, NCH2CH2), 3.03–
3.09 (1 H, m, NCH2CH2), 3.13–3.20 (2 H, m, CH2Ph), 3.22–3.26
(1 H, m, NCH2CH2), 3.38 (1 H, d, J 5.2, lactam CH2), 3.54 (1
H, br s, lactam CH2), 4.19–4.21 (2 H, m, Ala CH + Fmoc CH),
4.37–4.42 (2 H, m, Fmoc CH2), 4.96–4.99 (2 H, m, CH CH2),
5.21 (1 H, br s, NH), 5.51–5.59 (1 H, m, CH CH2), 6.64 (1 H, s,
NH), 7.23–7.77 (13 H, m, Fmoc, Ph); dC(125 MHz, CDCl3) 172.4,
167.4, 155.8, 143.7, 141.3, 134.7, 134.6, 130.2, 128.5, 127.7, 127.2,
127.0, 125.0, 120.0, 116.9, 67.6, 67.1, 50.8, 47.1, 40.8, 38.8, 31.4,
18.6; HRMS (ESI) calcd. for C32H33N3O4Na [M+Na]+ 546.2369,
found 546.2365.
This compound (0.20 mmol, 103 mg) was then dissolved i◦n a
mixture of CCl4–CH3CN–H2O (2 : 2 : 3 (v/v/v), 28 mL) at 0 C.
NaIO4 (2.95 mmol, 634 mg) and RuCl3 (3.94 mmol, 1 mg) were
added. The resulting emulsion was stirred at room temperature
for 2 h, after which time the organic phase of this reaction
mixture was diluted with CH2Cl2 and separated, and the aqueous
phase was acidified using 1 M HCl. After extraction of the
aqueous phase with CH2Cl2, the combined organic layers were
evaporated to dryness to give a residue, which was purified by flash
chromatography, eluting with MeOH–CH2Cl2 (1 : 10), to furnish 7
(92 mg, 87%) as a white solid, mp 116–117 ◦C. [a]D25 -0.2 (c 1.25 in
CH3OH); (Found: C, 68.26; H, 6.01; N, 7.76. C31H31N3O6 requires
C, 68.75; H, 5.77; N, 7.76%); nmax(KBr)/cm-1 3301 (NH), 1743
(C O); dH(500 MHz, CD3OD) 1.30 (3 H, d, J 7.0, CH3), 2.08 (2
H, br s, CH2COOH), 3.10–3.16 (2 H, m, CH2Ph), 3.22–3.28 (2 H,
m, NCH2), 3.38 (1 H, d, J 4.5, lactam CH2), 3.53 (1 H, br s, lactam
CH2), 4.14 (1 H, q, J 7.0, Ala CH), 4.20 (1 H, t, J 6.7, Fmoc CH),
4.36 (2 H, d, J 6.7, Fmoc CH2), 7.22–7.30 (5 H, m, Ph), 7.31 (2
◦
73%) as a yellow solid, mp 55–56 C. [a]2D5 -23.8 (c 1.05 in
CH2Cl2); nmax(KBr)/cm-1 3339 (NH), 1738 (C O), 1540 (NO2);
dH(500 MHz; CDCl3) 1.98–2.03 (2 H, m, NCH2CH2), 2.33–2.38
(1 H, m, NCH2CH2), 2.43–2.48 (1 H, m, NCH2CH2), 3.00 (1 H, d,
J 12.5, CH2Ph), 3.07 (1 H, d, J 12.5, CH2Ph), 3.31 (1 H, d, J 13.4,
CCH2NH), 3.43 (1 H, d, J 3.4, CCH2NH), 3.61 (3 H, s, OCH3),
4.95–4.98 (2 H, m, CH CH2), 5.56–5.65 (1 H, m, CH CH2),
7.25–7.29 (5 H, m, Ph), 7.70–7.77 (2 H, m, Ns), 7.94 (1 H, d, J 7.5,
Ns), 8.17 (1 H, d, J 7.5, Ns); dC(125 MHz; CDCl3) 171.7, 147.4,
136.7, 135.9, 134.8, 133.0, 132.7, 130.3, 129.9, 128.3, 127.2, 125.3,
116.3, 68.2, 53.5, 52.5, 48.5, 41.3, 34.1; HRMS (ESI) calcd. for
C18H20N3O6S [M - C3H5]+ 406.1073, found 406.1071.
