Microwave assisted solid phase synthesis of highly functionalized N-alkylated oligobenzamide α-helix mimetics

Article abstract of DOI:10.1016/j.bmc.2012.09.053

Protein-protein interactions (PPIs) mediate cellular pathways and are implicated in numerous aberrant conditions. α-Helix mimetics - small molecules that reproduce the spatial projection of key residues from an α-helix involved in a PPI - are attractive generic templates for development of screening libraries, however library syntheses of α-helix mimetics with diverse functionality are less established. This manuscript describes the automated, microwave assisted solid phase synthesis based on one such scaffold; an N-alkylated oligobenzamide.

Full text of DOI:10.1016/j.bmc.2012.09.053

Bioorganic & Medicinal Chemistry xxx (2012) xxx–xxx
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Microwave assisted solid phase synthesis of highly functionalized N-alkylated
oligobenzamide a-helix mimetics
Kérya Long a,b, Thomas A. Edwards b,c, Andrew J. Wilson a,b,
⇑
a School of Chemistry, University of Leeds, Woodhouse Lane, Leeds LS2 9JT, UK
b Astbury Centre for Structural Molecular Biology, University of Leeds, Woodhouse Lane, Leeds LS2 9JT, UK
c School of Molecular and Cellular Biology, University of Leeds, Woodhouse Lane, Leeds LS2 9JT, UK
a r t i c l e i n f o
a b s t r a c t
Article history:
Available online xxxx
Protein–protein interactions (PPIs) mediate cellular pathways and are implicated in numerous aberrant
conditions.
an -helix involved in a PPI—are attractive generic templates for development of screening libraries,
however library syntheses of -helix mimetics with diverse functionality are less established. This man-
a-Helix mimetics—small molecules that reproduce the spatial projection of key residues from
a
Keywords:
a
Aromatic oligoamide
Automated solid phase synthesis
Protein–protein interactions
Helix mimetic
uscript describes the automated, microwave assisted solid phase synthesis based on one such scaffold; an
N-alkylated oligobenzamide.
Ó 2012 Elsevier Ltd. All rights reserved.
1. Introduction
the regulation of apoptosis, a form a programmed cell death that
is crucial during the development of multicellular organisms and
Oligomers that adopt preferential conformations are named
foldamers;1–3 their stable and predictable folding into well-defined
structures have been exploited in various areas of research. Their
relative synthetic accessibility from monomer building blocks also
confers adaptability, versatility, and the possibility to modify their
inherent properties. These features make them attractive utensils
to modulate biological processes.3 Previously, we reported the
manual solid phase synthesis (SPS) of an N-alkylated aromatic oli-
goamide scaffold (Fig. 1) and demonstrated that it was suitable for
in avoiding tumourigenesis. Although small molecule inhibi-
tors19–24 and constrained helical peptides25–27 or oligomers retain-
ing the helical conformation28–30 have been used to target these
and related PPIs, designing scaffolds that allow mimicry of key
interfacial residues on an a-helix involved at a PPI interface offers
considerable potential for the elaboration of generic approaches.5
With this in mind, several such proteomimetic31 scaffolds have
been designed over the last decade; Hamilton’s terphenyl scaffold
was the first true helix mimetic, displaying IC50’s in the micromolar
range against several important PPI targets.20,31–33 Subsequently
several further scaffolds were described that were synthetically
more accessible and presented drug-like properties including
the terephthalamide,34 the 4,4-dicarboxamide,35 and the
oligo-benzoylurea36 templates amongst others.37–40 Aromatic
oligoamides41 represent an attractive class of foldamers,35,42–47 as
their constitutive units can be easily prepared and then assembled
into functional structures using modular syntheses. As they are
somewhat similar to natural peptides in that they are made of ami-
no acid subunits linked to one another by amide bonds, they are
investigated as structural peptide mimetics. Although methods
are already well-established for natural peptide synthesis, the
assembly of aromatic oligomers (using solid phase synthesis)48,49
has proven rather troublesome due to the reduced reactivity of
the monomers as compared to natural amino acids, making assem-
bly of screening libraries challenging.44,50 In the present manu-
script, we report an automated methodology for the preparation
of N-alkylated aromatic oligoamides using a state of the art micro-
wave assisted peptide synthesizer. This method provides enhanced
a
-helix mimicry through identification of low lM inhibitors of the
helix mediated p53/hDM2 interaction.4 In its extended all trans
conformation, substituents appended to the nitrogen atoms are
displayed in such a manner as to match the spatial presentation
of side chains located at the i, i + 4 and i + 7 positions of an
This subset of residues located along one helical face has been shown
to be a recurrent pattern in -helix mediated protein-protein inter-
a-helix.
