Serine-threonine protein phosphatase inhibitors derived from nodularin: Role of the 2-methyl and 3-diene groups in the Adda residue and the effect of macrocyclic conformational restraint

Article abstract of DOI:10.1039/b100402f

In order to probe the effect upon macrocycle conformation and PP1_cat enzyme inhibition of structural changes to nodularin, specific replacements for the Adda residue were introduced. Two new analogues, cyclo[-(3S,E)-3-phenylethenyl-3-aminopropa

Full text of DOI:10.1039/b100402f

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Serine–threonine protein phosphatase inhibitors derived from
nodularin: role of the 2-methyl and 3-diene groups in the Adda
residue and the effect of macrocyclic conformational restraint
1
Michael E. O’Donnell, Jonathan Sanvoisin and David Gani*
School of Chemistry, The University of Birmingham, Edgbaston, Birmingham, UK B15 2TT
Received (in Cambridge, UK) 9th January 2001, Accepted 24th May 2001
First published as an Advance Article on the web 4th July 2001
In order to probe the effect upon macrocycle conformation and PP1cat enzyme inhibition of structural changes
to nodularin, specific replacements for the Adda residue were introduced. Two new analogues, cyclo[-(3S,E)-3-
phenylethenyl-3-aminopropanoyl-α-(R)-Glu-α-OH-γ-Sar-(R)-Asp-α-OH-β-(S)-Phe-] 19a and cyclo[-(2S,3S,E)-2-
methyl-3-phenylethenyl-3-aminopropanoyl-β-(R)-Glu-α-OH-γ-Sar-(R)-Asp-α-OH-β-(S)-Phe-] 19b were prepared
incorporating previously optimised preparative protocols [see previous article, K. L. Webster, A. B. Maude,
M. E. O’Donnell, A. P. Mehrotra and D. Gani, J. Chem. Soc., Perkin Trans. 1 (DOI: 10.1039/b100401h)], and
these differed only at C-2 of the Adda residue. The presence of a (2S)-methyl group in compound 19b stabilised the
trans-rotameric form of the (2R)-Glu-γ-Sar amide bond in solution as determined by NMR spectroscopic analysis
(trans–cis; 10:1), and enhanced efficacy as a PP1cat inhibitor by 20-fold over compound 19a. The methyl homologue
displayed a competitive mode of inhibition, with respect to the substrate Ac-Arg-Arg-Thr(P)-Val-Ala and
displayed a Ki value of 206 30 µmol dmϪ3. Substitution of the Sar residue in the methyl homologue by (2S)-Pro
gave a competitive inhibitor of similar efficacy (Ki = 400 75 µmol dmϪ3). The proline analogue 22 existed as a
6:1 mixture of trans–cis rotamers. Evidently the trans-rotamer of the (2S)-Pro-containing compound differed in
conformational structure compared to the sarcosine-containing variant, only close to the site of the substitution.
A structural model for the inhibition of PP1cat and a strategy for the selective inhibition of PP1 over PP2A are
discussed within the context of the results.
molecules and/or ions as inhibitors and activators. Type 1
Introduction
PPase activities (PP1) are those that are inhibited by the small
thermostable, acid-resistant proteins inhibitor 1 (I-1), inhibitor
2 (I-2 or modulator) and DARPP-32.5 Type 2 activities
(PP2) are insensitive to inhibitors 1 and 2 and are further
sub-divided into three types, PP2A, PP2B (calcineurin) and
PP2C, according to their requirement for divalent cations and
modulating proteins and their primary structures. PP1, PP2A
and PP2B are structurally related to each other and possess
almost identical active sites.6 Each site contains two cat-
alytically essential divalent metal ions, usually two Mn2ϩ ions,
and shows considerable similarity to purple acid phosphatase.7
PP2B is differentiated from PP2A through its activation by
calmodulin while PP2C possesses an unrelated primary struc-
ture and requires Mg2ϩ ions.8
The native catalytic PP1cat and PP2Acat subunits show little
substrate specificity and are able to dephosphorylate a range
of substrates.9 However, they are highly regulated in vivo by
the action of the modulating subunits and endogenous protein
inhibitors described above, and by compartmentalisation,
through subcellular localisation. Our attentions have been
focussed in two major areas. The first has concerned the mode
of action of the highly toxic natural product inhibitors typified
by microcystin and nodularin, with the objective of developing
inhibitory analogues that are differentially selective for PP1cat
and PP2Acat, see preceding article.10 The second has striven to
understand the mechanism of catalytic hydrolysis and its
allosteric modulation. To these ends we have recently developed
a new and reliable assay for the catalytic subunits of both PP1
and PP2A using 14C-radiolabelled phosphopeptide substrates,
see following article.11 We have also shown that dephosphoryl-
ation occurs via a ternary complex mechanism in which water
directly attacks the phosphate ester moiety.12 The new assay
Reversible protein phosphorylation plays a major role in the
control of a wide range of metabolic processes. In eukaryotic
cells, the reversible phosphorylation–dephosphorylation of
serine, threonine and tyrosine residues serves as a switch in
turning on and off key enzyme activities within cells. This is
achieved by inducing changes in protein conformation and
charge that alter binding affinities in protein–protein and
protein–ligand interactions.1 The downstream effect is the
regulation of a host of cellular processes including glycogen
synthesis, cell differentiation and proliferation, gene expression,
muscle contraction and the control of signal transduction
pathways.2 While protein kinases as a group have been the focus
of attention for decades, recent years have witnessed enormous
advances in understanding the role, modulation and structure
of protein phosphatases (PPases).3
To date, eight types of Ser-Thr protein phosphatase have
been identified and four of these, PP1 and PP2A, B and C,
see below, have been studied in some detail. The catalytic
subunit of such phosphatases, for example PP1cat, is usually
associated with one of a number of modulating subunits.4
These modulating proteins are able either to enhance catalytic
activity and, simultaneously, modify substrate specificity, or
to inhibit activity. Given that the modulating protein may itself
be a phosphoprotein substrate for the same or a different phos-
phatase catalytic subunit, the situation is extremely complex.
In view of the overlapping specificity of different types of
catalytic subunit for given substrates and the effects of modu-
lation on altering the substrate specificity of a given catalytic
subunit, PPases cannot be classified by substrate alone. It
has been necessary to include consideration of the action of
the modulating protein subunits and, also, the action of small
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This journal is © The Royal Society of Chemistry 2001
DOI: 10.1039/b100402f

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protocol (described in full in the following article11) has facili-
tated a detailed analysis of the binding determinants for
substrates and inhibitors and has allowed a direct comparative
analysis of the activities of PP1cat and PP2Acat. Most pertinent
is the low affinity of the dephosphorylated product peptide for
the enzyme (PP1cat) and the predominant effect of inorganic
phosphate as a product inhibitor. In the current study, this
information, together with the preliminary data on the struc-
tural binding determinants for nodularin analogues, has been
utilised in the design of inhibitory probes for PP1cat to test the
role of key moieties in the macrocycle. Here we report on the
synthesis of such nodularin analogues and on their conforma-
tional properties and biological activities. Based upon these
results we provide a structural model for inhibitor binding
interactions at the active site of PP1cat. These interactions
suggest a strategy for the preparation of selective inhibitors that
can differentiate between PP1cat and PP2Acat.
Results and discussion
In previous work,10,13–15 a number of synthetic strategies for
preparing nodularin analogues were considered. The three key
requirements concerned facilitating the construction of linear
precursors to the nodularin macrocycle, performing macro-
lactamisation reactions, and introducing suitable lipophilic
moieties into Adda [(2S,3S,4E,6E,8S,9S)-3-amino-9-methoxy-
2,6,8-trimethyl-10-phenyldeca-4,6-dienoic acid] surrogates.
This work established that strategies based upon the gel-
phase construction of fully functionalised linear isopeptidic
precursors, already possessing preformed Adda surrogates,
and solution-phase, off-resin, macrolactamisation worked best.
Given these potentially robust preparative protocols, attention
could be turned to the molecular recognition issues under-
pinning the design of selective inhibitors for PP1cat and for
PP2Acat.
Before embarking on such, a comment on safety in relation
to research strategy is relevant. In previous work in which we
had started to explore the potential biological activity of PP1cat
inhibitors, we were acutely aware of the severe toxicity of the
natural nodularins and microcystins. Such compounds display
IC50 values for PP1cat of ca. 0.1 nM and are potent hepatotoxins
in a diverse array of species including mammals and including
man.16 We were concerned to the extent that we chose to
deliberately tune-down biological activity. This was achieved
by replacing the Adda side-chain in nodularin analogues by
smaller lipophilic side-chains, for example, as in compounds
1 and 2. The Adda side-chain in nodularin is thought to bind to
a hydrophobic groove formed by residues Ile-130, Ile-133, Tyr-
134 and Tyr-206 in PP1cat, as for microcystin. Such analogues
1 and 2 displayed Ki values in the low millimolar range and
were expected, on the basis of the correlation of hepatotoxicity
with efficacy as a PP1cat inhibitor, to be some 107 times less
toxic. This millimolar range for inhibitory activity seemed ideal
for inhibitor development in that both improvements and
impairments in binding could be detected easily without
exposing research personnel to highly toxic end products. Thus,
for the purposes of refining our understanding of how to dif-
ferentially inhibit PP1cat and PP2Acat in the present study, we
opted to continue to employ tuned-down non-toxic inhibitors.
It was also expected that for biochemical studies designed to
deconvolute the individual role of each enzyme, access to highly
selective inhibitors for each type was more important than
absolute inhibitory efficacy. Moreover, it was reasoned that the
absolute efficacy of any given inhibitor could be enhanced at
any stage simply by re-introducing a larger part of the Adda
residue, or all of it, should this be necessary.
synthetic analogues differ from the natural products in that the
α-methyl group of the Adda surrogate (at residue position 1)
is missing. Thus, we wished first to address the effect of the
methyl group on the conformation of the macrocycle and,
in turn, the correlation of changes in conformation with
biological activity. In order to relate our findings to unbiased
macrocycles, in the first instance, we chose to use sarcosine, an
unrestained N-alkyl amino acid residue, at position 3. We also
opted to prepare an Adda surrogate that would more closely
resemble the diene moiety appended to the β-position of the
Adda residue. The rigid (E)-phenethenyl moiety seemed ideal
for this purpose and, hence, homochiral 3-phenethenyl-3-
aminopropanoic acids were chosen for residue position 1.
This strategy was expected to increase the confidence level of
modelling the active-site interactions of PP1cat and also PP2Acat
with nodularin-type inhibitors on the basis of structure–
activity relationships.
Synthesis of 3-phenethenyl-3-aminopropanoic acids
Several groups have reported the total synthesis of Adda and its
derivatives.19 Typically, the construction of the Adda residue
has required 19–20 steps and has provided the product in 5%
overall yield. However, a very recent synthesis was reported in
which Adda was obtained in 40% overall yield over 13 steps.20
Many of the synthetic schemes share a common connective
strategy based on development of the (E,E)-diene framework
via Julia–Wittig type chemistry, which does not allow for
complete control of the stereochemistry of the double bonds.
Given our desire to produce tuned-down macrocyclic analogues
possessing homochiral 3-phenethenyl-3-aminopropanoic acids,
we looked to utilise an asymmetric conjugate addition reaction
of the type developed by Davies and Waters for the intro-
duction of the 3-amino group.21 Such reactions use a chiral
lithium amide, for example, that derived from (S)-benzyl-α-
methylbenzylamine, to control the absolute stereochemistry at
C-3 in the product. To prepare the 2-methyl analogue it was
expected that C-methylation of the enolate derived from the
addition product would proceed diastereoselectively to give
(2S)-absolute stereochemistry.
Accordingly, Horner–Wadsworth–Emmons reaction of
methyl diethylphosphonoacetate with cinnamaldehyde gave the
required (E,E)-dienoate precursor 3 in 93% yield (Scheme 1).22
Reaction of lithium (S)-N-benzyl-N-α-methylbenzylamide
with the dienoate 3 afforded the desired 1,4-addition product 4a
with high diastereoselectivity in 61% yield along with the 1,4-
and 1,2-bis-addition products in 15% yield. No 1,6-addition
product was detected. For non-methylated target residue 10a,
With respect to improving efficacy in the tuned-down
analogues, it was noted that compounds 1 and 2 did not exist as
single conformers or rotamers in solution, as determined by
NMR spectroscopy,10 unlike the natural nodularins.17,18 The
J. Chem. Soc., Perkin Trans. 1, 2001, 1696–1708
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the tertiary amine 4a was stored pending deprotection as
described below.
In view of these findings, it was decided to remove the N-
benzyl groups with concomitant reduction of the double bond,
as in formation of 5a, and to re-introduce the double bond later
on in the synthesis, through benzylic bromination followed by
the elimination of hydrogen bromide. To avoid potential side
reactions, the amine functionality in compounds 5a and 5b were
first protected as their tert-butyl urethane derivatives 7a and 7b
through reaction with Boc anhydride. The urethanes were then
irradiated in the presence of NBS using a tungsten lamp, to
afford the intermediate 5-bromides which upon treatment with
DBU at 70 ЊC for 10 min eliminated HBr to give the alkenes 8a
and 8b in moderate yield.27 Approximately 20% of the starting
material was also recovered in each case. The synthesis of each
β-amino acid was completed by removal of the methyl ester and
Boc protecting groups, using lithium hydroxide and then TFA,
respectively, to give compounds 10a and 10b, each in approxi-
mately 75% yield. In preparation for using standard solid-phase
peptide synthesis protocols, each free amino acid was converted
to its N-Fmoc derivative 11a and 11b.
