Solid-phase synthesis of cyclic peptide chitinase inhibitors: SAR of the argifin scaffold

Article abstract of DOI:10.1039/b815077j

A new, highly efficient, all-solid-phase synthesis of argifin, a natural product cyclic pentapeptide chitinase inhibitor, is reported. The synthesis features attachment of an orthogonally protected Asp residue to the solid support and assembly of the line

Full text of DOI:10.1039/b815077j

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PAPER
www.rsc.org/obc | Organic & Biomolecular Chemistry
Solid-phase synthesis of cyclic peptide chitinase inhibitors: SAR of
the argifin scaffold†
Mark J. Dixon,a Amit Nathubhai,a Ole A. Andersen,b Daan M. F. van Aaltenb and Ian M. Eggleston*a
Received 29th August 2008, Accepted 6th October 2008
First published as an Advance Article on the web 13th November 2008
DOI: 10.1039/b815077j
A new, highly efficient, all-solid-phase synthesis of argifin, a natural product cyclic pentapeptide
chitinase inhibitor, is reported. The synthesis features attachment of an orthogonally protected Asp
residue to the solid support and assembly of the linear peptide chain by Fmoc SPPS prior to cyclisation
and side-chain manipulation on-resin. Introduction of the key N-methyl carbamoyl-substituted Arg
side chain is achieved via derivatisation of a selectively protected Orn residue, prior to cleavage from the
resin and side-chain deprotection. A severe aspartimide side-reaction observed upon final deprotection
is circumvented by the use of a novel aqueous acidolysis procedure. The flexibility of the synthesis is
demonstrated by the preparation of a series of argifin analogues designed from the X-ray structure of
the natural product in complex with a representative family 18 chitinase.
nonetheless been identified. These have been implicated in a
number of major disease states including asthma,9 osteoarthritis10
Introduction
and lipid storage disease,11 and so chitinase inhibitors are therefore
also of interest as selective chemical probes to investigate the role
of such proteins in these disorders, and as potential drug leads.
So far, most of the chitinase inhibitors that have been identified
are either natural products,12 or have been inspired by natural
product leads. The pseudo-trisaccharide allosamidin 213 (Fig. 1),
has been widely studied, and is a potent inhibitor of a broad
range of family-18 chitinases, as well showing interesting biological
effects against fungal and insect pathogens.14,15 However, although
the total synthesis of 2 has been achieved by several laboratories,
the complexity of these syntheses limits both its availability and
the scope for preparing structural analogues.12,16,17
Chitinases catalyse the hydrolysis of chitin (1, Fig. 1), the natural
homopolymer of b(1,4)-linked N-acetyl-D-glucosamine. Chitin
is a key structural component of the cell walls, exoskeletons,
and eggshells of pathogenic fungi, insects, and nematodes,
respectively,1 which all rely on the ability to hydrolyse chitin at spe-
cific points in their life cycles. For this reason, chitinase inhibitors
are now attracting considerable interest as novel fungicides2 and
insecticides,3 as well as potential chemotherapeutic agents against
a variety of tropical diseases, such as filariases4 and malaria.5
Although chitin is not found in mammalian physiology, two hu-
man chitinases (chitotriosidase6 and acidic mammalian chitinase7)
and several chitin binding proteins (termed chi-lectins8) have
In this context, we have recently focussed our attention on two
natural product cyclic pentapeptides, that have been shown to
be nanomolar inhibitors of bacterial-type family 18 chitinases.
Argifin 318 and argadin 419 (Fig. 1), isolated from Gliocladium and
Clonostachys fungal cultures respectively, represent a new class of
potent chitinase inhibitors that are significantly more synthetically
accessible and amenable to rational structure-based optimisation
aWolfson Laboratory of Medicinal Chemistry, Department of Pharmacy and
Pharmacology, University of Bath, Claverton Down, Bath, BA2 7AY, UK.
E-mail: ie203@bath.ac.uk; Fax: ++ 44 01225 386114
bDivision of Molecular and Environmental Microbiology, University of
Dundee, Dundee, DD1 5EH, UK
† Electronic supplementary information (ESI) available: ES-MS data for
compounds 10a and 10b. See DOI: 10.1039/b815077j
Fig. 1 Chitin 1, allosamidin 2, argifin 3 and argadin 4.
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than allosamidin. The first syntheses of 3 and 4 were reported
by us,20,21 based on a combination of solid-phase and solution
techniques.
a representative family 18 chitinase (chitinase B1 from Aspergillus
fumigatus, Af ChiB1).
In our original synthesis of 3,20 the key N-methyl carbamoyl
modification of the Arg residue was effected in solution as the
final step (Scheme 1). However, acidolytic deprotection of the
preceding Arg-containing cyclic peptide precursor proved to be
problematic, necessitating a time-consuming HPLC purification
at this stage of the synthesis and a consequently reduced overall
yield. In order to improve the efficiency of production of 3,
and develop SAR around the argifin scaffold, we have therefore
developed a new synthesis of 3 and analogues based upon an
all-solid-phase approach, in the process identifying a significant
side reaction in our previous synthesis, that is eliminated now via
a novel side-chain deprotection procedure. The flexibility of the
synthetic strategy is demonstrated by the preparation of a series of
compounds inspired by the X-ray structure of 3 in complex22 with
Results and discussion
Improved solid-phase preparation of argifin
Our revised approach to argifin is outlined in Scheme 1, path
A. In contrast to our original approach (Scheme 1, path B),
assembly of the linear peptide chain is now achieved via attachment
to the solid-support (P2) through the a-carboxyl group of an
orthogonally protected Asp residue. Instead of releasing the linear
precursor into solution, on-resin cyclisation is now effected, after
C- and N-terminal deprotections (P4 and P5). This is followed by
introduction of the derivatised Arg side-chain through guanidina-
tion of a selectively protected Orn residue (P3), before final cleavage
from the resin and deprotection of the a-carboxyl group of the
Scheme 1 Synthetic approaches to argifin. Path A: All-solid-phase approach. Path B: Previous combined solid-phase–solution approach. (Path B
requires HPLC purification prior to the final acylation stage).20 For both synthetic routes, P5 corresponds to temporary N(a)-protection. For Path A, P1,
P3, P4 correspond to side-chain protection, P2 to the solid support. For Path B, P1, P2, P3 correspond to side-chain protection, P4 to the solid support.