This sulfonamide (0.62 mmol, 279 mg) was dissolved in THF
◦
(15 mL) at -10 C and a 1 M solution of LiHMDS (1.55 mmol,
1.6 mL) in THF was slowly added. The resulting solution was
stirred for 20 min, quenched with a saturated solution of NaHCO3
(15 mL) and extracted with CH2Cl2 (3 ¥ 30 mL). The combined
organic layers were dried over MgSO4 and evaporated to give a
5350 | Org. Biomol. Chem., 2010, 8, 5345–5353
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H, t, J 7.5, Fmoc), 7.39 (2 H, t, J 7.5, Fmoc), 7.65 (2 H, d, J 6.0,
Fmoc), 7.79 (2 H, d, J 7.5, Fmoc); dC(125 MHz, CD3OD) 175.4,
174.7, 170.1, 158.2, 145.2, 142.6, 135.7, 131.4, 129.4, 128.8, 128.3,
128.1, 126.2, 120.9, 68.7, 67.9, 51.7, 51.5, 48.4, 39.3, 38.0, 32.7,
18.2; HRMS (ESI) calcd. for C31H31N3O6Na [M+Na]+ 564.2111,
found 564.2128.
(1 : 10), to furnish the expected N-Fmoc-Ala-(b-lactam)-prop-2-
◦
ene (101 mg, 66%) as a white solid, mp 75–76 C. [a]2D5 -19.3 (c
2.3 in CH2Cl2); (Found: C, 72.82; H, 6.14; N, 8.66. C31H31N3O4
requires C, 73.06; H, 6.13; N, 8.25%); nmax(KBr)/cm-1 3298 (NH),
1748, 1720, 1672 (C O); dH(500 MHz, CDCl3) 1.37 (3H, d, J 6.0,
CH3), 3.16 (2H, s, CH2Ph), 3.33 (1H, d, J 5.7, lactam CH2), 3.50
(d, 1H, J 4.9, lactam CH2), 3.55 (1H, dd, J 15.5 and 6.2, CH2),
3.76 (1H, dd, J 15.4 and 4.5, CH2), 4.16–4.21 (1H, m, Ala CH),
4.27–4.36 (1H, m, Fmoc CH), 4.39 (2H, d, J 3.0, Fmoc CH2), 4.90
(1H, dd, J 28.5 and 2.0, CH2), 5.00 (1H, dd, J 15.5 and 1.5, CH2),
5.28–5.35 (1H, m, CH), 5.44 (1H, d, J 7.6, NH), 6.99 (1H, s, NH),
7.22–7.32 (7H, m, Fmoc Ph), 7.39 (2H, t, J 12.0, Fmoc), 7.57 (2H,
d, J 12.0, Fmoc), 7.75 (2H, d, J 12.5, Fmoc); dC(125 MHz, CDCl3)
172.4, 167.3, 155.9, 143.7, 141.3, 134.5, 130.9, 130.2, 128.5, 127.7,
127.2, 127.0, 125.0, 120.0, 118.4, 67.7, 67.1, 50.4, 50.2, 47.1, 44.1,
38.8, 18.6; HRMS (ESI) calcd. for C27H26N2O3 [M–C4H5NO]+
426.1943, found 426.1948.