The absolute stereochemistry at C-3 of the adduct 4a was
confirmed as 3R by converting the amino ester 4a to the known
amine, compound 12,28 for comparison of optical rotation
values (Scheme 2). Treatment of the ester 4a with DIBAL-H
followed by reaction with TBDPSCl in the presence of imid-
azole afforded the silyl ether 13 in 75% yield and subsequent
hydrogenation and debenzylation using Pearlman’s catalyst gave
amine 12 in 60% yield. Upon comparison of the specific rotation
{[α]2D4 Ϫ2.4 (c 1.45 in CHCl3)} to the literature value [α]2D4 Ϫ2.81
(c 1.63 in CHCl3) it was evident that the compound was the
(3S)-antipode. Note the priority change following reduction of
the double bond that reverses the absolute stereochemical
In order to prepare the 2-methyl derivative 10b, the tertiary
amine 4a was treated with LDA at Ϫ78 ЊC to generate the
lithium enolate and the resulting anion was alkylated with
methyl iodide to give a 4:1 ratio of anti to syn products.21 The
required anti product 4b was obtained in 57% yield (Scheme 1).
Removal of the benzyl groups, while keeping the double
bond intact, was not possible. Standard hydrogenolysis con-
ditions employing Pd(OH)2/C (Pearlman’s catalyst) cleanly
removed the N-benzyl groups from amino ester 4a but also
hydrogenated the double bond to give the saturated amine 5a,
as expected.23 Analysis of the reaction products after partial
reduction indicated that the double bond was reduced before
complete hydrogenolysis occurred. Dissolving metal reduc-
tions, which are widely used for debenzylations,24 were deemed
to be unsuitable due to the presence of the conjugated double
bond in compound 4a and were not attempted. Several
alternative protocols exist for debenzylation of amines under
non-reducing conditions including the use of trichloroethoxy-
carbonyl chloride (Treoc-Cl).25 However, when debenzylation
was attempted on compound 4a, only starting material was
recovered. Partial success was achieved using ceric ammonium
nitrate (CAN)26 and treatment of the dibenzylamine 4a with
3 equivalents of CAN in acetonitrile–water resulted in rapid
monodebenzylation to give the amine 6 in good yield (80–90%).
The partial deprotection was complete in 15–20 min and benz-
aldehyde was generated as the oxidised by-product. Reactions
performed using excess CAN and/or for extended reaction
periods were not successful and a variety of oxidised products
were formed.
Scheme 1 Reagents and conditions: i) (EtO)2POCH2CO2CH3, NaH, THF, 0 ЊC, then add cinnamaldehyde at Ϫ78 ЊC, 20 min, 93%; ii) (S)-N-benzyl-
α-methylbenzylamine, BuLi, THF, Ϫ78 ЊC, 2 h, 61%; iii) LDA, Ϫ78 ЊC, 45 min, MeI, Ϫ78 ЊC, 30 min, 57%; iv) Pd(OH)2, H2, CH3OH–H2O–
CH3COOH (20:3:3), rt, 3 h, 71–77%; v) Boc2O, NaHCO3, dioxane, H2O, rt, 4 h, 72–82%; vi)NBS, CCl4, 25 ЊC, 2 h, then DBU, 60 ЊC, 10 min, 42–45%;
vii) LiOH, THF, CH3OH, H2O, 1 h, 20 ЊC, 71–95%; viii) TFA, DCM, rt, 2 h, 79–83%; ix) Fmoc-Cl, K2CO3, dioxane, H2O, 0 ЊC→rt, 12 h, 52–66%.
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existed as a mixture of cis–trans rotamers/conformers. This was
in accord with expectations since it had been shown previously
that the natural product motuporin, an analogue of nodularin,
existed in a single conformation whereas the dimethyl ester
existed in multiple forms.29 Saponification of the methyl ester
protecting groups in compounds 18a and 18b was performed
using LiOH in aqueous THF. The acidified products were puri-
fied by reverse-phase HPLC to give the required diacids 19a
{(ES) 658 ([M ϩ Na]ϩ)} in 77% yield and 19b {(ES) 672
([M ϩ Na]ϩ)} in 72% yield respectively. As expected, the
1H-NMR spectra of the diacids were much less complex than
for the dimethyl ester precursors. The analogue 19a, which is
derived from the non-methylated β-amino acid 5a, possesses
two α-H-atoms. This compound existed as a 2.5:1 mixture of
two conformers in solution and detailed analysis revealed that
these were trans- and cis-rotamers at the N-acylsarcosine amide
bond, respectively. The more restrained macrocyclic methyl
1
Scheme 2 Reagents and conditions: i)DIBAL-H, THF, Ϫ78 ЊC, 2 h; ii)
TBDPSCl, imidazole, THF, rt, 6 h, 75% from 4a; iii) Pd(OH)2,
ammonium formate, CH3OH, 60 ЊC, 2 h, 60%.
homologue 19b displayed an even simpler H-NMR spectrum
corresponding to a 10:1 mixture of trans- to cis-rotamers at the
N-acylsarcosine amide bond. Thus, each macrocycle existed
predominantly in the trans-rotameric form. Interestingly, these
major forms displayed almost identical coupling constants and
chemical shift values to each other in 1H-NMR and 13C-NMR
spectra, for all of the conserved residues.
These findings suggest that the solution conformations of
the major forms are identical. Moreover, it appeared that the
conformations of the dominant trans-rotameric forms closely
matched that for the solution-phase conformation of nodularin
as determined by comparison of 1H-NMR data (see discussion
below).
assignment. 1H-NMR spectra of the crude precursors indicated
that the diastereomeric purity was greater than 95%.
The relative stereochemistry at C-2 and C-3 in compound 4b
was assigned by conversion of the Boc protected amine 8b to a
β-lactam, (Scheme 3).21 The Boc group was removed with TFA
(2S)-Proline analogue
The crystal structure of microcystin-LR bound to PP1cat shows
that the Adda residue, residue number 1, binds in a hydro-
phobic groove on the surface of the protein, while the Adda
carbonyl group and the glutamate α-carboxy group are
involved in water-mediated hydrogen bonding to the metal ion
site.30 Results to date indicate that the solution structure of
microcystin is highly preorganised for binding and shows a high
degree of similarity to the PP1cat-bound conformation. In our
earlier work to identify a stripped-down nodularin analogue,
we had shown that the N-methyldehydroalanine residue
in microcystin does not form a covalently bonded Michael
adduct with Cys-273 as part of the mechanism for tight-binding
inhibition.13 Indeed, we showed that each of the dihydromicro-
cystin epimers containing (2R)- and (2S)-N-methylalanine at
position 3 were equally potent inhibitors to the parent. We also
showed that the introduction of (2R)- and (2S)-proline at the
congruous 3-position in nodularin analogues gave restrained
macrocycles that displayed poor but significant inhibitor
activity against PP1cat.10 These compounds did not exist as
single conformers in solution and conformational analysis was
extremely difficult. It was however clear that each macrocycle
existed in both the trans- and cis-rotameric forms and that each
of these rotamers existed in more than one conformation that
interconverted slowly on the NMR time scale.
Given these difficulties, a fuller structural analysis was not
possible. However, the compounds did not contain an α-methyl
group in the Adda surrogate at position 1, the presence of
which might have reduced the number of populated conform-
ations and allowed a simplified analysis. In view of the results
obtained for the sarcosine containing analogues 19a and 19b,
above, it was determined that a comparison of the structures of
the identically substituted macrocycles possessing α-methylated
Adda analogues and differing only in the nature of the residue
at position 3 would be highly informative.
Scheme 3 Reagents and conditions: i) TFA, DCM, rt, 1 h; ii) LDA,
THF, Ϫ78 ЊC, 20 min.
to give the amino ester 14 which was cyclised to the β-lactam 15
through treatment with LDA at Ϫ78 ЊC. The observed H-2/H-3
coupling constant of ∼2 Hz indicates a trans-relationship, and
given that the β-lactam is derived from amino ester 4a which
possesses (3R)-stereochemistry before methylation and (3S)-
stereochemistry after methylation, due to a priority change, it is
evident that compound 4b and, therefore, compound 8b possess
(2S,3R)-absolute stereochemistry.
Solid-phase peptide synthesis (SPPS) of pentapeptides
The SPPS of the linear pentapeptides 17a and 17b from the
Fmoc-Asp-(α)-OMe-O-Wang 16 was carried out on a Rainin
PS3 automated peptide synthesiser using methods similar to
those as described previously (Scheme 4).10 The crude products
17a and 17b were obtained in quantitative recovery after
cleavage from the resin. Analysis by HPLC revealed one major
peak in each case (approx. 80% of total) with several close
running bands which were removed by HPLC. Each pure
material eluted as a single peak and gave the required ES mass
spectral data (704, [M ϩ Na]ϩ) for 17a and (718, [M ϩ Na]ϩ)
for 17b. Analysis of the 1H and 13C spectra revealed the
presence of two major rotomeric forms, in each case.
The macrocyclisations of compounds 17a and 17b were
carried out under high dilution conditions (1 mg cmϪ3), using
BOP-Cl in the presence of N,N-diisopropylethylamine
(DIPEA) in a CH2Cl2–DMF mixture over a period of five days
using conditions previously optimised in the preparation of
earlier analogues (see previous article).10 The crude products
were purified by flash chromatography on silica to give the
cyclic pentapeptides 18a {(ES) 686 ([M ϩ Na]ϩ)} in 28% yield
1
and 18b {(ES) 700 ([M ϩ Na]ϩ)} in 27% yield. H-NMR and
Accordingly, the (2S)-Pro analogue of macrocycle 19b was
prepared using the now established protocols starting with
the SPPS of the linear pentapeptide 20. Cyclisation to 21 was
mass spectral data indicated that each cyclisation had occurred
and analysis of the NMR data indicated that each diester
J. Chem. Soc., Perkin Trans. 1, 2001, 1696–1708
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performed using BOP-Cl in DMF–CH2Cl2 in the presence of
DIPEA and the macrocyclic diester 21 was obtained in 23%
yield (Scheme 5). Again the H-NMR spectrum of the diester
revealed the existence of multiple conformations. Gratifyingly,
saponification of the methyl ester groups gave diacid 22 which
1
1
existed in one predominant conformation as judged by H-
Scheme 4 Reagents and conditions: i) 20% piperidine in DMF; ii) Fmoc-Sar-OH, PyBOP, DMF; then 20% piperidine in DMF; iii) Fmoc-(2R)-Glu-
α-OMe-γ-OH, PyBOP, DMF; then 20% piperidine in DMF; iv) 11a or 11b, PyBOP, DMF; then 20% piperidine in DMF; v) Fmoc-(2S)-Phe-OH,
PyBOP, DMF; then 20% piperidine in DMF; vi) TFA–TES–H2O (95:2.5:2.5), 1 h, rt; vii) BOP-Cl, DIPEA, DCM, DMF, rt, 7 days, 27–28%; viii)
LiOH, THF, H2O, 0 ЊC, 1 h, 68–80%.
Scheme 5 Reagents and conditions: i) 20% piperidine in DMF; ii) Fmoc-(2S)-Pro-OH, PyBOP, DMF; then 20% piperidine in DMF; iii) Fmoc-(2R)-
Glu-α-OMe-γ-OH, PyBOP, DMF; then 20% piperidine in DMF; iv) 11b, PyBOP, DMF; then 20% piperidine in DMF; v) Fmoc-(2S)-Phe-OH,
PyBOP, DMF; then 20% piperidine in DMF; vi) TFA–TES–H2O (95:2.5:2.5), 1 h, rt, quantitative recovery from 16; (vii) BOP-Cl, DIPEA, DCM,
DMF, rt, 7 days, 23%; viii) LiOH, THF, H2O, 0 ЊC, 1 h, 85%.
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NMR spectroscopy. More thorough analysis revealed that the
major conformation corresponded to the trans-rotameric form
of the γ-Glu-Pro amide bond in a 6:1 mixture with a conformer
displaying signals consistent with its cis-rotameric form. Com-
pared to the trans-rotamer of the sarcosine containing macro-
cycle 19b, the trans-rotamer of macrocyclic diacid 22 showed
minor but significant changes in chemical shift values and
coupling constants for conserved residues. The differences were
most pronounced in the signals for the (2R)-Glu and (2R)-Asp
residues (residues 2 and 4) that flank the variant residue. These
results indicate that the introduction of (2S)-Pro at position 3
causes local changes, but not global changes, to the conform-
ation of the macrocycle, in accord with expectations. These
conclusions are supported by the biological activity of the
analogues, as is described below.
Thus, the perturbations in the conformation of the macrocycle
introduced through the inclusion of a pyrrolidine ring, as
determined by NMR spectroscopy (see above), did not under-
mine the enhanced affinity for the protein mediated by the extra
methyl group. It is interesting to recall that the macrocyclic
benzylpiperidine amides, which differed in the nature of the
residue at position 3 (described in the previous article10),
showed similar IC50 values of 2900 and 2700 µmol dmϪ3 respec-
tively. However, it was not possible to deconvolute the
rotameric and conformational contributions to the solution
structures of the macrocycles. It should be noted that the Sar
residue in 19b cannot adopt a low energy conformation
in which the N-methyl group and the 2-pro-S proton are
nearly eclipsed, as in a conformation that mimics that for (2S)-
proline. Thus, the two macrocycles must exist in different con-
formations about residues 2,3 and 4, as was observed by NMR
spectroscopy.
