Solid-phase methodology for synthesis of O-alkylated aromatic oligoamide inhibitors of α-helix-mediated protein-protein interactions

Article abstract of DOI:10.1002/chem.201204098

Rapid access to rigid rods: A method is described for the synthesis of 3-O-alkylated aromatic oligobenzamide foldamers that could be used for assembly of libraries of α-helix mimetic inhibitors of protein-protein interactions (see scheme; Fmoc=9-fluorenyl

Full text of DOI:10.1002/chem.201204098

DOI: 10.1002/chem.201204098
Solid-Phase Methodology for Synthesis of O-Alkylated Aromatic Oligoamide
Inhibitors of a-Helix-Mediated Protein–Protein Interactions
Natasha S. Murphy,[a, b] Panchami Prabhakaran,[a, b] Valeria Azzarito,[a, b]
Jeffrey P. Plante,[a] Michaele J. Hardie,[a] Colin A. Kilner,[a] Stuart L. Warriner,[a, b] and
Andrew J. Wilson*[a, b]
Dedicated to Prof. Andrew D. Hamilton on the occasion of his 60th birthday
A major effort in modern bio-organic chemistry focuses
on the design, synthesis and structural characterisation of
foldamers:[1] non-natural oligomers that adopt well-defined
secondary, tertiary and quaternary structures.[2–5] One ulti-
mate objective of such studies is to recapitulate the func-
tional behaviour of biomacromolecules.[6] Particular empha-
sis has been placed on inhibitors[7–12] of a-helix-mediated[13]
protein–protein interactions[14]—an endeavour that in its
own right represents a major challenge.[15,16] The develop-
ment of synthetic methodologies that allow access to small-
to-medium sized libraries of foldamers incorporating diverse
side chains, represents the cornerstone upon which such
studies are pursued. In this regard, it is noteworthy that the
Figure 1. a) a-Helix (taken from protein database ID: 1YCR) with i, i+4
most robust methodology exists for peptoids,[17] b-peptides[18]
and i+7 side chains highlighted; b) chemical structure of 3-O-alkylated
and more recently oligoureas;[19] templates that have seen
oligoamide helix mimetic c) energy minimised structure of a helix mimet-
the most significant use in a biological context.[7,20] We[21–24]
and others[25–29] have recently reported on the use of aromat-
ic oligoamides[5] as potential a-helix mimetics.[30,31] Rather
than topographical mimicry of the a-helix (as is the case for
b,[32] a/b[7,9,12] and other foldamers[8]), these compounds
mimic an a-helix by presenting key side chains from a rod-
like template in a spatial orientation that matches that of
the a-helix (Figure 1).[33] Although solution methods for as-
sembly of very large[34] and long aromatic oligoamides[35]
have been described, a significant advance in this area
would be the ready availability of solid-phase methods toler-
ant to a diverse array of side chains; this would facilitate li-
ic with R3 =R2 =R3 =iPr; d) idealized a-helix superimposed onto mini-
mised aromatic oligoamide.
brary generation and ease of purification. Other than our
own preliminary report on N-alkylated aromatic oligoa-
mides,[22] only a limited number of reports have been descri-
bed on the synthesis of benzanilides[36,37] and related aromat-
ic oligoamides[38,39] that meet the criteria outlined above.
Herein, we describe such a method that can be used for syn-
thesis of 3-O-alkylated aromatic oligobenzamides. Using mi-
crowave irradiation, trimers can be assembled on a solid
support in 2.5 h in sufficient purity for screening purposes.
The methodology is tolerant to a large and diverse collec-
tion of monomers and amenable to synthesis of significantly
longer oligomers. The approach and our observations in de-
veloping it should have general applicability for synthesis of
aromatic oligoamide foldamers.
[a] N. S. Murphy, Dr. P. Prabhakaran, V. Azzarito, Dr. J. P. Plante,
Prof. M. J. Hardie, C. A. Kilner, Dr. S. L. Warriner, Prof. A. J. Wilson
School of Chemistry, University of Leeds
Woodhouse Lane, Leeds, LS29 JT (UK)
Fax : (+44)1133431409
In developing our approach we sought to avoid imple-
mentation of novel protecting group chemistries and con-
strained ourselves to use of Fmoc (Fmoc=fluorenylme-
thyoxycarbonyl) as a semi-permanent protecting group and
permanent acid labile protecting groups on the side chains.
On this basis a four-step synthesis of a broad array of mono-
mers 1a–r was developed (Scheme 1) exploiting either alky-
lation of the intermediate phenol at the diversification point
using alkyl halides or alcohols under Mitsunobu conditions
E-mail: A.J.Wilson@leeds.ac.uk
[b] N. S. Murphy, Dr. P. Prabhakaran, V. Azzarito, Dr. S. L. Warriner,
Prof. A. J. Wilson
Astbury Centre for Structural Molecular Biology,
University of Leeds, Woodhouse Lane, Leeds, LS2 9JT (UK)
Supporting information for this article is available on the WWW
under http://dx.doi.org/10.1002/chem.201204098.
Re-use of this article is permitted in accordance with the Terms and
Conditions set out at http://onlinelibrary.wiley.com/journal/10.1002/
TRENNUNG(ISSN)1521-3765/homepage/2111_onlineopen.html.
ACHTUNG-
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Chem. Eur. J. 2013, 19, 5546 – 5550