a
actions (PPIs),5–7 where they account for most of the binding energy
and are referred to as ‘hot-spot’ residues.8,9 PPIs possess features
that largely differentiate them from more conventional enzyme-
substrate interactions that are traditionally targeted by medicinal
chemistry efforts.10 Such interactions include the gp41 fusion pro-
tein, responsible for the fusion process enabling HIV infection;11
the interaction between p53—a tumor suppressor involved in 50%
of all human cancers—and its negative regulators hDM2 and
hDMX;12–15 and the Bcl-2 family of interactions16–18 involved in
⇑
Corresponding author.
E-mail address: A.J.Wilson@leeds.ac.uk (A.J. Wilson).
0968-0896/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmc.2012.09.053
Please cite this article in press as: Long, K.; et al. Bioorg. Med. Chem. (2012), http://dx.doi.org/10.1016/j.bmc.2012.09.053

2
K. Long et al. / Bioorg. Med. Chem. xxx (2012) xxx–xxx
Figure 1. (a) Mimicry of the spatial presentation of residues i, i + 4, i + 7 (coloured spheres) in an
the exploitation of -helix mimicry in achieving competitive inhibition.
a-helix by our N-alkylated proteomimetic scaffold; (b) cartoon representing
a
yields and purities as well as reduced handling and coupling times.
The tolerance of our coupling method towards functionalities
encountered in proteinogenic amino acids is demonstrated, along
with some more tailored, unnatural side chains.
semi-permanent protecting group strategy, hence the prime syn-
thetic objective was to optimize amide bond formation; however
the deactivated secondary aniline is significantly less nucleophilic
than a primary amine. Coupling agents that are classically used in
peptide synthesis (HCTU, PyBOP, TFFH, EDCI, etc.) proved ineffi-
cient when applied to our scaffold under microwave heating (as
had been the case in our earlier manual SPS study).4 Different cou-
pling conditions were thus necessary and have been carefully opti-
mized. Previous studies on the solution phase synthesis of our
trimers revealed that the monomers required activation as acyl
chlorides in order for the coupling reaction to take place. Acyl chlo-
rides of our monomers could be obtained by reaction with dichlo-
rotriphenylphosphorane55 (Cl2PPh3) at elevated temperatures and
with Ghosez’s reagent56 at room temperature. We thus investi-
gated both reagents under microwave heating conditions. As the
activation using the phosphorane required heating, both the
monomer and the coupling agent had to be dissolved in the desired
solvent and placed on the synthesizer’s manifold; the acyl chloride
was then obtained in situ within the reaction vessel. Because Gho-
sez’s reagent could generate the reactive species at room temper-
ature, the acyl chloride could either be generated in situ, or
obtained by pre-activation. Due to the water sensitivity of these
coupling agents and the low reactivity of the aniline towards acyl
chlorides at room temperature, significant care was required to ex-
clude water from the pre-activation step and coupling reaction. At
first, coupling was optimized for the formation of dimers using
dichlorotriphenylphosphorane, as it is easier to handle than Gho-
sez’s reagent. Standard coupling conditions were set to allow the
study of each parameter (base, solvent, temperature and coupling
time) independently. Temperature and coupling time were set to
60 °C and 30 min respectively, and 4 equiv of monomer were used
per coupling. Different solvents were tested, with the pre-requisite
that all reagents should be soluble in the considered solvent. Single
couplings in dioxane, tetrahydrofuran or dichloromethane pro-
vided no or low conversion to dimer, whereas conversions from
55% to 60% were obtained in chloroform or DMF. In DMF, conver-
sion could be somewhat enhanced by raising the coupling temper-
ature to 90 °C. Further optimization was carried out in parallel for
both solvents. Prolonging the coupling time to 45 min or 1 h did
not improve the conversion, but reducing the coupling time to
20 min was not detrimental either. Finally, switching to a double
coupling improved conversion to the dimer from 60% with a single
coupling, to quantitative. Despite these encouraging preliminary
results, dichlorotriphenylphosphorane proved to be unreliable
from batch to batch and in subsequent studies on the functional
2. Results and discussion
Our strategy for automated solid phase peptide synthesis (SPPS)
of N-alkylated oligobenzamides centres on use of a microwave as-
sisted peptide synthesizer and the well established Fmoc strat-
egy,51 providing orthogonality with side chain protecting groups.