Biological activity
These results are exciting for two reasons. First, it would
appear that the structures of the Adda and (2R)-Glu and (2R)-
Asp residues, residues 1,2 and 4, respectively, are largely
responsible for conferring inhibitory activity. As such, it
appears that anything that would fit in the active site at position
3 will allow binding. [It is useful to recall that the structure at
position 5 varies considerably in known natural products and
is not important for activity.13] The fact that microcystin is a
heptaisopeptidic macrolactam in which the two extra residues
occur between residues 3 and 4 in nodularin is consistent
with this analysis. Moreover, the observation that the (2S)- and
(2R)-N-methylalanine13 epimers of dihydromicrocystin were as
active as the parent microcystin further suggests that the nature
of the residue at and around position 3 is not crucial. Second,
the (2S)-proline structure itself provides a conformationally
stable and well-defined substructure to which additional func-
tional groups can be appended. There are large differences
in size and charge between the two target phosphatases, PP1
and PP2A, in the vicinity of the region adjacent to nodularin
residue 3 in modelled bound complexes. The results of the
work presented here provide confidence to the validity of these
modelled complexes and indicate that selectively modified (2S)-
Pro analogues might give specific inhibitors of both PP1 and
PP2A. Work towards these ends is in progress.
Each of the three synthetic macrocyclic diacids, 19a, 19b, and
22 were tested as inhibitors for recombinant PP1cat using a new
assay protocol involving the dephosphorylation of the synthetic
phosphopeptide [acetyl-14C]-Ac-Arg-Arg-Ala-Thr(P)-Val-Ala
(described in the following article). The assay allows the
determination of V and KM under a wide range of conditions
and, importantly, for the purposes of evaluating inhibitors,
allows the mode of inhibition to be determined. Previously
it has been common practice to report only IC50 values and,
unfortunately, such values are of limited use in the determination
of inhibition mechanism or in allowing comparisons of enzyme
activity under differing conditions. A full consideration is given
in the following article.11
All three nodularin analogues were competitive inhibitors, in
accord with the results obtained for nodularin (see following
article11). The non-methylated macrocycle containing Sar at
position 3, 19a displayed a Ki value of 4000 1000 µmol dmϪ3.
This compares with an IC50 value of 2900 µmol dmϪ3 obtained
for the 4-benzylpiperidine amide analogue 1, described previ-
ously. Thus, the phenethenyl side chain at C-3 in 19a appears
to confer a similar level of binding affinity to the macrocycle
as the 4-benzylpiperidine amide, given that detailed analysis
is complicated by differing conformational preferences. The
methylated homologue 19b was 20-fold more active than macro-
cycle 19a and gave a Ki value of 206 30 µmol dmϪ3. Thus, the
presence of the methyl group significantly enhances binding to
PP1cat. The methyl homologue and the non-methylated macro-
cycle possess very similar solution-phase structures and both
exist predominantly in the trans-rotameric form at the γ-Glu-
Sar amide bond. The large differences in inhibitory efficacy,
therefore, appear to be largely due to solvent exclusion, rather
than due to conformational preferences alone. Interestingly
there is a well-defined hydrophobic methyl group binding
pocket at the active site of PP1cat formed by Ile-130 and Tyr-134
in the X-ray crystal structures of microcystin- PP1cat complexes.
It would appear, therefore, that nodularin does bind in a highly
conserved manner where the hydrophobic Adda contacts,
emanating from C-2 and C-3, and the bridged water molecule
binding interactions with the carboxylate moieties and the
metal ions are similar to those for microcystin. This same
methyl binding pocket also appears to be utilised in conferring
substrate specificity to the phosphatases PP1 and PP2A. While
no structural information is yet available for substrate binding,
it is known that phosphothreonine residues are preferred over
phosphoserine residues in small peptides.31 Moreover, our
recent mechanistic studies,12 which indicate that phospho-
threonyl peptide hydrolysis should occur by direct attack of
a µ-bound hydroxide ion, with inversion of configuration at
phosphorus, place the threonine 3-methyl group in the same
hydrophobic pocket.
Experimental
Elemental microanalyses were performed in the departmental
micro-analytical laboratory. NMR spectra were recorded on a
Bruker AM-300 spectrometer (1H, 300 MHz; 13C, 75.4 MHz),
a Bruker AMX-400 spectrometer (1H, 400 MHz; 13C, 100.6
MHz), or a Bruker DRX 500 spectrometer (1H, 500 MHz; 13C,
125 MHz). Chemical shifts are described in parts per million
downfield shift from SiMe4 and are reported consecutively as
position (δH or δC), relative integral, multiplicity (s = singlet,
d = doublet, t = triplet, q = quartet, dd = double of doublets,
sep = septet, m = multiplet, and br = broad), coupling constant
(J/Hz) and assignment (numbering according to the IUPAC
nomenclature for the compound).1H-NMR spectra were
2
referenced internally on HOH (δ 4.68), CHCl3 (δ 7.27) or
(CH3)2SO (δ 2.47). 13C-NMR spectra were referenced on
C2HCl3 (δ 77.5) or (CH3)2SO (δ 39.70). Pyrrolidine ring carbons
and hydrogens are assigned in NMR spectra as α,β,γ,δ, going
anticlockwise from the ring nitrogen, according to normal
convention. Where more than one conformational isomer could
be detected in the NMR spectrum due to the presence of a
tertiary amide moiety, these are assigned as c (cis) or t (trans),
according to the rotameric state of the amide bond. Assign-
ments for proline were aided by COSY, TOCSY and HSQC
NMR experiments and by comparison with well established
13C-NMR differences for the β- and γ-CH2 signals.32,33 Sarco-
sine cis/trans isomers were assigned on the basis of their 1H/13C
NMR chemical shifts, where the trans isomer showed upfield
The (2S)-proline containing analogue 22 was also a com-
petitive inhibitor and gave a Ki value of 400 75 µmol dmϪ3.
J. Chem. Soc., Perkin Trans. 1, 2001, 1696–1708
1701

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resonances relative to the cis isomer in the 1H-NMR spectra. IR
spectra were recorded on Perkin-Elmer 1710 or Nicolet Avatar
360 FT-IR spectrometers. The samples were prepared as Nujol
mulls, solutions in chloroform or thin films between sodium
chloride discs. The frequencies (ν) as absorption maxima are
given in wavenumbers (cmϪ1) relative to a polystyrene standard.
Mass spectra and accurate mass measurements were recorded
on a VG 70-250 SE, a Kratos MS-50, VG Zabspec or VG
Prospec spectrometer. Fast atom bombardment spectra were
recorded using glycerol as a matrix. CI spectra were recorded
using ammonia as a reagent. Major fragments were given
as percentages of the base peak intensity (100%). Flash
chromatography was performed according to the method of
Still et al.34 using Fluka Kieselgel C60 (40–60 µm mesh) silica
gel. Analytical thin layer chromatography was carried out on
0.25 mm precoated silica gel plates (Whatman PE SIL G/UV)
and compounds were visualised using UV fluorescence or
ethanolic phosphomolybdic acid. Melting points were taken
on an Electrothermal melting point apparatus and are uncor-
rected. Optical rotations were measured at 24 ЊC on a Optical
Activity AA-1000 polarimeter using 10 or 20 cm path length
cells and are given in units of 10Ϫ1 deg cm2 gϪ1.
(156 mg, 0.66 mmol) was added and the mixture stirred at 0 ЊC
for 6 h. The solution was allowed to warm to room temperature
and then stirred for a further 7 days. The reaction mixture
was concentrated under reduced pressure, redissolved in ethyl
acetate (50 cm3), and then washed with 10% citric acid (2 ×
25 cm3), 5% sodium bicarbonate (2 × 25 cm3), distilled water
(2 × 25 cm3) and then brine (1 × 25 cm3). The organic phase
was dried (MgSO4) and the solvent removed under reduced
pressure to a volume of 1 cm3. The product was obtained as a
white solid after trituration with diethyl ether (10 cm3).
Methyl (2E,4E)-5-phenylpenta-2,4-dienoate 3
To an ice-cooled suspension of hexane-washed sodium hydride
(60% dispersion; 4 g, 0.1 mol) in THF (150 cm3) was slowly
added methyl diethylphosphonoacetate (19 cm3, 0.1 mol) and
the mixture was then stirred for 15 min. The mixture was cooled
to Ϫ78 ЊC and after the dropwise addition of cinnamaldehyde
(12.6 cm3, 0.1 mol) stirring was continued for a further 20 min
and then the mixture was allowed to warm up to room tem-
perature. The resulting mixture was quenched with saturated
aqueous NH4Cl (100 cm3) and then extracted with diethyl
ether (2 × 100 cm3). The organic phase was washed with brine
(50 cm3), dried (MgSO4) and concentrated under reduced pres-
sure to give the crude product as an off-white solid. Purification
by flash silica chromatography (petroleum ether–ethyl acetate;
9:1) gave the dienoate 3 as a white solid (17.5 g, 93%), mp 68–
70 ЊC (lit.,35 67–68 ЊC); νmax(thin film)/cmϪ1 1709 (CO, ester)
Preparative RP HPLC was carried out using a PerSeptive
BioCAD SPRINTTM Perfusion® Chromatography System.
Preparative RP HPLC was performed on a Luna C-18(2) 10 µm
column (150 × 21.2 mm) or on a Luna C-18(2) 10 µm column
(250 × 21.2 mm) fitted with a Luna C-18(2) 10 µm column
(60 × 21.2 mm).
and 1626 (C᎐C, alkene); δ (300 MHz; C2HCl ) 3.78 (3 H, s,
᎐
H
3
Protected amino acid precursors were purchased from
Calbiochem-Novabiochem (UK) Ltd (Beeston, Nottingham).
All other chemicals were of analytical grade or were recrystal-
lised or redistilled before use. The solvents used were either
distilled or of Analar quality and petroleum ether refers to
that portion boiling between 40–60 ЊC. Solvents were dried
according to literature procedures. Ethanol and methanol
were dried using magnesium turnings. DMF, CH2Cl2 and diiso-
propylamine were distilled over CaH2. THF and diethyl ether
were dried over sodium–benzophenone and distilled under
nitrogen.
OCH3), 6.02 (1 H, d, J 16.0, 2-H), 6.87 (2 H, m, 4-H and 5-H)
and 7.25–7.60 (6 H, m, Ar-H and 3-H); δC(75.4 MHz; C2HCl3)
51.6 (OCH3), 120.8, 126.2, 127.2, 128.8 and 129.1 (Ar-CH and
C᎐C), 136.0 (Ar-C quaternary), 140.6 and 144.8 (Ar-CH)
᎐
and 167.5 (CO, ester); m/z (EI) 188 (60%, Mϩ), 157 (30,
[M Ϫ OCH3]ϩ) and 129 (100, [M Ϫ CO2CH3]ϩ).
Methyl (3R,4E,ꢀS)-3-(N-benzyl-N-ꢀ-methylbenzylamino)-5-
phenylpent-4-enoate 4a
A stirred solution of (S)-N-benzyl-N-α-methylbenzylamine
(3.34 cm3, 16 mmol) in dry THF (40 cm3) was cooled to Ϫ78 ЊC
and butyllithium (15 mmol, 6 cm3 of 2.5 mol dmϪ3 solution in
hexanes) was slowly added. The resulting pink solution of the
lithium amide was stirred for 30 min, and then a solution of
methyl (2E,4E)-5-phenylpenta-2,4-dienoate 3 (2.1 g, 11 mmol)
in THF (15 cm3) was added dropwise. Stirring was continued
for 2 h at Ϫ78 ЊC and then the reaction was quenched by the
slow addition of saturated aqueous NH4Cl (5 cm3). The
mixture was allowed to warm up to room temperature and then
partitioned between brine (30 cm3) and diethyl ether (30 cm3).
The organic layer was separated, dried (MgSO4), and then con-
centrated under reduced pressure to furnish the crude conjugate
adduct as a 3:1 ratio of the required 1,4 addition product and
1,2-1,4 bis-addition product respectively. Purification by flash
silica chromatography (petroleum ether–ethyl acetate; 9:1) gave
the conjugate adduct 4a as a colourless oil (2.7 g, 61%) (Found
C, 81.0; H, 7.5; N, 3.3. C27H29NO2 requires C, 81.2; H, 7.3; N,
3.5%) (HRMS: found [M ϩ Na]ϩ, 422.2084. C27H29NO2Na
requires 422.2096); [α]D ϩ89 (c 10 in CH2Cl2); νmax(thin film)/
cmϪ1 1733 (CO, ester) and 1626 (C᎐C, alkene); δ (300 MHz;
General solid-phase peptide synthesis and peptide removal from
Wang resin
Solid-phase synthesis of the pentapeptides was carried out
using a Ranin PPS automated peptide synthesiser. The syn-
thesis employed Fmoc chemistry and the C-terminal and amino
acids were linked to α-methyl (2R)-N-(fluoren-9-ylmethoxy-
carbonyl)aspartyl-Wang resin. A four-fold excess of the amino
acid was used for each coupling procedure. The N-α-Fmoc
group was deprotected using a 20% piperidine–DMF solution
and the activation was achieved using a 5% NMM–DMF solu-
tion. Once SPPS was complete the resin was placed in a sintered
glass funnel washed sequentially with DMF, acetic acid,
CH2Cl2 and finally methanol. The resin was then dried in vacuo
for several hours. The dried resin was then treated with a
mixture of 95% TFA–2.5% water–2.5% TES (5 cm3) for 1 h.
The resin was filtered and washed through with TFA (5 cm3).
The filtrate was concentrated under reduced pressure until an
oil was obtained. The flask was placed in an ice bath and after
the addition of diethyl ether (15 cm3) the mixture was stirred for
10 min. The suspension was allowed to settle and the diethyl
ether was decanted off using a pipette. The last traces of diethyl
ether were removed by flushing the flask with argon for a few
minutes. The peptide was then lyophilised to yield the product
as a white solid.
