The effect of N-methylation on transition state mimetic inhibitors of the Plasmodium protease, plasmepsin v

Article abstract of DOI:10.1039/c4md00409d

N-Methylation of the N-Cα peptide bond is a known strategy to overcome the liabilities inherently associated with peptide-like molecules. Here, we apply this strategy to transition state mimetics that are potent inhibitors of the malarial protease, plasme

Full text of DOI:10.1039/c4md00409d

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The effect of N-methylation on transition state
mimetic inhibitors of the Plasmodium protease,
plasmepsin V†
Cite this: DOI: 10.1039/c4md00409d
Michelle Gazdik,ab Matthew T. O'Neill,ab Sash Lopaticki,ab Kym N. Lowes,ab
ab
Brian J. Smith,c Alan F. Cowman,ab Justin A. Boddeyab and Brad E. Sleebs
*
N-Methylation of the N–Ca peptide bond is a known strategy to overcome the liabilities inherently
associated with peptide-like molecules. Here, we apply this strategy to transition state mimetics that are
potent inhibitors of the malarial protease, plasmepsin V, with the aim of enhancing their activity against
Plasmodium parasites. We demonstrate that independent N-methylation of each N–Ca bond of the
mimetics interferes with binding interactions to plasmepsin V, resulting in reduced affinity for the
protease. We provide evidence that N-methylation improves proteolytic stability and slightly improves
lipophilicity. However, the observed parasite activity of the N-methyl compounds had little correlation
with on-target plasmepsin V activity, indicating non-specific activity. This study underscores the benefits
and the drawbacks of the N-methylation strategy, and provides further evidence that plasmepsin V is
highly sensitive to substrate modification.
Received 15th September 2014
Accepted 24th November 2014
DOI: 10.1039/c4md00409d
www.rsc.org/medchemcomm
protease.11 Processing by PMV is an essential step for export of
PEXEL-containing proteins and for parasite survival in human
erythrocytes.12 PMV and PEXEL proteins are conserved in all
Introduction
Malaria is caused by infection with Plasmodium parasites. Each
year, Plasmodium parasites cause several hundred million
infections and over 650 000 human deaths.1 Combination drug
therapies have proved effective in reducing the disease burden.
However, the current arsenal of clinically used artemisinin
combination therapies, and promising drug candidates under-
going clinical assessment, may not be sufficient in effectively
eliminating the disease due to the threat of emerging resis-
tance. This highlights an urgent need for the development of
new antimalarial therapies.
To survive within erythrocytes, parasites deliver proteins into
the host cell, using protein export mechanisms, to remodel the
infected erythrocyte and its surface (reviewed in ref. 2–4). The
majority of proteins destined for export to the erythrocyte
possess an N-terminal pentameric motif known as the Plasmo-
dium export element (PEXEL; RxLxQ/E/D)5 or vacuolar transport
signal.6 The PEXEL motif is proteolytically processed in the
parasite's endoplasmic reticulum (ER)7,8 by Plasmepsin V
(PMV),9,10 an ER-resident, membrane-bound aspartic acid
Plasmodium spp.13
Cleaved PEXEL proteins exit the ER and are trafficked across
the parasite membrane and parasitophorous vacuole
membrane through the PTEX complex (the Plasmodium trans-
locon of exported proteins)14–16 to reach the host cell.
Recently, transition state (TS) mimetics of the natural PEXEL
substrate were shown to be potent inhibitors of PMV from
P. falciparum and P. vivax, and to impair protein export in
P. falciparum-infected erythrocytes, killing the parasites.12,17 The
most potent PMV inhibitor to date, WEHI-916, has high affinity
for the PMV enzyme (19 nM IC50), and an EC50 of 2.5 mM against
P. falciparum cultured in human erythrocytes.12,17 The marked
decrease in efficacy in culture may be linked to the peptide-like
nature of WEHI-916.
One strategy to overcome the liabilities associated with
peptide-like compounds is N-methylation of the backbone
amide bonds.18–20 This approach has been shown to reduce
proteolytic degradation21–24 and aid in cell membrane perme-
ability.24–26 In some instances, this has led to improved cellular
potency.24,27–29 Herein, we describe the generation of a set of
analogues delineated from an N-methyl scan of the peptide
backbone of previously described TS mimetics that are potent
inhibitors of PMV. We used the approach to independently
probe the amide bond conformational rigidity and hydrogen
bonding requirements of the mimetics with PMV. The
analogues provide evidence that highlight the advantages and
disadvantages of N-methylation as a strategy to overcome the
aThe Walter and Eliza Hall Institute of Medical Research, Parkville, 3052, Australia.