260 | Org. Biomol. Chem., 2009, 7, 259–268
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second Asp residue (P1). Although either of the Asp a-carboxyl
groups would be a suitable candidate for attachment of the solid
support, the residue adjacent to MePhe was initially chosen so
as to provide an analogous cyclisation precursor sequence to that
employed in solution. Indirect introduction of the Arg residue,
via Orn, was envisaged primarily to circumvent use of acid-labile
protection for the former, and also to facilitate a fully on-resin
approach.
The necessary level of orthogonality between the solid-phase
linker and the other protecting groups required was planned
as follows. Acid-labile 2-chlorotrityl chloride23 polystyrene resin
was chosen as solid support (P2), with the base-labile Fmoc
group as temporary Na-protection (P5) and C-terminal allyl ester
protection, removable under neutral conditions, selected for P4.
The Dde group, normally cleaved by hydrazinolysis24 and stable
to both Fmoc and allyl ester deprotection conditions, was chosen
to protect the Orn side chain (P3), and acid-labile tert-butyl ester
protection selected for the a-carboxyl group of the second Asp
residue (P1).
For our synthesis of 3 by this approach, Fmoc-Asp-OAll
5 was loaded onto 2-chlorotrityl chloride polystyrene resin to
give the orthogonally protected Asp resin 6 with a loading of
0.4 mmol/g (Scheme 2). The desired resin-bound linear peptide 7
was assembled using standard Fmoc SPPS conditions, followed
by removal of the C-terminal allyl ester (Pd(Ph3P)4/PhSiH3)25
and N-terminal Fmoc protection to give cyclisation precursor 8.
Cyclisation was then effected upon the solid support by treatment
with PyBOP/DIPEA for 2 ¥ 2 h. Cleavage of a small sample
of resin at this stage with TFA/DCM (1:99) and analysis by
HPLC and ES-MS confirmed the success of this transformation,
with essentially quantitative conversion to the expected cyclic
peptide 10a being observed (Fig. 2a). Surprisingly, when the
same material was exposed to a higher concentration of TFA, to
effect simultaneous side-chain deprotection (Fig 2b), the product
profile was more complex, mirroring the results obtained on
attempted deprotection of the Arg side chain of the cyclic peptide
in our original synthesis, and with ES-MS analysis also indicating
the presence of aspartimide by-products (see ESI†). That these
Scheme 2 Reagents and conditions: a) 2-Chlorotrityl chloride polystyrene resin, DIPEA, DCM, 60 min; b) Fmoc SPPS; c) Pd(Ph3P)4, PhSiH3, DCM,
3 ¥ 20 min; d) piperidine/DMF (1:4), 4 ¥ 3 min; e) PyBOP, DIPEA, DCM, 2 ¥ 60 min; f) TFA/DCM (1:99), for 10a, TFA/DCM (80:20) for 10b;
g) H2NNH2/DMF (1:49), 2 ¥ 15 min; h) 15, DIPEA, DMF, 2 ¥ 16 h; i) 1H-pyrazole-1-carboxamidine hydrochloride, DIPEA, DMF, 16 h; j) N-succinimidyl
N-methylcarbamate, DBU, DMF, 2 h; k) TFA/DCM (1:99), 10 ¥ 2 min; l) 1 M HCl, 60 ◦C, 90 min.
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during linear assembly, based upon our previous observation that
the use of Asp(OBn) was ineffective in this context.20 Similarly,
elimination of the cyclised product from the solid support, via
aspartimide formation during the basic conditions of the final
acylation step (see below), was totally suppressed by the use of
2-chlorotrityl resin for the synthesis.28
Removal of Dde protection from the resin-bound cyclic pen-
tapeptide 9 was achieved by brief treatment with hydrazine
monohydrate in DMF to give 11 ready for introduction of the
derivatised Arg side-chain. The Orn→Arg(MC) transformation
was attempted in two ways. Firstly, one-step conversion using the
known reagent 1529 was explored, by analogy with our recently
reported synthesis of argadin. However, 15 was found to be
rather unreactive and gave only moderate conversion even when
used in a large excess and for extended periods (e.g. approx.
15% conversion with 10 eq. 15, 12 eq. DIPEA in DMF for 2 ¥
16 h). An alternative, two-step procedure was therefore adopted
involving initial guanidination of the Orn residue to Arg, followed
by an acylation reaction to insert the N-methylcarbamoyl moiety.
Thus, treatment of 11 with 10 eq of 1H-pyrazole-1-carboxamidine
hydrochloride30 for 16 h gave a quantitative conversion to the Arg
derivative 12, which was in turn acylated with N-succinimidyl N-
methylcarbamate in the presence of DBU to give the advanced
intermediate 13. Using the same conditions as those applied in
the previous solution synthesis, (6 eq. DBU, 3 eq. N-succinimidyl
N-methylcarbamate for 2 h)20 70% of the desired mono-acylated
compound was obtained, according to HPLC analysis on cleaved
material, with only minor amounts of unreacted starting material
and the di-acylated product 18 detectable.
In order to complete the synthesis, it was necessary to cleave
the cyclic peptide from the solid support, and remove the
remaining tert-butyl ester protection, without aspartimide for-
mation. However, despite considerable experimentation, varying
acid concentration and contact time, it was not possible to effect
concomitant resin cleavage and side-chain deprotection using
TFA/DCM without the formation of some aspartimide products.
Indeed, intermediates 11 and 12 also proved equally prone to this
side reaction upon attempted cleavage.