᎐
H
C2HCl3) 1.40 (3 H, d, J 7.0, CH3), 2.42 (1 H, dd, J 7.7 and 14.3,
1 H of CH2CO), 2.59 (1 H, dd, J 7.2 and 14.4, 1 H of CH2CO),
3.48 (3 H, s, OCH3), 3.72 and 3.77 (2 H, AB system, J 11.6,
PhCH2N), 3.97 (1 H, q, J 7.0, PhCHCH3N), 4.08 (1 H, m,
NCHCH2), 6.30 (1 H, dd, J 7.5 and 16.0, 4-H), 6.43 (1 H, d,
J 16.0, 5-H) and 7.20–7.40 (15 H, m, Ar-H); δC(75.4 MHz;
C2HCl3) 17.12 (CH3), 39.11 (CH2CO), 50.77 (CH2N), 51.96
(OCH3), 57.01 and 57.36 (2 × CHN), 121.25 (ArCHCH),
126.80, 127.20, 127.65, 127.99, 128.34, 128.49, 128.65, 128.94,
129.04, 129.26, 129.53, 130.27 and 131.74 (Ar-CH), 137.44,
141.33 and 144.48 (Ar-C quaternary) and 172.39 (CO, ester);
General cyclisation procedure using BOP-Cl
To a stirred solution of the pentapeptide (350 mg, 0.6 mmol)
in CH2Cl2–DMF (9:1) (350 cm3) was added DIPEA (150 mm3,
1.5 mmol) and the mixture was cooled down to 0 ЊC. BOP-Cl
1702
J. Chem. Soc., Perkin Trans. 1, 2001, 1696–1708

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m/z (CI) 400 (100%, [M ϩ H]ϩ), 296 (10, [M ϩ H Ϫ
liquid (0.9 g, 71%). For analytical purposes a small amount of
PhCHCH3]ϩ).
the amine was chromatographed on silica gel (CH2Cl2–
methanol; 95:5) (HRMS: found [M ϩ Na]ϩ, 244.1311. C13-
H19NO2Na requires 244.1313); [α]D Ϫ2.4 (c 10 in CH2Cl2);
νmax(thin film)/cmϪ1 3387 (NH2) and 1728 (CO, ester); δH(300
MHz; C2HCl3) 1.18 (3 H, d, J 7.0, CH3CH), 1.40 (2 H, s, NH2),
1.47–1.65 (1 H, m, 1 H of PhCH2CH2), 1.75–1.90 (1 H, m,
1 H of PhCH2CH2), 2.49 (1 H, dq, J 6.8 and 7.0, CHCO), 2.65–
2.85 (1 H, m, 1 H of PhCH2), 2.89–2.95 (1 H, m, NH2CH), 3.69
(3 H, s, OCH3) and 7.20–7.32 (5 H, m, Ar-H); δC(75.4 MHz;
C2HCl3) 14.57 (CH3), 32.86 (ArCH2CH2), 37.26 (ArCH2CH2),
46.45 (CHCO), 51.83 (OCH3), 53.96 (CHNH), 126.20 and
128.73 (Ar-CH), 142.27 (Ar-C quaternary) and 176.20 (CO,
ester); m/z (CI) 222 (100%, [M ϩ H]ϩ).
Methyl (2S,3S,4E,ꢀS)-2-methyl-3-(N-benzyl-N-ꢀ-methylbenzyl-
amino)-5-phenylpent-4-enoate 4b
A solution of methyl (3R,4E,αS)-3-(N-benzyl-N-α-methyl-
benzylamino)-5-phenylpent-4-enoate 4a (5.5 g, 14 mmol) in
THF (10 cm3) was added dropwise to a stirred solution of
lithium diisopropylamide (18 mmol) in THF (35 cm3) at
Ϫ78 ЊC. The reaction mixture was stirred for 1 h at Ϫ78 ЊC and
then methyl iodide (6 cm3, 90 mmol) was added. The reaction
mixture was allowed to warm up to room temperature over-
night and then quenched with saturated aqueous NH4Cl
solution (40 cm3). The mixture was then partitioned between
diethyl ether (40 cm3) and brine (40 cm3). The organic layer was
separated, dried (MgSO4) and solvent removed under reduced
pressure to yield the crude product as a 4:1 ratio of the anti and
syn diastereoisomers respectively. Flash silica chromatography
(petroleum–ethyl acetate; 30:1) gave the unwanted (2R,3S)
diastereoisomer product (800 mg), followed by product 4b as a
oil (3.2 g, 57%). Recrystallisation from a cold methanol–water
(9:1) mixture gave 4b as a crystalline solid. Major diastereo-
isomer 4b: mp 80–82 ЊC (HRMS: found [M ϩ Na]ϩ, 436.2243.
C28H31NO2Na requires 436.2252); [α]D ϩ128 (c 10 in CH2Cl2);
Methyl (3S)-3-(N-tert-butoxycarbonylamino)-5- phenyl-
pentanoate 7a
To
a stirred solution of methyl (3S)-3-amino-5-phenyl-
pentanoate 5a (4.6 g, 22 mmol) in dioxane (8 cm3) was a solu-
tion of Na2CO3 (2.3 g, 0.6 mol) in water (16 cm3) added. The
mixture was cooled on an ice bath and di-tert-butyl dicarbonate
(5.5 g, 25 mmol) was slowly added. The solution was allowed
to warm up to room temperature and stirred for 3 h after
which time a heavy white precipitate had appeared. Most of the
solvent was removed under reduced pressure and the residue
dissolved in ethyl acetate (40 cm3). The organic layer was
washed successively with water (50 cm3), 0.5 mol dmϪ3 HCl
solution (50 cm3) and brine (50 cm3), dried (MgSO4) and con-
centrated under reduced pressure. The residue was purified by
flash silica chromatography (petroleum ether–ethyl acetate; 9:1)
to give the Boc-protected amine 7a as a colourless oil which
solidified upon drying in vacuo to a white solid (5.5 g, 82%).
Rf = 0.35; mp 55–57 ЊC (lit.,36 64–66 ЊC) (Found C, 66.7; H, 8.5;
N, 4.5. C17H25NO4 requires C, 66.4; H, 8.2; N, 4.6%) (HRMS:
found [M ϩ Na]ϩ, 330.1695 C17H25NO4Na requires 330.1681);
[α]D Ϫ9.5 (c 1.8 in CHCl3); νmax(CH2Cl2)/cmϪ1 3360 (NH,
urethane), 1732 (CO, ester) and 1712 (CO, urethane); δH(300
MHz; C2HCl3) 1.42 [9 H, s, C(CH3)3], 1.77–1.91 (2 H, m,
PhCH2CH2), 2.50–2.85 (4 H, m, PhCH2CH2 and CH2CO), 3.68
(3 H, s, OCH3), 3.90–4.01 (1 H, m, NHCH), 4.97 (1 H, d, J 9.9,
NH) and 7.13–7.31 (5 H, m, Ar-H); δC(75.4 MHz; C2HCl3)
28.42 [C(CH3)3], 32.62 (ArCH2CH2), 36.48 (ArCH2CH2), 39.21
(CH2CO), 47.43 (CHNH), 51.70 (OCH3), 79.34 [OC(CH3)3],
125.98, 128.38 and 128.45 (Ar-CH), 141.47 (Ar-C quaternary),
155.38 (CO, urethane) and 172.08 (CO, ester); m/z (ES) 330
(100%, [M ϩ Na]ϩ), 274 (90, [M ϩ Na ϩ H Ϫ C4H9]ϩ) and
230 (10, [M ϩ H ϩ Na Ϫ C5H9O2]ϩ).
νmax(thin film)/cmϪ1 1733 (CO, ester) and 1626 (C᎐C, alkene);
᎐
δH(300 MHz; C2HCl3) 0.90 (3 H, d, J 6.8, CH3CHCO), 1.37
(3 H, d, J 6.8, PhCHCH3), 2.79 (1 H, dq, J 6.9 and 10.7,
CHCO), 3.43 (3 H, s, OCH3), 3.47 (1 H, dd, J 10.2 and 10.3,
NCHCH), 3.60 and 3.84 (2 H, AB system, J 11.6, PhCH2N),
4.14 (1 H, q, J 6.8, PhCHCH3), 6.11 (1 H, dd, J 9.5 and 16.0,
4-H), 6.32 (1 H, d, J 16.0, 5-H) and 7.20–7.40 (15 H, m, Ar-H);
δC(75.4 MHz; C2HCl3) 16.20 and 16.52 (2 × CH3), 43.70
(CHCO), 50.22 (CH2N), 51.84 (OCH3), 56.03 (ArCHN), 64.15
(CHCHNH), 126.80, 127.23, 127.40, 128.07, 128.27, 128.51,
129.15 and 129.49 (Ar-CH and C᎐C), 137.36, 140.58 and
᎐
144.72 (Ar-C quaternary) and 176.00 (CO, ester); m/z (CI) 414
(100%, [M ϩ H]ϩ) and 310 (10, M ϩ H Ϫ PhCHCH3]ϩ).
Methyl (3S)-3-amino-5-phenylpentanoate 5a
To a stirred solution of methyl (3R,4E,αS)-3-(N-benzyl-N-
α-methylbenzylamino)-5-phenylpent-4-enoate 4a (6.0 g, 20
mmol) in methanol (100 cm3) was added Pearlman’s catalyst
(1.0 g) and ammonium formate (1.5 g, 48 mmol). The mixture
was heated under reflux for 45 min, allowed to cool down,
filtered through a plug of Celite and the filtrate concentrated
under reduced pressure to give a viscous oil. This oil was dis-
solved in 10% aqueous NaHCO3 (30 cm3) and extracted with
CH2Cl2 (50 cm3). The organic layer was dried (MgSO4) and
concentrated under reduced pressure to yield the product amine
5a as a colourless liquid (2.4 g, 77%). For analytical purposes a
small amount of the amine was chromatographed on silica gel
(CH2Cl2–methanol; 95:5). Rf = 0.2 (HRMS: found [M ϩ H]ϩ,
208.1343. C12H17NO2 requires 208.1338); [α]D Ϫ1.6 (c 1 in
CHCl3); νmax(CH2Cl2)/cmϪ1 3375 (NH2) and 1732 (CO, ester);
δH(300 MHz; C2HCl3) 1.55 (2 H, br s, NH2), 1.61–1.85 (2 H, m,
PhCH2CH2), 2.29–2.55 (2 H, m, CH2CO), 2.63–2.83 (2 H, m,
PhCH2), 3.18–3.29 (1 H, m, NH2CH), 3.68 (3 H, s, OCH3)
and 7.20–7.32 (5 H, m, Ar-H); δC(75.4 MHz; C2HCl3) 32.17
(ArCH2CH2), 39.06 (ArCH2CH2), 42.20 (CH2CO), 47.70
(CHNH2), 51.25 (OCH3), 125.6, 128.05 and 128.16 (Ar-CH),
141.56 (Ar-C quaternary), and 172.54 (CO, ester); m/z (FAB)
208 (100%, [M ϩ H]ϩ).
Methyl (2S,3R)-3-(N-tert-butoxycarbonylamino)-2-methyl-5-
phenylpentanoate 7b
This compound was prepared in a manner identical to that
described for 7a using methyl (2S,3S)-3-amino-2-methyl-5-
phenylpentanoate 5b (2.7 g, 12 mmol) to yield the Boc-
protected amine 7b as a colourless oil which solidified upon
drying in vacuo to a white solid (2.8 g, 72%), mp 62–64 ЊC
(Found C, 67.2; H, 8.4; N, 4.3. C18H27NO4 requires C, 67.3;
H, 8.5; N, 4.4%) (HRMS: found [M ϩ Na]ϩ, 344.1833
C18H27NO4Na requires 44.1838); [α]D ϩ2.1 (c 4 in CH2Cl2);
νmax(CH2Cl2)/cmϪ1 3433 (NH, urethane) and 1729 (CO, ester
and Boc); δH(300 MHz; C2HCl3) 1.21 (3 H, d, J 7.2, CH3CH),
1.47 [9 H, s, C(CH3)3], 1.70–1.80 (2 H, m, PhCH2CH2), 2.60–
2.78 (3 H, m, PhCH2CH2 and CHCO), 3.67 (3 H, s, OCH3),
3.75–3.81 (1 H, m, NHCH), 5.21 (1 H, d, J 9.9, NH) and 7.20–
7.32 (5 H, m, Ar-H); δC(75.4 MHz; C2HCl3) 15.07 (CH3), 28.82
[C(CH3)3], 33.12 (ArCH2CH2), 36.61 (ArCH2CH2), 43.27
(CHCO), 52.07 (OCH3), 52.88 (CHNH), 79.51 [OC(CH3)3],
126.30 and 128.81 (Ar-CH), 142.12 (Ar-C quaternary), 156.47
(CO, urethane) and 176.20 (CO, ester); m/z (ES) 344 (100%,
[M ϩ Na]ϩ), 288 (48, [M ϩ Na Ϫ C4H9]ϩ).
