Design and preparation of serine-threonine protein phosphatase inhibitors based upon the nodularin and microcystin toxin structures. Part 3

Article abstract of DOI:10.1039/b100401h

Nodularin and microcystins are complex natural cyclic isopeptidic hepatotoxins that serve as subnanomolar inhibitors of the eukaryotic serine-threonine protein phosphatases PP1 and PP2A, enzymes that are intimately involved in controlling cellular metabolism. Previously we described a solution-phase synthesis of stripped-down nodularin analogues; cyclo[-β-Ala-(R)-Glu-α-OMe-γ-Sar-(R)-Asp-α-OMe- β-(S)-Phe-] 3 and cyclo[-(3R)-3-hydroxymethyl-β-Ala-(R)-Glu-α-OMe-γ-Sar-(R)- Asp-α-OMe-β-(S)-Phe-] 5. The synthetic strategy was designed to allow post-macrocyclisation elaboration. Here we examine alternative methods for introducing diversity and achieving macrolactamisation and compare the relative efficiency of solution- vs. solid-phase peptide syntheses of the macrocycles. Syntheses and the biological activities of the macrocycles cyclo{-[(2R)-α-4-benzylpiperidinylamido-Asp]-β-[(R)-Glu]-γ- Sar-[(R)-Asp]-β-(S)-Phe-} 29 and cyclo{-(2S)-Phe-[(2R)-α-4-benzylpiperidinylamido-Asp]-(R)-Glu-γ- (S)-Pro-β-(R)-Asp-} 65 are compared. Both compounds contain sufficient side-chain functionality to interact with a hydrophobic groove at the enzyme active site. The proline containing analogues 30, 31 (R³ = CH₃) where sarcosine is replaced in macrocycles 3 and 4, were also synthesised in order to correlate conformational properties with biological activity. In accord with predictions macrocycles 29 and 65 were found to be weak inhibitors of PP1 with IC₅₀ 2.9 and 2.7 mM respectively.

Full text of DOI:10.1039/b100401h

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Design and preparation of serine–threonine protein phosphatase
inhibitors based upon the nodularin and microcystin toxin
structures. Part 3†
1
Kerri L. Webster,a Antony B. Maude,a Michael E. O’Donnell,b Amit P. Mehrotrab and
David Gani*b
a School of Chemistry and Centre for Biomolecular Sciences, The Purdie Building,
The University, St. Andrews, Fife, UK KY16 9ST
b 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
Nodularin and microcystins are complex natural cyclic isopeptidic hepatotoxins that serve as subnanomolar
inhibitors of the eukaryotic serine–threonine protein phosphatases PP1 and PP2A, enzymes that are intimately
involved in controlling cellular metabolism. Previously we described a solution-phase synthesis of stripped-down
nodularin analogues; cyclo[-β-Ala-(R)-Glu-α-OMe-γ-Sar-(R)-Asp-α-OMe-β-(S)-Phe-] 3 and cyclo[-(3R)-3-hydroxy-
methyl-β-Ala-(R)-Glu-α-OMe-γ-Sar-(R)-Asp-α-OMe-β-(S)-Phe-] 5. The synthetic strategy was designed to allow
post-macrocyclisation elaboration. Here we examine alternative methods for introducing diversity and achieving
macrolactamisation and compare the relative efficiency of solution- vs. solid-phase peptide syntheses of the macro-
cycles. Syntheses and the biological activities of the macrocycles cyclo{-[(2R)-α-4-benzylpiperidinylamido-Asp]-β-
[(R)-Glu]-γ-Sar-[(R)-Asp]-β-(S)-Phe-} 29 and cyclo{-(2S)-Phe-[(2R)-α-4-benzylpiperidinylamido-Asp]-(R)-Glu-γ-
(S)-Pro-β-(R)-Asp-} 65 are compared. Both compounds contain sufficient side-chain functionality to interact with a
hydrophobic groove at the enzyme active site. The proline containing analogues 30, 31 (R3 = CH3) where sarcosine is
replaced in macrocycles 3 and 4, were also synthesised in order to correlate conformational properties with biological
activity. In accord with predictions macrocycles 29 and 65 were found to be weak inhibitors of PP1 with IC50 2.9 and
2.7 mM respectively.
Introduction
The natural cyclic isopeptide toxins microcystin 1 and nodu-
larin 2 are known to inhibit the catalytic subunit of the mam-
malian serine–threonine protein phosphatases PP1 and PP2A
and communication.2–4 The toxin sensitive enzymes, PP1cat and
PP2Acat, are highly homologous and display ∼50% identity in
their primary structure.5,6 The microcystins 1 and nodularin 2
differ considerably from other cyclic peptides. Both groups are
cyclic triisopeptides and contain two free carboxylic acid
groups, an N-methyl dehydro amino acid moiety and a large
rigid lipophilic side-chain which forms part of the Adda residue
[(2S,3S,4E,6E,8S,9S)-3-amino-9-methoxy-2,6,8-trimethyl-10-
phenyldeca-4,6-dienoic acid]. These five motifs are the only
structural features common to the two toxin families.