To overcome this problem, a two-step cleavage/deprotection
procedure was ultimately devised. Firstly, the resin-bound cyclic
peptide 13 was treated with TFA/DCM (1:99) for 10 ¥ 2 min
to effect release of the cyclic peptide 14 only, without side-
chain deprotection. As acid-catalysed aspartimide formation upon
Fig. 2 (a) HPLC of crude cyclisation precursor (lower trace) and
crude cyclic peptide 10a (upper trace) following cleavage from the solid
support with TFA/DCM (1:99). Conditions: Dionex C-18 column (see
experimental), 5–95% solvent B in 10 min. (b) HPLC of crude cyclic
peptide 10b following cleavage from the solid support with TFA/DCM
(80:20). Conditions: as for Fig. 2a.
products were generated only upon acidolysis and not by base-
induced aspartimide formation26,27 (Scheme 3) during Fmoc
synthesis, was confirmed by the observation that cleavage of 7
with 80% TFA gave rise only to the expected linear pentapeptide in
high purity. We had chosen tert-butyl protection for the non-resin-
linked Asp residue specifically to eliminate aspartimide formation
Scheme 3 Formation of aspartimides from b-peptide derivatives.
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strong acid treatment is known to occur even with unprotected
Asp residues,31 we speculated that in the latter case, aspartimide
formation involving the “side-chain” a-Asp carboxyls might be
avoidable if the tert-butyl ester protection could be effectively
cleaved and scavenged first under very mild conditions. The use
of aqueous mineral acids has occasionally been proposed as a
green alternative to TFA for the removal of tert-butyl protection
in peptide synthesis,32 as water is known to be an effective scavenger
for Me3C+ and other carbenium ion species,33 and so to this end
14 was exposed to 1 M aq HCl at room temperature. Under
these conditions, a clean conversion to 3 was observed in 5 days,
without any detectable aspartimide products. Furthermore, when
the reaction was conducted at 60 ◦C, deprotection was now
complete in only 90 min, with no loss in purity (Fig. 3).34
Fig. 3 HPLC of purified tert-butyl ether-protected cyclic peptide 14
(lower trace); following treatment with 1 M HCl (60 ◦C, 90 min) to generate
3 (upper trace). Conditions: Dionex C-18 column, 5–60% solvent B in
20 min.
Fig. 4 (a) HPLC of crude argifin 3, following solid-phase assembly and
two stage deprotection. Conditions: as for Fig. 3. (b) HPLC of argifin 3,
following single final purification. Conditions: as for Fig. 3.
With the incorporation of the two step acidolysis protocol into
the synthetic route, 3 was now obtained in >98% purity and an
overall yield of 18% over the 17-step sequence, following a single
final HPLC purification (see Figs 4a, b). The isolated product gave
1H and 13C NMR spectra which were identical to those originally
reported by Arai et al18 for the natural product, and material
previously synthesised by us, confirming that no aspartimide
mediated b→a isomerisation of the Asp linkages had occurred
during the second phase of deprotection.
failed completely at the cyclisation step, with no product observed.
Similarly, the conservative mutation of the N-terminal bAsp to
bHse for the synthesis of 26 had a severe impact, with only minor
amounts of cyclised material being detectable upon cleavage. In
contrast, when the point of attachment to the solid phase was
Table 1 Details of synthetic yields and enzyme inhibition data for
analogues
Cyclisation
yield (%)a
Yield
(%)b
IC50
Compound Mutation
(mM)c
Analogue synthesis
3
None
Quant.
42
55
n/a
n/a
90
34
72
81
53
18
2
9
n/a
n/a
16
7
3
10
8
11
n/a
n/a
0.029
0.280
0.182
50.0
>1000
>1000
>1000
0.011
0.144
60.0
With an improved synthetic route in hand, a group of argifin
analogues were designed (Fig. 5a), based on the high resolution
X-ray crystal structure of argifin in complex with Af ChiB1
(Fig. 5b).22 The synthesis of the analogues using the newly
developed all-solid-phase route generally proceeded smoothly.
HPLC and MS analysis at each stage of the syntheses indicated
that the critical step was the on-resin cyclisation, which appeared
to vary quite significantly in efficiency depending on the analogue
in question, and was the main reason for the variation in overall
yields observed (Table 1). In particular, the cyclisation reaction
was highly sensitive to variations in the N-terminal residue of the
linear cyclisation precursor 8. For example, attempted synthesis
of 27, in which bAla was substituted for bAsp at this position,
16
17
18
19
20
21
22
23
24
25
26
27
D-Ser for D-Ala
D-Thr for D-Ala
Arg(MC)2 for Arg(MC)
Arg for Arg(MC)20
Orn(MC) for Arg(MC)
His for Arg(MC)
MeTyr(Bn) for MePhe
MeTyr for MePhe
Phe for MePhe
bHse for bAsp
62
nd
n/a
1.200
n/a
n/a
bHse for bAsp
bAla for bAsp
a From HPLC analysis. b Final isolated yield following HPLC. c Measured
against Af ChiB1.
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a
b
Fig. 5 (a) Compounds investigated in this study. By-product in synthesis of 3. Intermediate in synthesis of 3. (b) X-ray structure of argifin 3 in
complex with Af ChiB1, showing key residues for SAR development (model extracted from previously published complex22a, PDB entry 1W9V).
moved to the other Asp residue, in order to allow mutation of the
residue adjacent to MePhe in 3, an efficient on-resin cyclisation
was observed, along with an efficient overall conversion to the
desired analogue 25 (Scheme 4).
All the analogues were obtained in pure form after a single
HPLC purification, following the two-step resin cleavage and
aqueous side-chain deprotection protocol. For the D-Ser, D-Thr
and MeTyr analogues 16, 17 and 23 respectively, the tert-butyl
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21 was inspired by the observation that 4, which interacts with
conserved active site residues through this residue, is able to
bind deeper into the active site of Af CHiB1 than 3 and forming
significantly more interactions, by virtue of its more compact cyclic
peptide backbone.22a However, this substitution once more led to
almost total loss of activity, highlighting again the critical role of
the Arg(MC) residue.