Methyl (2S,3S)-3-amino-2-methyl-5-phenylpentanoate 5b
This compound was prepared in a manner identical to that
described for 5a using methyl (2S,3S,4E,αS)-2-methyl-3-
(N-benzyl-N-α-methylbenzylamino)-5-phenylpent-4-enoate 4b
(2.4 g, 6 mmol) to give the product amine 5b as a colourless
J. Chem. Soc., Perkin Trans. 1, 2001, 1696–1708
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Methyl (3R,4E)-3-(N-tert-butoxycarbonylamino)-5-phenylpent-
4-enoate 8a
removed under reduced pressure to give an oily residue. Purifi-
cation by flash silica column chromatography (ethyl acetate–
petroleum ether; 70:30) gave the free acid 9a as a white solid
(0.95 g, 71%), Rf = 0.15, mp 123–125 ЊC (Found C, 66.0; H, 7.3;
N, 4.75. C16H21NO4 requires C, 65.95; H, 7.25; N, 4.8%)
(HRMS: found [M ϩ Na]ϩ, 314.1374. C16H21NO4Na requires
314.1368); [α]D ϩ36.6 (c 1.1 in CHCl3); νmax(CH2Cl2)/cmϪ1 3322
(NH, urethane) and 1711 (CO, acid and urethane); δH(300
MHz; C2HCl3) 1.46 [9 H, s, C(CH3)3], 2.65–2.82 (2 H, m,
CH2CO), 4.62–4.78 (1 H, m, NHCH), 5.20–5.38 (1 H,
m, NH), 6.21 (1 H, dd, J 6.25 and 15.8, 4-H), 6.57 (1 H, d,
J 15.8, 5-H) and 7.20–7.42 (5 H, m, Ar-H); δC(75.4 MHz;
C2HCl3) 28.40 [C(CH3)3], 39.48 (CH2CO), 49.50 (CHNH),
79.78 [OC(CH3)3], 126.55, 127.81, 128.23, 128.58 and 131.11
Under an atmosphere of argon, a solution of N-Boc ester 7a
(4.0 g, 13 mmol) in dry carbon tetrachloride (50 cm3) was
treated with N-bromosuccinimide (2.6 g, 15 mmol). The reac-
tion mixture was illuminated for 3 h under a 60 W tungsten
lamp held ∼2 cm away from the flask while maintaining the
temperature at less than 10 ЊC. The reaction was judged com-
plete when the initial red–brown colour due to bromine faded
to a pale yellow colour. The precipitated succinimide was
filtered off and the filtrate was concentrated under reduced
pressure to give the crude bromide as a mixture of epimers. To
the resulting crude bromide was then added DBU (6.0 cm3,
38 mmol) and the mixture heated to 70 ЊC for 15 min. The
mixture was partitioned between diethyl ether (30 cm3) and
water (30 cm3). The organic layer was washed successively with
water (3 × 40 cm3) and brine (40 cm3), dried (MgSO4) and con-
centrated under reduced pressure to give the alkene as a dark
oil. Purification by flash silica column chromatography (ethyl
acetate–petroleum ether; 9:1) gave the alkene product 8a as an
oil which slowly solidified to a white solid upon drying in vacuo
(1.70 g, 42%), mp 65–67 ЊC (Found C, 67.0; H, 7.7; N, 4.4.
C17H23NO4 requires C, 66.9; H, 7.6; N, 4.6%) (HRMS: found
[M ϩ Na]ϩ, 328.1531. C17H23NO4Na requires 328.1525);
[α]D ϩ22.7 (c 2.2 in CHCl3); νmax(CH2Cl2)/cmϪ1 3353 (NH,
urethane), 1736 (CO, ester) and 1712 (CO, urethane); δH(300
MHz; C2HCl3) 1.46 [9 H, s, C(CH3)3], 2.67–2.74 (2 H, m,
CH2CO), 3.70 (3 H, s, OCH3), 4.62–4.78 (1 H, m, NHCH),
5.23–5.37 (1 H, m, NH), 6.19 (1 H, dd, J 6.2 and 15.7, 4-H),
6.55 (1 H, d, J 15.7, 5-H) and 7.20–7.38 (5 H, m, Ar-H); δC(75.4
MHz; C2HCl3) 28.41 [C(CH3)3], 39.54 (CH2CO), 49.13
(CHNH), 51.83 (OCH3), 79.68 [OC(CH3)3], 126.52, 127.75,
128.55, and 130.89 (Ar-CH and alkene C), 136.46 (Ar-C
quaternary), 155.07 (CO, urethane) and 171.63 (CO, ester);
m/z (ES) 328 (100%, [M ϩ Na]ϩ) and 272 (45, [M ϩ
Na ϩ H Ϫ C4H9]ϩ).
(Ar-CH and CH᎐CH), 136.39 (Ar-C quaternary), 155.34
᎐
(CO, urethane) and 174.63 (CO, acid); m/z (ES) 338 (20%,
[M ϩ 2Na Ϫ H]ϩ), 314 (100, [M ϩ Na]ϩ) and 258 (45, [M ϩ
Na ϩ H Ϫ C4H9]ϩ).
(2S,3S,4E)-3-(N-tert-Butoxycarbonylamino)-2-methyl-5-
phenylpent-4-enoic acid 9b
This compound was prepared in a manner identical to that
described for 9a using methyl (2S,3S,4E)-3-(N-tert- butoxy-
carbonylamino)-2-methyl-5-phenylpent-4-enoate 8b (1.2 g, 11
mmol) to give the free acid 9b as a colourless oil (1.1 g, 95%)
(HRMS: found [M ϩ Na]ϩ, 328.1523. C17H23NO4Na requires
328.1525); [α]D ϩ50.4 (c 4 in CH2Cl2); νmax(CH2Cl2)/cmϪ1 3421
(NH, urethane) and 1706 (CO, acid and urethane); δH(300
MHz; C2HCl3) 1.28 (3 H, d, J 7.17, CH3CH), 1.47 [9 H, s,
C(CH3)3], 2.79–2.90 (3 H, m, CH3CH), 4.45–4.55 (1 H, m,
NHCH), 5.45 (1 H, br d, NH), 6.13 (1 H, dd, J 9.0 and 15.8,
4-H), 6.57 (1 H, d, J 15.8, 5-H) and 7.20–7.35 (5 H, m, Ar-H);
δC(75.4 MHz; C2HCl3) 15.01 (CH3CH), 28.82 [C(CH3)3],
43.25 (CHCO), 56.25 (CHNH), 79.85 [OC(CH3)3], 126.98,
128.16, 128.99 and 131.84 (Ar-CH and CH᎐CH), 136.94 (Ar-C
᎐
quaternary), 156.66 (CO, urethane) and 180.44 (CO, acid);
m/z (ES) 350 (30%, [M ϩ 2Na Ϫ H]ϩ), 328 (100, [M ϩ Na]ϩ)
and 272 (20, [M ϩ Na Ϫ C4H8]ϩ).
Methyl (2S,3S,4E)-3-(N-tert-butoxycarbonylamino)-2-methyl-5-
phenylpent-4-enoate 8b
(3R,4E)-3-Amino-5-phenylpent-4-enoic acid 10a
This compound was prepared in a manner identical to that
described for 7a using methyl (2S,3S)-3-(N-tert-butoxy-
carbonylamino)-2-methyl-5-phenylpentanoate 7b to give the
product alkene 8b as an oil which slowly solidified upon drying
in vacuo to a white solid (1.25 g, 45%), mp 52–56 ЊC (HRMS:
found [M ϩ Na]ϩ, 342.1687. C18H27NO4Na requires 342.1681);
[α]D ϩ38.8 (c 10 in CH2Cl2); νmax(CH2Cl2)/cmϪ1 3433 (NH,
urethane) and 1712 (CO, ester and urethane); δH(300 MHz;
C2HCl3) 1.27 (3 H, d, J 7.0, CH3CH), 1.46 [9 H, s, C(CH3)3],
2.75–2.85 (1 H, m, CHCO), 3.70 (3 H, s, OCH3), 4.47 (1 H, m,
NHCH), 5.41–5.45 (1 H, m, NH), 6.11 (1 H, dd, J 6.4 and 16.0,
4-H), 6.56 (1 H, d, J 16.0, 5-H) and 7.20–7.35 (5 H, m, Ar-H);
δC(75.4 MHz; C2HCl3) 15.02 (CH3), 28.87 [C(CH3)3], 44.24
(CHCO), 52.26 (OCH3), 55.03 (CHNH), 79.96 [OC(CH3)3],
126.96, 127.10, 128.72, 128.98 and 131.57 (Ar-CH and alkene
C), 137.04 (Ar-C quaternary), 156.02 (CO, urethane) and
175.66 (CO, ester); m/z (ES) 342 (100%, [M ϩ Na]ϩ) and 286
(31, [M ϩ Na Ϫ C4H9]ϩ).
To an ice-cooled solution of the N-Boc unsaturated acid 9a
(1.8 g, 6.2 mmol) in dry CH2Cl2 (30 cm3) was added TFA
(20 cm3). The solution was allowed to warm up to room
temperature, stirred for a further 4 h and then concentrated
under reduced pressure to an oil. CH2Cl2 (25 cm3) was added
and the mixture concentrated again under reduced pressure and
the resulting residue was thoroughly dried in vacuo for several
hours. The residue was dissolved in water (30 cm3) and the
resulting aqueous solution was washed with ether (30 cm3). The
aqueous layer was lyophilised to give the β-amino acid 10a as
a foamy white solid (1.55 g, 83%); mp 123–125 ЊC; [α]D Ϫ7.6
(c 1 in water); νmax(KBr disc)/cmϪ1 1698 (NH) and 1650 (CO,
2
acid); δH(300 MHz; H2O) 2.79 (2 H, d, J 6.6, CH2CO), 4.20–
4.30 (1 H, m, NHCH), 6.12 (1 H, dd, J 8.1 and 16.0, 4-H), 6.69
(1 H, d, J 16.0, 5-H) and 7.18–7.40 (5 H, m, Ar-H); δC(75.4
2
MHz; H2O) 39.30 (CH2CO), 52.54 (CHNH2), 125.11 (4-C),
129.43 and 131.54 (Ar-CH), 137.80 (Ar-C quaternary), 138.29
(5-C) and 176.08 (CO, acid); m/z (CI) 192 (100 %, [M ϩ H]ϩ),
175 (45, [M Ϫ NH2]ϩ) and 132 (40, [M Ϫ CO2H Ϫ NH2]ϩ).
(3R,4E)-3-(N-tert-Butoxycarbonylamino)-5-phenylpent-4-enoic
acid 9a
(2S,3S,4E)-3-Amino-2-methyl-5-phenylpent-4-enoic 10b
To a cooled solution of N-Boc unsaturated ester 8a (1.4 g,
4.5 mmol) in methanol–water–THF (3:1:1) (45 cm3) was added
a solution of lithium hydroxide (800 mg, 31 mmol) in water
(3 cm3) and the mixture stirred at 0 ЊC for 3 h. The solution was
concentrated under reduced pressure to 2–3 cm3, diluted with
water (40 cm3), acidified to pH 2 with HCl (0.1 mol dmϪ3) and
then extracted with ethyl acetate (45 cm3). The organic layer
was washed with brine (30 cm3), dried (MgSO4) and solvent
This compound was prepared in a manner identical to that
described for 10a using (2S,3S,4E)-3-N-(tert-butoxy-
carbonylamino)-2-methyl-5-phenylpent-4-enoic acid 9b (1.1 g,
5.3 mmol) to give the β-amino acid 10b as a foamy white solid
after lyophilisation (870 mg, 79%) (HRMS: found [M ϩ Na]ϩ,
206.1188. C12H16NO2 requires 206.1181); [α]D Ϫ24.9 (c 8.6 in
water); νmax(KBr disc)/cmϪ1 1696 (NH) and 1650 (CO, acid);
1704
J. Chem. Soc., Perkin Trans. 1, 2001, 1696–1708

View Article Online
2
δH(300 MHz; H2O) 1.08 (3 H, d, J 7.17, CH3CH), 2.75–2.95
over 25 minutes; flow rate 2 cm3 minϪ1] and the peak corre-
(3 H, m, CH3CH), 4.00–4.05 (1 H, m, NHCH), 6.03 (1 H, dd,
J 9.0 and 15.8, 4-H), 6.67 (1 H, d, J 15.8, 5- H) and 7.20–7.35
(5 H, m, Ar-H); δC(75.4 MHz; C2H3O2H) 14.21 (CH3), 42.87
(CHCO), 56.45 (CHNH), 122.40, 127.91 and 130.00 (Ar-CH
and ArCHCH), 138.24 (Ar-C quaternary) and 178.01 (CO,
acid); m/z (CI) 206 (100%, [M ϩ H]ϩ), 160 (17, [M Ϫ CO2H]ϩ)
and 145 (15, [M Ϫ CO2H Ϫ NH2]ϩ).
sponding to retention time 19.6 min was collected and the
solvent removed under reduced pressure and by lyophilisation
to give the desired peptide. Mp 100–105 ЊC (softening point)
(HRMS: found [M ϩ Na]ϩ, 704.2903. C34H43N5O10Na requires
704.2908); [α]D ϩ20 (c 0.2 in water); νmax(KBr disc)/cmϪ1
3411 br (COOH), 1737 (CO, esters) and 1672 (amides); δH[500
MHz; (C2H3)2SO mixture of rotamers] 1.78–2.01 [2 H, m,
β-CH2(Glu)], 2.25–2.45 [4 H, m, γ-CH2 (Glu) and α-CH2 (pro-
panoyl)], 2.60–2.88 [2 H, m, β-CH2(Asp)], 2.77 and 2.85 (3 H,
2 × s, NCH3), 2.97–3.12 [2 H, m, β-CH2(Phe)], 3.55, 3.57 and
3.65 (6 H, 3 × s, 2 × CH3), 3.89–4.02 [2 H, m, CH2 (Sar)], 4.05–
4.12 [1 H, m, α-H, (Phe)], 4.23–4.32 [1 H, m, α-H, (Glu)], 4.61–
4.70 [1 H, m, α-H, (Asp)], 4.80–4.89 [1 H, m, β-H, (propanoyl)],
6.20–6.27 [1 H, m, γ-H (propanoyl)], 6.50 [1 H, d, J 18.0, δ-H
(propanoyl)], 7.16–7.44 (10 H, m, Ar-H), 8.18–8.24 [1 H, m,
NH (Phe)], 8.34 [0.6 H, d, J 8.5, NH (Asp)], 8.42 [0.4 H, d,
J 8.5, NH (Glu)], 8.46 [0.6 H, d, J 8.5, NH (Glu)], 8.56 [0.4 H,
d, J 8.5, NH (Asp)] and 8.60 [0.6 H, d, J 8.5, NH (Asp)]; δC[125
MHz; (C2H3)2SO mixture of rotamers] 26.4 [β-CH2 (Glu)], 28.3
and 28.5 [γ-CH2 (Glu)], 35.7 [β-CH2 (Asp)], 37.0 [β-CH2 (Asp)],
39.6 [α-CH(propanoyl)], 34.1 and 35.5 (NCH3), 48.4 [α-C
(Asp)], 47.8 [β-C (propanoyl)], 49.5 and 51.3 [α-C (Sar)], 51.4
[α-C (Glu)], 51.4 and 51.6 (2 × CH3), 53.5 [α-C (Phe)], 126.1,
127.1, 127.5, 128.1, 128.4 and 128.7 (Ar-CH and Ar-C quater-
nary), 128.6 and 129.4 [γ-and δ-C (propanoyl)], 134.8 [γ-C
(Phe)] and 165.0, 166.9, 168.2, 169.3, 171.3, 171.8 and 172.3
(CO, amides and esters); m/z (ES) 726 (17%, [M ϩ 2Na Ϫ H]ϩ),
704 (100, [M ϩ Na]ϩ) and 682 (30, [M ϩ H]ϩ).