This compound (0.098 mmol, 50 mg) was then dissolved i◦n a
mixture of CCl4–CH3CN–H2O (2 : 2 : 3 (v/v/v), 14 mL) at 0 C;
NaIO4 (0.98 mmol, 210 mg) and RuCl3 (2 mmol, 0.4 mg) were
added. The resulting emulsion was stirred at room temperature
for 2 h, after which time the organic phase of this reaction mixture
was diluted with CH2Cl2 and separated, and the aqueous phase
was acidified with 1 M HCl. After extraction with CH2Cl2 and
EtOAc, the combined organic layers were evaporated to give a
residue, which was purified by flash chromatography, eluting with
MeOH–CH2Cl2 (1 : 7), to furnish 8 (35 mg, 68%) as a white solid,
mp 120–121 ◦C. [a]D25 -7.4 (c 1.5 in CH3OH); nmax(KBr)/cm-1 3391
(NH, OH), 1743, 1614 (C O). dH(500 MHz, CD3OD) 1.28 (3H,
d, J 7.0, CH3), 3.31 (2H, m, PhCH2), 3.60 (1H, d, J 5.5, lactam
CH2), 3.62–3.71 (3H, m, lactam CH2 + NCH2), 4.14 (1H, q, J 6.8,
Ala CH), 4.21 (1H, t, J 6.0, Fmoc CH), 4.31–4.38 (2H, m, Fmoc
CH2), 7.15–7.33 (7H, m, Fmoc + Ph), 7.39 (2H, t, J 7.5, Fmoc),
7.67 (2H, d, J 7.0, Fmoc), 7.80 (2H, d, J 7.5, Fmoc); dC(125 MHz,
CD3OD) 175.3, 170.0, 158.1, 145.2, 142.6, 136.7, 131.2, 129.3,
128.8, 128.2, 128.0, 126.2, 120.9, 69.1, 67.9, 53.5, 51.7, 39.4, 18.2;
HRMS (ESI) calcd. for C30H29N3O6Na [M+Na]+ 550.1954, found
550.1942.
(R)-3-Benzyl-1-(prop-2-enyl)-3-(2-nitrophenylsulfonamido)-aze-
tidin-2-one (6). To a solution of methyl (R)-2-benzylserinate
45 (1.9 mmol, 398 mg) in CH3CN (40 mL) were added NsCl
(4.18 mmol, 926 mg) and KHCO3 (9.50 mmol, 951 mg), and
the reaction mixture was stirred overnight under reflux. The
◦
temperature was decreased to 50 C, allylic amine hydrochloride
(2.87 mmol, 0.215 mL) and KHCO3 (2.87 mmol, 287 mg)
were added and the solution was allowed to stir at the same
temperature until full consumption of the N-nosyl aziridine. The
reaction mixture was concentrated and the residue was purified
by flash chromatography, eluting with Hex–EtOAc (3 : 1), to
furnish the expected methyl (R)-2-benzyl-3-(prop-2-enylamino)-2-
(2-nitrophenylsulfonamido)-propanoate (570 mg, 69%) as a yellow
◦
solid, mp 53–55 C. [a]D25 -21.8 (c 1.00 in CH2Cl2); (Found:
C, 55.72; H, 5.29; N, 9.42. C20H23N3O6S requires C, 55.42; H,
5.35; N, 9.69%); nmax(KBr)/cm-1 3340 (NH), 1738 (C O), (NO2);
dH(500 MHz, CDCl3) 2.92–2.98 (2H, m, CH2CHCH2), 3.05–3.08
(2H, m, NCH2C), 3.39 (2H, dd, J 13.8 and 30.2, CCH2Ph), 3.62
(3H, s, OCH3), 4.95–5.03 (2H, m, CH2CHCH2), 5.54–5.73 (1H,
m, CH2CHCH2), 7.14–7.26 (5H, m, Ph), 7.66–8.17 (4H, m, Ns);
dC(125 MHz, CDCl3) 171.6, 147.3, 136.2, 136.0, 134.6, 133.1,
132.7, 130.2, 129.8, 128.2, 127.1, 125.3, 115.8, 68.4, 52.9, 52.6,
51.7, 41.4; m/z (ESI) 434 (MH+, 100%).