Analogues 22–24 designed to probe the role of the MePhe
residue also proved highly informative. The binary Af ChiB1–3
crystal structure reveals the presence of a relatively large pocket,
lined by residues 219–220 and 243–247 and the side chains of
Tyr178, Lys224 and Phe273, which is situated next to the MePhe
benzyl group and occupied by approximately eight ordered water
molecules. 22 (MeTyr(Bn) for MePhe) and 23 (MeTyr for MePhe)
were designed to expand the inhibitor side chain into this pocket.
The extra hydroxyl group of 23 is potentially favourable by
allowing a hydrophilic group to face the ordered water structure of
the pocket; however, in the event, the addition of a hydroxyl group
did not have any beneficial effect, indicating a possible conflict
between this hydrophilic modification and the nearby hydrophobic
groups of Trp137 and Phe251, although the reduction in activity is
fairly modest, compared to substitutions at Arg(MC). Extension
of the hydrophobic side chain in 22 did, however, lead as predicted
to an increase in potency, presumably due to favourable contacts
with residues in the available pocket, lowering the IC50 two-fold
relative to 3 itself.
A very significant feature of the Af ChiB–3 complex, and indeed
complexes with other family 18 chitinases, is the presence of a
b-turn centred on the Arg(MC)-MePhe motif, with a cis-amide
bond between these residues that occupy respectively the i + 1 and
i + 2 turn positions. This conformation allows the Arg(MC) side
chain to occupy the -1 pocket of the enzyme, whilst providing
a favourable orientation of the hydrophobic MePhe side chain.
Removal of the N-methyl group in compound 24, led to a 1000-
fold drop in activity, suggesting the removal of the preferred
cis Arg(MC)-MePhe amide bond configuration. To confirm this
detail, the conformation of 24 was studied in detail, both in
solution and bound to the enzyme. ROSEY NMR experiments in
D2O revealed an absence of correlations between the a-protons of
the Arg and Phe residues, or between the Arg b methylene protons
and the Phe aromatic protons, thus confirming the predominantly
trans-configuration of the Arg-Phe amide bond in 24 in solution.
It should be noted that 3 and also all the analogues prepared
(apart from 21) do show these diagnostic features, as well as an
Arg b-methylene proton appearing at unusually low field in the
1D spectrum (dH = -0.43 ppm for 3), which is significantly shifted
(dH = 1.52 ppm) in 24. To probe the configuration of this amide
bond when bound to the enzyme, a complex of 24 and Af ChiB1
was obtained by soaking, and the resulting high-resolution
Scheme 4 Synthesis of 25 via alternative Asp attachment point.
ether side-chain protections used were also smoothly cleaved under
the aqueous acidolysis procedure.
Enzymology and SAR studies
The inhibitory properties of the analogues against Af ChiB1 were
investigated using a fluorometric assay with 4-methylumbelliferyl-
b-N,N¢-diacetylchitobiose as substrate as previously reported.22
The IC50 values are shown in Table 1. Substitution in the D-Ala
position was fairly well tolerated, with relatively small decreases in
potency observed for both 16 and 17. This residue appears to form
no significant interactions with the protein in the binary Af ChiB1–
3 complex. Although it was predicted that introduction of an
additional hydroxyl could provide a favourable hydrogen bonding
interaction with the nearby Thr138 residue, this was not achieved
for either compound. In contrast, replacement of the bAsp residue
following MePhe with bHse in 25 led to over an order of magnitude
increase in IC50. The binary Af ChiB1–3 complex reveals a
hydrogen bond between the a-carboxylate of the Asp residue
in question and the indole ring of Trp137. The apparent loss of
this interaction through the relatively conservative bAsp→bHse
mutation highlights the importance of this interaction for the
binding of argifin-like molecules. Although the same mutation
at the other Asp position of the scaffold could not be achieved
in this study, it seems likely that it would have a similar impact,
since the a-carboxyl group of this Asp residue forms three water-
mediated hydrogen bonds with the carboxyl group of Glu322, and
the backbone N and amide group of Asn323, respectively.
As expected, given the number of binding interactions that
are observed involving the Arg(MC) residue, the argifin scaffold
was highly sensitive to modifications at this position. We have
indeed recently demonstrated that the methylguanylurea side-
chain fragment from the Arg(MC) residue is the minimum
pharmacophore derivable from 3.22b Inclusion of a second methyl
carbamoyl group in the Arg side chain (18) caused a large decrease
in activity, presumably due to steric clashes of the second acyl
group with active site residues Asp246, Tyr299 and Trp384, while
truncation of Arg(MC) to Arg as in 19, or Orn(MC), i.e. citrulline,
in 20, totally abolished activity. The Arg(MC)→His mutation
˚
(1.9 A) X-ray crystal structure investigated (data not shown). This
revealed that 24 does in fact bind with the key amide bond in
question still adopting the cis-configuration. The poor affinity of
24 must therefore result from the necessity of an unfavourable
trans-to-cis isomerisation occurring prior to binding.
Conclusions
We have devised an efficient all-solid-phase route to the potent
chitinase inhibitor argifin. All the steps proceed with high
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efficiency, which should therefore allow the approach to be adapted
to automation and scale-up. The formation of aspartimide prod-
ucts upon TFA-based cleavage of side-chain protecting groups
is avoided by indirect introduction of the Arg residue, and the
use of aqueous acidolysis to achieve final removal of tert-butyl
protection. Argifin analogues obtained by this protocol provide
valuable SAR concerning the cyclic peptide scaffold, and in
particular highlight the key role played by the Arg(MC)-MePhe
dipeptide in binding. Further studies are ongoing to apply these
lessons to the design of potent peptidomimetic inhibitors.
