Cyclic tetrapeptides via the ring contraction strategy: Chemical techniques useful for their identification

Article abstract of DOI:10.1039/b800464a

Cyclic tetrapeptides are a class of natural products that have been shown to have broad ranging biological activities and good pharmacokinetic properties. In order to synthesise these highly strained compounds a ring contraction strategy had previously been reported. This strategy was further optimised and a suite of techniques, including the Edman degradation and mass spectrometry/mass spectrometry, were developed to enable characterisation of cyclic tetrapeptide isomers. An NMR solution structure of a cyclic tetrapeptide was also generated. To illustrate the success of this strategy a library of cyclic tetrapeptides was synthesised. The Royal Society of Chemistry.

Full text of DOI:10.1039/b800464a

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PAPER
www.rsc.org/obc | Organic & Biomolecular Chemistry
Cyclic tetrapeptides via the ring contraction strategy: chemical techniques
useful for their identification
Douglas A. Horton,†a Gregory T. Bourne,†a,b Justin Coughlan,a,b Sonya M. Kaiser,a,b Carolyn M. Jacobs,a
Alun Jones,c Andreas Ru¨hmann,a Jill Y. Turnerb and Mark L. Smythe*a,b
Received 10th January 2008, Accepted 5th February 2008
First published as an Advance Article on the web 28th February 2008
DOI: 10.1039/b800464a
Cyclic tetrapeptides are a class of natural products that have been shown to have broad ranging
biological activities and good pharmacokinetic properties. In order to synthesise these highly strained
compounds a ring contraction strategy had previously been reported. This strategy was further
optimised and a suite of techniques, including the Edman degradation and mass spectrometry/mass
spectrometry, were developed to enable characterisation of cyclic tetrapeptide isomers. An NMR
solution structure of a cyclic tetrapeptide was also generated. To illustrate the success of this strategy a
library of cyclic tetrapeptides was synthesised.
A ring contraction approach was reported by Meutermans et al.
that enables the synthesis of several cyclic tetrapeptides from their
Introduction
linear precursor.19,20 The strategy utilizes an auxiliary, 2-hydroxy-
6-nitrobenzylaldehyde (HnB) (Fig. 1). Due to the success of this
strategy, the objective of this work was to further optimise this
procedure, develop a suite of characterisation techniques for cyclic
tetrapeptides, and to synthesise a cyclic tetrapeptide library.
Cyclic tetrapeptides are a class of naturally occurring privileged
substructures, providing many potent molecules in a diverse range
of therapeutic areas.1,2 Typical examples, all derived through
extraction from culture broths, are the cytotoxic and antimitogenic
agents HC toxin3,4 and chlamydocin,5,6 the antitumor agent
trapoxin,7 the tyrosinase inhibitor cyclo-[(L)Pro-(L)Tyr-(L)Pro-
(L)Val]8 and the antimalarial apicidins.9 One compound from
the apicidin family is reported to show in vivo activity by both
parenteral and oral administration in mice against Plasmodium
berghei.9,10
Head-to-tail cyclic tetrapeptides differ from their larger macro-
cyclic counterparts in that they are highly constrained, to the point
that typically the planarity of the amide bonds is twisted.3,5,11–18
This ensures that they display their substituents in a highly
spatially defined manner, which makes them very useful as
molecular toolkits to probe a receptor and then develop smaller
organics with the requisite functionality.
Fig. 1 HnB auxiliary.
Results and discussion
However, because of these characteristics cyclic tetrapeptides
are difficult to synthesise. So-called “difficult” sequences produce
linear and cyclic oligomers in preference to the monocyclic target,
even when cyclisation is performed at high dilution,19,20 making
them generally inaccessible. The few cyclic tetrapeptides that have
been synthesised carry either D-residues, postmodified or unnatu-
ral residues, or at least one tertiary amide in the sequence.11,15,16,21–29
Cyclic tetrapeptides are also difficult to characterize. NMR
spectra of these molecules are complicated as they are often a
mixture of slowly interconverting conformers, which results in
peak broadening.
Synthesis of cyclic tetrapeptides
A photolabile cyclisation auxiliary (HnB, Fig. 1) was recently
reported that permits the synthesis of difficult cyclic pep-
tides sequences including cyclic tetrapeptides from their linear
precursors.19,20 The strategy is outlined in Scheme 1. The auxiliary
was first reductively aminated onto the N-terminus of the resin-
bound peptide. After cleavage from resin and concomitant removal
of the side-chain protecting groups to produce 1, cyclisation can be
accomplished to produce the cyclic nitrophenyl ester intermediate
2. Upon heating at 70 ◦C, this product ring contracts through
an O-to-N acyl transfer to generate the desired cyclic peptide 3.
Photolytic removal of HnB then provides the target cyclic product
4. Using the described method several tetrapeptide sequences
have been successfully synthesized, including cyclo-[Tyr-Arg-Phe-
Ala].20
aInstitute for Molecular Bioscience, The University of Queensland,
St. Lucia, Qld, 4072, Australia. E-mail: m.smythe@imb.uq.edu.au;
Web: http://www.imb.uq.edu.au/; Fax: +61-7-3346-2101; Tel: +61-7-3346-
2222
bProtagonist Pty. Ltd., Level 7 Queensland Bioscience Precinct, 306
Carmody Rd, St Lucia, Qld, 4072, Australia; Web: http://www.
protagonist.com.au.; Fax: +61-7-3346-2101; Tel: +61-7-3346-2222
The versatility of this synthetic route for each of the synthetic
steps was explored for a number of cyclic tetrapeptides. In parallel
with this, the development of a suite of analytical tools to aid in
the characterisation of these compounds and their intermediates
cARC Special Research Centre for Functional and Applied Genomics,
† Co-first authors. Equally contributed
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Scheme 1 Synthesis of difficult sequences through a ring contraction strategy.
was very important. Having accomplished this, a library of cyclic
tetrapeptides was synthesized.
diisopropylethylamine (this method had been used to synthesize
other cyclic tetrapeptides).20
The initial sequences chosen for the study were based on the
(D)Phe-Arg-Trp and Arg-Phe-Phe sequences. The Trp, Arg and
Phe residues were selected as they are often found as common
recognition motifs. Glycine was appended to the C-terminus of
these sequences as prior experience had shown that the C-terminal
residue is capable of racemizing during synthesis.20 Initially, no
side chain protecting groups were used. It was believed that the
presence of protecting groups was unnecessary and could possibly
hinder cyclisation-ring contraction yields based on steric effects.