This sulfonamide (1.31 mmol, 570 mg) was dissolved in THF
◦
(15 mL) at -10 C and a 1 M solution of LiHMDS (3.3 mmol,
3.3 mL) in THF was slowly added. The resulting solution was
stirred for 20 min, quenched with a saturated solution of NaHCO3
(15 mL) and extracted with CH2Cl2 (3 ¥ 30 mL). The combined
organic layers were dried over MgSO4 and evaporated to give a
residue, which was purified by flash chromatography, eluting with
Hex–EtOAc (2 : 1), to furnish 6 (493 mg, 94%) as a yellow oil.
[a]2D5 -88.6 (c 0.75 in CH2Cl2); nmax(KBr)/cm-1 3350 (NH), 1745
(C O), 2922 (NO2); dH(500 MHz, CDCl3) 3.12 (2H, s, CH2Ph),
3.38 (1H, d, J 5.6, lactam CH2), 3.68 (1H, d, J 5.4, lactam CH2),
3.68–3.70 (2H, m, CH2CHCH2), 5.02–5.09 (2H, m, CH2CHCH2),
5.39–5.45 (1H, m, CH2CHCH2), 5.90 (1H, s, NH), 7.22–7.27 (5H,
m, Ph), 7.69–8.17 (4H, m, Ns); dC(75 MHz, CDCl3) 166.0, 147.4,
135.4, 133.5, 133.4, 133.0, 131.0, 130.9, 130.1, 128.8, 127.7, 125.3,
118.7, 70.1, 52.3, 44.2, 40.7; m/z (ESI) 402 (MH+, 100%).
Ns-(b-Lactam)-Aib-OBn (10). To a solution of Ns-(b-lactam)-
Aib-OH 95 (1.07 mmol, 479 mg) in CH2Cl2 (15 mL) at 0 ◦C
were added oxalyl chloride (1.60 mmol, 140 mL) and a catalytic
◦
amount of DMF. The solution was stirred at 0 C for 1 h, then
at room temperature for another 1 h. The reaction mixture was
concentrated and kept under vacuum for 1 h. This freshly prepared
acyl chloride was then diluted with CH2Cl2 (15 mL) and benzyl
alcohol (3.21 mmol, 0.34 mL) and triethylamine (6.43 mmol,
N-Fmoc-Ala-(b-lactam)-Gly-OH (8). To a solution of 6
(0.30 mmol, 120 mg) in CH3CN (10 mL) were added K2CO3
(0.30 mmol, 41 mg) and PhSH (0.60 mmol, 0.06 mL). The mixture
was allowed to stir overnight at room temperature. The suspension
was filtered through a celite pad and the filtrate was concentrated
to dryness. The residue was used without further purification.
It was dissolved in CH2Cl2 (10 mL) at -10 ◦C. N-Fmoc-Ala-
OH (0.45 mmol, 140 mg) and EEDQ (0.54 mmol, 134 mg) were
successively added. The solution was then allowed to warm up
to room temperature and stirring was maintained overnight. The
reaction mixture was then diluted with CH2Cl2, washed with 1 M
HCl, dried over MgSO4 and evaporated to give a residue, which
was purified by flash chromatography, eluting with MeOH–DCM
◦
0.9 mL) were added at 0 C. The mixture was allowed to warm
up to room temperature and stirring was maintained overnight.
The mixture was then acidified with 1 M HCl, extracted with
CH2Cl2, dried over MgSO4 and evaporated to give a residue,
which was purified by flash chromatography, eluting with EtOAc–
Hex (1 : 3), to furnish 10 (506 mg, 88%) as a white solid, mp
◦
92–93 C. [a]2D5 -77.1 (c 0.99 in CH2Cl2); (Found: C, 60.01; H,
4.58; N, 7.95. C27H27N3O7S requires C, 60.32; H, 5.06; N, 7.82%);
n
max(KBr)/cm-1 3357 (NH), 1759, 1738 (C O), 1540 (NO2);
dH(500 MHz, CDCl3) 1.45 (3H, s, CH3), 1.46 (3H, s, CH3), 3.05
(1H, d, J 14.0, CHPh), 3.09 (1H, d, J 14.0, CHPh), 3.48 (1H, d,
J 5.3, lactam CH2), 3.75 (1H, d, J 5.3, lactam CH2), 5.15 (1H, d,
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J 12.3, OCHPh), 5.17 (1H, d, J 12.3, OCHPh), 5.85 (1H, s, NH),
7.17–7.34 (10H, m, 2Ph), 7.67–8.12 (4H, m, Ns); dC(125 MHz,
CDCl3) 172.2, 165.1, 147.3, 135.3, 133.5, 133.4, 132.9, 131.0, 130.0,
128.7, 128.6, 128.4, 128.3, 128.1, 127.6, 125.2, 68.1, 67.3, 59.0, 51.5,
40.4, 23.7, 23.5; m/z (ESI) 538 (MH+, 100%).