(2 eq) and DIPEA (4 eq) in DCM for 2 h. The resin was drained and
the procedure repeated. The resin was then drained and washed
sequentially with DMF, DCM, MeOH and Et2O (5¥ each). Dde
deprotection: The resin was swollen in DCM for 20 min, drained
and treated with a solution of hydrazine monohydrate/DMF (v/v
1:49) for 15 min. The resin was drained and the procedure repeated.
The resin was then drained and washed sequentially with DMF,
DCM, MeOH and Et2O (5¥ each). Guanidination: The resin was
swollen in DCM for 20 min, drained and treated with a solution of
1H-pyrazole-1-carboxamidine hydrochloride (10 eq) and DIPEA
(12 eq) in DMF for 16 h. The resin was drained into 1 M aqueous
CuSO4 and washed sequentially with DMF, DCM, MeOH and
Et2O (5¥ each). The whole procedure was then repeated. Acylation:
The resin was swollen in DCM for 20 min, drained and treated
with a solution of N-succinimidyl-N-methylcarbamate (3 eq) and
DBU (6 eq) in DMF for 2h. The resin was drained and washed
sequentially with DMF, DCM, MeOH and Et2O (5¥ each). The
whole procedure was then repeated. Cleavage from the resin: The
resin was swollen in DCM for 20 min, drained and treated with
TFA/DCM (1:99) for 2 min. The resin was drained into a solution
of pyridine/MeOH (v/v 1:9) and the procedure repeated a further
9 times. The combined filtrates were evaporated to dryness to
give the partially protected cyclic peptides. Aqueous side-chain
acidolysis: The crude cyclic peptides were dissolved in 1 M aq
Experimental
General information
NMR spectra were acquired on a Varian Mercury VX400 MHz
spectrometer, operating at 400 MHz for 1H and 100 MHz
for 13C, or a Varian Unity INOVA 600 MHz spectrometer,
operating at 600 MHz for 1H and 150 MHz for 13C. All coupling
constants (J values) were measured in Hertz. High resolution mass
spectrometry was performed using a Bruker MicroTOF autospec
electrospray ionisation mass spectrometer. Analytical RP-HPLC
was performed on a Dionex HPLC system equipped with a Dionex
Acclaim 3 mm C-18 (150 ¥ 4.6 mm) column with a flow rate of
1 mL/min. Preparative RP-HPLC was performed on a Dionex
HPLC system equipped with a Phenomenex Gemini 5 mm C-18
(250 ¥ 30 mm) column with a flow rate of 22.5 mL/min. Mobile
phase A was 0.1% TFA in water, Mobile phase B was 0.1% TFA
in acetonitrile.
◦
HCl (1.5 mg/mL) and heated at 60 C for 90 min. Evaporation
of the solvent, followed by purification by preparative HPLC and
lyophilisation gave the final deprotected peptides as TFA salts in
>95% purity.
Argifin (3)
Synthesis of argifin 3 and analogues 16–18 and 20–25
1
Scale: 0.094 mmol. Yield: 13.0 mg, 18%. H NMR (600 MHz,
Resin loading: 2-Chlorotrityl chloride polystyrene resin
(1.2 mmol/g loading) was treated with Fmoc-Asp(OAll)-OH
(1 eq) and DIPEA (4 eq) in DCM for 60 min. The resin was filtered
and treated with DCM/MeOH/DIPEA (17:2:1) for 15 min,
drained, and washed sequentially with DMF, DCM, MeOH, and
Et2O (5¥ each). Resin loading was measured after the first Fmoc
deprotection step using the Fmoc method. Fmoc deprotection:
The resin was swollen in DCM for 20 min, drained and treated
with piperidine/DMF (1:4 v/v, 3 mL) for 3 min. The resin was
drained and the procedure repeated a further 3 times. The resin was
drained and washed sequentially with DMF, DCM, MeOH and
Et2O (5¥ each). Peptide couplings: The resin was swollen in DCM
for 20 min, drained and treated with a solution of Fmoc-amino
acid (2 eq), PyBOP (1.9 eq), and DIPEA (4 eq) in DCM/DMF
(v/v 4:1) for 60 min, except in the case of coupling to MePhe where
PyBrOP35 (2 eq) was used. The resin was drained and washed with
DMF, DCM, MeOH, and Et2O (5¥ each). Solid phase reactions
were monitored by the qualitative Kaiser test for the detection of
primary amines and the chloranil test for detection of secondary
amines. Allyl ester cleavage: The resin was swollen in degassed
DCM for 20 min, drained, and treated with DCM/PhSiH3 (v/v
3:1) for 2 min, prior to the addition of Pd(Ph3P)4 (20 mg). After
20 min reaction, the resin was drained and washed with dry
DCM (10¥). The procedure was repeated twice, then the resin was
drained and washed sequentially with DMF, DCM, MeOH and
Et2O (5 ¥ each). Peptide backbone cyclisation: The resin was swollen
in DCM for 20 min, drained, and treated with a solution of PyBOP
D2O): d 7.26–7.10 (5H, m, MePhe 2 ¥ dCH, 2 ¥ eCH, zCH),
5.01 (1H, dd, J = 12, 3, MePhe aCH), 4.66 (1H, m, Asp aCH),
4.42 (1H, dd, J = 12, 2.5, Asp aCH), 4.18 (1H, m, Arg aCH),
4.06 (1H, q, J = 7, Ala aCH), 3.08–2.84 (6H, m, MePhe bCH2,
Arg dCH2, Asp bCH2), 2.76 (3H, s, MePhe NCH3), 2.67 (1H, m,
Asp bCHH), 2.63 (3H, s, MeCbm CH3), 2.39 (1H, t, J = 13, Asp
bCHH), 1.29 (1H, m, Arg gCHH), 1.19 (3H, d, J = 7, Ala bCH3),
1.10–0.94 (2H, m, Arg gCHH, Arg bCHH), -0.44 (1H, m, Arg
bCHH); 13C NMR (100 MHz, D2O): d 175.3, 174.3, 171.5, 171.4,
170.3, 155.2, 137.5, 129.7, 129.2, 127.3, 62.3, 50.6, 50.0, 49.6, 48.8,
40.7, 37.8, 35.1, 33.4, 29.9, 26.6, 26.1, 24.0, 16.8; Analytical RP-
HPLC (5–60% B in 20 min, l = 220 nm): Rt: 13.0 min; MS (ES+)
calcd for C29H42N9O10: 676.3055; found: 676.3018 [M + H]+.