Other naturally occurring competitive inhibitors of PP1cat
and PP2Acat include the powerful tumour promoter okadaic
(but not PP2B or 2C) at subnanomolar levels, as determined
for IC50 values.1 Each of these catalytic activities, together with
serine–threonine protein kinases, are involved in the main-
tenance of a delicate balance of pools of phosphorylated and
dephosphorylated proteins which affect cellular metabolism
acid, which is responsible for diarrhetic shellfish poisoning,
tautomycin,7 cantharidin8 and the calyculins.9 However, these
compounds show little specificity towards either of the two
enzyme types. This lack of specificity can be rationalised by the
analysis of the aligned amino acid sequences for the enzymes
within the context of the published X-ray crystal structures
10,11
available for PP1cat
.
Essentially this analysis indicates that
† For Part 2, see ref. 21.
DOI: 10.1039/b100401h
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the structure and composition of the active-site cleft is
extremely highly conserved and that the inhibitors only interact
with the conserved regions.12
free amino group of the (2S)-phenylalanine residue proceeded
in excellent yield (89%).18,19 A similar approach was employed
in the synthesis of the nodularin analogue motuporin.20 Exten-
sion of this chemistry allowed us to prepare the 25-membered
microcystin macrocycle, cyclo[-β-Ala-(R)-Glu-α-OMe-γ-NMe-
Gly-(R)-Ala-(S)-Leu-(R)-Asp-α-OMe-β-(S)-Phe-], 4 (69% yield)
and the functionalised 19-membered nodularin macrocycle,
cyclo[-(3R)-hydroxymethyl-β-Ala-(R)-Glu-α-OMe-γ-NMeGly-
(R)-Asp-α-OMe-β-(S)-Phe-], 5 (41% yield).21 We believed that
the lengthy sequences and relatively low overall yield obtained
for the preparation of macrocycle 5 warranted further investi-
gation. In essence we wished to assess the potential advantages
offered by use of such a preformed functionalised macrocycle
for post-cyclisation elaboration against linear and divergent
solid-phase methods where the cyclisation step is performed
either off-resin, in solution, or on-resin in the gel-phase.
It has been shown that the macrocyclic structure, the Adda
residue and the two free carboxylic acids are essential for the
inhibitory effect.13–16 Sodium borohydride reduction of the
dehydroalanine residue in microcystin-LR gives dihydro-
microcystin-LR diastereomers containing either (2R)- or (2S)-
alanine.17 Each of these diastereoisomers was found to be
equipotent with the parent compound when tested against
PP2A.18 Using this information, and data available for natural
variants, we were able to identify a simplified macrocycle which
should serve as a framework for attaching specific func-
tionalities.18 Since one of our goals was to synthesise minimal
analogues to probe the active-site binding interactions and
then identify specific inhibitors for each catalytic subunit type,
PP1cat and PP2Acat, we opted initially for a convergent synthetic
strategy. Here it was expected that the macrocycle and the
lipophilic side-chain precursor could be separately preformed
and then brought together towards the end of the synthesis. It
was reasoned that such a strategy would enable the preparation
of libraries of both macrocycles and side-chains which could be
connected to give a diverse array of synthetic toxin analogues.
A pre-formed functionalised nodularin macrocycle
In the synthesis of the model nodularin macrocycle, cyclo-
[-β-Ala-(R)-Glu-α-OMe-γ-Sar-(R)-Asp-α-OMe-β-(S)-Phe-]
3
described earlier,18,19 the lipophilic Adda side-chain was totally
omitted through the use of a β-alanine residue. Since we wished
to introduce a group into the 3-position of β-alanine that could
be easily modified to provide a range of lipophilic side-chains,
including those containing diene functionalities, to provide
rigidity, and lipophilic amides, we chose to use a 3-formyl
group, the aldehyde 6, or a 3-carboxy group, compound 7.
A similar strategy using appropriately protected aspartic α-
semialdehydes and Wittig or Julia chemistry has been success-
fully employed in the synthesis of the Adda residue by other
groups in the recent past.16,20,22–27
As it was important to retain the stereochemical integrity at
the (2R)-α-centre of the aspartic α-semialdehyde residue and,
therefore, to avoid racemisation through enol formation, a
route to the reduced macrocyclic analogue 5 was devised
in which the (3R)-3-amino-4-hydroxybutanoate residue was
incorporated directly at the alcohol oxidation level. In order to
stream-line the synthesis and provide both the aldehyde 6 and
the carboxylic acid 7 a new pathway was devised to give direct
access to the mono acid diester 7. This approach required the
incorporation of a (2R)-aspartic acid residue in place of the
β-Ala residue in the peptidic macrocycle 3 and was expected
only to present challenges in selecting suitably orthogonal ester
group protections. It was seen as a potential additional advan-
tage to introduce lipophilic side-chain surrogates for the Adda
residue last through exo-macrocyclic amide formation with the
α-carboxy group of such a (2R)-aspartic acid residue. Such a
strategy was expected to give access to the aldehyde 6 and its
ene derivatives and, at the same time, allow efficient access to
a diverse range of exocyclic amide derivatives. Moreover, our
modelling work had indicated that the natural protein sub-
strates, which are of course polyamides, should employ the
Adda binding pocket for substrate recognition. Any inform-
ation on substrate or inhibitor binding was worthy of pursuit
given that no structural information is yet available to define the
manner in which the substrates recognise the Ser-Thr protein
phosphatase 1 and 2A enzymes. While such information is
always inaccessible directly due to the catalytic lability of the
substrate, it was anticipated that the same information might be
obtained, indirectly, through the preparation and evaluation
of exocyclic amide derivatives of the natural inhibitors. Given
these objectives we set out to prepare precursors for exocyclic
ene and amide derivatives. We wished, within the current study,
to assess both post- and pre-macrocyclisation elaboration
strategies and also the potential for using solid-phase synthesis
in the construction of the peptide and for macrocyclisation. It
was also intended to gain sufficient biological information
on structural variants to focus future synthetic work on the
preparation of selective inhibitors for PP1 and PP2A.