to swell for 20 min. After filtration, the N-Fmoc-Val anhydride
solution and DMAP (0.1 equiv. relatively to the anhydride) were
added, and the resin was shaken 4 h. After filtration, the resin
was successively washed twice with DMF, MeOH, and CH2Cl2
and again with DMF. The Fmoc protecting group was removed
by using twice a solution of piperidine in DMF (1 : 4, v/v) for
3 min, then for 7 min. After filtration, the resin was successively
washed as before. The SPPS was continued using N-Fmoc amino
acids (3 equiv.) in the presence of HBTU (3 equiv.) and DIEA (5
equiv.) in DMF. Each coupling reaction was performed for 45 min,
after which time the resin was washed as described above. After
final deprotection and cleavage from the resin, all peptides were
checked for purity by RP-HPLC and purified by semi-preparative
RP-HPLC.
N-Fmoc-Ala-(b-lactam)-Aib-OH (11). To a solution of 10
(0.75 mmol, 403 mg) in CH3CN (10 mL) were added K2CO3
(0.75 mmol, 103.6 mg) and PhSH (1.5 mmol, 0.15 mL). The
mixture was allowed to stir for 3 h at room temperature. The
suspension was filtered through a celite pad and the filtrate was
concentrated to give a residue, which was used without further
purification. It was dissolved in CH2Cl2 (50 mL) at -10 ◦C. N-
Fmoc-Ala-OH (0.90 mmol, 280 mg) and EEDQ (1.35 mmol,
333 mg) were successively added. The solution was then allowed
to warm up to room temperature and stirring was maintained
overnight. The reaction mixture was then diluted with CH2Cl2,
washed with 1M HCl, dried over MgSO4 and evaporated to give a
residue, which was purified by flash chromatography, eluting with
MeOH–CH2Cl2 (1 : 10), to furnish the expected N-Fmoc-Ala-(b-
Solid-phase synthesis of peptides 12–14. The H-(D)ILTV pep-
tide construct on Wang resin (300 mg, 0.054 mmol) was split into
three equal fractions that were pre-swelled in DMF and to which
were respectively added a solution of the b-lactam scaffolds 7, 8 or
11 (1.5 equiv. relatively to the resin loading) in DMF, a solution of
DIEA (2.2 equiv.) in DMF and a solution of HBTU (1.5 equiv.)
in DMF. The resins were shaken overnight and were washed as
described in the general procedure. After standard removal of the
Fmoc-protecting group and introduction of the two last amino
acids (N-Fmoc-Leu-OH and N-Boc-bAla-OH), final deprotection
and cleavage from the resins was carried out using a TFA-H2O-TIS
mixture (95 : 2.5 : 2.5, v/v/v) for 4 h. Peptides were precipitated
using cold Et2O and then lyophilized. After purification by semi-
preparative RP-HPLC, peptides 12, 13 and 14 were obtained
in correct yields: 31%, 8% and 32%, respectively (see ESI† for
characterization data).
◦
lactam)-Aib-OBn (390 mg, 80%) as a white solid, mp 98–99 C.
[a]2D5 -20.1 (c 1.01 in CH2Cl2); (Found: C, 72.73; H, 6.306; N, 6.56.