D-Ala→D-Ser (16)
Scale: 0.094 mmol. Yield: 1.8 mg, 2%. 1H NMR (600 MHz, D2O):
d 7.32–7.16 (5H, m, MePhe 2 ¥ dCH, 2 ¥ eCH, zCH), 5.08
(1H, dd, J = 11.5, 3, MePhe aCH), 4.69 (1H, m, Asp aCH),
4.47 (1H, dd, J = 11.5, 2.5, Asp aCH), 4.28–4.22 (2H, m, Arg
aCH, Ser aCH), 3.71 (2H, d, J = 5.5, Ser bCH2), 3.11 (1H, m,
MePhe bCHH), 3.02–2.92 (4H, m, MePhe bCHH, Arg dCH2,
Asp bCHH), 2.86–2.80 (4H, m, MePhe NCH3, Asp bCHH), 2.76
(1H, m, Asp bCHH), 2.69 (3H, s, MeCbm CH3), 2.53 (1H, t, J =
13, Asp bCHH), 1.35 (1H, m, Arg gCHH), 1.14–1.03 (2H, m,
Arg gCHH, Arg bCHH), -0.34 (1H, m, Arg bCHH); 13C NMR
(100 MHz, D2O): d 175.5, 174.4, 172.1, 171.7, 171.4, 170.5, 137.5,
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129.7, 129.2, 127.3, 62.3, 61.3, 55.6, 50.6, 50.2, 48.9, 40.7, 38.0,
35.1, 33.3, 29.9, 26.6, 26.1, 24.0; Analytical RP-HPLC (5–60%
B in 20 min, l = 220 nm): Rt: 12.5 min; MS (ES+) calcd for
C29H42N9O11: 692.2998; found: 692.2967 [M + H]+.
4.65 (1H, m, Asp aCH), 4.50 (1H, dd, J = 11, 3.5, His aCH), 4.45
(1H, dd, J = 11.5, 2.5, Asp aCH) 3.93 (1H, q, J = 7.5, Ala aCH),
3.08 (1H, dd, J = 14, 3, MePhe bCHH), 2.98–2.90 (2H, m, MePhe
bCHH, His bCHH), 2.68 (1H, dd, J = 13.5, 3, Asp bCHH), 2.84–
2.71 (5H, m, His bCHH, Asp bCHH, MeCbm CH3), 2.42–2.35
(2H, m, 2 ¥ Asp bCHH), 1.04 (3H, d, J = 7.5, Ala bCH3); 13C
NMR (100 MHz, D2O): d 175.3, 175.2, 174.4, 172.5, 171.5, 171.1,
170.4, 137.4, 133.5, 129.6, 129.2, 128.9, 127.4, 115.9, 62.7, 50.7,
50.1, 49.6, 46.9, 37.7, 35.3, 33.4, 30.1, 24.8, 16.5; Analytical RP-
HPLC (5–60% B in 20 min, l = 220 nm): Rt: 11.4 min; MS (ES+)
calcd for C27H34N7O9: 600.2413; found 600.2394 [M + H]+.
D-Ala→D-Thr (17)
Scale: 0.107 mmol. Yield: 7.7 mg, 9%. 1H NMR (400 MHz, D2O):
d 7.28–7.12 (5H, m, MePhe 2 ¥ dCH, 2 ¥ eCH, zCH), 5.06 (1H, dd,
J = 11, 3, MePhe aCH), 4.63 (1H, dd, J = 13, 3, Asp aCH), 4.45
(1H, dd, J = 11.5, 2.5, Asp aCH), 4.23 (1H, m, Arg aCH), 4.03–
3.92 (2H, m, Thr aCH, Thr bCH), 3.06 (1H, m, MePhe bCHH),
3.00–2.81 (4H, m, MePhe bCHH, Arg dCH2, Asp bCH2), 2.77
(3H, s, MePhe NCH3), 2.75–2.67 (2H, m, 2 ¥ Asp bCHH), 2.65
(3H, s, MeCbm CH3), 2.54 (1H, t, J = 13, Asp bCHH), 1.31 (1H,
m, Arg gCHH), 1.09–0.98 (5H, m, Arg gCHH, Thr gCH3, Arg
bCHH), -0.43 (1H, m, Arg bCHH); 13C NMR (100 MHz, D2O): d
175.2, 174.3, 174.2, 172.2, 171.8, 171.4, 170.5, 137.5, 129.7, 129.2,
127.3, 67.4, 62.3, 59.6, 50.4, 50.1, 48.9, 40.7, 37.9, 35.1, 33.3, 29.9,
26.6, 26.1, 24.2, 19.2; Analytical RP-HPLC (5–60% B in 20 min,
l = 220 nm): Rt: 12.7 min; MS (ES+) calcd for C30H44N9O11:
706.3155; found: 706.3149 [M + H]+.