The viability of this strategy rested on our ability to prepare
stripped down macrocycles lacking the side chain function-
alities. Various cyclisation protocols were tested on a model
nodularin macrocycle target, cyclo[-β-Ala-(R)-Glu-α-OMe-γ-
NMeGly-(R)-(Asp)-α-OMe-β-(S)-Phe-], 3.18,19 These studies
revealed that while macrolactamisation between an activated
sarcosine (or glycine) carboxy group and the amino group
of the (2R)-aspartic acid residue in suitably protected linear
peptides did not occur, displacement of the β-pentafluoro-
phenyl ester of the (2R)-aspartate α-methyl ester residue by the
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In order to refer to specific residues within the macrocycle of
nodularin analogues, the surrogate for the Adda residue will be
referred to as residue number 1. Other residues are then num-
bered in a conventional sense from the C-terminal of the Adda
surrogate such that for nodularin analogues, there is an amide
bond between residues 5 and 1.
availability of aspartic acid β-allyl ester.34–36 With respect to the
α-carboxy groups, it was important to be able to selectively
deprotect the α-carboxy group of one of these (2R)-Asp
residues without affecting the protection of either the other
(2R)-Asp residue or the (2R)-Glu residue. Methyl ester protec-
tion had been employed in the precursor Boc-(2R)-Glu-α-OMe-
γ-NMeGlyOH 16 for macrocycles 3 and 5.18,19,21 Therefore,
we chose to use the same protection for the α-carboxy
group of the (2R)-Asp residue, number 4, since this would be
coupled to the peptide fragment 16. Orthogonal benzyl ester
protection could then be used for the α-carboxy of the
other (2R)-Asp residue, number 1. Thus, access to both (2R)-
Asp-α-OBn-β-Oallyl and (2R)-Asp-α-OMe-β-Oallyl would be
required.
Accordingly, (2R)-aspartic acid was esterified by treatment
with HCl in allyl alcohol in a modification of the procedure
of Lajoie36 to give (2R)-aspartic acid β-allyl ester hydrochloride
12 (mp 176–178 ЊC). The amino group was protected as its N-
tert-butoxycarbonyl derivative, under standard conditions, to
give N-Boc-(2R)-aspartic acid β-allyl ester 13 (Scheme 2). The
Results and discussion
Solution-phase synthesis
Disconnection of cyclo[-(R)-Asp-α-OH-β-(R)-Glu-α-OMe-γ-
Sar-(R)-Asp-α-OMe-β-(S)-Phe-] 7 at the peptide bond between
the amino group of the (S)-Phe residue and the β-carboxy
group of the (2R)-aspartate α-methyl ester, residues 4 and 5,
using the same strategy as employed previously,18,19,21 gives the
linear triisopentapeptide 8. The three C-terminal residues
(residues 2, 3 and 4) had been incorporated into macrolactams
3 and 5 previously as the tripeptide triester (R)-Glu-α-
OMe-γ-NMeGly-(R)-Asp-α-OMe-β-OBn 9 without incident.
Therefore, the isopentapeptide (S)-Phe-(R)-Asp-α-OH-β-(R)-
Glu-α-OMe-γ-Sar-(R)-Asp-α-OMe-γ-OH 8 was disconnected
between the (R)-Asp-α-OH and (R)-Glu residues to give the
tripeptide diester 10 and the dipeptide (2S)-Phe-(2R)-Asp
α-benzyl ester 11 (Scheme 1).
Scheme 1
The success of this strategy hinged on the availability of an
ester protecting group which is orthogonal to the Boc, benzyl
and methyl ester or urethane groups that might be employed
in the preparation of molecules based upon the routes used
for the macrolactams 3 and 5. Allyl ester groups can be
selectively removed by the use of tetrakis(triphenylphos-
phine)palladium(0) in the presence of a suitable allyl acceptor
or by treatment with tris(triphenylphosphine)rhodium()
chloride.28–33 However, whilst an allyl ester might be removed
without affecting a benzyl ester in the same molecule, catalytic
hydrogenolysis of a benzyl ester would give concomitant hydro-
genation of the allyl group. It was decided, therefore, to protect
the β-carboxylic acid groups of the two (2R)-aspartic acid
residues (residues 1 and 4) as their allyl esters, given the ready
Scheme 2 Reagents and conditions: i) HCl, allyl alcohol, 0 ЊC→rt,
89%; ii) Boc2O, Et3N, H2O, dioxane, 91%; iii) CH2N2, Et2O, 0 ЊC,
94%; iv) HCl, EtOAc, 0 ЊC, 96%; v) (2R)-N-(tert-butoxycarbonyl)-
[α-methylglutamyl]-γ-sarcosine 16, IBCF, NMM, THF, Ϫ40 ЊC→rt,
69%; vi) HCl, EtOAc, 0 ЊC, 100%.