COMMUNICATION
a ketene intermediate. We also synthesised an Fmoc-pro-
tected monomer mimicking glycine; our design positions the
phenolic oxygen as the atom mimicking the a position of
the amino acid within the helix and therefore this represents
a poor mimic of glycine and would require protection
during synthesis. We therefore protected commercially avail-
able 3-methyl-4-aminobenzoic acid with Fmoc to furnish the
glycine mimic 1s.
A general outline of the solid-phase synthesis (SPS)
method is illustrated in Scheme 2. In terms of developing
this methodology, the amide bond forming reaction is chal-
Scheme 1. Synthesis of Fmoc-protected monomers for SPS.
(i.e., 2 to 3 in Scheme 1). As is shown, a full array of peptide
based side chains covering the entirety of functionality
found in native peptide side chains is accessible (with the
exception of cysteine, arginine and histidine), whilst several
non-natural side chains and chiral side chains can also be in-
corporated. There are several noteworthy points as follows:
1) for benzylic side chains (e.g., 3e–k) it was necessary to
use tin(II) chloride for nitro group reduction (3 to 4 in
Scheme 1) as opposed to palladium on charcoal, so as to
avoid cleavage of the side chain, 2) for the hydrolysis step
(4 to 5 in Scheme 1), care was be required to avoid cleavage
of the side chain, requiring use of lithium hydroxide and
mild conditions (e.g., room temperature). Cleavage of the
side chain by elimination of the phenol occured under forc-
ing conditions—a feature that prevented us from obtaining
a monomer mimicking histidine. Additionally, for side
chains possessing an electron-withdrawing group g to the
phenol, E1CB was promoted (not shown). Finally, for the
tert-butyl ester side chain 1m we observed deprotection of
the tert-butyl group using sodium hydroxide presumably via
Scheme 2. Solid-phase synthesis protocol.
lenging as the substrate is a deactivated aniline. We selected
acid labile Wang resin for these studies and achieved resin
loading using thionyl chloride or Ghosezꢁs reagent either di-
rectly to the resin or to glycine loaded resin. We attempted
a series of screening experiments using the isopropyl mono-
mer 1a, chloroform as solvent and microwave assistance
(using a CEM peptide synthesiser) to identify suitable cou-
pling regents. From our screening experiments, only activat-
ing agents that form acid chlorides proved successful (i.e.,
thionyl chloride and Ghosezꢁs reagent). This was not entire-
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5547