One of the key features of this oligoamide backbone is its synthetic
accessibility. The scaffold is based on trimers of N-alkylated p-ami-
nobenzoic acid units linked by sequentially installed amide bonds.
The preferred cis geometry of the amide bond in these molecules52
has been shown to facilitate the cyclisation of such trimers.53
2.1. Monomer synthesis
In order to develop a method amenable to the generation of a
library of diversely functionalized tribenzamides, a range of mono-
mers was prepared following the previously reported route
(Scheme 1a).4 Although our prior study confirmed that hydropho-
bic side chains are privileged for the design of inhibitors of the p53/
hDM2 interaction (and a considerable number of others),6 we
sought to incorporate a broad range of functionalities encountered
in natural amino acids into the monomer set so as to scope the
functional group tolerance of our method. In the case of amine con-
taining side chains where the starting aldehydes were not com-
mercially available, Swern oxidation54 was performed on the
parent alcohol (Scheme 1b), whilst one further side chain, contain-
ing an acidic functionality, was prepared using a different route
(Scheme 1c).
2.2. Solid phase peptide synthesis
The trimeric oligoamides are built from sequential coupling of
N-alkylated p-aminobenzoic acid monomers via SPS using the well
established Fmoc strategy, widely employed in natural peptide
synthesis. The previously reported SPS on this scaffold4 was
adapted for a CEM LibertyÒ automated microwave assisted peptide
synthesizer. Our experiments focused on the use of Wang resin
preloaded with Gly as the solid support, although it should be
noted that we also performed a limited number of experiments
using unfunctionalized Wang resin. The same protection strategy
as for
a-amino acid coupling was sought for our scaffold so as to
group tolerance of the method. Further studies on Ghosez’s
a-chlo-
avoid the development of an entirely new permanent and
roenamine were then performed in chloroform; couplings proved
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K. Long et al. / Bioorg. Med. Chem. xxx (2012) xxx–xxx
3
F
N
Boc
a
g
d
j
Fmoc
(a)
(b)
CF3
NH2
RCHO
Pic.BH3
MeOH
HN
R
N
R
Fmoc-Cl
CHCl3
R =
S
b
c
e
f
h
i
k
l
70-90%
75-90%
O
OH
O
OH
O
OH
O
Cl
1
2
3
Fmoc
1. (COCl)2, CH2Cl2,
-78 °C
N
R
O
1. Pic.BH3, MeOH
2. DMSO, -78 °C
m
R =
R
O
HO
R
O
N
H
O
2. Fmoc-Cl, CHCl3
60-75% (2 steps)
3. NEt3, r.t.
70-95%
4
O
O
OH
3
Fmoc
O
N
O
(c)
(d)
O
1. Pic.BH3, MeOH
AgNO3
NaOAc
O
O
O
N+
Br
-O
2. Fmoc-Cl, CHCl3
60-75% (2 steps)
DMSO, r.t.
60%
MeCN, r.t.