MePhe →MeTyr(Bn) (22)
Scale: 0.075 mmol. Yield: 2.2 mg, 3%. 1H NMR (600 MHz,
CD3CN): d 7.41–7.29 (5H, m, 5 ¥ ArCH), 7.11 (2H, d, J = 8.5,
MeTyr, 2 ¥ ArCH), 6.94 (2H, d, J = 8.5, MeTyr, 2 ¥ ArCH), 5.03
(2H, s, OCH2Ar), 4.93 (1H, m, MeTyr aCH), 4.62 (1H, m, Asp
aCH), 4.42 (1H, m, Asp aCH), 4.20–4.10 (2H, m, Arg aCH, Ala
aCH), 3.11–2.62 (13H, m, MeTyr bCH2, Arg dCH2, Asp bCH2,
Asp bCHH, MeTyr NCH3, MeCbm CH3), 2.42 (1H, t, J = 13,
Asp bCHH), 1.43 (1H, m, Arg gCHH), 1.24 (3H, d, J = 7.5, Ala
bCH3), 1.18–1.04 (2H, m, Arg gCHH, Arg bCHH), -0.22 (1H,
m, Arg bCHH); 13C NMR (100 MHz, CD3CN): d 175.5, 174.3,
171.1, 158.5, 137.9, 131.8, 131.1, 129.5, 129.0, 128.6, 116.2, 70.7,
63.0, 51.1, 50.9, 50.1, 49.5, 41.5, 38.8, 36.1, 33.5, 30.2, 27.8, 26.7,
25.3, 17.8; Analytical RP-HPLC (5–60% B in 20 min, l = 220 nm):
Rt: 17.2 min; MS (ES+) calcd for C36H48N9O11: 782.3468; found
782.3476 [M + H]+.
Arg(MC) →Arg(MC)2 (18) – minor diacylated product from
argifin synthesis
1H NMR (600 MHz, D2O): d 7.26–7.11 (5H, m, MePhe 2 ¥ dCH,
2 ¥ eCH, zCH), 5.02 (1H, m, MePhe aCH), 4.55 (1H, m, Asp
aCH), 4.34 (1H, dd, J = 12, 2, Asp aCH), 4.23 (1H, m, Arg
aCH), 4.05 (1H, q, J = 7, Ala aCH), 3.13–3.02 (4H, m, MePhe
bCHH, MeCbm CH3), 2.97–2.87 (4H, m, MePhe bCHH, Arg
dCH2, Asp bCHH), 2.76–2.62 (8H, m, MePhe NCH3, MeCbm
CH3, 2 ¥ Asp bCHH), 2.35 (1H, t, J = 13, Asp bCHH), 1.34
(1H, m, Arg gCHH), 1.20 (3H, d, J = 7, Ala bCH3), 1.13 (1H,
m, Arg gCHH), 1.01 (1H, m, Arg bCHH), -0.44 (1H, m, Arg
bCHH); Analytical RP-HPLC (5–60% B in 20 min, l = 220 nm):
Rt: 13.5min; MS (ES+) calcd for C31H45N10O11: 733.3269; found:
733.3282 [M + H]+.
MePhe →MeTyr (23)
Scale: 0.075 mmol. Yield: 6.2 mg, 10%. 1H NMR (400 MHz, D2O):
d 7.35 (2H, d, J = 8.5, MeTyr 2 ¥ ArCH), 7.09 (2H, d, J = 8.5,
MeTyr 2 ¥ ArCH), 5.31 (1H, dd, J = 11.5, 3, MeTyr aCH), 5.03
(1H, m, Asp aCH), 4.78 (1H, dd, J = 12, 2.5, Asp aCH), 4.55 (1H,
m, Arg aCH), 4.43 (1H, q, J = 7, Ala aCH), 3.35–3.02 (10H, m,
MeTyr bCH2, Arg dCH2, Asp bCH2, Asp bCHH, MeTyr NCH3)
2.99 (3H, s, MeCbm CH3), 2.76 (1H, t, J = 13, Asp bCHH), 1.73
(1H, m, Arg gCHH), 1.56 (3H, d, J = 7, Ala bCH3), 1.47–1.38
Arg(MC) →Orn(MC) (20)
(2H, m, Arg gCHH, Arg bCHH), 0.00 (1H, m, Arg bCHH); 13
C
1
Scale: 0.094 mmol. Yield: 11.1 mg, 16%. H NMR (600 MHz,
NMR (100 MHz, D2O): d 175.4, 175.3, 174.4, 171.5, 170.2, 154.9,
131.1, 129.1, 115.9 62.5, 50.6, 50.0, 49.6, 48.9, 40.8, 37.8, 35.1,
32.4, 29.8, 26.6, 26.0, 24.3, 16.8; Analytical RP-HPLC (5–60%
B in 20 min, l = 220 nm): Rt: 11.2 min; MS (ES+) calcd for
C29H42N9O11: 692.2998; found 692.3003 [M + H]+.
D2O): d 7.58–7.39 (5H, m, MePhe 2 ¥ dCH, 2 ¥ eCH, zCH), 5.25
(1H, dd, J = 11, 3, MePhe aCH), 4.92 (1H, dd, J = 12.5, 2.5,
Asp aCH), 4.68 (1H, m, Asp aCH), 4.40–4.32 (2H, m, Orn aCH,
Ala aCH), 3.34–3.12 (3H, m, MePhe bCH2, Orn dCHH), 3.01
(3H, s, MePhe NCH3), 2.98–2.88 (4H, m, Orn dCHH, Asp bCH2,
Asp bCHH,), 2.83 (3H, s, MeCbm CH3), 2.69 (1H, t, J = 13, Asp
bCHH), 1.50–1.41 (4H, m, Ala bCH3, Orn gCHH), 1.24–1.16
MePhe →Phe (24)
(2H, m, Orn gCHH, Orn bCHH), -0.25 (1H, m, Orn bCHH); 13
C
Scale: 0.101 mmol. Yield: 6.3 mg, 8%. 1H NMR (400 MHz, D2O):
d 7.24–7.05 (5H, m, Phe 2 ¥ dCH, 2 ¥ eCH, zCH), 4.91 (1H, dd,
J = 12, 4.5, Asp aCH) 4.59 (1H, dd, J = 12.5, 3.5, Asp aCH),
4.46 (1H, dd, J = 14, 4, Phe aCH), 4.17 (1H, q, J = 7, Ala aCH),
4.07 (1H, m, Arg aCH), 3.30 (1H, dd, J = 14, 4, Phe bCHH),
2.95–2.84 (4H, m, Asp bCHH, Asp bCHH, Arg dCH2), 2.77–2.57
(6H, m, Phe bCHH, 2 ¥ Asp bCHH, MeCbm CH3), 1.39 (2H,
m, Arg gCH2), 1.23 (3H, d, J = 7, Ala bCH3), 1.17 (1H, m, Arg
bCHH), 0.79 (1H, m, Arg bCHH); 13C NMR (100 MHz, D2O): d
177.2, 174.8, 173.9, 173.8, 172.9, 171.6, 171.1, 137.5, 129.2, 128.8,
126.9, 55.8, 53.9, 50.4, 49.9, 49.0, 40.5, 36.5, 36.4, 35.8, 26.9, 26.1,
NMR (100 MHz, D2O): d 175.5, 175.4, 175.0, 174.5, 171.7, 171.6,
170.3, 161.5, 138.2, 130.4, 129.9, 128.2, 62.9, 51.0, 50.5, 50.1, 49.7,
40.0, 38.6, 35.8, 34.2, 30.3, 27.6, 27.2, 17.7; Analytical RP-HPLC
(5–60% B in 20 min, l = 220 nm): Rt: 12.2min; MS (ES+) calcd
for C28H40N7O10: 634.2831; found: 634.2831 [M + H]+.