α-carboxylic acid was then converted to its methyl ester by
treatment with ethereal diazomethane to give fully protected α-
methyl β-allyl N-tert-butoxycarbonyl (2R)-aspartate diester 14.
The amino protecting group was removed by treatment with
a solution of HCl in ethyl acetate to afford α-methyl β-allyl
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(2R)-aspartate diester hydrochloride 15. This was coupled to
α-methyl N-Boc-(R)-γ-glutamyl-N-methylglycine 16, prepared
as described previously,18,19,21 to give the tripeptide, N-Boc-(R)-
Glu-α-OMe-γ-NMeGly-(R)-Asp-α-OMe-β-Oallyl 17, in 69%
yield after purification by flash chromatography on silica.
Acidolytic removal of the N-Boc protecting group gave a
suitably protected form of the 2,3,4-tripeptide, [α-methyl (2R)-
γ-glutamyl]-N-methylglycyl-[α-methyl β-allyl (2R)-aspartate]
triester hydrochloride 18 (Scheme 2), which was ready for
coupling to the 5,1-dipeptide.
To prepare the 5,1-dipeptide fragment, (S)-Phe-(R)-Asp-α-
OBn-β-OH 22, the doubly protected (2R)-aspartic mono acid
derivative 13 was neutralised by treatment with 20% caesium
carbonate solution in aqueous methanol. The resulting caesium
salt was dissolved in dry DMF and was reacted with benzyl
bromide according to the procedure of Gisin and co-workers.37
The required protected diester N-Boc-(2R)-Asp-α-OBn-β-
Oallyl 19 was obtained as a colourless oil in 91% yield and the
N-Boc group was removed using HCl in ethyl acetate to afford
α-benzyl β-allyl (2R)-aspartate diester hydrochloride 20. The
free amine was coupled to N-Boc-(2S)-Phe to give the 5,1-
dipeptide diester, N-Boc-(2S)-phenylalanyl-[α-benzyl β-allyl
(2R)-aspartate] 21 in 83% yield after flash chromatography
on silica. This compound was isolated as a viscous oil which
solidified on prolonged standing. The allyl ester was selectively
cleaved through reaction with catalytic quantities of tetrakis-
(triphenylphosphine)palladium(0) and pyrrolidine in dichloro-
methane to give the required 5,1-dipeptide free acid 22 in 56%
yield after flash chromatography on silica (Scheme 3). This
24 was esterified with pentafluorophenol, using 1-(3-dimethyl-
aminopropyl)-3-ethylcarbodiimide methiodide (EDCI) activ-
ation, to give the N-Boc-pentapeptide pentafluorophenyl (PFP)
ester 25 in 86% yield over the two steps, after chromatographic
purification. The N-terminal tert-butoxycarbonyl protecting
group was removed by treatment with trifluoroacetic acid in
dichloromethane and the resulting ammonium trifluoroacetate
salt 26 was thoroughly dried under high vacuum. Treatment
with N,N-diisopropylethylamine (DIPEA), under conditions
of high dilution in dichloromethane, allowed cyclisation to pro-
ceed. After 7 days at room temperature, when periodic TLC
analysis showed that the conversion was complete, the reaction
was terminated to afford the fully protected macrocyclic
pentapeptide 27 in 75% yield as a white solid (Scheme 4). The
Scheme 4 Reagents and conditions: i) IBCF, NMM, THF, DMF,
Ϫ40 ЊC→rt, 81%; ii) (Ph3P)4Pd, pyrrolidine, CH2Cl2; iii) EDCI,
pentafluorophenol, CH2Cl2, 0 ЊC→rt, 86% over two steps; iv) TFA,
CH2Cl2; v) DIPEA, CH2Cl2, 75% over two steps.
Scheme 3 Reagents and conditions: i) Cs2CO3, H2O, MeOH; ii) BnBr,
DMF, 0 ЊC→rt, 91%; iii) HCl, EtOAc, 0 ЊC, 89%; iv) IBCF, NMM,
Boc-(S)-Phe, THF Ϫ40 ЊC→rt, 83%; v) (Ph3P)4Pd(0), pyrrolidine,
CH2Cl2, 95%.
compound existed as a 3:1 mixture of conformers/rotoisomers
in DMSO solution, in accord with the properties of other
synthetic nodularin analogues lacking an α-methyl group in the
first β-amino acid residue, see below.
purification was not routinely repeated. Instead the crude
material (isolated in 95% recovery) was carried forward directly
to the next stage, with no significant diminution of the yield for
the coupling of the two peptide fragments.
The two protected peptides 18 and 22 were coupled using
mixed anhydride methodology in a mixture of dry DMF and
THF to give the fully protected linear pentapeptide 23 in 81%
yield after flash chromatography on silica. The allyl ester was
selectively removed using a similar protocol to that described
above, to give the free acid 24 which displayed the expected
signals in its 1H-NMR spectrum. Without purification acid
Catalytic hydrogenolysis of the fully protected macrocyclic
pentapeptide 27 in glacial acetic acid–methanol (50:50) gave
the macrocyclic dimethyl ester mono acid 7. The 1H-NMR
spectrum of the acid showed the expected omissions in the
aromatic region and the presence of multiple conformations
in methanol, but in DMSO, one conformer was dominant.
Without further purification the acid 7 was coupled with 4-
benzylpiperidine in a mixture of dry THF and DMF, via the
intermediacy of the mixed isobutyl carbonic anhydride.