E-mail: sleebs@wehi.edu.au; Tel: +61 3 9345 2718
bDepartment of Medical Biology, The University of Melbourne, Parkville, 3010,
Australia
cDepartment of Chemistry, La Trobe University, 3086, Australia
† Electronic supplementary information (ESI) available: Experimental for
chemistry, molecular modeling and biology. Compound dose response curves
for the PMV biochemical assay, the P. falciparum viability assay and results of
the serum stability assay. See DOI: 10.1039/c4md00409d
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liabilities inherently associated with peptide-like small
molecules.
Results and discussion
Chemistry
We selected compound 1 (Fig. 1), previously described in Sleebs
et al.,17 as the progenitor compound on which to conduct the N-
methyl scan. The mimetic 1 inhibited PMV with an IC50 value of
0.35 mM, and in addition reduced parasite viability in culture
with an EC50 of 15 mM. It is known from previous studies12,17
that compound 1 possesses key attributes that are required for
inhibition of PMV. The P3 Arg of 1 makes key interactions in the
S3 pocket of PMV and is critical for binding affinity. The P1 Leu
that occupies the S1 pocket of PMV was found to be highly
sensitive to modication. The S2 pocket of PMV tolerates a
variety of amino acids, however, mimetics with a Val, Ile or Leu
were shown to give the highest binding affinity.12,17 For the
purpose of this study, the N-methyl compounds 2–5 were
proposed, based on the parent compound 1 that possessed a P2
Ala, as depicted in Fig. 1. In addition to 1, a more potent
inhibitor of PMV that possessed a P2 Val, 6, (analogous to
WEHI-916 (ref. 12 and 17)) and its N-methyl surrogate, 7, were
also prepared (Fig. 1).
There are a plethora of methods to synthesise N-methyl
amino acids present in the literature,30 however, many of these
methods result in racemization of the a-centre of the amino
acid, are not compatible with peptide protection group strate-
gies, or result in incomplete N-methylation. A high yielding
approach that overcomes these shortfalls is the use of the 5-
oxazolidinone scaffold. This methodology has not only allowed
the generation of all proteinaceous a-amino acids, but also a
range of unnatural amino acids.31–38 We sought to apply this
technology to the synthesis of N-methyl amino acids used in the
N-methyl scan.
Scheme 1 Synthesis of analogues 1 and 5. Reagents and conditions:
(a) HCl$NH2–Ala–OEt, HBTU, DIPEA, DMF, 18 h; (b) LiOH, THF, H2O, 5
min; (c) 2-(4-chlorophenyl)-ethylamine for 11; 2-(4-chlorophenyl)-N-
methyl ethanamine for 12; phenylethylamine for 13; HBTU, DIPEA,
DMF, 18 h; (d) 4 N HCl in dioxane, 1 h; (e) 14 for 17; 15 for 18 HBTU,
DIPEA, DMF, 18 h; (f) TFA, DCM, 18 h.
give the respective products 17 and 18. Finally, Boc-depro-
tection gave the desired compounds 1 and 5.
The N-methyl Ala and N-methyl Val methyl ester, 19 and 20,
were produced using the previously described oxazolidinone
strategy31–34 and were independently coupled to Cbz-Orn(N-
Boc)-OH 21. The resulting methyl esters 23 and 25 were
hydrolysed to obtain 27 and 29, respectively, which were
coupled to the statine amide 14 to give the respective products
30 and 32. The N-Boc protection group was then removed under
acidic conditions, and reacted with N,N0-bis-Boc-1-guanylpyr-
azole to form the guanidine moieties 33 and 35. Boc-depro-
tection gave rise to the desired N-methyl Ala analogue 3 and the
N-methyl Val analogue 7. The synthesis of the non-methylated
To construct the peptide-like analogues that incorporated
the N-methyl amino acids, solution phase synthesis was
employed. The synthesis of the progenitor compound 1 and the
N-methyl C-terminal N–H compound, 5, were produced using a
similar synthetic scheme to that of Sleebs et al.,17 as shown in
Scheme 1. Briey, Cbz-Arg(N,N-diBoc)-OH 8, was coupled to
alanine ethyl ester. The ester 9 was then hydrolysed to obtain 10.