(3R,4E)-3-[N-(Fluoren-9-ylmethoxycarbonyl)amino]-5-phenyl-
pent-4-enoic acid 11a
To an ice-cooled solution of (3R,4E)-3-amino-5-phenylpent-4-
enoic 10a (1.7 mg, 5.6 mmol) in dioxane–water (60 cm3; 3:1)
was added potassium carbonate (1.4 g, 10 mmol). A solution of
Fmoc-Cl (2.0 g, 6.6 mmol) in dioxane (10 cm3) was added. The
resulting solution was warmed to room temperature and stirred
for 12 h. The solution was concentrated under reduced pressure
to one third of the original volume and water (35 cm3) was
added. The aqueous phase was acidified to pH 2 with 1 mol
dmϪ3 HCl and then extracted with ethyl acetate (2 × 25 cm3).
The organic layers were combined, washed with brine (5 cm3),
dried (MgSO4) and the solvent was removed under reduced
pressure to give the crude product as an oil. Purification by
flash silica chromatography (petroleum ether–ethyl acetate;
60:40) gave the Fmoc protected amine 11a as a white solid
(1.2 g, 52%), mp 167–171 ЊC (HRMS: found [M ϩ Na]ϩ,
436.1529. C26H23NO4Na requires 436.1525); [α]D ϩ22.4 (c 8.6
in CHCl3); νmax(CH2Cl2)/cmϪ1 3310 (NH, urethane) and 1690
(CO, urethane); δH(300 MHz; C2HCl3) 2.60–2.85 (2 H, m,
CH2CO), 4.17 (1 H, t, J 6.60, Fmoc-CH), 4.30–4.60 (2 H, m,
Fmoc-CHCH2), 4.61–4.75 (1 H, m, NHCH), 5.39–5.47 (1 H,
m, NH), 6.08–6.20 (1 H, m, 4-H), 6.50 (1 H, d, J 15, 5-H) and
7.15–7.75 (13 H, m, Ar-H); m/z (ES) 436 (100%, [M ϩ Na]ϩ).
(2S)-Phenylalanyl-[(2S,3S,E)-2-methyl-3-phenylethenyl-3-
aminopropanoyl]-[ꢀ-methyl (2R)-glutamyl]-ꢁ-sarcosyl-[ꢀ-methyl
(2R)-aspartate] diester 17b
This compound was synthesised on the peptide synthesiser
from Fmoc-Asp-(α)-OMe-O-Wang 16 (0.75 g, ∼0.6 mmol
loading) using the general SPPS method described above,
followed by cleavage of the peptide from the resin to give the
crude peptide 17b as a white solid in quantitative yield after
(2S,3S,4E)-3-[N-(Fluoren-9-ylmethoxycarbonyl)amino]-2-
methyl-5-phenylpent-4-enoic acid 11b
This compound was prepared in a manner identical to that
described for 11a using (2S,3S,4E)-3-amino-2-methyl-5-
phenylpent-4-enoic 10b (800 mg, 3.2 mmol) to give the Fmoc-
protected amine 11b as a white solid (1.1 g, 66%), mp 147–
151 ЊC (Found C, 75.8; H, 6.0; N, 3.2. C27H25NO4 requires C,
75.9; H, 5.9; N, 3.3%) (HRMS: found [M ϩ Na]ϩ, 704.2903.
C27H25NO4Na requires 704.2908); [α]D ϩ41.5 (c 6.5 in EtOH);
νmax(CH2Cl2)/cmϪ1 3321 (NH, urethane) and 1712 (CO, ester
and urethane); δH(300 MHz; C2HCl3) 1.30 (3 H, d, J 6.80,
CH3CH), 2.85–2.95 (1 H, m, CH3CH), 4.17 (1 H, t, J 6.62,
Fmoc-CH), 4.30–4.60 (3 H, m, Fmoc-CHCH2 and NHCH),
5.56 (1 H, d, J 10.0, NH), 6.08 (1 H, dd, J 6.40 and 15.7, 4-H),
6.50 (1 H, d, J 15.63, 5-H) and 7.15–7.55 (15 H, m, Ar-H);
δC(75.4 MHz; C2HCl3) 15.22 (CH3), 44.10 (CHCO), 47.70
(Fmoc-CHCH2), 55.47 (CHNH), 67.36 (Fmoc-CHCH2),
120.43, 125.49, 127.04, 127.53, 127.75, 128.15, 128.34, 129.01
and 132.38 (Ar-CH and 2 × alkene), 136.64, 141.75 and 144.30
(Ar-C quaternary), 156.69 (CO, urethane) and 178.01 (CO,
acid); m/z (ES) 450 (100%, [M ϩ Na]ϩ).
lyophilisation. For analytical purposes
a small amount
(∼10 mg) was further purified by preparative RP HPLC using
a Luna C-18 10 µm column (150 × 21.1 mm). The column was
eluted under gradient conditions [100%A– 0%B to 40%A–
60%B (solvent A: 0.1% aqueous TFA; solvent B: 80:20 CH3-
CN–H2O with 0.1% TFA) over 25 min; flow rate 2 cm3 minϪ1]
and the peak corresponding to retention time 20.4 min was
collected and the solvent removed under reduced pressure to
give the desired peptide, mp 90–95 ЊC (softening point)
(HRMS: found [M ϩ Na]ϩ, 718.3054. C35H45N5O10Na requires
718.3064); νmax(KBr disc)/cmϪ1 3426 br (COOH), 1737 (CO,
esters) and 1672 (amides); δH[500 MHz; (C2H3)2SO mixture of
rotamers] 0.90–0.95 [3 H, m, CH3 (propanoyl)], 1.79–1.98 [2 H,
m, β-CH2 (Glu)], 2.22–2.40 [2 H, m, γ-CH2 (Glu)], 2.60–2.80
[3 H, m, α-H (propanoyl) and β-CH2 (Asp)], 2.73 (3 H, s,
NCH3), 2.93–3.13 [2 H, m, β-CH2 (Phe)], 3.65 (6 H, s, 2 × CH3),
3.85–4.00 [2 H, m, CH2 (Sar)], 4.20–4.28 [2 H, m, α-H (Phe) and
α-H, (Glu)], 4.62 [2 H, m, α-H (Asp) and β-H (propanoyl)],
6.20–6.26 [1 H, m, γ-H (propanoyl)], 6.55 [1 H, d, J 18.0, δ-H
(propanoyl)], 7.23–7.44 (10 H, m, Ar-H), 8.16–8.20 [2 H, m,
NH2 (Phe)], 8.32 [1 H, d, J 8.5, NH (Asp)], 8.38 [1 H, d, J 8.5,
NH (Glu)] and 8.46 [1 H, d, J 8.5, NH (propanoyl)]; δC[125
MHz; (C2H3)2SO mixture of rotamers] 14.3 [CH3 (propanoyl)],
26.1 [β-CH2 (Glu)], 28.7 [γ-CH2 (Glu)], 35.8 [β-CH2 (Asp)], 35.0
(NCH3), 37.1 [β-CH2 (Phe)], 42.8 [α-CH (propanoyl)], 48.4 [α-C
(Asp)], 49.4 and 52.0 [α-C (Sar)], 51.5 [α-C (Glu)], 52.1 (OCH3),
53.0 [β-C (propanyl)], 53.4 [α-C (Phe)], 126.1, 127.1, 127.5,
127.9, 128.5, 128.6 and 129.4 (Ar-CH and Ar-C quaternary),
134.8 [γ-C (Phe)], 136.3 [γ-C (propanoyl)] and 167.3, 171.3,
172.0 and 173.3 (CO, amides and esters); m/z (ES) 718 (100%,
[M ϩ Na]ϩ) and 696 (30, [M ϩ H]ϩ).
(2S)-Phenylalanyl-[(3S,E)-3-phenylethenyl-3-aminopropanoyl]-
[ꢀ-methyl (2R)-glutamyl]-ꢁ-sarcosyl-[ꢀ-methyl (2R)-aspartate]
diester 17a
This compound was synthesised on the peptide synthesiser
from Fmoc-Asp-(α)-OMe-O-Wang 16 (1 g, 0.54 mmol loading)
using the general SPPS method described above, followed by
cleavage of the peptide from the resin to give the crude peptide
17a as a white solid after lyophilisation (280 mg). For analytical
purposes a small amount (∼10 mg) was further purified by
preparative RP HPLC using a Luna C-18 10 µm column
(150 × 21.1 mm). The column was eluted under gradient
conditions [100%A–0%B to 50%A–50%B (solvent A: 0.1%
aqueous TFA; solvent B: 80:20 CH3CN–H2O with 0.1% TFA)
J. Chem. Soc., Perkin Trans. 1, 2001, 1696–1708
1705

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Cyclo[-(3S,E)-3-phenylethenyl-3-aminopropanoyl-ꢀ-(R)-Glu-ꢀ-
OMe-ꢁ-Sar-(R)-Asp-ꢀ-OMe-ꢂ-(S)-Phe-] 18a
vigorously stirred for 1 h and then partitioned between ethyl
acetate (3 cm3) and 1 mol dmϪ3 NaHSO4 solution (1.5 cm3). The
organic layer was separated, dried (MgSO4) and concentrated
under reduced pressure to give the diacid as a white solid
in quantitative recovery. The crude diacid was then purified
by preparative RP HPLC using a Luna C-18 10 µm column
(150 × 21.1 mm). The column was eluted under gradient
conditions [100%A–0%B to 40%A–60%B (solvent A: 0.1%
aqueous TFA; solvent B: 100% CH3CN) over 25 min; flow rate
2 cm3 minϪ1] and the peak corresponding to retention time 20.4
min was collected and the solvent removed under reduced
pressure to give the desired peptide (7 mg, 68%), mp ∼130 ЊC
(softening point); νmax(KBr disc)/cmϪ1 3444 br (NH), 1737 (CO,
esters) and 1639 (amides); δH[500 MHz; (C2H3)2SO mixture of
rotamers] 1.35–1.85 [2 H, m, β-CH2(Glu)], 1.95–2.60 [4 H,
m, α-CH2 (Glu) and β-CH2 (Asp)], 2.65 and 2.72 (3 H, 2 × s,
NCH3), 2.75–2.97 [2 H, α-CH2 (propanoyl)], 3.05–3.30 [2 H, m,
β-CH2 (Phe)], 3.78–4.18 [2 H, m, CH2 (Sar)], 4.39–4.44 [1 H,
m, α-H, (Glu)], 4.51–4.59 [1 H, m, α-H, (Phe)], 4.78–4.84 [1 H,
m, α-H, (propanoyl)], 4.88–4.92 [1 H, m, α-H, (Asp)], 6.18–6.24
[1 H, m, γ-H (propanoyl)], 6.46 [1 H, d, J 18.0, δ-H (pro-
panoyl)], 7.16–7.44 (10 H, m, Ar-H), 7.76 [1 H, d, J 8.5, NH
(Asp)], 8.19 [1 H, m, NH (Phe)], 8.40 [1 H, d, J 8.5, NH (pro-
panoyl)] and 8.78 [1 H, d, J 8.5, NH (Glu)]; δC[125 MHz;
(C2H3)2SO mixture of rotamers] 26.3 [β-CH2 (Glu)], 27.5 [γ-
CH2 (Glu)], 35.4 (NCH3), 35.7 [β-CH2 (Phe)], 35.9 [β-CH2
(Asp)], 37.0 [α-CH (propanoyl)], 46.4 [β-C (propanoyl)], 48.2
[α-C (Asp)], 49.8 [α-C (Glu)], 50.9 [α-C (Sar)], 53.6 [α-C (Phe)],
125.8, 127.1, 128.1 and 128.7 (Ar-CH and Ar-C quaternary),
129.0 and 129.4 [γ- and δ-C (propanoyl)] and 168.4, 169.2,
170.6, 171.5 and 172.3 (CO, amides and esters); m/z (ES) 658
(100%, [M ϩ Na]ϩ).