A small number of unprotected Phe-Arg-Trp-Gly sequences
were synthesized on solid-support (Table 1, 1aa, 1ac, 1ae). After
cleavage and purification, the cyclisation and ring contraction
steps were performed using 1 equiv. BOP at 70 ◦C with 10 equiv. of
Although the linear starting material could still be observed
two products were also identified by LC/MS for each of the
sequences cyclised. In addition, after purification both products
appeared to have the mass corresponding to the cyclised material.
Characterisation at this point was found to be problematic due
to the instability of the HnB cyclised products. Hence, the HnB
auxilary was immediately removed by photolysis. NMR character-
isation of the final products was found to be very labour intensive,
primarily due to the slowly interconverting conformations. It thus
became apparent that other characterisation techniques would
be necessary in order to quickly identify the products. This is
especially important when generating large numbers of cyclic
tetrapeptides as a possible discovery project.
Characterisation techniques: Edman degradation and
mass-spectrometry/mass-spectrometry (MS/MS)
Table 1 Cyclisation of linear tetrapeptides 1 and corresponding yields of
the cyclic product 3 after photolysis to give product 4
Characterisation of cyclic tetrapeptides is very challenging. In
many cases, multiple conformers of the same molecule may be
observed by both NMR and HPLC. Conceptually, there are
three possible cyclisation products: the desired N- to C-terminal
cyclised product (4, Scheme 1), a side-chain to C-terminal cyclised
product (for example, when cyclisation occurs through the amino
group of lysine or the guanidyl group of arginine) (6, Scheme 1)
or an amide bond to C-terminal cyclised product (for example,
producing a diketopiperazine). Two different analytical techniques
were selected to assist in characterisation: the Edman degradation
and mass-spectrometry/mass-spectrometry (MS/MS).
The Edman degradation was selected as the reaction will
proceed for side-chain cyclised peptides, but not head-to tail
cyclised peptides. Furthermore, the reaction may be accomplished
on very small quantities and analysis merely requires a mass
spectrometer.
Linear peptide
1
Yield (%)a
3
4
aa
ab
ac
ad
ae
af
ag
ah
ai
[HnB](D)Phe-(D)Arg-Trp-Gly-OH
[HnB](D)Phe-(D)Arg(Pbf)-Trp(Boc)-Gly-OH
[HnB](D)Phe-Arg-(D)Trp-Gly-OH
46b
29b
77b
34c
54b
36c
28b
40b
30b
29b
65b
12e,g
18e,h
<5e,g
11h
[HnB](D)Phe-Arg(Pbf)-(D)Trp(Boc)-Gly-OH
[HnB]Phe-(D)Arg-(D)Trp-Gly-OH
25e,g
72d,g
19e,h
26e,h
19e,h
21e,h
10e,h
[HnB]His-(D)Phe-Arg-Trp-OHf
[HnB](D)Phe-(D)Arg(Pbf)-(D)Trp(Boc)-Gly-OH
[HnB]Phe-Arg(Pbf)-(D)Trp(Boc)-Gly-OH
[HnB]Phe-(D)Arg(Pbf)-Trp(Boc)-Gly-OH
[HnB](D)Phe-Arg(Pbf)-Trp(Boc)-Gly-OH
[HnB]Arg(Pbf)-Phe-Phe-Gly-OH
aj
ak
a Yield of purified product after RP-HPLC. b 1 eq HATU, 2 eq DIEA,
1 mM in DMSO, 3h/rt; ii) 10 eq DIEA, 15 h, 70 ◦C. c 1 eq BOP, 2 eq
◦
DIEA, 1 mM in DMSO, 3h/rt; ii) 10 eq DIEA, 15 h, 70 C. d ht, 1 mM
peptide in 1% HOAc in DMSO, 5 h. e ht, 0.1 mM peptide in 1% HOAc in
DMSO, 5 h. f Chiral amino acid analysis showed that the C-terminal Trp
a-carbon inverted 99.7% during cyclic tetrapeptide synthesis. g Yield after
photolysis. h Yield after photolysis of protected peptide followed by TFA
treatment. Note: 4aa and 4ab are equivalent; 4ac and 4ad are equivalent.
The Edman degradation proceeds through the reaction of
an N-terminal amino group with phenyl isothiocyanate under
mildly alkaline conditions to form a thiourea. This product then
rearranges under acidic conditions to produce the thiohydantoin,
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Table 2 Edman degradation of selected cyclic tetrapeptides
Observed Peaks after Treatmenta
Observed Mass of
Peptide-thiourea
complex
Peptide
Starting Materiala
Starting Material
Thiohydantoin
Des-R1 Peptide
ACP (65–74)b
1063
—
547.4
—
547.6
547.6
547.6
—
—
627.4
760.5
235.1 (Val)
—
283.1 (Phe)
—
Not observed
—
283.1 (Phe)
—
—
—
964.7
—
400.3
—
400.3
—
400.3
—
—
—
—
—
—
—
—
535.3
—
4aa
6aa
4ac
6ac
4ae
6ae
Cyclo-[(D)Phe-(D)Arg-Trp-Gly]
(D)Phe-Cyclo[(D)Arg-Trp-Gly]
Cyclo-[(D)Phe-Arg-(D)Trp-Gly]
(D)Phe-Cyclo-[Arg-(D)Trp-Gly]
Cyclo-[Phe-(D)Arg-(D)Trp-Gly]
Phe-Cyclo[(D)Arg-(D)Trp-Gly]
Cyclo[(D)Arg-(D)Trp-Gly]
Cyclo-[His-(D)Phe-Arg-Trp]c
Cyclo-[Arg(Pbf)-Phe-Phe-Gly)
547.4
547.3
547.6
547.3
547.6
547.3
400.3
627.4
760.5
4af
4ak
—
—
—
a Peaks are for [M + H] ions, [M + Na] ions were also often observed. b Acetyl Carrier Protein ACP(65–74) was used as a positive control. c It was later
confirmed that (L)Trp had inverted to (D)Trp in 99.7% purity.
leaving the peptide chain without the N-terminal residue. If the
N-terminus is not a primary amine, the reaction will not proceed.