A. J. Wilson et al.
ly surprising given that our prior studies,[21,22,24] in the ab-
sence of microwave, indicated to us that strongly activated
acids (e.g., acid chlorides) would be necessary to mediate
amide bond formation. We then proceeded to develop an
optimised method by attempting oligomer synthesis and
broadening the monomer set. Unfortunately, the majority of
monomers were found to be poorly soluble in chloroform,
so we resorted to the use of DMF. Using in situ formation
of the acid chloride from Ghosezꢁs reagent and microwave
irradiation, no anilide formation was observed. Similarly,
with pre-activation or isolation of the acid chloride followed
by microwave assistance, we were unable to effect the ani-
lide formation. An explanation for these results was ob-
tained from LC-MS analysis of the reaction mixture, which
revealed capping of the immobilised aniline by both DMF
and Ghosezꢁs reagent to give a stable amidine (Figure 2).
alised monomers that tended not to precipitate upon reac-
tion with thinoyl chloride it was preferable to use the in situ
method (these highly functionalised monomers tended to be
less stable as acid chlorides). We found that for direct addi-
tion of acid chlorides, a single cycle of coupling at 508C for
30 min in the absence of base was sufficient to achieve high
conversion, however, for longer oligomers we used double
couplings. For Fmoc removal no special optimisations were
required and 20% piperidine in NMP was sufficient
(Scheme 2). Care was required with the global deprotection
reaction, which we performed off-line from the synthesiser;
certain side chains (see below) were found to be susceptible
to cleavage by elimination with the indole side chain a nota-
ble example, thus this stage of the procedure requires care-
ful monitoring.
With these observations and optimisations established, we
demonstrated the versatility of the method by synthesising a
sufficient number of trimers 7–24 (Table 1) so as to demon-
Table 1. Oligomers synthesised.[a]
Trimer
R1
R2
R3
Final purity [%]
Yield [%]
Precipitate
HPLC
7
8
9
a
e
e
a
e
g
e
a
a
a
a
a
a
a
a
a
a
a
a
e
h
h
i
j
g
s
a
e
a
e
a
a
a
a
a
a
a
a
a
a
a
a
a
a
95
95
90
90
99
99
95
99
99
73
92
71
82
86
99
78
64
73
79
N/A
N/A
69
–
–
–
–
–
–
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
–
–
32
35
21
N/A
N/A
22
19
22
32
28
17
d[b]
f[b]
k
99
N/A
N/A
99
99
99
90
99
99
Figure 2. Mechanism for capping of anilines during SPS via Vilsmeier in-
termediates.
l
m[b]
n[b]
o[c]
p
–
69
–
This capping reaction, which is observed even when the acid
chloride is used directly, indicates that the solvent reversibly
reacts with the acid chloride to generate the Vilsmeier inter-
mediate, which can then cap the aniline. We did not observe
this behaviour for synthesis of N-alkylated aromatic oligoa-
mides—one explanation is that capping of an N-alkylated
aniline results in an unstable intermediate, which cannot
lose a proton to form the amidine (Figure 2). With these re-
sults in hand we performed a solvent screen to identify
more polar solvents, which would not lead to such side reac-
tions.
q[a]
r[b]
–
Oligomer
Sequence
Final purity [%]
Yield [%]
Precipitate
HPLC
25
26
27
28
acbb(G)
acca(G)
abd(I)
95
95
99
95
91
93
–
–
–
35
–
aaaaaa(G)
87
[a] For an explanation of the R groups, see Scheme 1. [b] By using in situ
activation of monomer with thionyl chloride. [c] By using in situ activa-
tion of monomer with Ghosezꢁs reagent.
From our solvent screen we identified N-methylpyrrolidi-
none (NMP) as a suitable solvent with which to perform
solid-phase coupling to give the aromatic benzamides. After
further screening and optimisation we established that direct
use of the acid chlorides obtained from thionyl chloride or
pre-activation using Ghosezꢁs reagent prior to coupling in
the microwave synthesiser could effect coupling in high
yield (Scheme 2). The use of acid chlorides was preferable
for the majority of alkyl/aryl Fmoc-protected monomers 1
as these could be precipitated and stored for at least one
month with no decomposition. For the more highly function-
strate that each monomer in the set could couple and be
coupled to. In addition we also illustrated that amino acids
(other than glycine could be appended to the C-terminus 27
(through use of different amino acid loaded Wang resins)
and to the N-terminus. The only problematic monomer was
1k with the resulting oligomer undergoing cleavage of the
benzylic phenol under the standard deprotection conditions
required to cleave the phenolic tert-butyl protecting group.
In addition, whilst we observed reasonable coupling with
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Aromatic Oligoamide Inhibitors
COMMUNICATION
To illustrate the potential for longer oligomers to act as
mimics of extended helices, we performed molecular model-