This compound was synthesised according to the general
cyclisation procedure using 17a (210 mg, 0.3 mmol) as starting
material to give the desired cyclic peptide 18a, after flash silica
chromatography (methanol–CH2Cl2; 2:98), as a white solid
(45 mg, 28%), Rf = 0.5, mp >230 ЊC (decomp.) (HRMS: found
[M ϩ Na]ϩ, 686.2797. C34H41N5O9Na requires 686.2802);
[α]D ϩ5.6 (c 1 in CH3OH); νmax(KBr disc)/cmϪ1 3318 br (NH),
1742 (CO, esters) and 1652 (amides); δH[500 MHz; (C2H3)2SO
mixture of rotamers] 1.75–2.02 [2 H, m, β-CH2 (Glu)], 2.15–
2.35 [2 H, m, γ-CH2 (Glu)], 2.40–2.80 [4 H, m, β-CH2 (Asp) and
α-CH2 (propanoyl)], 2.78 and 2.85 (3 H, 2 × s, NCH3), 3.10–
3.30 [2 H, m, β-CH2 (Phe)], 3.60, 3.63 and 3.65 (6 H, 3 × s,
2 CH3), 3.89–4.18 [2 H, m, CH2 (Sar)], 4.23–4.32 [1 H, m, α-H,
(Glu)], 4.37–4.50 [2 H, m, α-H, (Phe) and α-H (Asp)], 4.78–4.86
[1 H, m, β-H (propanoyl)], 6.28–6.33 [1H, m, γ-H (propanoyl)],
6.52 [1 H, d, J 18.0, δ-H (propanoyl)], 7.18–7.44 (10 H, m, Ph)
and 7.80–8.74 [4 H, m, NH of Phe, Asp, Glu and propanoyl];
δC[125 MHz; (C2H3)2SO mixture of rotamers] 26.2 [β-CH2
(Glu)], 28.0 [γ- CH2 (Glu)], 34.1 (NCH3), 35.1 [β-CH2 (Asp)],
36.4 [β-CH2 (Phe)], 38.6 [α-CH (propanoyl)], 47.3 [β-C (pro-
panoyl)], 48.7 [α-C (Asp)], 51.1 [α-C (Glu)], 51.1 [α-C (Sar)],
51.4 and 51.6 (2 × CH3), 53.6 [α-C (Phe)] and 125.8, 127.1,
127.5, 128.1 and 128.7 (Ar-CH and Ar-C quaternary); m/z (ES)
686 (100%, [M ϩ Na]ϩ).
Cyclo[-(2S,3S,E)-2-methyl-3-phenylethenyl-3-aminopropanoyl-
ꢀ-(R)-Glu-ꢀ-OMe-ꢁ-Sar-(R)-Asp-ꢀ-OMe-ꢂ-(S)-Phe-] 18b
This compound was synthesised according to the general
cyclisation procedure using 17b (310 mg, 0.45 mmol) as starting
material to give the desired cyclic peptide 18b after flash silica
chromatography (methanol–CH2Cl2; 2:98), as a white solid
(80 mg, 27%), Rf = 0.5, mp > 150 ЊC (softening point) (HRMS:
found [M ϩ Na]ϩ, 700.2964. C35H43N5O9Na requires 700.2958);
νmax(KBr disc)/cmϪ1 3446 br (NH), 1740 (CO, esters) and 1645
(amides); δH[500 MHz; (C2H3)2SO mixture of rotamers] 1.00–
1.10 [3 H, m, CH3 (propanoyl)], 1.30–1.36 [1 H, m, 1 H of
β-CH2 (Glu)], 1.70–1.75 [1 H, m, 1 H of β-CH2 (Glu)], 1.90–
1.96 [1 H, m, 1 H of γ-CH2 (Glu)], 2.15–2.20 [1 H, m, 1 H of
γ-CH2 (Glu)], 2.63 and 2.72 (3 H, 2 × s, NCH3), 2.72– 2.80 [3 H,
m, β-CH2 (Asp) and α-H (propanoyl)], 2.96–3.14 [2 H, m,
β-CH2 (Phe)], 3.20–3.24 [1 H, m, 1 H of CH2 (Sar)], 3.50, 3.58,
3.62 and 3.67 (6 H, 4 × s, 2 × OCH3), 4.25–4.31 [1 H, m, α-H,
(Phe)], 4.32–4.45 [2 H, m, α-H, (Glu) and α-H, (Asp)], 4.48–
4.55 [1 H, m, 1 H of CH2 (Sar)], 4.64–4.68 [1 H, m, β-H (pro-
panoyl)], 6.22 [1 H, dd, J 7.5, 18.0, γ-H (propanoyl)], 6.52 [1 H,
d, J 18.0, γ-H (propanoyl)], 7.18–7.44 (10 H, m, Ar-H) and
8.00–8.82 (4 H, m, NH of Phe, Asp, Glu, propanoyl); δC[125
MHz; (C2H3)2SO mixture of rotamers] 14.83 and 15.97 [CH3
(propanoyl)], 26.24, 26.69, 27.91 and 29.36 [β-CH2 (Glu) and
γ-CH2 (Glu)], 33.61 and 35.54 (NCH3), 35.78, 36.43 and 37.15
[β-CH2 (Asp) and β-CH2 (Phe)], 41.53 and 41.95 [α-CH
(propanoyl)], 48.86 [α-C (Asp)], 49.99 [α-C (Glu)], 50.08 [α-C
(Sar)], 51.25, 51.35, 51.76, 52.02, 52.18 and 52.24 (2 × OCH3),
52.56 [β-C (propanoyl)], 54.78 and 55.79 [α-C (Phe)] and
126.19, 126.30, 128.05, 128.22, 128.61, 128.66, 129.01, 129.09,
129.82 and 129.89, [Ar-CH, γ-C (propanoyl) and δ-C (pro-
panoyl)], 136.17, 136.47, 138.24 and 128.34 (Ar-C quaternary)
and 167.57, 168.15, 169.32, 170.13, 170.52, 171.13, 171.56,
171.67, 171.92, 172.33, 174.82 and 175.52 (CO, amides and
esters); m/z (ES) 700 (100%, [M ϩ Na]ϩ).
Cyclo[-(2S,3S,E)-2-methyl-3-phenylethenyl-3-aminopropanoyl-
ꢂ-(R)-Glu-ꢀ-OH-ꢁ-Sar-(R)-Asp-ꢀ-OH-ꢂ-(S)-Phe-] 19b
This compound was prepared in a manner identical to that
described for 19a using 18b (5 mg, 0.077 mmol) as starting
material to give the crude product as a white solid. The crude
diacid 19b was then purified by preparative RP HPLC using a
Luna C-18 10 µm column (150 × 21.1 mm).The column was
eluted under gradient conditions [100%A–0%B to 30%A–70%B
(solvent A: 0.1% aq. TFA; solvent B: 100% CH3CN) over
25 min; flow rate 2 cm3 minϪ1] and the peak corresponding to
retention time 16.9 min was collected and the solvent removed
under reduced pressure and by lyophilisation to give the desired
peptide (4 mg, 80%), mp >150 ЊC (decomp.); [α]D ϩ24 (c 0.1 in
CH3OH); νmax(KBr disc)/cmϪ1 3430 br (NH), 1732 (CO, esters)
and 1637 (amides); δH[500 MHz; (C2H3)2SO] 1.04–1.08 [3 H, m,
CH3 (propanoyl)], 1.25–1.33 [1 H, m, 1 H of β-CH2 (Glu)],
1.80–1.83 [1 H, m, 1 H of β-CH2 (Glu)], 2.08–2.16 [2 H, m,
1 H of γ-CH2 (Glu) and 1 H of β-CH2 (Asp)], 2.22–2.32 [1 H,
m, 1 H of γ-CH2 (Glu)], 2.56 [1 H, m, 1 H of β-CH2 (Asp)],
2.63 (3 H, s, NCH3), 3.02–3.10 [1 H, α-CH (propanoyl)], 3.14–
3.24 [3 H, m, β-CH2 (Phe) and 1 H of CH2 (Sar)], 4.39–4.24
[1 H, m, α-H, (Glu)], 4.56–4.62 [2 H, m, α-H, (Phe) and β-H
(propanoyl)], 4.63–4.68 [1 H, m, 1 H of α-CH2 (Sar)], 4.88–5.01
[1 H, m, α-H, (Asp)], 6.25 (1 H, dd, J 4.5 and 16.0, γ-H
(propanoyl)], 6.38 (1 H, d, J 16.0, δ-H (propanoyl)], 7.26–7.42
(10 H, m, Ar-H), 7.68 [1 H, d, J 8.5, NH (Asp)], 8.02 [1 H,
d, J 8.5, NH (propanoyl)], 8.16 [1 H, m, J 8.5, NH (Phe)]
and 8.80 [1 H, d, J 8.5, NH (Glu)]; δC[125 MHz; (C2H3)2SO]
14.3 [CH3 (propanoyl)], 26.3 [β-CH2 (Glu)], 27.5 [γ-CH2 (Glu)],
35.2 (NCH3), 35.6 [β-CH2 (Phe)], 35.9 [β-CH2 (Asp)], 37.0
[α-CH (propanoyl)], 48.0 [α-C (Asp)], 49.7 [α-C (Glu)], 50.7
[α-C (Sar)], 52.1 [β-C (propanoyl)], 53.6 [α-C (Phe)], 125.8,
127.1, 128.1 and 128.7 (Ar-CH and Ar-C quaternary), 129.0
[γ- and δ-C (propanoyl)] and 168.0, 170.2, 170.6, 171.5 and
176.0 (CO, amides and esters); m/z (ES) 672 (100%,
[M ϩ Na]ϩ).
Cyclo[-(3S,E)-3-phenylethenyl-3-aminopropanoyl-ꢀ-(R)-Glu-ꢀ-
OH-ꢁ-Sar-(R)-Asp-ꢀ-OH-ꢂ-(S)-Phe-] 19a
To a cooled solution of diester 18a (11 mg, 0.16 mmol) in
a mixture of THF (2 cm3) and water (0.75 cm3) was added
10 drops of 1 mol dmϪ3 LiOH solution.The mixture was
1706
J. Chem. Soc., Perkin Trans. 1, 2001, 1696–1708

View Article Online
(2S)-Phenylalanyl-[(2S,3S,E)-2-methyl-3-phenylethenyl-3-
aminopropanoyl]-[ꢀ-methyl (2R)-glutamyl]-ꢁ-[(2S)-prolyl]-[ꢀ-
methyl (2R)-aspartate] diester 20
56.7 [α-CH (Phe)], 59.6 [α-CH (Pro)], 126.1, 126.2, 127.6, 129.9,
128.1, 128.5, 128.6, 126.9, 129.0, 129.1, 129.7 and 128.1 (γ-
and δ-C propanoyl, and Ar-CH), 136.4 and 138.8 (Ar-C
quaternary) and 169.6, 170.1, 170.7, 171.3, 171.6, 172.0 and
175.5 (ester and amides); m/z (ES) 726 (100%, [M ϩ Na]ϩ).
This compound was synthesised on the peptide synthesiser
from Fmoc-Asp-(α)-OMe-O-Wang 16 (0.8 g, ∼0.6 mmol
loading) using the general SPPS method described above, fol-
lowed by cleavage of the peptide from the resin to give the crude
peptide 20 as a white solid in quantitative recovery (433 mg)
Cyclo[-(2S,3S,E)-2-methyl-3-phenylethenyl-3-aminopropanoyl-
ꢂ-(R)-Glu-ꢀ-OH-ꢁ-(S)-Pro-(R)-Asp-ꢀ-OH–ꢂ-(S)-Phe-] 22
1
after lyophilisation. H NMR spectroscopy showed that the
This compound was prepared in a manner identical to that
described for 19a using 21 (4.5 mg, 0.07 mmol) as starting
material to give the crude product as a white solid. The crude
product was purified by preparative RP HPLC using a Luna
C-18 10 µm column (150 × 21.1 mm). The column was eluted
under gradient conditions [100%A–0%B to 40%A–60%B
(solvent A: 0.1% aq. TFA; solvent B: 100% CH3CN) over
25 min; flow rate 2 cm3 minϪ1] and the peak corresponding to
retention time 17.4 min was collected and the solvent removed
under reduced pressure and by lyophilisation to give the desired
peptide as a white solid (3.5 mg, 85%), mp ∼130 ЊC (softening
point) (HRMS: found [M ϩ Na]ϩ, 698.2822. C35H41N5O9Na
requires 698.2802); νmax(KBr disc)/cmϪ1 1742 (CO, esters) and
1635 (amides); δH[500 MHz; (C2H3)2SO] 1.08–1.12 [3 H, m,
CH3 (propanoyl)], 1.25–1.35 [2 H, m, β-CH2 (Glu)], 1.56–1.75
[2 H, m, γ-CH2 (Pro)], 1.76–1.80 [1 H, m, 1 H of γ-CH2 (Glu)],
1.92–2.06 [2 H, m, β-CH2 (Pro)], 2.08–2.20 [1 H, m, 1 H of
β-CH2 (Asp)], 2.21–2.26 [1 H, m, 1 H of γ-CH2 (Glu)], 2.51
[1 H, m, 1 H of β-CH2 (Asp)], 2.77–2.82 [1 H, m, 1 H of δ-CH2
(Pro)], 2.92–2.98 [1 H, m, 1 H of δ-CH2 (Pro)], 3.00–3.05 [1 H,
α-H (propanoyl)], 3.20–3.23 [2 H, m, β-CH2 (Phe)], 4.25–4.30
[1 H, m, α-H (Pro)], 4.40–4.50 [1 H, m, α-H, (Glu)], 4.54–4.62
[2 H, m, α-H, (Phe) and β-H (propanoyl)], 4.88–4.92 [1 H, m,
α-H, (Asp)], 6.15–6.20 (1 H, m, γ-H (propanoyl)], 6.38 (1 H, d,
J 16.0, δ-H (propanoyl)], 7.26–7.42 (10 H, m, Ar-H), 7.80–7.85
[2 H, m, NH (Asp and Phe)], 8.07 [1 H, d, J 8.5, NH (prop-
anoyl)] and 8.62–8.65 [1 H, m, NH (Glu)]; δC[100.6 MHz;
(C2H3)2SO, predominately trans rotamer] 14.4 [β-CH3 (prop-
anoyl)], 21.8 [γ-CH2 (Pro)], 25.5 [β-CH2 (Glu)], 26.5 and 27.0
[β-CH2 (Pro) and γ-CH2 (Glu)], 32.9 [β-CH2 (Phe)], 33.8 [β-CH2
(Asp)], 39.5 [α-CH (propanoyl)], 44.0 [δ-CH2 (Pro)], 46.9 [α-CH
(Asp)], 48.0 [α-CH (Glu)], 50.1 [β-CH (propanoyl)], 51.9 [α-CH
(Phe)], 57.7 [α-CH (Pro)], 124.1, 124.2, 124.6, 125.5, 125.7,
126.2, 126.4, 126.7, 126.9, 127.1, 127.4 and 127.8 (γ and δ-C
propanoyl, and Ar-CH), 134.1 and 135.7 (Ar-C quaternary),
167.9, 168.6, 168.8, 169.5, 169.7, 170.9 and 174.5 (amides and
acids); m/z (ES) 698 (100%, [M ϩ Na]ϩ).
crude peptide was at least 90% pure. For analytical purposes a
small amount (∼10 mg) was further purified by preparative
RP HPLC using a Luna C-18 10 µm column (150 × 21.1 mm).