After development on the positive control peptide Acyl Carrier
Protein (ACP) (65–74), all of the cyclic peptides prepared above
(Table 1) were photolysed and purified by RP-HPLC (Scheme 1).
The peptides were then tested (Table 2). As can be observed, ACP
(65–74) works very well as a control, producing only the desired
thiohydantoin and cleaved peptide. Next, pairs of Phe-Arg-Trp-
Gly photolysed products from the [HnB]peptide cyclisation were
tested (4 and 6). In each case, one of these failed to react at
all with the phenyl isothiocyanate, confirming its identity as the
desired N- to C-terminal cyclised product. The other compound
produced the N-terminal thiohydantoin and a cleaved peptide.
This product could therefore either be an arginine side-chain
cyclised product, or could be cyclised through one of the amide
bonds. As a result, the crude Edman degradation mixture from
Phe-cyclo[(D)Arg-(D)Trp-Gly] was re-subjected to the Edman
degradation. Gratifyingly, the cyclo[(D)Arg-(D)Trp-Gly] thiourea
complex was observed, but no thiohydantoin corresponding to the
arginine appeared. Therefore, this product had cyclised through
the arginine side-chain. Only one product was obtained from the
cyclisation and photolysis of [HnB]His-(D)Phe-Arg-Trp-OH and
[HnB]Arg(Pbf)-Phe-Phe-Gly-OH. These were examined by the
Edman degradation, which verified that they had cyclised through
the N- and C-termini.
Many of these products were also subjected to mass-
spectrometry/mass-spectrometry (MS/MS) analysis. This con-
firmed the results from the Edman degradation. For example,
head-to-tail cyclised products such as cyclo[His-(D)Phe-Arg-Trp]
4af exhibited fragments corresponding to a N- and C- terminal
residue dipeptide fragment (Trp-His). The side-chain cyclised
product (D)Phe-Cyclo[(D)Arg-Trp-Gly] 6ac exhibited no fragment
corresponding to Phe-Gly or Arg-Gly (Arg-Gly is unlikely, as three
bonds must be broken and cleavage at the guanidyl group would
be favoured), although fragments corresponding to the loss of Phe
were observed.
Development and optimisation
Having identified all three materials obtained from the cyclisation,
optimisation of the yield of the head-to-tail cyclised product was
attempted. Five different coupling agents were trialled over the
standard conditions (13 h at 70 ◦C) (Table 3). The ratio of the
products did not appreciably change after 13 hours, except in the
case of BOP and DIC cyclisations. For cyclisation with activating
agent DIC, very little cyclic material (either head to tail or head
to side chain) was obtained. Treatment with HATU resulted in a
much higher yield of the desired cyclic product.
It was observed that the best coupling agent to form the head-
to-tail cyclic material was HATU and the best to form the side-
chain cyclised material was BOP. However it became clear that
due to hydrolysis and slow decomposition, immediate purification
and photolysis was required. Interestingly, while conducting these
tests it was observed that 3 could be retreated with coupling agent
to obtain 5.
Analysis using mass spectrometry indicated that by-products of
HnB photolysis were reacting with the cyclic peptide. Photolysis of
o-nitrobenzylamines is known to proceed through the elimination
of o-nitrosobenzaldehyde, which can then break down to other
products when irradiated at wavelengths higher than 350 nm.42 To
try and limit the effect of these products, a number of conditions
were tested, including the addition of scavengers, using different
solvents, cooling the solution, diluting the solution and utilising
different workup conditions. Unfortunately, no improvement on
Table 3 Cyclisation yields and products of [HnB]Phe-(D)Arg-(D)Trp-
Gly-OH 1aea
Coupling
Reagent
Yieldb (%)
(1)
Yieldc (%)
(3)
Yieldd (%)
(5)
BOP
DIC
HBTU
PyBOP
HATU
11
80
40
8
37
12
27
51
63
52
8
33
41
21
Thus, three products were obtained through cyclisation with
the HnB auxiliary. The first of these products to elute by RP-
HPLC was the linear peptide, followed by the arginine-side chain
cyclised product and then the desired N- to C-termini cyclised
product.
16
a Relative yields calculated by HPLC Data after 13 hours heating at
70 ◦ C. b [HnB]Phe-(D)Arg-(D)Trp-Gly-OH 1ae c [HnB]Cyclo-[Phe-(D)Arg-
(D)Trp-Gly] 3ae. d [HnB]Phe-Cyclo-[(D)Arg-(D)Trp-Gly] 5ae.
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the 1% acetic acid/DMF solution could be found, except for the
dilution of the DMF solution to 0.1 mM.
It therefore seems that the substantial increase in yield for
both the cyclisation and photolysis of Hnb-Cyclo[His-(D)Phe-
Arg-(D)Trp] is the result of slow hydrolysis of this product. This
indicates that the stereoinversion to the (D)Trp product is a
thermodynamic sink.
As another confirmation of the structure of the head-to-tail
cyclic peptides 4aa and 4ac these products were re-synthesised
with all side-chain protecting groups 4ab and 4ad (producing
3, Scheme 1, Table 1). Overall yields for the cyclisation ring
contraction step for the protected peptide were up to half that
of the analogous reaction with the unprotected peptide. This
illustrates that the presence of protecting groups may hinder
cyclisation-ring contraction yields based on steric effects. However,
as expected the yields for the photolysis of the protected cyclic
peptides were much higher. Subsequent protecting group cleavage
and purification reduced the yields for this step as shown in Table 1,
so that the overall yield for the complete synthesis of the cyclic
tetrapeptide was similar when functional groups were protected or
unprotected. Five more peptides (4ag-4ak) were also synthesized
with all side-chain protecting groups giving comparable yields.