The acid 10 was then coupled to the statine amides 14 and 15, to
Scheme 2 Synthesis of 3, 6 and 7. Reagents and conditions: (a)
HCl$NH2–Ala–OMe for 22; HCl$NH(CH3)–Ala–OMe 19 for 23;
HCl$NH2–Val–OMe for 24; HCl$NH(CH3)–Val–OMe 20 for 25; HBTU,
DIPEA, DMF, 18 h; (b) LiOH, THF, H2O; (c) 14 for 30; 16 for 31 and 32;
Fig. 1 The P3 to P20 regions of 1 and the associated binding pockets of HBTU, DIPEA, DMF, 18 h; (d) 4 N HCl in dioxane, 1 h; (e) N,N0-bis-Boc-
0
PMV (S3–S2 ). The inset table describes the proposed N-methyl scan.
1-guanylpyrazole, Et3N, DCM, 18 h; (f) TFA, DCM, 18 h.
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valine analogue 6 was also produced following this pathway, as
shown in Scheme 2.
We next wanted to generate the N-methyl Arg analogue.
However, directly accessing N-methyl Arg from arginine using
the oxazolidinone technology is problematic, and therefore a
modied synthesis starting from a protected Orn was utilised,
previously described by Aurelio and Sleebs et al.31,33 Cbz-Orn(N-
Phth)-OH 36, was converted to the N-methyl amino acid 38 via
the oxazolidinone 37. This allowed for coupling of the dipeptide
40 to the protected N-methyl Orn 36 generating the tripeptide
41. The N-Phth protection group was removed from 41 using
hydrazine hydrate and the resultant free amine was subse-
quently reacted with N,N0-bis-Boc-1-guanylpyrazole to install the
protected guanidine moiety. Boc deprotection of 42 yielded the
desired N-methyl Arg analogue 2 (Scheme 3).
The synthesis of the N-methyl statine has been previously
described by Wagner et al.39,40 This method is similar to that
utilising the oxazolidinone to produce N-methyl amino acids,
however, the key intermediate is an oxazolidine. The synthesis
was initiated with protecting the carboxylic acid of Boc-statine
43 as a benzyl ester; this step prevented the preferential
formation of the lactone when forming the oxazolidine. The
oxazolidine intermediate 44 was formed smoothly using para-
formaldehyde with a catalytic quantity of PTSA. The benzyl ester
was then hydrolysed to form the acid 45, which was then
coupled to 2-(4-chlorophenyl)-ethylamine to produce the amide
46. Concomitant Boc-deprotection and reductive cleavage was
then performed generating the N-methyl statine 47. However, in
Scheme 4 Synthesis of the N-methyl statine analogue 4. Reagents
and conditions: (a) Benzyl bromide, K2CO3, DMF, 3.5 h; (b) PTSA,
paraformaldehyde, toluene, 90 ꢀC, 4 h; (c) LiOH, THF, H2O, 18 h; (d) 2-
(4-chlorophenyl)-ethylamine, HBTU, DIPEA, DMF, 18 h; (e) Et3SiH, TFA,
DCM, 18 h; (f) 47, HBTU, DIPEA, DMF, 18 h; (g) 4 N HCl in dioxane,
5 h; (h) N,N0-bis-Boc-1-guanylpyrazole, Et3N, DCM, 18 h; (i) DCM,
TFA, 18 h.
guanidine functionality affording 49. Removal of the Boc-
protection gave the desired N-methyl statine analogue 4
(Scheme 4).