The column was eluted under gradient conditions [100%A–
0%B to 40%A–60%B (solvent A: 0.1% aq. TFA; solvent B:
80:20 CH3CN–H2O with 0.1% TFA) over 25 min; flow rate
2 cm3 minϪ1] and the peak corresponding to retention time of
approx. 21 min was collected and the solvent removed under
reduced pressure and by lyophilisation to give the desired pep-
tide. Mp 114–117 ЊC (softening point); νmax(KBr disc)/cmϪ1
1740 (CO, esters) and 1672 (amides); δH[500 MHz; (C2H3)2SO
mixture of rotamers] 0.86–0.90 [3 H, m, CH3 (propanoyl)],
1.68–1.96 [6 H, m, β-CH2 (Glu), β-CH2 (Pro) and γ-CH2 (Pro)],
2.12–2.26 [2 H, m, γ-CH2 (Glu)], 2.60–2.80 [3 H, m, α-H (prop-
anoyl) and β-CH2 (Asp)], 2.90–3.13 [3 H, m, 1 H of δ-CH2
(Pro) and β-CH2 (Phe)], 3.20–3.26 [1 H, m, 1 H of δ-CH2 (Pro)],
3.55–3.64 (6 H, m, 2 × CH3), 4.16–4.26 [3 H, m, α-H (Phe), α-H
(Glu) and α-H (Pro)], 4.55–4.60 [1 H, m, α-H (Asp)], 4.64–4.69
[1 H, m, β-H (propanoyl)], 6.18–6.24 [1 H, m, γ-H (propanoyl)],
6.50–6.56 [1 H, m, δ-H (propanoyl)], 7.21–7.40 (10 H, m,
Ar-H), 8.06–8.16 [2 H, m, NH2 (Phe)], 8.18 [1 H, d, J 8.5, NH
(Asp)], 8.38 [1 H, d, J 8.5, NH (Glu)] and 8.44 [1 H, d, J 8.5,
NH (propanoyl)]; δC[125 MHz; (C2H3)2SO mixture of rotamers]
14.06 and 14.30 [CH3 (propanoyl)], 22.01 and 23.82 [γ-CH2
(Pro)], 26.05 [β-CH2 (Glu)], 29.29 [β-CH2 (Pro)], 30.19 [γ-CH2
(Glu)], 35.98 [β-CH2 (Asp)], 37.47 [β-CH2 (Phe)], 42.77 [α-CH
(propanoyl)], 46.37 [δ-CH2 (Pro)], 48.65 [α-C (Asp)], 51.68 [α-C
(Glu)], 51.85, 52.02 and 52.08 (OCH3), 53.01 [β-C (propanyl)],
53.42 [α-C (Phe)], 59.00 [α-CH2 (Pro)], 126.13, 127.14, 127.57,
127.99, 128.56, 128.61, 129.42, 130.34, 134.86 and 136.34 [Ar-
CH, Ar-C quaternary, γ-C (propanoyl) and δ-C (propanoyl)]
and 169.76, 171.20, 171.45, 172.24 and 173.74 (CO, amides
and esters); m/z (ES) 766 (20%, [M ϩ 2Na Ϫ H]ϩ), 744 (100,
[M ϩ Na]ϩ) and 722 (35, [M ϩ H]ϩ).
Cyclo[-(2S,3S,E)-2-methyl-3-phenylethenyl-3-aminopropanoyl-
ꢂ-(R)-Glu-ꢀ-OMe-ꢁ-(S)-Pro-(R)-Asp-ꢀ-OMe–ꢂ-(S)-Phe-] 21
This compound was synthesised according to the general
cyclisation procedure using pentapeptide 20 (250 mg, 0.35
mmol) as starting material to yield the cyclic peptide 21 as
a white solid after column chromatography (55 mg, 23%)
(HRMS: found [M ϩ Na]ϩ, 726.3123. C37H45N5O9Na requires
726.3115); δH[500 MHz; (C2H3)2SO] 1.11 [3 H, d, J 7, CH3
(propanoyl)], 1.30–1.36 [1 H, m, 1 H of β-CH2 (Glu)], 1.55–1.62
[1 H, m, 1 H of β-CH2 (Glu)], 1.72–1.98 [5 H, m, 1 H of γ-CH2
(Glu), β-CH2 (Pro), γ-CH2 (Pro)], 2.15–2.24 [1 H, m, 1 H of
γ-CH2 (Glu)], 2.66–2.78 [3 H, m, 1 H of β-CH2 (Asp) and
δ-CH2 (Pro)], 2.86–2.98 [2 H, m, 1 H of β-CH2 (Asp) and δ-H
(propanoyl)], 3.16–3.30 [2 H, m, β-CH2 (Phe)], 3.57, 3.58 and
3.66 [6 H, 3 × s, OCH3 (Asp) and OCH3 (Asp)], 4.05–4.65 [5 H,
α-H of Glu, Asp, Phe and Pro, and β-CH (propanoyl)], 6.19
[1 H, dd, J 3.5 and 16, γ-H (propanoyl)], 6.41 [1 H, dd, J 16,
δ-H (propanoyl)], 7.15–7.42 (10 H, m, Ar-H), 7.75–8.50 (3 H,
m, NH of propanoyl, Phe and Asp) and 8.67 [1 H, d, J 9.5, NH
(Glu)]; δC[125.8 MHz; (C2H3)2SO, predominately trans rotamer]
15.8 [β-CH3 (propanoyl)], 23.6 [γ-CH2 (Pro)], 26.9 [β-CH2
(Glu)], 28.9 and 30.0 [β-CH2 (Pro) and γ-CH2 (Glu)], 34.8
[β-CH2 (Phe)], 36.7 [β-CH2 (Asp)], 41.7 [α-CH (propanoyl)],
45.8 [δ-CH2 (Pro)], 49.6 [α-CH (Asp)], 50.2 [α-CH (Glu)], 52.1
and 52.2 [OCH3 (Asp) and (Glu)], 52.6 [β-CH (propanoyl)],
Acknowledgements
The authors would like to thank Dr R. Beri for providing a
recombinant strain of E. coli expressing PP1 and Drs M.
Akhtar and A. P. Mehrotra for scientific support. We thank the
BBSRC Biomolecular Sciences Committee for grant B/49/
B07992 and AstraZeneca for a Strategic Research Fund.
References
1 (a) L. N. Johnson and M. O’Reilly, Curr. Opin. Struct. Biol., 1996, 6,
762; (b) L. N. Johnson and D. Barford, Annu. Rev. Biophys. Biomol.
Struct., 1993, 22, 1056.
2 (a) N. Berndt, Front. Biosci., 1999, 4, d22; (b) H. Y. L. Tung,
W. Wang and C. S. M. Chain, Mol. Cell. Biol., 1995, 15, 6064;
(c) G. A. Nimmo and P. Cohen, Eur. J. Biochem., 1978, 87, 353.
3 (a) S. Wera and B. A. Hemmings, Biochem. J., 1995, 311, 17; (b)
E. Y. C. Lee, L. Zhang, S. Zhao, Q. Wei, J. Zhang, Z. Q. Qi and
E. R. Belmonte, Front. Biosci., 1999, 4, d270.
4 J. B. Aggen, A. C. Nairn and A. R. Chamberlin, Chem. Biol., 2000,
7, R13.
5 P. Cohen, Annu. Rev. Biochem., 1989, 58, 453.
6 D. L. Lohse, J. M. Denu and J. E. Dixon, Structure (London), 1995,
3, 987.
J. Chem. Soc., Perkin Trans. 1, 2001, 1696–1708
1707

View Article Online
7 J. Uppenberg, F. Lindqvist, C. Svensson, B. Ek-Rylander and
G. Anderson, J. Mol. Biol., 1999, 290, 201.
8 C. C. Fjeld and J. M. Denu, J. Biol. Chem., 1999, 274, 20336.
9 P. Agostinis, J. Goris, E. Waelkens, L. A. Pinna, F. Marchiori and
W. Merlevede, J. Biol. Chem., 1987, 262, 1060.
22 A. G. M. Barrett, W. W. Doubleday and G. J. Tustin, Tetrahedron,
1996, 52, 15325.
23 R. G. Hiskey and R. C. Northrop, J. Am. Chem. Soc., 1961, 83, 4798.
24 S. A. Hart, M. Sabat and F. A. Etzkorn, J. Org. Chem., 1998, 63,
7580.
10 K. L. Webster, A. B. Maude, M. E. O’Donnell, A. P. Mehrotra and
D. Gani, J. Chem. Soc., Perkin Trans. 1 (DOI: 10.1039/b100401h).
11 J. Sanvoisin, J. R. Pollard, P. Hormozdiari, W. H. J. Ward and
D. Gani, J. Chem. Soc., Perkin Trans. 1 (DOI: 10.1039/b100403o).
12 J. Sanvoisin and D. Gani, Bioorg. Med. Chem. Lett., 2001, 11, 471.
13 A. P. Mehrotra, K. L. Webster and D. Gani, J. Chem. Soc., Perkin
Trans. 1, 1997, 2495.
25 V. H. Rawal, R. J. Jones and M. P. Cava, J. Org. Chem., 1987, 52, 19.
26 F. L. Buenadicha, M. T. Bartolome, M. J. Aguirre, C. Avendano and
M. Sollhuber, Tetrahedron: Asymmetry, 1998, 9, 483.
27 R. A. Barrow, T. Hemscheidt, J. Liang, S. Paik, R. E. Moore and
M. A. Tius, J. Am. Chem. Soc., 1995, 117, 2479.
28 N. Asao, N. Tsukada and Y. Yamamoto, J. Chem. Soc., Chem.
Commun., 1993, 1660.
14 A. B. Maude, P. Mehrotra and D. Gani, J. Chem. Soc., Perkin
Trans. 1, 1997, 2513.
29 R. J. Valentekovich and S. L. Schreiber, J. Am. Chem. Soc., 1995,
117, 9069.
15 K. L. Webster, T. J. Rutherford and D. Gani, Tetrahedron Lett.,
1997, 38, 5713.
30 J. Goldberg, H. B. Huang, Y. G. Kwon, P. Greegard, A. C. Nairn and
J. Kuriyan, Nature (London), 1995, 376, 745.
31 P. Agostinis, J. Goris, L. A. Pinna, F. Marchiori, J. W. Perich,
H. E. Meyer and W. Merlevede, Eur. J. Biochem., 1990, 189,
235.
32 M. M. Lenman, A. Lewis and D. Gani, J. Chem. Soc., Perkin
Trans. 1, 1997, 2297.
33 (a) R. E. London, N. A. Matwiyoff, J. M. Stewart and J. R. Cann,
Biochemistry, 1978, 17, 2277; (b) R. E. London, J. M. Stewart,
R. Williams, J. R. Cann and N. A. Matwiyoff, J. Am. Chem. Soc.,
1979, 101, 2455.
34 W. C. Still, M. Kahn and A. Mitra, J. Org. Chem., 1978, 43,
2923.
35 J. Drew, M. Letellier, P. Morand and A. G. Szabo, J. Org. Chem.,
1987, 52, 4047.
36 G. A. Molander and P. J. Stengel, Tetrahedron, 1997, 53, 8887.
16 (a) R. E. Honkanen, J. Zwiller, S. L. Daily, B. S. Khatra,
M. Dukelow and A. L. Boynton, J. Biol. Chem., 1991, 266, 6614;
(b) W. W. Carmichael, J. Appl. Bacteriol., 1992, 72, 445.
17 A. Annila, J. Lehtimaki, K. Mattila, J. E. Eriksson, K. Sivonen,
T. T. Rantala and T. Drakenberg, J. Biol. Chem., 1996, 271, 16695.
18 (a) S. Rudolph-Bohner, D. F. Mierke and L. Moroder, FEBS Lett.,
1994, 349, 319; (b) D. F. Mierke, S. Rudolph-Bohner, G. Muller and
L. Moroder, Biopolymers, 1995, 36, 811.
19 For recent synthesis, see D. L. Cundy, A. C. Donohue and
T. D. McCarthy, J. Chem. Soc., Perkin Trans. 1, 1999, 559.
20 C. Pearson, K. L. Rinehart, M. Sugano and J. R. Costerison, Org.
Lett., 2000, 2, 2901.
21 S. G. Davies and I. A. S. Waters, J. Chem. Soc., Perkin Trans. 1, 1994,
1129.
1708
J. Chem. Soc., Perkin Trans. 1, 2001, 1696–1708