Interestingly, while the photolysis yield normally varied from
3–26%, one cyclic peptide displayed quite different results. Cyclo-
[His-(D)Phe-Arg-Trp] 4af photolysed in 72% yield. As the cyclisa-
tion only yielded one product in similar quantity to that obtained
for the other peptides and it was known that epimerisation of the
C-terminal residue of these peptides can be problematic,20 these
results were suspicious, even though Edman degradation proved
that the product was a head-to-tail cyclic peptide. Chiral amino
acid analysis revealed that the tryptophan had inverted in 99.7%
purity.
It was presumed that the stereoinversion must have occurred at
the cyclisation step. Cyclisation proceeds through the formation of
the C-terminal residue activated benzotriazole ester (from BOP),
followed by the nucleophilic substitution of the benzotriazole with
the phenolic oxygen of HnB. An O- to N- acyl transfer then
occurs, contracting the ring system. This product is known to
be unstable and the reaction reversible. Although epimerisation
of the C-terminal residue can occur prior to the formation of the
benzotriazole ester, this is unlikely given the rate of this intermolec-
ular reaction (analogous amide bond forming intermolecular
reactions are completed in minutes at room temperature). As
a result, stereoinversion most likely occurred from the phenolic
or benzotriazole esters or from the amide, i.e. immediately
prior to or during cyclisation. As cyclisation and stereoinversion
are reversible, this indicates that the LDLL cyclic tetrapeptide
is significantly more sterically constrained than the LDLD
peptide, effectively trapping the reaction after complete stereo-
inversion.
LDLD cyclic peptides are known to behave very differently to
other cyclic peptides. Ngu-Schwemlein et al. synthesized a series
of LDLD cyclic tetrapeptides in which every second residue was
(D)-alanine.11 In this case, the synthesis proceeded at elevated
temperature with an activated ester and no ring contraction
auxiliary was necessary. It has also been reported that cyclic LD
peptides can adopt flat conformations, in which the backbone
amides are orientated perpendicular to the side chains and the
plane of the ring system.30,31 The products (head-to-tail cyclo-
octapeptides) have also been known to form cylindrical b-sheet
peptide assemblies, although a dimerisation is more difficult
in smaller cyclic systems and is dependent upon amino acid
composition.30,31
NMR structure of cyclo[(L)His-(D)Phe-(L)Arg-(D)Trp]
The majority of cyclic tetrapeptides synthesized (1aa–1ak) ex-
hibited a great deal of line broadening by 1H NMR, making
structural analysis very difficult. Cyclo[Phe-(D)Arg-(D)Trp-Gly]
was a typical example (Fig. 2). However, cyclo[His-(D)Arg-Phe-
(D)Trp] exhibited a very sharp spectrum, and only one set of
signals. A solution structure was generated from this product.
A DQF-COSY, TOCSY, ECOSY and ROESY was run on
cyclo[His-(D)Arg-Phe-(D)Trp] in d6-DMSO and a TOCSY and
ROESY were run in two mixtures of acetonitrile/water/deuterium
oxide, in an effort to resolve all cross-peaks. As can be seen in
Table 4 a small change in chemical shift for the NH’s, Ha’s and
Hb’s could be observed. This is due to the change in hydrogen
bonding ability of the solvent systems, and may also indicate
a change in conformational state in aqueous media. However,
a solution structure was only examined in d6-DMSO. Analysis
of the spectrum indicated that the cyclic peptide possessed only
trans amide bonds. The ROESY spectrum exhibited very strong
inter-residue rOe’s between all NH (i) and Ha (i+1), but much
weaker intra-residue rOe’s between NH and Ha (Fig. 3) indicating
that these atoms are close in space, which is illustrative of a
trans configuration.3,11,14 In addition, no inter-residue cross-peaks
could be observed between any Ha’s, which is usually a sign of
cis amide bonds.14 According to the Karplus equation, the large
3
repeating JHN-Ha pattern of 9.19 Hz and 9.56 Hz indicates an
Fig. 2 1H NMR Spectrum (500 MHz), 9–6 ppm and 5–1 ppm, of Cyclo[Phe-(D)Arg-(D)Trp-Gly] 4ae and Cyclo]His-(D)Arg-Phe-(D)Trp] 4af.
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Table 4 Proton chemical shifts for cyclo[His-(D)Phe-Arg-(D)Trp]
Solvent
Proton
d6-DMSO
H2O/d3-MeCN/D2O (76.5:13.5:10)
d3-MeCN/D2O (9:1)
Random Coil Values32
Histidine
NH
Ha
8.034, J 9.56 Hz
4.612
8.407
4.865
8.41
4.63
4.870
Hb
ArH
2.983, 2.784
7.165, 8.827
3.235, 3.149
7.290, 8.220
3.361, 3.147
3.26, 3.20
7.14, 8.12
Phenylalanine
NH
7.962, J 9.19 Hz
8.311
8.23
Ha
Hb
2,6H
3,5H
4H
4.540
2.978, 2.792
7.15–7.23
4.917
3.308, 3.180
7.46–7.57
4.901
3.303, 3.208
7.49–7.57
4.66
3.22, 2.99
7.30
7.39
7.34
Arginine
NH
Ha
7.761, J 9.56 Hz
4.305
8.323
4.580
8.27
4.38
4.583
Hb
Hc
Hd
1.590, 1.462
1.364, 1.284
3.041
1.915, 1.826
1.631, 1.526
3.239
1.953, 1.837
1.717, 1.619
3.331
1.89, 1.79
1.70
3.32
NHe
Tryptophan
NH
7.429
7.290
7.17, 6.62
7.928, J 9.19 Hz
4.634
8.373
5.030
8.09
4.70
Ha
4.967
Hb
ArH
NH1
3.139, 2.810
6.942, 7.025, 7.047, 7.315, 7.476
10.717
3.491, 3.283
7.359, 7.470, 7.717, 7.884
10.261
3.525, 3.258
7.315,7.333, 7.442, 7.681, 7.858
3.32, 3.19
7.17, 7.24, 7.50, 7.65
10.22
away from each other as possible. In addition, identical ECOSY
cross-peaks between Ha and Hb indicate that rotation about this
bond is largely unrestrained.