The macrocyclic amide dimethyl ester 28 was obtained in
an optimum isolated recovery of 44% following an aqueous
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work-up, after very many attempts. No other coupling pro-
cedures offered any improvement. The 1H-NMR spectrum
of the amide 28 showed the presence of the benzylpiperidine
moiety and an extremely complex mixture of conformers in
chloroform. However, in DMSO solution the 1H-NMR showed
the presence of two major conformers in a 1:1 ratio. In view of
the very poor yields for the reaction and the amount of material
consumed in attempting to optimise the reaction, and the need
to assess biological activity, the methyl ester groups were
removed using lithium hydroxide in aqueous methanol, as
described by Chamberlain.24 The free diacid 29 was obtained
in approximately 50% isolated yield after HPLC purification
(Scheme 5). The 1H-NMR spectrum of the diacid showed
solid-phase protocols for construction of the linear peptide
precursors and also for macrocyclisation.
Preparation of nodularin type macrocycles via solid-phase
synthesis
The principal advantages of solid-phase peptide synthesis
(SPPS) over solution-phase approaches are well established and
lead to shorter synthesis times and higher yields of products
requiring minimal chromatographic purification. This effi-
ciency is achieved by using excess reagents in both the peptide
coupling reactions and in the deprotection steps. Thus, yields
are high, and by supporting the growing peptide on a polymer
resin, unreacted reagents are easily removed by washing. Given
our requirements to prepare a range of nodularin analogues
as potential protein phosphatase inhibitors, a solid-phase strat-
egy designed to allow the introduction of structural diversity
into the lipophilic side-chain of residue 1, as well as into the
macrocycle itself seemed ideal.
We had consistently been able to perform macrolac-
tamisation reactions between the N- and C-terminals of
5,1,2,3,4-pentapeptides of the type H-(2S)-Phe-X1aa-[(2R)-Glu
α-OMe]-γ-X3aa-[(2R)-Asp α-OMe β-OH] where X1aa is either
β-alanine, (3R)-3-amino-4-hydroxybutyrate, or (2R)-aspartate
α-benzyl ester (see above) and X3aa is sarcosine.18,19,21 Since we
could rationalise why some other points of disconnection had
failed when macrocyclisations were attempted,18 by examining
the lowest energy conformations for activated esters of the pre-
cursors, we opted to continue to employ the formation of the
residue 4 to residue 5 amide bond in the macrocyclisation step.
In principle, for strategies that allow both the linear peptide
precursor to be constructed and the precursor to be cyclised
on the resin, there were two obvious ways of connecting the
peptide to the polymer support. Specifically, either through the
α-carboxy group of the (2R)-Asp residue (No. 4) or through
the α-carboxy group of the (2R)-Glu residue (No. 2). We opted
to use the α-carboxy group of the (2R)-Asp residue, residue 4,
for our initial experiments. We chose to incorporate a Pro
residue in place of sarcosine at position 3 for three reasons.
First, to increase the populations of conformers in the linear
precursors which might possess proximal reacting termini. The
intrinsic conformational restraint provided by the pyrrolidine
ring in the Pro residue was expected to increase the ease of
macrocycle formation. Second, the constraints provided by the
Pro residue were expected to restrict the number of accessible
conformations in the ground state of the macrocycle such that
conformational analysis would be much easier. Third, our work
on the differences of the two enzymes, PP1 and PP2A, in the
vicinity of the sarcosine binding pocket close to Cys-273 in
PP1 suggested that Pro derivatives at position 3 might allow
differentially selective interactions with the two enzymes (see
following article). As a prelude to solid-phase studies, a solu-
tion synthesis of each diastereomer of the target macrocycle,
the proline epimers, 30 and 31 was undertaken. The targets
contained a β-alanine residue at position 1 to facilitate com-
parison of the ease of macrolactamisation with the previously
prepared sarcosine derivatives 3, 5 and 27.18,19,21
Scheme 5 Reagents and conditions: i) H2, Pd/C, MeOH–AcOH (1:1),
85%; ii) IBCF, NMM, THF, DMF Ϫ40 ЊC, then 4-benzylpiperidine,
THF, Ϫ40 ЊC→rt, 44%; iii) 0.1 M LiOH(aq), MeOH, 50%.
the absence of methyl ester groups and two predominant
Sar N-methyl signals consistent with the existence of two
major conformers. The diacid 29 displayed the required mass
spectrum but no other data were obtained in order to use
the remaining material for biological testing. [Note that a
1
comparison of the H-NMR spectrum of 29, with that for an
analogue in which the Sar residue was replaced by (2S)-Pro
(compound 63), using a different synthesis, see below, was
completely consistent with the expected structure.] Diacid 29
was tested as an inhibitor for PP1cat, as described below, and
was found to be weakly active, in accord with expectations.
A disappointing and unsatisfactory feature of the synthesis
of the macrocyclic peptide 29 was the poor optimised yield
(44%) obtained for the formation of the exocyclic amide 28 in
the penultimate step. Analogous reactions using acyclic aspartic
acid derivatives gave much better yields (see below) and it
seemed probable that the poor yield of amide 28 resulted from
inaccessibility of the electrophilic α-carboxy group within the
3-D conformational structure of the macrocyclic activated acid.