C39H39N3O6 requires C, 72.54; H, 6.09; N, 6.51%); nmax(KBr)/cm-1
3292 (NH), 1734, 1717 (C O); dH(500 MHz, CDCl3) 1.26–1.33
(3H, m, Ala CH3), 1.38 (3H, s, Aib CH3), 1.46 (3H, s, Aib CH3),
3.12–3.17 (2H, m, CH2Ph), 3.42 (1H, d, J 5.0, lactam CH2), 3.59
(1H, br d, J 3.0, lactam CH2), 4.14–4.21 (2H, m, Fmoc CH + Ala
CH), 4.35–4.41 (2H, m, Fmoc CH2), 5.09–5.14 (3H, m, OCH2Ph +
Ala NH), 6.47 (1H, s, NH), 7.22–7.25 (5H, m, Ph), 7.31–7.33 (7H,
m, Ph, Fmoc), 7.40 (2H, t, J 7.5, Fmoc), 7.57 (2H, d, J 7.0,
Fmoc), 7.77 (2H, d, J 7.5, Fmoc); dC(125 MHz, CDCl3) 172.4,
172.2, 166.6, 155.8, 143.8, 141.3, 135.4, 134.9, 130.2, 128.6, 128.4,
128.3, 128.1, 127.7, 127.2, 127.1, 125.0, 120.0, 67.2, 67.1, 65.8,
59.0, 49.7, 47.1, 38.7, 23.6, 23.55, 18.5; HRMS (ESI) calcd. for
C39H40N3O6 [M+H]+ 646.2917, found 646.2938.
Solid-phase synthesis of b-lactam peptide hybrids 1–3. These
peptidomimetics were synthesized on an amino-PEGA resin
(0.4 mmol g-1). Briefly, N-Fmoc-Gly-OH (3 equiv.) and para-
hydroxymethylbenzoic acid (3 equiv.) were successively intro-
duced, followed by the two first amino-acids (N-Fmoc-Val-OH
and N-Fmoc-Thr(tBu)-OH) as described above. The resin was
then suspended in a solution of DCM-TFA (50 : 50, v/v) for
2 h. After filtration, the resin was washed with DCM, MeOH
and again with DCM. A solution of pyridine–acetic anhydride
(1 : 1, v/v) was then added and the resin was shaken for 30 min.
After filtration, the resin was washed as described in the general
procedure. After introduction of the two next amino acids (N-
Fmoc-Leu-OH and N-Fmoc-(D)-Ile-OH), the resin was split into
three equal fractions. Again, after Fmoc protecting group removal,
solutions of 7, 8 or 11 (1.2 equiv.) in DMF (3 mL), HBTU (1.2
equiv.) and DIEA (3 equiv.) were added to each resin and let
react overnight. After washings, the two last amino acids were
successively introduced (N-Fmoc-Leu-OH and N-Fmoc-b-Ala-
OH). The last Fmoc protecting group was finally removed and
cleavage from the resins was carried out using a 0.1 M NaOH
aqueous solution for 3 h. Each resin was filtrated off and washed
once with 0.1 M NaOH. The filtrates were then acidified with
acetic acid until pH = 4 and lyophilized. After purification by
semi-preparative RP-HPLC without any acid in the eluent system
to prevent b-lactam ring opening, peptidomimetics 1–3 were
respectively obtained in 3%, 3% and 10% yields (see ESI† for
characterization data).
This benzyl ester (0.14 mmol, 90 mg) was added to a suspension
of 10% Pd/C in MeOH under H2, which was stirred for 1 h.