Biological evaluation
this reaction we observed that 20% de-formylation of the imi- To assess whether N-methylation of the mimetics affected the
nium intermediate had occurred shown by the integration of inhibitory activity of PMV, the N-methyl analogues were
1
the N–Me peak in the H-NMR spectrum. Despite this, 47 was assessed and then compared to the activity of 1. Inhibitory
then used in the coupling reaction with the dipeptide 26 to activity was determined using the previously described uoro-
produce the tripeptide 48. The side chain of the ornithine in the genic substrate processing assay that employs a substrate con-
tripeptide 48 was then deprotected under acidic conditions, and taining the PEXEL sequence RTLAQ from the exported knob-
reacted with N,N0-bis-Boc-1-guanylpyrazole to install the associated histidine-rich protein (KAHRP) with the DABCYL –
EDANS uorophore combination.9
The evaluation of compounds against P. falciparum PMV
revealed that N-methylation of every peptide bond N–H was
detrimental to activity when compared to 1. In particular,
analogue 3, which possessed P2 N-methyl Ala, had the greatest
reduction in inhibition, with an IC50 value of 48 mM. This
dramatic reduction in affinity for PMV was also reected when
comparing 7, which possessed P2 N-methyl Val, to the parent
non-methylated analogue 6. A 50-fold decrease in inhibitory
activity was observed with N-methylation of the P1 Arg (2),
compared to the non-methylated compound 1. The N-methyla-
tion of the C-terminal amide (5) exhibited a 20-fold reduction in
inhibitory activity. However, the N-methylation of statine (4) was
somewhat tolerated with an IC50 of 890 nM, and only slightly
less potent than the N–H comparator 1 (300 nM IC50) (Table 1).
The reduced inhibitory activity of the compounds in this
study likely results from either the loss of essential hydrogen
bond interactions of the N–H amide with hydrogen-bond
acceptors in the active site of PMV (necessary for the target
protein to recognize its ligand) – the concomitant steric clash
with the N–Me group – or, restriction of the conformational
exibility about the N–Ca and Ca–C(O) bonds.41 In a model of
Scheme 3 Synthesis of the N-methyl arginine analogue 2. Reagents
and conditions: (a) PTSA, paraformaldehyde, toluene, 90 ꢀC, 3 h; (b)
Et3SiH, TFA, DCM, 18 h; (c) 14, HBTU, DIPEA, DMF, 18 h; (d) 4 N HCl in
dioxane, 1 h; (e) 40, TFFH, DIPEA, DMF, 18 h; (f) N2H4$H2O, EtOH, 18
h; (g) N,N0-bis-Boc-1-guanylpyrazole, Et3N, DCM, 18 h; (h) DCM,
TFA, 18 h.
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Table 1 Biological activity of the N–H and N-methyl analogues
#
R3
R2
R1
R4
R5
PMVa (ꢁSEM) (mM)
Parasite viabilityb (ꢁSEM) (mM)
HepG2c (mM)
c log Pd
1
2
3
4
5
6
7
H
Me
H
H
H
H
H
Me
H
H
H
H
H
Me
H
H
H
H
H
Me
H
H
Me
Me
Me
Me
Me
iPr
iPr
0.30 (0.13)
15.6 (4.1)
48.0 (7.6)
0.89 (0.33)
5.35 (3.3)
0.026 (0.01)
>50
15.3 (0.09)
67.8 (0.25)
23.2 (0.52)
29.2 (0.45)
20.3 (0.21)
3.97 (0.17)
5.89 (0.46)
>50
>50
>50
>50
>50
>50
N.d.
2.34
2.57
2.57
2.57
2.57
2.63
2.85
H
H
H
Me
H
H
a
IC50 Data represents means ꢁ SEMs for three uorogenic substrate cleavage experiments. A 9-point dilution series of each compound was
b
incubated (37 ꢀC) with P. falciparum ipPMV-HA. WEHI-916 (ref. 12 and 17) IC50 24 nM. EC50 data represents means ꢁ SEMs for three
c
experiments. 7-point dilution series of 1–7 were incubated with P. falciparum 3D7 parasites for 72 h. Chloroquine EC50 0.01 mM. EC50 data
d
represents means for three experiments in an 11-point dilution series over 48 h. Etoposide EC50 9.8 mM. Calculated using Chemaxon
soware.41 N.d. – no data. R1 to R5 refers to groups in Fig. 1.
the progenitor non-methylated compound 1 bound to PMV The dramatic loss of activity from N-methylation of both the P3
(Fig. 2), Thr312 forms a hydrogen-bond interaction with the and P2 residues further highlights the high sensitivity PMV
N–H of the P3 Arg of 1, and may explain why N-methylation of possesses toward substrate modication.
the P3 (2) is not tolerated by PMV. Glu136, that makes a key
The improvement in potency of 6 compared to 1, is sug-
interaction with the guanidine side-chain, forms a hydrogen gested by the homology model of PMV in complex with
bond with the N–H of the P2 alanine of 1; this P2 N–H group lies compound 1 (Fig. 2). The model shows that the replacement of
in close proximity to the side-chains of both the P1 Leu and P3 the R5 Me (1) with iPr (6) would place this group in close prox-
Arg. N-Methylation of the P2 alanine (3) caused a 160-fold loss in imity to the hydrophobic side-chains of Cys178 and Phe370;
potency. We propose that this loss is not only attributed to the improved hydrophobic packing of iPr relative to Me at this site
disruption of the N–H interaction with Glu136, but also to may account for the increased affinity of 6 compared to its
perturbing the critical interaction that Glu136 forms with the P3 parent 1.