Thus, the post-macrocyclisation elaboration strategy seemed
flawed. In light of the result, and in order to assess the synthetic
efficiency of alternative strategies, we turned our attention to
Using solution-phase methods similar to those employed
for the synthesis of the sarcosine containing macrocycle 3,18
the (2S)-Pro containing linear peptide analogue 32 was pre-
pared starting from Boc-(2R)-Asp-α-OMe-β-OBn 33 and
Boc-(2S)-Phe-β-AlaOH 34, both of which have been described
previously.18 Thus Boc-(2S)-ProOH was activated as the mixed
isobutyl carbonic anhydride and was treated with (2R)-Asp-
α-OMe-β-OBn 35 (which was obtained by acidolysis of Boc-
urethane 33) to give the required dipeptide diester 36 in 78%
yield. The amino protecting group of the Pro residue was
removed using HCl in ethyl acetate and the resulting hydro-
chloride salt 37 was added to the mixed anhydride of Boc-(2R)-
Glu-α-OMe-γ-OH,38 in the presence of N-methylmorpholine to
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give the required tripeptide 38 in 82% yield (see Scheme 6).
Again the Boc protection was removed from tripeptide 38
under acidic conditions and the resulting hydrochloride salt 39
was reacted with activated Boc-(2S)-Phe-β-AlaOH 34 to afford
the fully protected 5,1,2,3,4-pentapeptide 32 in 92% recovery.
Recrystallisation from ethyl acetate–hexane gave the pure linear
precursor in 62% yield. The compound 32 gave satisfactory
analytical data and existed in both the trans- and cis-γ-Glu-Pro
amide rotameric forms. Catalytic hydrogenolysis of the benzyl
ester protection gave the free acid 40 in quantitative recovery
and this was esterified with pentafluorophenol to give the
triester 41 in the presence of EDCI in 62% yield after chroma-
tography on silica. Treatment of the protected triester 41 with
TFA, followed by thorough drying, and then treatment with
DIPEA, as described above, gave the required macrocyclic
diester 42 after 7 days at room temperature. As before, the
reaction was monitored by TLC analysis. After purification by
column chromatography on silica, the macrocyclic diester 42
was obtained in 52% yield, 13% from Boc-(2S)-ProOH. This
yield is at least as good as those obtained for derivatives 3, 5
and 27 containing sarcosine at position 3 in the macrocycle
and indicates that the presence of a (2S)-Pro residue does not
impede cyclisation.
The epimer 43 (of the macrocyclic diester 42), containing a
(2R)-Pro residue, was prepared similarly and was obtained in
16% overall yield and the macrocyclisation step afforded 50%
of the required lactam, after chromatographic purification on
silica. For each macrocycle the replacement of the Sar residue
by Pro allowed partial analysis of the 3-D structures of each
conformer (see discussion below). After saponification of the
methyl ester groups each epimer 30 and 31 (R3 = H) was tested
for biological activity as an inhibitor for PP1cat, as is described
below.
in which cyclisation was achieved through the reaction of a
(2S)-Phe residue (No. 4) with an activated (2R)-Asp β-PFP
ester in the linear precursor 41,18,38 was adapted for use with
Wang resin, Fmoc protection and benzotriazol-1-yloxytris-
pyrrolidinophosphonium
activation protocols.
hexafluorophosphate
(PyBOP)
Accordingly, N-Fmoc (2R)-Asp β-allyl ester 44 [mp 110 ЊC,
[α]D ϩ3.04 (MeOH)] was prepared in 94% yield starting
from (2R)-Asp β-allyl ester 12. The free α-carboxy group was
activated as its 2,6-dichlorobenzoic anhydride,38 and was then
reacted with the 4-hydroxymethyl group of Wang resin to give
the immobilised aspartate diester 45 (Scheme 7). Preparation of
α-methyl N-fluorenylmethoxycarbonyl-(2R)-glutamate 49 was
similar and treatment of (2R)-glutamic acid with chloro-
trimethylsilane in allyl alcohol according to the procedure
of Belshaw,36 gave the γ-allyl ester hydrochloride 46. This
was sequentially N- and C-protected to give the fully protected
(2R)-glutamate 48 which was selectively deprotected using
(Ph3P)4Pd(0) to give the required (2R)-glutamate derivative 49
in 53% overall yield ready for use in SPPS (Scheme 8).
Loadings of the immobilised aspartate diester 45 ranged
from 0.6 to 0.8 mmol gϪ1 resin. Removal of the Fmoc group
using 20% piperidine in DMF followed by reaction with
PyBOP-activated Fmoc-(2S)-Pro gave the resin-bound diester
50 which was treated with piperidine to remove the Fmoc
group. Fmoc-(2R)-Glu-α-OMe-γ-OH 4938 was activated with
PyBOP then added to the free amine derived from diester 50 to
give the immobilised Fmoc-tripeptide triester 51 (Scheme 9).
Removal of the Fmoc group followed by reaction with PyBOP-
activated Fmoc-β-alanine gave the tetrapeptide triester 52
which was deprotected and reacted with PyBOP-activated
Fmoc-(2S)-Phe to give the Fmoc-pentapeptide triester 53.