After filtration through a celite pad and evaporation of the
filtrates, the b-lactam 11 (77 mg, 100%) was obtained as a white
◦
solid, degradation at 101 C. [a]2D5 -0.3 (c 0.49 in CH3OH);
n
max(KBr)/cm-1 3404 (OH, NH), 1728, 1710, 1690, 1679 (C O);
dH(500 MHz, CD3OD) 1.22 (3H, s, Aib CH3), 1.32 (3H, d, J 6.8,
Ala-CH3), 1.36 (3H, s, Aib CH3), 3.16 (2H, s, CH2Ph), 3.44 (1H,
d, J 5.0, lactam CH2), 3.60 (1H, d, J 5.0, lactam CH2), 4.16 (1H,
q, J 6.8, Ala CH), 4.21 (1H, t, J 6.2, Fmoc-CH), 4.36 (2H, d,
J 6.2, Fmoc-CHCH2), 7.21–7.33 (7H, m, Fmoc+Ph), 7.39 (2H,
t, J 7.5, Fmoc), 7.66–7.67 (2H, m, Fmoc), 7.80 (2H, d, J 7.5,
Fmoc); dC(125 MHz, CD3OD) 176.1, 175.5, 168.9, 158.2, 145.2,
142.6, 136.2, 131.5, 129.4, 128.8, 128.2, 126.2, 120.9, 68.0, 67.1,
60.1, 51.7, 50.3, 48.5, 48.4, 39.4, 24.0, 18.3; HRMS (ESI) calcd.
for C32H33N3O6Na [M+Na]+ 578.2267, found 578.2258.
General procedure for solid-phase peptide synthesis (SPPS).
The synthesis of all peptides was carried out on solid phase, fol-
lowing a Fmoc strategy and standard SPPS protocols. Briefly, to a
solution of N-Fmoc-Val-OH (10 equiv. relatively to resin loading)
in CH2Cl2 (a few drops of DMF were required to ensure complete
dissolution) under N2 was added di-iso-propylcarbodiimide (DIC,
5 equiv.). The reaction mixture was stirred for 20 min at 0 ◦C. At
the same time, the resin was suspended in CH2Cl2 and allowed
5352 | Org. Biomol. Chem., 2010, 8, 5345–5353
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Peptide-MHC binding assay
4 C. Palomo, J. M. Aizpurua, A. Benito, J. I. Miranda, R. M. Fratila, C.
Matute, M. Domercq, F. Gago, S. Martin-Santamaria and A. Linden,
J. Am. Chem. Soc., 2003, 125, 16243.
5 C. Palomo, J. M. Aizpurua, E. Balentova, A. Jimenez, J. Oyarbide, R.
M. Fratila and J. I. Miranda, Org. Lett., 2007, 9, 101.
6 S. G. E. Marsh, P. Parham, L. D. Barber, The HLA Facts Book,
Academic Press, London, 2000.
7 S. Beismann-Driemeyer and R. Tampe´, Angew. Chem., Int. Ed., 2004,
43, 4014.
8 M. G. Rudolph, R. L. Stanfield and I. A. Wilson, Annu. Rev. Immunol.,
2006, 24, 419.
The mutant LCL T-lymphoblastoid hybrid cell line 174xCEM.T2
(referred to as T2 cells) is an antigen-presentation mutant line.
The cells express stable HLA-A2 molecules on their surface upon
addition of an exogenous HLA-A2-binding peptide. T2 cells were
incubated with pseudopeptides 1–3 or 12–14 in AIM V serum
media (Invitrogen Co., Carlsbad, California) at 26 ◦C for 16 h,
◦
followed by incubation at 37 C for 2 h. Quantification of stable
9 G. A. Weiss, E. J. Collins, D. N. Garboczi, D. C. Wiley and S. L.
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T cell stimulation
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Thermal stability measurement
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Acknowledgements
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The authors thank the Servier laboratories and the Socie´te´
Franc¸aise de Chimie The´rapeutique for financial support and
M.T.’s research assistantship, the Ministerio de Educacio´n y
Ciencia (MEC, Spain) (Grant CTQ2006-13891/BQU) and Go-
bierno Vasco (ETORTEK inanoGUNE IE-08/225) for financial
support and I.A.’s research assistantship. J.J.M is supported by an
NHMRC Biomedical Fellowship. Mathew Clement and Kristin
Ladell are gratefully acknowledged for their contribution to the
production of T cell lines and FACS analysis.
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