Variable temperature experiments were conducted on the cyclic
tetrapeptide in d6-DMSO between 298 and 338 K (Fig. 4). It
appeared that there are no strong hydrogen bonding interactions
as all NH’s exhibited a change in chemical shift with temperature
of between 3 and 5 ppb/K. When a D2O exchange experiment
was conducted (Fig. 5) it was clear that the amide NH’s of Arg,
Trp and Phe all exchange very slowly (approximately 11.5, 11
and 4.5 h, respectively for half of the NH’s to exchange), but
the His NH is fully exchanged after approximately 5 h. When
these pieces of data are examined together it becomes clear that
none of the amide bond NH’s are involved in hydrogen bonding,
although they all appear to be quite buried in the molecule. This
concurs with a previously reported LDLD cyclic peptide structure
(cyclo[Leu-(D)Ala-Leu-(D)Ala]) by Ngu-Schwemlein et al., in
Fig. 3 NH-Ha region of ROESY spectrum in d6-DMSO.
almost antiperiplanar relationship between NH and Ha, which
corresponds to a ␾ angles of around 120◦ (CO-NH-Ha-CO
torsion angle) and also illustrates a symmetry to the peptide
backbone. These coupling constants are typical of extended
peptide units, such as a b-sheet32 and is therefore indicative of
trans amide bonds.
The amino acid side chains in this cyclic tetrapeptide exhibited
very few inter-residue rOe’s. This would be expected, as the
orientation of the backbone forces the side chains to be as far
Fig. 4 Variable temperature experiment: Dd amide nitrogen 1H NMR
shift versus temperature (d6-DMSO).
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Fig. 5 D2O exchange experiment (5% D2O in d6-DMSO).
which an open b-turn structure not stabilized by intramolecular
hydrogen bonding was proposed.11
From the information collected, a solution structure was
generated using X-PLOR. This resulted in x angle (Ca-CO-NH-Ca
torsion angles) violations. X-ray structures of cyclic tetrapeptides
indicate that the x angles (Ca-CO-NH-Ca torsion angles) are
quite commonly twisted from planarity, regardless of whether the
amide bonds are cis or trans.12,13,15–18,33 In fact, the all-trans cyclic
tetrapeptides commonly possess a x angle twist of 15–25 ◦.3,5,12,13
As a result, all of the energy restraints in parallhdg5.2.pro (the X-
PLOR parameter file) on the x torsion angle were relaxed from
their default value of 500. Solution structures were then calculated
at energy values of 500, 250, 150 and 50 (Table 5, Fig. 6).
As can be seen in Table 5 and Fig. 6, the x dihedral angles
twist to approximately 20◦ when the energy restraint is relaxed to
50, which correlates well with twist seen in X-ray structures. This
structure also displays a relatively small change in energy over
the 20 lowest energy structures, and the smallest RSMD. Another
factor of note is that as the x twist energy restraint is relaxed, the
direction of planarity of the amide bond becomes more and more
perpendicular to the plane of the peptide backbone.
Fig. 6 Superimposition of the peptide backbone and bC (purple) of the
20 lowest energy minimised structures of cyclo-[His-(D)Phe-Arg-(D)Trp],
in which the x torsion angle energy restraint is relaxed to 50.
Replacement of thermal heat with microwave irradiation resulted
in shorter reaction times. Isolation of 3 was not carried out, as the
crude mixture was photolysed immediately to generate the desired
cyclic tetrapeptide 4.
Due to the difficulties in synthesising these highly constrained
macrocyles, in combination with the well known “sequence depen-
dency” on cyclisation, it was not expected that every compound
Synthesis of the cyclic-tetrapeptide library
Having explored the synthesis and characterisation of a small
number of cyclic-tetrapeptides, a general synthesis of a 44 member
library was conducted (Table 6). This synthesis was carried out
using HATU cyclisation and microwave assisted ring contraction.
Table 5 Solution structure information from the relaxation of the x angle energy restraints on cyclo[His-(D)Phe-Arg-(D)Trp]
Restraint energy of Omega bond (dihedrals)
500 (default)
250
150
50
DE for top 20 structures
11.7 kcal/mol
8.4 kcal/mol
10.0 kcal/mol
5.4 kcal/mol
RSMD for backbone atoms (N,C,CA) of top 20 structures
0.069
−174.72
175.10
0.109
−171.48
170.47
0.097
−166.88
166.48
0.075
−160.45
159.56
xHis-Phe
xPhe-Arg
xArg-Trp
xTrp-His
−175.53
−171.52
−167.17
−160.49
173.98
169.33
165.66
159.70
␾
−139.19
144.37
−131.07
141.79
−125.01
129.18
−100.70
106.11
His
␾
Phe
␾
−142.25
−134.13
−125.97
−101.12
Arg
␾
Trp
149.56
143.59
130.90
105.29
wHis
wPhe
wArg
wTrp
39.53
40.71
47.87
55.77
−39.91
−44.60
−49.51
−59.79
34.96
38.75
46.02
56.75
−42.35
−47.19
−50.02
−59.95
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Table 6 Cyclic tetrapeptide library synthesis
Linear peptidea (1)
Yield (4)
Mass Theoryc
Mass Foundc
al
[HnB]Ala-Arg-Tyr-Gly-OH
[HnB]Ala-Ile-Ile-Gly-OH
[HnB]Ala-Asn-His-Gly-OH
[HnB]Phe-Gly-Asn-Gly-OH
[HnB]Phe-His-Ala-Gly-OH
[HnB]Phe-Asn-Phe-Gly-OH
[HnB]Phe-Arg-His-Gly-OH
[HnB]Tyr-Ala-Ile-Gly-OH
[HnB]Tyr-Asn-Ile-Gly-OH
[HnB]Tyr-Trp-Phe-Gly-OH
[HnB]Tyr-Trp-Ile-Gly-OH
[HnB]Ile-Ala-Val-Gly-OH