95%
O
O
O
5
6
O
OH
3n
Fmoc
Fmoc
N
R
N
Rn
1. pre-activation
HN
R3
O
Cl
O
O
OH
Fmoc
N
R
4. coupling
H
MocF
R
R2
N
O
N
N
R
5. cleavage
O
Cl
Fmoc
N
H
2. loading
O
R1
O
O
N
O
n
n
N
H
N
H
Cl
O
3. deprotection
N
O
NHFmoc
N
H
7-18
COOH
O
Ghosez's
reagent
Fmoc-Gly loaded
Wang resin
Scheme 1. (a) Synthesis of Fmoc protected N-alkylated p-aminobenzoic acid monomers; (b) preliminary Swern oxidation of basic side chain; (c) synthetic route for the
preparation of the aldehyde precursor of the acidic monomer and (d) outline of procedure used for solid phase synthesis of N-alkylated aromatic oligoamide -helix mimetics.
a
1. Pre-activation: monomer (5 equiv to resin, 0.5 mmol) in anhydrous CHCl3 (5.5 mL), pre-activated with Ghosez’s reagent (0.95 equiv to the monomer, 0.48 mmol) for a
minimum of 1 h; 2. Loading: resin (0.1 mmol, 127 mg) swelled in 5 mL DMF, monomer loaded using a double coupling method (2 Â 20 min, 60 °C); 3. Coupling: same method
as for the loading; 4. Deprotection: standard microwave method used (two cycles, 75 °C, 3 min, cf. experimental section); 5. Manual cleavage: DCM-TFA (1–1, 0.7 mL),
2 Â 20 min, r.t.
to be much more reproducible than with dichlorotriphenylphos-
phorane and it was therefore established as the reagent of choice
for these monomers. Further optimization of the coupling time,
temperature, number of equivalents of monomer to the resin and
solvent led to complete conversion to trimers in good yields
(Scheme 1d). Because double couplings are required, using 4 equiv
of monomer per coupling means that a large amount of monomer
is consumed. After further optimization, the minimum number of
equivalents of monomer to the resin was identified as 2.5, and
the coupling time was maintained at 20 min. Standard microwave
assisted deprotection was used and didn’t require any optimiza-
tion. Including the wash cycles, this new method therefore yields
trimers in 4 h, which is a dramatic improvement on the 4 days pre-
viously required using our manual SPS method.4 Although cleavage
from the resin can be performed on the synthesizer, this would re-
quire additional and extensive washes of the reactor. We therefore
chose to carry out the cleavages manually (cf. Experimental sec-
tion), which also allows to optimize the productivity of the
synthesizer.
For side chains containing a reactive functionality that needs to
be protected during the coupling, our strategy was to use acid-
labile protecting groups. These protecting groups would thus
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4
K. Long et al. / Bioorg. Med. Chem. xxx (2012) xxx–xxx
remain untouched during the basic Fmoc deprotection step, but
would be removed during acidic cleavage from the resin. During
coupling however, one equivalent of hydrochloric acid is generated
that can potentially deprotect the side chains. To quench this
acidity, a base needed to be present during coupling and the base
tolerance of our method was thus assessed. Firstly, N-methyl imid-
azole was tested, but this base presented the inconvenience of
being highly hygroscopic, thus requiring distillation and storage
under strictly anhydrous conditions prior to use; the reagent was
therefore considered incompatible with the microwave synthe-
consistent data, we performed the majority of experiments using
the same monomers at the N- and C-termini for each trimer,
respectively the benzyl and isobutyl monomers. The optimized
coupling conditions afforded the desired trimers with good conver-
sion and yield (Table 1) and the crude material was in most cases
more than 90% pure as observed on the LC–MS spectrum. When
the purity of the crude material was judged unsatisfactory, purifi-
cation was performed. Several methods were used including col-
umn chromatography, preparative TLC, preparative HPLC, mass
directed preparative HPLC or precipitation, which improved purity
but considerably affected the isolated yields of pure compounds. In
the range of monomers that were submitted to coupling, the indole
containing monomer was problematic, in agreement with previous
observations.4 LC–MS analysis of the product indicated that the
coupling occurs, but that the indole functionality is lost, presum-
ably due to acid catalyzed elimination of the Boc-protecting group
during the TFA-cleavage step, followed by cascade elimination of
the indole ring (Scheme 2). This issue was addressed by using
milder conditions for the cleavage step. The cleaving cocktail was
diluted from a 1–1 mixture of dichloromethane-TFA down to a
10% TFA in dichloromethane solution. For side chains containing
a functional group that is prone to elimination, it might be neces-
sary to adjust cleaving mixture and cleaving time to achieve neat
cleavage from the resin as well as complete deprotection whilst
preserving the side chain. The small library demonstrates the toler-
ance of our method towards: aliphatic chains and hence analogues
sizer. For this reason,
a solid inorganic base seemed more
reasonable and we tested couplings in the presence of sodium
bicarbonate, which was introduced in excess at the same time as
the resin. Using a solid base presents the additional advantage that
it is retained in the reaction vessel after each round of filtered
washes following couplings and is therefore introduced only once,
again minimizing entry points for water. The bicarbonate is readily
removed by rinsing the resin with water prior to the cleavage from
the resin.