Arg of the substrate, and steric clash with the side-chains of
these two critical substrate residues.
A key benet of N-methylation is improved proteolytic
stability. To test whether this was indeed the case with our
The reduced activity arising from N-methylation of the C- compounds, the N-methyl analogues 2, 3, 4 and 5 were
terminal N–H (5) can be attributed to the loss of the C-terminal compared to their progenitor non-methylated compound 1, in
N–H interaction with the backbone carbonyl of Gly77; steric the presence of a pancreatic extract containing active peptidases
clash of the N-methyl with this carbonyl will also likely cause and proteases (Fig. 3). We used the pancreatic extract as a
displacement of the amide plane and subsequent loss of the surrogate model for emulating the peptidases and proteases
hydrogen bond between the statine carbonyl and the backbone that are found in Plasmodium parasites. Each compound was
amide of Cys135. The model also suggests that the statine N–H incubated in the presence of the pancreatic extract at 37 ꢀC and
interacts with the backbone carbonyl of Gly310; notably though, monitored for proteolytic degradation by LCMS at several time
N-methyl substitution of the statine (4) was shown to retain points over 24 h. The study showed that the N-methyl analogues
some activity, suggesting this interaction may not be critical.
Fig. 3 Stability of the non-methylated compound 1 compared to the
N-methyl analogues 2, 3, 4 and 5, incubated in the presence of
Fig. 2 An interaction diagram showing the key interactions of the pancreatin in phosphate buffer (pH 7.4) at 37 ꢀC. Data presented are
backbone amides of 1 (cyan) with PMV (tan), based on the homology averages of duplicate measurements and expressed as percentages
model. Hydrogen bonds between the substrate (1) and PMV are relative to the average concentration of the initial time point.
highlighted with yellow-dotted lines.
Error + SD.
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2, 3 and 5 exhibited improved stability relative to the non-
methylated compound 1. However, the N-methyl statine
analogue was less stable compared to 1 over 24 h. The stability
study suggests that compounds 2, 3 and 5 may have improved
stability in the presence of proteases and peptidases harboured
by Plasmodium parasites in culture. Analogues 1–5 were all
found to be stable in human serum over 55 h (ESI Table S1†).
Another benet of N-methylation is the possibility of
enhanced lipophilicity and membrane permeability. c log P
values are shown in Table 1 and demonstrate that there is
negligible difference between the lipophilicity of the non-
methylated 1 in comparison to the N-methyl compounds 2–5
and 6 in comparison to 7. From the c log P values, it is unlikely
that a small change in lipophilicity would enhance the
membrane permeability of the mono N-methylated analogues
2–5 and 7.
There is published evidence that N-methylation can improve
cellular potency.24,27–29 As we observed no increase in inhibitory
activity against PMV, we predicted that our N-methylated
compounds were unlikely to have increased potency against
parasites in culture, if the compounds were truly specic to
PMV. To examine this, compounds 1–7 were assessed in a
parasite viability assay described previously.12,17 P. falciparum-
infected human erythrocytes were treated with the compounds
in 9-point titrations and parasitaemia determined aer 72 h by
ow cytometry and compared to vehicle-treated controls.
Compared to the non-methylated compounds, 1 and 6, the
Fig. 4 Activity of selected transition state mimetics against PMV in
cultured parasites. P. falciparum trophozoites expressing PfEMP3-GFP
in infected erythrocytes were treated with 20 mM of compounds 1–7,
WEHI-916 (ref. 12 and 17) or vehicle control (DMSO) and processing of
respective mono N-methylated analogues 2–5 and 7, all exhibi- the PEXEL in PfEMP3 was assessed by immunoblotting with anti-GFP
antibodies. A schematic of the GFP protein and its cleavage positions is
shown at the top. Uncleaved (black arrow), PEXEL-cleaved (blue
arrow), and “GFP only” (a degraded remnant of the GFP reporter in the
food vacuole) species of PfEMP3-GFP are indicated next to the
ted weaker inhibition of parasite growth. This was not unex-
pected as a loss in PMV affinity was observed for 2–5 and 7
compared to 1 and 6. It is noted that analogues 2, 3 and 7 killed
parasites (67.8, 23.2 and 5.9 mM EC50, respectively) despite
immunoblot. PEXEL R > A mutant PfEMP3-GFP was included as a size
having very weak activity against PMV (15.6, 48.0 and >50 mM control, and the blot was probed with parasite anti-HSP70 as a loading
control. Densitometry of the uncleaved band in each lane is shown
beneath the blot.