[HnB]Ile-Trp-Phe-Gly-OH
[HnB]Asn-His-Phe-Gly-OH
[HnB]Asn-Arg-Phe-Gly-OH
[HnB]Asn-Ser-Trp-Gly-OH
[HnB]Arg-His-Asn-Gly-OH
[HnB]Arg-Ile-His-Gly-OH
[HnB]Thr-His-Phe-Gly-OH
[HnB]Trp-Ala-His-Gly-OH
[HnB]Trp-Asn-His-Gly-OH
[HnB]Trp-Arg-Phe-Gly-OH
[HnB]Tyr-Ile-Gln-Gly-OH
[HnB]Phe-Tyr-Ala-Gly-OH
[HnB]Ala-Arg-Tyr-Gly-OH
[HnB]Ile-Arg-Tyr-Gly-OH
[HnB]Ile-Trp-Tyr-Gly-OH
[HnB]Tyr-Ile-Ile-Gly-OH
[HnB]Tyr-Asn-Arg-Gly-OH
[HnB]Arg-His-Ile-Gly-OH
[HnB]Phe-Arg-Ile-Gly-OH
[HnB]Arg-Arg-Gly-Gly-OH
[HnB]Asn-Arg-Tyr-Gly-OH
[HnB]Arg-Tyr-Val-Gly-OH
[HnB]Trp-Arg-Gly-Gly-OH
[HnB]Gln-Tyr-Trp-Gly-OH
[HnB]Arg-Tyr-Arg-Gly-OH
[HnB]Ala-Trp-Ile-Gly-OH
[HnB]Arg-Trp-Val-Gly-OH
[HnB]Ser-Ile-Ile-Gly-OH
[HnB]Arg-Ser-Ile-Gly-OH
[HnB]Phe-Arg-Trp-Gly-OH
[HnB]Tyr-Val-Phe-Gly-OH
[HnB]Phe-Val-Tyr-Gly-OH
29%
5%
9%
8%
9%
4%
12%
9%
—
4%
5%
—
—
—
448.23
355.24
380.29
376.16
413.20
466.21
498.26
405.22
448.21
554.24
520.26
341.21
504.39
456.20
475.24
445.19
465.23
464.28
443.21
452.21
495.21
547.28
462.24
439.20
448.23
490.28
420.26
447.26
491.24
464.28
474.29
427.26
491.24
476.26
457.23
535.23
533.30
428.23
499.28
371.23
414.25
547.28
467.23
467.23
448.23
355.22
380.28
376.28
413.22
466.34
498.39
405.41
—
554.40
520.23
—
—
—
am
an
ao
ap
aq
ar
as
at
au
av
aw
ax
ay
az
ba
bb
bc
bd
be
bf
bg
bh
bi
bj
bk
bl
bm
bn
bo
bp
bq
br
bs
bt
bu
bv
bw
bx
by
bz
ca
cb
cc
6%
—
474.50
—
10%
15%
10%
7%
5%
11%
—
11%
19%
11%
—
—
—
11%
16%
—
7%
9%
12%
28%
9%
14%
11%
7%
12%
5%
7%
6%
465.25
464.27
444.18
452.20
495.28
547.29
—
439.22
478.23
490.30
—
—
—
464.31
474.40
—
491.32
476.35
457.22
535.22
333.69
428.25
499.34
371.20
414.26
547.33
467.42
467.32
a All naturally occurring amino acids are the L-isomer. b % Yield over two steps from 1 without purification of the intermediate material; i) 1 eq HATU,
2 eq DIEA, 1 mM in DMSO, 3h/rt; ii) 10 eq DIEA, 3h/microwave; iii) AcOH added to make a 1% solution, ht, 3 h. c Mass of the purified cyclic
tetrapeptide. Characterisation was based upon mass spectroscopy results.
could be successfully prepared. Surprisingly, around 70% of all of
these peptides were successfully synthesised.
yields were low and this is probably a reflection of the photolysis
rather than the ring contraction.
Some unsuccessful syntheses contained b-branched amino
acids (Val, Ile), indicating that steric problems were a possible
cause. Cyclo[Tyr-Ala-Ile-Gly] 4as was successful, whilst related
compound cyclo[Ile-Ala-Val-Gly] 4aw was not. Further, cyclo[Tyr-
Trp-Phe-Gly] 4au succeeded, whilst cyclo[Ile-Trp-Phe-Gly] 4ax did
not. When sequences were changed, dramatic effects were also
observed. Cyclo[Ile-Arg-Tyr-Gly] 4bk succeeded, whilst cyclo[Ile-
Trp-Tyr-Gly] 4bl could not be prepared. Strikingly cyclo[Gln-
Tyr-Trp-Gly] 4bu was synthesised in relatively high yield, whilst
the related compound cyclo[Asn-Ser-Trp-Gly] 4ba could not be
prepared. This sequence dependent effect on cyclisation had been
noted previously, and consequently HnB had been incorporated
into the backbone of cyclic tetrapeptides to further facilitate
cyclisation of these more difficult sequences.19 Overall, isolated
Edman degradation was performed on the successfully synthe-
sised library members. In most cases the correct cyclic material was
observed. The only exception was when arginine was present in the
4th position (Table 6). The library contained 7 such sequences 4bb,
4bc, 4bo, 4bq, 4bs, 4bv, 4bx, 4bz. Of these only 3 of the cyclised
products contained both the head-to-tail cyclic material and the
sidechain-to-head cyclic material 4bo, 4bs, 4bz. Interestingly when
Asn was placed in the 4th position no side chain cyclic product
was observed. In addition, side chain cyclised products were not
identified by mass spectrometry when nucleophilic sidechains were
positioned in the 2nd or 3rd position. This is probably an indication
that 9 to 11 membered rings would not form due to the constrained
nature of these systems, and consequently that the formation of
the cyclic tetrapeptides would be favoured. This library is currently
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being screened against a number of G-protein coupled receptors
and protein kinases.
Separations were achieved using linear gradients of buffer B in
A (A = 0.1% aqueous TFA; B = 90% CH3CN, 10% H2O, 0.09%
TFA) at a flow rate of 1 mL/min (analytical) and 20 mL/min
(preparative). Microwave irradiation of library members was per-
formed sing the Ethos Microwave Labstation (Milestone Inc., CT,
USA). Abbreviations: MeCN, acetonitrile; TFA, trifluoroacetic
acid; DCM, dichloromethane; petrol, petroleum spirit (bp 40–
60 ◦C); EtOAc, ethyl acetate; DIEA, diisopropylethyl amine;
AcOH, acetic acid; MeOH, methanol; Et2O, diethyl ether; DMSO,
dimethylsulfoxide; DMF, N,N -dimethylformamide; HBTU,
O-benzotriazol-1-yl-N,N,N’,N’-tetramethyluronium hexafluoro-
phosphate; HATU, O-(7-azabenzotriazol-1-yl)-N,N,N’,N’-tetra-
methyluronium hexafluorophosphate; BOP, benzotriazol-1-yl-
oxytris(dimethyamino)phosphonium hexafluorophosphate; DIC,
diisopropylcarbodiimide; PyBOP, benzotriazole-1-yloxytripyr-
rolidinophosphonium hexafluorophosphate.