ling on the hexamer 28 as is illustrated in Figure 3b (see the
Supporting Information for more details). With all side
chains located on one face, hexamer 28 can project side
chains in such an orientation so as to mimic five consecutive
side chains along an a-helical surface, whilst rotation around
À
the terminal Ar CO bond permits the hexamer 28 to mimic
a sixth chain. This demonstrates that such oligomers could
find use in the inhibition of more extended a-helix mediated
protein–protein interactions. Finally we obtained several
crystal structures of a representative trimer 29 (described
previously)[21,24] comprising isopropyl monomers and with a
C-terminal methyl ester and N-terminal nitro group (Fig-
ure 3c–e). These exemplify the salient points of our previ-
ously published analysis of the conformational preference of
these oligomers,[21,23,40] that is, they adopt a rod-like confor-
À
mation with free rotation around the Ar CO axes and rota-
À
tion around the Ar NH axes restricted through S(5) intra-
molecular hydrogen bonding. Critically, side chains adopt
both syn and anti orientations with respect to one another,
whilst variations along the backbone permit the side chains
to project in subtly different orientations.[23]
In conclusion, we have developed a method for synthesis
of aromatic oligoamides containing a diverse array of natu-
ral and non-natural amino acid side chains using a micro-
wave-assisted automated peptide synthesiser. A four-step
monomer synthesis allows generation of Fmoc-protected
building blocks for SPS with trimers accessible in 2.5 h in
sufficient purity for screening. These foldamers represent
good templates to act as mimetics of the a-helix and hence
as inhibitors of protein–protein interactions. Our method
represents a useful tool with which to obtain protein–protein
interaction inhibitors by sequence based design[9] and for li-
brary generation to screen against unknown targets. Our
own future efforts in this area will describe detailed studies
on aromatic oligoamide helix mimetics targeted against a
broad range of protein–protein interactions.
Figure 3. Characterisation of O-alkylated aromatic oligoamides a)
1H NMR spectrum (500 MHz, [D6]DMSO, 608C) of a hexamer b) molec-
ular model of a hexamer with all side chains on one face together with
an overlay onto an a-helix illustrating mimicry of more extended surfaces
c) solid-state structure I of trimer 29 (from THF) d) solid-state structure
II of trimer 29 (from chloroform/ethanol) e) solid-state structure III of
trimer 29 (from chloroform/cyclohexane).
Experimental Section
General procedure for oligomer formation—single coupling: Fmoc-pro-
tected pre-loaded Wang resin (127 mg, 0.1 mmol, 1 equiv) was loaded
onto a CEM microwave peptide synthesiser after being swelled for a
total of 30 min in NMP and CH2Cl2 solutions. A series of washes (3ꢂ
NMP), deprotection (2ꢂ20% Piperidine/NMP, total of 3.5 min at 758C)
and further washes (5ꢂNMP) prepared the resin for coupling. Fmoc pro-
tected acyl chloride of 1a–s (0.4 mmol, 4 equiv) obtained by pre-activa-
tion or prepared separately was dissolved in NMP (2.5 mL), delivered to
the reaction vessel and submitted to microwave irradiation at 508C for
30 min. A final series of filtered washes of the reaction vessel (3ꢂNMP)
finishes a coupling cycle.
monomer 1l, we were unable to isolate and characterise the
resulting trimer (18). Finally we also illustrated the versatili-
ty and power of the method through synthesis of longer
oligomers 25, 26 and 28 (up to a hexamer). This foldamer
was obtained in 10 h using double couplings and the NMR
spectrum is given in Figure 3a for the product obtained di-
rectly from the resin. This spectrum is typical of the spectral
data that is obtained immediately following resin cleavage
and indicates that the oligomers are obtained in sufficient
purity for preliminary screening. In several instances, this
was not the case, however, cleaner material could be ob-
tained by preperative HPLC.
Acknowledgements
This work was supported by the European Research Council [ERC-StG-
240324] and the Engineering and Physical Sciences Research Council
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5549

A. J. Wilson et al.
[EP/D077842/1 and EP/G022569/1]. N.S.M acknowledges the University
of Leeds for the award of a Mary & Alice Smith Ph. D. Scholarship.
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Org. Biomol. Chem. 2012, 10, 6469.
Keywords: amides · foldamers · helical structures · protein–
protein interactions · solid-phase synthesis
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Received: November 15, 2012
Published online: March 18, 2013
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Chem. Eur. J. 2013, 19, 5546 – 5550