The β-allyl ester group of Asp residue (No. 4) was unmasked
using (Ph3P)4Pd(0) to give 5,1,2,3,4-pentapeptide diester 54
(R = Wang) and the N-terminal Fmoc group of this material
was removed to give the resin-bound amino acid 55 (R =
Wang). Treatment with PyBOP and HOBt in the presence
Having compared the influence of proline on the course of
the macrocyclisation relative to sarcosine, the utilities of solid-
phase synthetic approaches were assessed using the (2S)-Pro
epimer as a model. Accordingly, the generic route (Scheme 6)
Scheme 6 Reagents and conditions: i) IBCF, NMM, THF, DMF, Ϫ15 ЊC, then (2R)-Asp-α-OMe-β-OBnؒHCl 35, NMM, THF, DMF, Ϫ15 ЊC→rt,
12 h, 78%; ii) HCl(g), EtOAc, 0 ЊC→rt, 4 h, 93%; iii) Boc-(2R)-Glu-α-OMe-γ-OH, IBCF, NMM, THF, DMF, Ϫ15 ЊC→rt, 12 h, 82%; iv) HCl(g)
,
EtOAc, 0 ЊC→rt, 2 h, 95%; v) Boc-(2S)-Phe-β-AlaOH 34, IBCF, NMM, THF, DMF, Ϫ15 ЊC→rt, 12 h, 62%; vi) H2, Pd/C, EtOH, rt, 12 h, 99%; vii)
EDCl, C6F5OH, CH2Cl2, 0 ЊC→rt, 12 h, 62%; viii) TFA, CH2Cl2, rt, 45 min, 100%; ix) DIPEA, CH2Cl2, rt, 7 days, 52%.
1678
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several close running peaks which were subsequently removed
by HPLC. The pure material (obtained in 30% yield) eluted
as a single peak and gave the expected ES mass spectrum
1
(574 Da, [M ϩ H]ϩ). H- and 13C-NMR spectra recorded in
DMSO and analysed using COSY, TOCSY and HSQC tech-
niques revealed the presence of two major conformers, as
expected,38 corresponding to the trans- and cis-Glu-γ-Pro
rotamers. Saponification of the monoester 57 (R = H) using
sodium hydroxide in aqueous methanol gave a diacid after
acidification, which displayed an identical ES mass spectrum
(560 Da, [M ϩ H]ϩ) to that of the product derived from
the saponification of the diester 42 (R = Me). Treatment of a
sample of the crude material 3 (R = H) with diazomethane gave
the crude diester 3 (R = Me) which displayed several 1H-NMR
spectral signals coincident with those for the pure solution-
phase synthesised material (Scheme 6) and further analysis
indicated that crude 42 (R = Me) was ca. 50% pure in keeping
with the HPLC analysis of the precursor 57 (R = H).
In an attempt to improve upon the overall yield of 30%, the
resin-bound diester 54 was converted to the PFP triester 58,
through treatment with pentafluorophenol and EDCI. The tri-
ester product 58, was treated briefly with piperidine to remove
the Fmoc protection, and the free amine 59 was immediately
washed (to minimise piperidine amide formation) and then
treated with DIPEA to give the resin-bound macrocycle 56
(R = Wang resin, Scheme 9). Removal from the resin gave crude
57 (R = H) in 80% recovery which was of similar purity to the
material 57 (R = H) prepared using PyBOP-activation. In order
to discover the cause of the low purity of the cyclised materials,
the Fmoc-pentapeptide triester 53 was removed from the resin
using TFA and the free Asp α-carboxy group was methylated to
give the linear pentapeptide triester 60 (Scheme 9). The crude
product 60 was >90% pure and gave the expected analytical and
spectroscopic data, showing that the solid-phase cyclisation
itself was the cause of the problem. The allyl and Fmoc
groups were then sequentially removed (Scheme 9) to give the
amino peptide acid 61 (R = Me) which was cyclised through
activation of the carboxy group with PyBOP to afford crude
42 (R = Me) in quantitative recovery. HPLC, MS and NMR
spectroscopic analysis indicated that this material was at least
85% pure and identical to the material obtained from the
solution-phase synthesis (Scheme 6). Thus, it appeared that the
resin-based synthesis gives low yields for the cyclisation step,
compared to the situation in solution, but offers significant
advantages in the construction of the linear isopentapeptide
precursor.
Scheme 7 Reagents and conditions: i) K2CO3, Fmoc-Cl, H2O, dioxane,
94%; ii) Wang resin (0.6–0.84 mmol gϪ1), 2,6-dichlorobenzoyl chloride,
pyridine, DMF, 70% loading.
A solid-phase synthesis of an analogue 64 containing the
isoasparagine derivative, 4-benzylpiperidyl α-aspartic acid
amide (see 68f in Scheme 11, below) at position 1 and (2S)-Pro
at position 3 was performed using an on-resin cyclisation
protocol identical to that employed in the preparation of
macrolactam 57 (Scheme 10). The required material 64
was obtained in 17% overall yield, after reverse-phase HPLC
purification and was saponified to give the diacid 65, for bio-
logical evaluation, see below. Indeed, several isoasparagine
analogues 66a–g were prepared starting from β-allyl N-fluor-
enylmethoxycarbonyl-(2R)-aspartate 44 (Scheme 11). Some of
these are described below together with phenyl substituted
(3-R/S)-N-Fmoc-3-amino-3-phenylpropanoic acids 67 (R = H,
OMe, Br) (Scheme 12).