IC50, respectively). This is most likely a result of non-specic
off-target activity.
To determine whether the inhibition of PMV contributed to
the observed parasite activity of compounds 1–7, we assessed
the ability of compounds 1–7 to block processing of the Plas-
The N-methylated compounds 2–5 and 7 did not have an
effect in the PfEMP3 processing assay (Fig. 4). Although The
N-methylated compounds 2–5 and 7 were shown to kill para-
sites in culture (Table 1), this was not as a result of engaging
PMV, demonstrated by the inability these compounds to effect
processing of PfEMP3 (Fig. 4). This demonstrates that the N-
methylation strategy was not only detrimental to PMV activity
and processing of PfEMP3, but also resulted in an increase in
non-specic activity as shown by the weak correlation of data
from the biochemical and parasite viability assays (Table 1).
Additionally, it is difficult to draw conclusions as to whether the
marginal improvement in metabolic stability and lipophilicity
actually improved parasite activity, due to the lack of affinity of
the N-methylated compounds, 2–5 and 7, for PMV.
modium falciparum erythrocyte membrane protein
3
(PfEMP3) fused to green uorescent protein (GFP) in transgenic
P. falciparum parasites in culture, following an established
protocol.12,17 The processing of the PEXEL in PfEMP3 was
assessed by immunoblotting with anti-GFP antibodies. The
western blot in Fig. 4 shows that only compounds 1 and 6 were
shown to have an effect on PEXEL processing of PfEMP3.
Compound 6 was the most effective at blocking processing,
which correlated well with compound 6 possessing the most
potent biochemical inhibition of PMV (Table 1). Compound 6
was also seen to have a similar effect on processing as the
previously described and structurally similar compound, WEHI-
916.12,17 The level of PEXEL-cleaved protein observed did not
quantitatively reect the degree of PMV inhibition by 1, 6 or
WEHI-916, as the inhibitors were added well aer PEXEL pro-
Conclusions
cessing and export of PfEMP3-GFP had initiated. This process- N-Methylation of the peptide backbone amides is favourably
ing experiment does however, demonstrate target engagement viewed with regard to improving proteolytic stability and
of PMV in P. falciparum parasites.
permeability in a cellular context.18–20 However, this study
demonstrates that N-methylation had profound effects on the
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binding affinity for PMV and, hence, inhibitory activity. The
PMV homology model provides evidence that reduced inhibi-
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actions with PMV, due to the combination of conformational
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Acknowledgements
This work was funded by the National Health and Medical
Research Council of Australia (Project Grant 1010326 to J.A.B), a
CASS Foundation Science and Medicine grant (SM.12.4348 to 17 B. E. Sleebs, M. Gazdik, M. T. O'Neill, P. Rajasekaran,
JAB), the Australian Cancer Research Foundation, the Victorian
State Government Operational Infrastructure Support and
Australian Government NHMRC IRIISS. We thank the Univer-
S. Lopaticki, K. Lackovic, K. N. Lowes, B. J. Smith,
A. F. Cowman and J. A. Boddey, J. Med. Chem., 2014, 57,
7644–7662.
sity of Melbourne for the provision of an Australian Post- 18 J. Chatterjee, C. Gilon, A. Hoffman and H. Kessler, Acc.
graduate Award to M.G. A.F.C. is Howard Hughes Chem. Res., 2008, 41, 1331–1342.
International Scholar and an Australia Fellow of the NHMRC. 19 J. Chatterjee, F. Rechenmacher and H. Kessler, Angew.
J.A.B is an Australian Research Council QEII Fellow. We thank
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Guillaume Lessene, Janet Weinstock and Andrew Powell for 20 S. Sagan, P. Karoyan, O. Lequin, G. Chassaing and
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