We then sought to test thoroughly the functional group toler-
ance of the method by assembly of a small library. These studies
were carried out in chloroform, as it is much less hygroscopic than
DMF. The present library was designed to assess the ability of our
monomers to be coupled to another monomer already attached to
the resin to form a dimer, as well as the ability of these dimers to
couple to another activated monomer to provide trimers. To obtain
Table 1
Library of N-alkylated tribenzamides with the side chains sequence
Trimer number
R3
R2
R1
Purity
90%
Yield
85%
b
a
d
7
a
a
d
d
d
d
8
95%
100%
ꢀ95%
ꢀ90%
100%
9
ꢀ85%
90%
e
i
a
a
10
11
CF3
Cl
d
d
h
f
95%
a
a
12
13
14
95%a (crude 80%)
80%
7%a
k
l
d
d
f
ꢀ85%
22%b
S
a
a
95%b (crude 75%)
OH
j
15
95%c
10%
Cl
N
H
a
a
m
a
16
17
NH2
OH
90%a (crude 80%)
95%
ꢀ6%a
d
n
100%
O
c
CF3
h
h
18
95%
100%
Cl
Unless otherwise specified, the reported purity and yield correspond to crude compounds.
a
Purified by precipitation.
Purified by MS-directed preparative HPLC.
Purified by column chromatography.
b
c
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K. Long et al. / Bioorg. Med. Chem. xxx (2012) xxx–xxx
5
H
H
O
NH
O
NH
O
N
O
N
N
N
CO2
+
O
O
O
O
N
Scheme 2. Cascade elimination of the indole functionality during acidic cleavage from the resin.
H2N
HN
HN
O
N
F
O
N
O
N
O
N
F
n
O
N
O
N
O
N
F
O
O
NH
O
NH
OH
O
NH
OH
OH
O
O
21
n = 3
19
20
22 n = 8
Figure 2. Compatibility of the method with natural amino acids both at the N- and C-terminus (compounds 19 and 20) and extension of the method to the synthesis of longer
oligomers (21 and 22).
of Ala, Val, Leu, Ile residues (trimers 7 and 18, Table 1), aromatic
analogues of Phe and halogenated aromatics (8–12), sulfur con-
taining side chains (13), tyrosine analogues (14), tryptophan ana-
logues (15), basic and acidic chains analogues of Lys, Asp and Glu
(16 and 17). We also investigated the incorporation of an aliphatic
sulfur containing side chain (i.e. a true Met analogue), but the syn-
thesis and purification of the corresponding monomer was prob-
lematic, and its limited solubility contributed to inefficient
coupling. In the case of an imidazole bearing monomer (i.e. a His
analogue), two protecting groups were considered, (Boc and Trt),
but in both cases the Fmoc protection step proved cumbersome
and fully protected monomer was not successfully isolated. Func-
tional tolerance towards a few other remaining proteinogenic
functionalities still remains to be demonstrated; notably proline,
cysteine, arginine and asparagine/glutamine, however the method
we have developed is sufficiently powerful for exploration of SAR
space. In addition, we have shown that amino acids can be coupled
to the N-terminus of the oligobenzamide; Val has been coupled to
the N-terminus (i.e. 19) rendering the approach entirely compati-
ble with standard oligopeptide synthesis (noting that amino acid
loaded resins demonstrate this feature for the C-terminus Gly for
7–18 and Ile for 20, Fig. 2).
icry of longer helical domains. The optimized coupling conditions
described in Scheme 1d have been applied in this study, using up
to ten rounds of deprotection-coupling cycles. Test cleavages on
small aliquots of resin were performed at different stages. To date,
pentamers for example 21 have been made with purity confirmed
by NMR as well as HRMS (cf. Supplementary data) and we have
been able to extend the synthesis as far as the decamer (Fig. 2,
compound 22).