Arg(MC) →His (21)
Scale: 0.094 mmol. Yield: 4.4 mg, 7%. 1H NMR (400 MHz, D2O):
d 8.47 (1H, s, His eCH), 7.18–7.04 (5H, m, MePhe 2 ¥ dCH, 2 ¥
eCH, zCH), 6.92 (1H, s, His dCH), 5.08 (1H, m, MePhe aCH),
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22.9, 16.1; Analytical RP-HPLC (5–60% B in 20 min, l = 220 nm):
Rt: 12.5 min; MS (ES+) calcd for C28H40N9O10: 662.2893; found
662.2868 [M + H]+.
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bAsp → bHse (25)
Scale: 0.099 mmol. Yield: 8.4 mg, 11%. 1H NMR (400 MHz, D2O):
d 7.78 (1H, s, Hse OH), 7.26–7.08 (5H, m, MePhe 2 ¥ dCH, 2 ¥
eCH, zCH), 4.95 (1H, dd, J = 12, 2.5, MePhe aCH), 4.69 (1H,
m, Asp aCH), 4.15–4.03 (2H, m, Ala aCH, Arg aCH), 3.96 (1H,
m, Hse aCH), 3.66–3.55 (2H, m, Hse CH2OH), 3.28 (1H, m,
MePhe bCHH), 2.92–2.55 (12H, m, MePhe bCHH, Asp bCH2,
Arg dCH2, Asp bCHH, MePhe NCH3, MeCbm CH3), 2.39 (1H,
dd, J = 14, 12, Asp bCHH), 1.46 (1H, m, Arg gCHH), 1.24–0.98
(5H, m, Ala bCH3, Arg gCHH, Arg bCHH), -0.28 (1H, m, Arg
bCHH); 13C NMR (100 MHz, D2O): d 175.1, 174.7, 174.2, 172.5,
171.5, 170.5, 165.1, 163.3, 163.0, 155.3, 153.6, 137.7, 129.7, 129.2,
128.8, 127.21, 117.9, 63.6, 61.8, 50.0, 49.6, 49.4, 48.4, 40.7, 38.2,
37.0, 34.2, 33.7, 30.9, 27.1, 26.1, 23.8, 16.8; Analytical RP-HPLC
(5–60% B in 20 min, l = 220 nm): Rt: 13.0 min; MS (ES+) calcd
for C29H44N9O9: 662.3257; found 662.3248 [M + H]+.
16 A. Berecibar, C. Grandjean and A. Siriwardena, Chem. Rev., 1999, 99,
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Enzymology
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Inhibition of Af ChiB1 was determined using the fluorogenic
substrate
4-methylumbelliferyl-b-D-N,N¢-diacetylchitobiose
(Sigma). In a final volume of 50 mL, 2 nM of enzyme was
incubated with 20 mM substrate in McIlvain buffer (100 mM citric
acid, 200 mM sodium phosphate, pH 5.5) containing 0.1 mg/mL
BSA, for 10 min at 37 ◦C in the presence of different inhibitor
concentrations. After the addition of 25 mL of 3 M glycine-NaOH,
pH 10.3, the fluorescence of the liberated 4-methylumbelliferone
was quantified using a Flx 800 microtitreplate fluorescence
reader (Bio-Tek Instruments Inc.), with excitation and emission
wavelengths of 360 nm and 460 nm, respectively, using 40 mm
slits. Experiments were performed in triplicate. Production of
4-methylumbelliferone was linear with time for the incubation
period used, and less than 10% of available substrate was
hydrolysed.
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Acknowledgements
27 (a) M. Mergler and F. Dick, J. Peptide Sci., 2005, 11, 650–657;
(b) Formation of aspartimides with b-Asp peptides: B. K. Handa and
C. H. Hassall, J. Chem. Soc. Perkin Trans 1, 1976, 2014–2019.
28 When the synthesis was conducted via side-chain anchoring of Asp to
p-alkoxybenzyl alcohol (Wang) resin (see J. Tulla-Puche and G. Barany,
J. Org. Chem., 2004, 69, 4101–4107), treatment with DBU was found
to result in complete elimination of cyclised material from the solid
support.
This work was supported by Wellcome Trust project grant 074337
(to IME and DvA) and a BBSRC studentship (AN). DvA
is supported by a Wellcome Trust Senior Research Fellowship
and the EMBO Young Investigator Programme. We thank Dr
T. Woodman for assistance with the NMR analyses.
29 Y. Nakayama and Y. Sanemitsu, J. Heterocycl. Chem., 1984, 21, 1553–
1556.
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