Conclusion
Despite three decades of discovery of numerous biologically active
cyclic tetrapeptides from various microbial sources and their
reported oral activity, these products have been largely unexplored
in the pharmaceutical industry. Primarily this is due to their
synthetic inaccessibility.
A photolabile ring-contraction auxiliary was used for the
synthesis of number of cyclic tetrapeptides and the cyclisation
and photolysis steps were examined and optimised. In parallel
with this, a suite of characterisation techniques was developed,
including Edman degradation and mass-spectrometry/mass-
spectrometry. An NMR solution structure of cyclo[His-(D)Phe-
Arg-(D)Trp] was also generated, which displayed all trans amide
bonds. The alternating L and D amino acids allowed the cyclic
peptide to adopt a relatively flat configuration in which the
backbone amides are orientated perpendicular to the ring system.
This product was synthesised from cyclo[His-(D)Phe-Arg-Trp] and
a complete stereoinversion at the C-terminal residue occurred
during the synthesis, illustrating that the LDLL cyclic peptide is
significantly more sterically constrained than the LDLD peptide.
A small number of cyclic tetrapeptides were synthesised to find
the optimal conditions for cyclisation. Following this, a larger
library of 34 compounds was synthesised and these are currently
being tested for biological activity aginst a number of GPCRs.
HnB is an ideal auxiliary for this procedure due to its superior
acyl transfer properties, its ready accessibility and its photolability.
Using this auxiliary, a range of cyclic tetrapeptides may be
synthesised which would be inaccessible by other means.
Materials
Aminomethyl polystyrene resin (sv = 0.41 mmol/g) and all
Na-Fmoc-amino acids were peptide synthesis grade purchased
from Auspep (Melbourne Australia) or Novabiochem (San
Diego, USA). Dichloromethane, diisopropylethylamine, N,N -
dimethylformamide, and trifluoroacetic acid were obtained from
Auspep (Melbourne, Australia). HPLC grade acetonitrile was
purchased from BDH (Brisbane, Australia). 2-(1H-Benzotriazol-
1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate (HBTU)
was purchased from Richelieu Biotechnologies (Quebec, Canada).
1H NMR spectrometry
All NMR experiments were recorded on a Bruker ARX 500
spectrometer equipped with a z-gradient unit at 298 K except
for variable temperature experiments. Peptide concentrations were
∼3 mM. Cyclo[His-(D)Arg-Phe-(D)Trp] was examined in d6-
DMSO, H2O/d3-MeCN/D2O (76.5:13.5:10) and d3-MeCN/D2O
Materials and methods
Thin Layer Chromatography (TLC) was performed on silica gel
60 F254 plates (Merck). The chromatograms were viewed under
U.V. light. Flash column chromatography was performed with
flash silica gel 60 (0.063–0.200 mm, Merck). Nuclear Magnetic
Resonance spectra were recorded at 300 MHz (1H)/75 MHz (13C)
or 500 MHz (1H) on a Varian Gemini-300 or a Bruker ARX 500
spectrometer, respectively. 1H and 13C chemical shifts (d) are given
in parts per million (ppm) using residual protonated solvent as
an internal standard. Coupling constants are given in Hertz (Hz).
The following abbreviations are used: s = singlet, d = doublet,
t = triplet, m = multiplet. Low resolution mass spectral data were
recorded on a Micromass LCT (TOF MS ES+) instrument. High
resolution mass spectral data was obtained on a PE Sciex API
QSTAR Pulsar (ES-QqTOF) instrument using ACP (acyl carrier
protein) (65–74) (C47H75N12O16 (M + H), 1063.5424) and reserpine
(C33H40N2O9 (M + H), 609.2812) as internal references. Resolution
for the instrument was set between 10,000 and 12,000 for all stan-
dards. Melting points were determined on a Bausch and Lomb hot
stage. Chiral amino acid analysis was performed by C.A.T. GmbH
& Co. Chromatographie und Analysentechnik KG, Tu¨bingen,
Germany. Analytical reversed-phase HPLC was performed on a
Vydac C18 column (4.6 × 250 mm). Preparative reversed phase
HPLC was performed on a Vydac C18 column (22 × 250 mm) or
1
(9:1). H NMR experiments recorded were ROESY with mixing
time of 400 ms, TOCSY, DQF-COSY and E-COSY. All spectra
were run over 5555 Hz with 4 K data points, 16–24 scans. Spectra
was processed using XWIN-NMR and 1H chemical shifts are given
in parts per million (ppm) using residual protonated solvent as an
internal standard. Coupling constants are given in Hertz (Hz).
3JNH-Ha coupling constants were measured on the 1D spectrum.
Distance restraints and structure calculations
Peak volumes in ROESY spectra were classified as strong,
medium, weak and very weak, corresponding to upper bounds
˚
on interproton distances of 2.7, 3.5, 5.0 and 6.0 A, respectively.
Appropriate pseudoatom corrections were made,34 and distances
˚
of 0.6 and 2.0 A were added to the upper limits of restraints
involving methyl and phenyl protons, respectively. 3JNH-Ha coupling
constants were used to determine ␾dihedral angle restraints,35 and
as all coupling constants exceeded 9.0 Hz, ␾ was restrained to +
or-120 30 ◦ for L- and D- amino acids, respectively.
Structures were calculated using the torsion angle dynam-
ics/simulated annealing protocol in X-PLOR version 3.851 using
a modified geometric force field based on parallhdg5.2.pro.36
Structure refinements were performed using energy minimisa-
tion (2000 steps) using parallhdg5.2.pro. Structure modelling,
˚
Phenomonex Jupiter 10 l 300 A C18 column (21.2 × 100 mm).