3. Conclusions
We have established a robust automated SPS methodology for
the synthesis of N-alkylated oligobenzamides using a microwave
assisted peptide synthesizer, allowing facile library generation of
diversely functionalized compounds. A wide range of monomers
has been prepared and incorporated into trimers to demonstrate
the reproducibility and functional tolerance of our method. Side
chains compatible with this method cover the whole range of
hydrophobic side chains, which are the most commonly involved
in PPI interfaces. Combinations of side chains give access to a wide
variety of oligomers, and thus allow mimicry of numerous poten-
tial helix surfaces. We have also reported the synthesis of oligo-
mers up to the decamer, which broadens the range of possible
applications for both this method and the present scaffold. Our
We have also investigated the robustness of our method to-
wards synthesizing longer oligomers, which might allow the mim-
Please cite this article in press as: Long, K.; et al. Bioorg. Med. Chem. (2012), http://dx.doi.org/10.1016/j.bmc.2012.09.053

6
K. Long et al. / Bioorg. Med. Chem. xxx (2012) xxx–xxx
ongoing efforts are directed towards the synthesis of a library and
from the CH2 group are numbered FHa, the neighbouring CH is
its screening against a diverse range of
a-helix mediated PPIs.
FHb, and the aromatic protons are FH2 to FH5.
4. Experimental section
1-α-CO2H
OH
2-HAr3
1-Hα
HN
2-HAr2
4.1. General considerations
2-HAr4
O
2-Hα
3-Hγ
3-Hβ
1-NH
All reagents were obtained from Aldrich, Alfa Aesar, Acros or
Fluka and used without further purification. All solvents used were
HPLC grade. Analytical TLC was performed using 0.2 mm silica gel
60 F254 pre-coated aluminium sheets (Merck) and visualised using
UV irradiation or, in the case of amine intermediates, by staining
with a ninhydrin solution. Flash column chromatography was car-
ried out on silica gel 60 (35–70 micron particles, FluoroChem). Sol-
vent ratios are described where appropriate. Solvents were
removed under reduced pressure using a Büchi rotary evaporator
at diaphragm pump pressure. Samples were freed of remaining
traces of solvents under high vacuum. 1H and 13C NMR spectra
were measured on a Bruker DPX300 or a Bruker Avance 500 spec-
trometer using an internal deuterium lock. Chemical shifts are re-
ported in parts per million (ppm) downfield from TMS in d units
and coupling constants are given in hertz (Hz). Coupling constants
are reported to the nearest 0.1 Hz. TMS is defined as 0 ppm for 1H
NMR spectra and the centre line of the triplet of CDCl3 was defined
as 77.10 ppm for 13C NMR spectra. When describing 1H NMR data
the following abbreviations are used; s = singlet, d = doublet,
t = triplet, q = quartet, m = multiplet. Melting points were deter-
mined using a Griffin D5 variable temperature apparatus and are
uncorrected. Microanalyses were obtained on a Carlo Erba Elemen-
tal Analyser MOD 1106 instrument, found composition is reported
to the nearest 0.05%. Infrared spectra were recorded on a Perkin–
Elmer FTIR spectrometer and samples analysed as solids (unless
stated). Mass spectra (HRMS) were recorded in house using a
Micromass GCT Premier, using electron impact ionisation (EI) or
a Bruker Daltonics micrOTOF, using electron spray ionisation
(ES). LC–MS experiments were run on a Waters Micromass ZQ
spectrometer, samples ionised by electrospray and analysed by a
time-of-flight mass spectrometer, or a Bruker Daltronics HCT Ul-
traTM series spectrometer, samples ionised by electrospray. All
experiments were run through a C18 column on an acetonitrile–
water gradient (typically 0–95% acetonitrile over 3 min). A repre-
sentative example of each synthetic procedure is provided with
all examples described in the ESI.