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visualisation and superimpositions were done using Insight II
(MSI). RMS deviations, and the quality of the solution structure
was determined in X-PLOR.
acetonitrile (50%). The product was then purified by reversed-
phase HPLC.
Peptide cyclisation
Edman degradation
1 equivalent of BOP or HATU and 2 equivalents of DIEA were
added to a 1 mM solution of the linear peptide in DMSO and
stirred for 3 h at rt. 10 equivalents of DIEA were then added
and the solution stirred overnight at 70 ◦C. The DMSO was then
removed under vacuum, the residue dissolved in a solution of
aqueous acetonitrile (50%) and lyophilised before purification by
reversed-phase HPLC.
Edman degradation studies were performed according to the
method of Pitt et al.,37 with the following modifications. Less
than 0.01 mg cyclic peptide was treated with 25 lL of a
phenylisothiocyanate/pyridine/H2O (1:1.5:1.5) solution. The so-
lution was left at room temperature for 1.5 h under argon then the
aqueous phase was extracted twice with 400 lL hexane/EtOAc
(2:1). Trifluoroacetic acid (20 lL) was then added to the aqueous
layer, which was subsequently left at room temperature for 1 h.
Finally, 60 lL of water was added and the resulting solution
analysed by ES-MS.
General procedure for photolysis
Cyclic peptides were dissolved in 1% acetic acid/DMF to make a 1
to 0.1 mM concentration. The solution was placed in a beaker and
photolysis carried out for 5 hours using a UV lamp (350–365 nm,
20 W, Black/White/Blue). The DMF was removed under vacuum,
the residue dissolved in a solution of aqueous acetonitrile (50%)
and lyophilised, before purification by reversed-phase HPLC.
The Pbf protecting group was then removed by treating the
product with 400 lL cleavage mixture (92.5% TFA, 5% H2O, 2.5%
TiPS) for 1 hour. The TFA solution was then blown off with
argon and the residue dissolved in a small amount of acetonitrile
and lyophilised. The product was then purified by reversed-phase
HPLC.
Synthesis
2-Hydroxy-6-nitrobenzaldehyde (HnB)38. meta-Nitrophenol
(10.0 g, 71.9 mmol) was dissolved in 57.1 mL 75% poly-phosphoric
acid and was heated to 100 ◦C. Hexamine (10.2 g, 72.4 mmol)
was then slowly added to this stirred solution. After the addition
of the hexamine, the solution was stirred for a further 50 minutes
before it was cooled and dissolved in water. This aqueous solution
was then extracted several times with ethyl acetate, before drying
the organic layer with MgSO4 and removing the ethyl acetate
under vacuum. The crude product was then chromatographed
over silica gel (10% EtOAc/petrol) and dried to produce a yellow
solid (1.25 g, 7.48 mmol). Yield: 10.4%. Melting point: 51–52 ◦C
Library synthesis of peptides
◦
1
All reactions were performed on a Bohdan MiniBlockTM using
1.5 mL of solvent for all reactions and washings. All linear
peptides were chemically synthesised stepwise as before using
Fmoc protecting groups and in situ HBTU activation protocols as
previously described.39,40 Coupling efficiencies were determined by
the quantitative ninhydrin test41 and recoupled where necessary to
obtain >99.5% efficiency. HnB was introduced as reported above.
Cleavage was performed using TFA 95% with 5% H2O, 1h at
RT. The resin was filtered and filtrate evaporated under reduced
pressure. The residue was washed with ether (2 × 10 mL) and
dissolved in aqueous acetonitrile (50%). The product was then
purified by reverse-phase HPLC (Table 6).
(lit. 53–54 C). H NMR (300 MHz, CDCl3) d 7.30 (m, 1H),
7.55 (m, 1H), 7.63 (t, 1H), 10.31 (s, 1H), 12.09 (s, 1H); 13C NMR
(75 MHz, CDCl3) d 112.3, 116.0, 124.1, 135.9, 151.2, 163.2, 193.8.
General procedure for the synthesis for peptides
Synthesis of linear peptides on resin. All linear peptides were
chemically synthesised stepwise using Fmoc protecting groups and
in situ HBTU activation protocols as previously described.39,40
Coupling efficiencies were determined by the quantitative nin-
hydrin test41 and recoupled where necessary to obtain >99.5%
efficiency. These peptides are displayed in Table 1.
HATU (1 eq) and DIEA (2 eq) were added to a 1 mM solution of
the linear peptide in DMSO and stirred for 1 h at RT. Excess DIEA
(10 eq) was then added and the solution stirred for 3 h at 100 ◦C
using the Ethos Microwave Labstation. After ring contraction,
the solution was placed in a beaker (2 mL). AcOH was added and
photolysis was carried out for 3 hours using a UV lamp (350–
365 nm, 20 W, Black/White/Blue). The DMSO was removed
under vacuum, the residue dissolved in aqueous acetonitrile (50%)
and the product purified by reversed-phase HPLC.
Introduction of HnB
The 2-hydroxy-6-nitrobenzaldehyde (2 eq) was dissolved in
MeOH/DMF (1:1) (0.1 M) and added to the amino peptide resin.
After 5 min the resin was filtered and a second portion of aldehyde
(2 eq) added. After another 5 min the resin was filtered and washed
with MeOH/DMF (1:1). 0.1M NaBH4 (10 eq) in MeOH/DMF
(1:3) was then added and the reaction mixture left standing for
5 min. The resin was again filtered and washed with MeOH/DMF
(1:3), DMF, MeOH/DCM (1:1) and air-dried.
Acknowledgements
Cleavage
We would like to acknowledge the Australian Research Council
(ARC) and the Biotechnology Infrastructure Fund (BIF) for their
financial support. We would also like to thank David Craik,
Christina Schroeder, Martin Stoermer, Sue Kydd, Peter Seale, John
Murray and Mike Hann.
Peptides on chlorotrityl resin were cleaved using TFA (either 95%
with 5% H2O or 1% in DCM, 10 mL/500 mg resin, 1h at RT). The
resin was filtered and solvents evaporated in vacuo. The residue
was washed with ether (2 × 10 mL) and dissolved in aqueous
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