N
3-Hα
N
1-H3
3-Hγ
1-H2
O
2-H3
2-H2
O
O
3-H3
3-H2
4-Hα
HN
4-NH
4-CO2H
CO2H
4.3. General procedure for reductive amination
To a stirred solution of primary aniline (1 equiv, 36.4 mmol) and
aldehyde (1.05 equiv, 38.2 mmol) in methanol (120 mL) under an
atmosphere of nitrogen, was added borane–picoline57 (1.2 equiv,
43.7 mmol). The reaction mixture was stirred at room temperature
for 4–36 h, until TLC indicated reaction completion. Concentration
and either direct purification by column chromatography or pre-
cipitation gave the target material which was dried under vacuum.
4.4. Solid phase synthesis experimental
127 mg of Fmoc-Gly-Wang resin (0.79 mmol gÀ1
,
100–
200 mesh; carrier: polystyrene, crosslinked with 1% DVB) from
Novabiochem, was used throughout. All solvents used were HPLC
grade. 1-Chloro-N,N,2-trimethyl-1-propenylamine (Ghosez’s re-
agent) was purchased from Sigma–Aldrich. The reagent was stored
at À20 °C and the bottle was used under a nitrogen atmosphere to
prevent degradation. For each coupling reaction, 5 equiv (to the re-
sin, spread over two couplings) of fully protected monomer and
0.95 equiv of Ghosez’s reagent to the monomer were used. The re-
sin was swelled in DMF for at least 15 min prior to coupling. The
reactions were all carried out on a CEM LibertyÒ microwave as-
sisted automated peptide synthesiser. The software interface al-
lows the creation of personalised loading, deprotection and
coupling cycles (order, number and volumes of additions of mono-
mer, DMF and CH2Cl2 washes), as well as the setting of parameters
for the microwave method (temperature, temperature gradient,
heating time, power, etc.). Up to 12 methods can be run in daisy-
chain on the synthesizer with no external intervention.
4.2. Naming and numbering of N-alkylated aromatic
oligoamides
To simplify the numbering and NMR assignment of our trimers,
we have devised a sequential nomenclature (based upon conven-
tional peptide naming), where each monomer building block is
considered separately. The monomers are numbered from 1 up-
wards starting from the N-terminus, (and for the current series gly-
cine is numbered 4). Within each monomer, the numbering is the
same: carbons from the aminobenzoic acid are numbered using the
standard system (the aromatic carbon bearing the carboxylic acid
is C1, the one bearing the amine is C4). Then, the lateral chain is
4.5. General coupling
For each coupling, the volume of pre-activated monomer solu-
tion added to the microwave reactor was set to 2.5 mL. Each fully
protected monomer was thus dissolved in anhydrous chloroform
(ꢀ5.5 mL), in VWRÒ tubes that fit on the CEMÒ synthesizer and
Ghosez’s reagent added. This mixture was then allowed to incubate
at room temperature for at least 1 h to allow acyl chloride forma-
tion. Each coupling cycle was carried out at 60 °C for 20 min.
numbered: the carbon attached to the nitrogen is the Ca, and the
numbering of the aliphatic part of the side chain continues with
Cb, etc. In the case of aromatic side chains, the aromatic carbons
are numbered CAr1, CAr2, etc. The numbering of the protons is
based on the carbon numbering. To differentiate each individual
carbon/proton, the monomer number is added as a prefix to the
carbon/proton number. The same nomenclature is applied to
the Fmoc-protected monomers, and the protons corresponding to
the Fmoc system are differentiated by the prefix F. The protons
4.6. General Fmoc deprotection
After each coupling reaction, the contents of the microwave
reactor were automatically drained and the resin washed three
times with CH2Cl2 and three times with DMF (7 mL each).
Please cite this article in press as: Long, K.; et al. Bioorg. Med. Chem. (2012), http://dx.doi.org/10.1016/j.bmc.2012.09.053

K. Long et al. / Bioorg. Med. Chem. xxx (2012) xxx–xxx
7
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Following the final DMF wash, 6 mL of 25% piperidine in DMF was
added to the reaction vessel and two deprotection cycles were car-
ried out (microwave heating to 75 °C for 3 min). The reactor was
then drained and the resin washed with neat DMF three times
(5 mL each). Finally, the deprotected resin was washed twice with
CH2Cl2 (5 mL each).
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Supplementary data
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