Preparation of the isoasparagine analogues was achieved by
the slow addition of a solution of the appropriate amine to a
solution of the mixed isobutyl carbonic anhydride derived
from β-allyl N-fluorenylmethoxycarbonyl-(2R)-aspartate 44 in
dry THF at 0 ЊC. Although the fluorenylmethoxycarbonyl
protecting group is base sensitive, it was found that under care-
fully controlled conditions good yields of fully protected N-
substituted and N,N-disubstituted isoasparagines 66a–g could
be prepared. Selective removal of the allyl ester was achieved by
the procedure of Guibe and co-workers39 using tetrakis
Scheme 8 Reagents and conditions: i) Me3SiCl, allyl alcohol 0 ЊC→rt,
65%; ii) K2CO3, Fmoc-Cl, H2O, dioxane, 100%; iii) CH2N2, EtOAc,
Et2O, 100%; iv) PhSiH3, (Ph3P)4Pd, CH2Cl2, 81%.
of DIPEA in DMF for 7 days gave the cyclic peptide 56
(R = Wang) which was removed from the resin with TFA solu-
tion to afford the crude macrocyclic monoester 57 (R = H) in
78% overall recovery, (Scheme 9). This displayed one major
peak by reverse-phase HPLC analysis (ca. 50% of the total) and
J. Chem. Soc., Perkin Trans. 1, 2001, 1673–1695
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Scheme 9 Reagents and conditions: i) 20% piperidine in DMF; ii) Fmoc (2S)-Pro, PyBOP, DMF; iii) Fmoc (2R)-Glu-α-OMe-γ-OH 49, PyBOP,
DMF; iv) Fmoc-β-Ala, PyBOP, DMF; v) Fmoc (2S)-Phe, PyBOP, DMF; vi) (Ph3P)4Pd(0), DMSO, THF, 0.5 M HCl, NMM (2:2:1:0.1), pH 6;
vii) PyBOP, HOBt, DIPEA, DMF, 7 days; viii) CH2Cl2–TFA–H2O–TES (53:40:5:2); ix) C6F5OH, EDCl, CH2Cl2; x) DIPEA, CH2Cl2; xi) CH2N2,
EtOAc, Et2O, 100%; xii) PhSiH3, (Ph3P)4Pd(0), CH2Cl2; xiii) 1.5 eq. piperidine, CH2Cl2.
(triphenylphosphine)palladium(0) and phenylsilane in dichloro-
methane and gave the required (2R)-isoasparagines 68a–g,
(Scheme 11).
Given the problems associated in using the on-resin cyclis-
ation strategy in which the α-carboxy group of the Asp residue
was used to attach the growing peptide to the resin, attention
was turned to the solid-phase preparation of appropriate linear
5,1,2,3,4-pentapeptides for solution-phase lactamisation. We
wished to form the 5–4 amide bond last, in the cyclisation step,
because we were confident that the reaction should work on
the basis of our previous results18,38 (see above). Since we had
already assessed use of the Asp α-carboxy group in attaching
the growing polypeptide to the resin in the preparation of the
linear pentapeptide 53 (see above), we reconsidered using the
Asp β-carboxy group for attachment. This option became
viable because it was no longer necessary to be able to activate
The 3-amino-3-phenylpropanoic acids 69 (R = H, OMe, Br)
were each prepared using Rodionov chemistry to form the
parent β-amino acid40–42 (Scheme 12). Again, it was expected
that such Adda surrogates would provide information on the
role of the Adda residue in binding to the enzymes. However,
the poor yields obtained for the desired macrocycles 57 and 64
prepared using the on-resin cyclisation protocol prompted a
final examination of the most useful preparative strategy for
analogues before fully addressing the requirements for bio-
logical activity and selectivity.
1680
J. Chem. Soc., Perkin Trans. 1, 2001, 1673–1695

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Scheme 12
the β-carboxy group to facilitate cyclisation whilst the peptide
remained bound to the resin. Such a strategy also allows the
resin alcohol to be used as a protecting group, thus reducing the
number of preparative steps, where removal of the penta-
peptide from the resin would unmask the β-carboxy group for
subsequent macrolactamisation. In order to remove the protec-
tion at the α-carboxy groups of (2R)-Glu and (2S)-Asp residues
after cyclisation, to allow biological evaluation, it seemed
expedient to use the same ester protecting groups and it
appeared that methyl esters would be most useful.
Scheme 10 Reagents and conditions: i) Pd(PPh3)4, DMSO–THF–
0.5 M HCl–NMM (2:2:1:0.1), pH 6; ii) PFP, EDCl, CH2Cl2, 18 h;
iii) 20% piperidine–DMF; iv) DIPEA, CH2Cl2; v) CH2Cl2–TFA–H2O–
TES (53:40:5:2), 17% from 45; vi) 1 M NaOH, MeOH, 83%.
Accordingly, to anchor the β-carboxy group, β-allyl N-
fluorenylmethoxycarbonyl-(2R)-aspartate 44 was treated with
diazomethane to furnish the fully protected aspartate diester
70. Selective removal of the allyl ester was achieved as before39
to give α-methyl N-fluorenylmethoxycarbonyl-(2R)-aspartate
ester 71 in 60% yield after purification by column chromato-
graphy. The compound was attached to the hydroxymethyl
derivative of Wang resin, using the method of Sieber,43 as for
the α-acid β-ester 46 above, to give the required Fmoc-protected
resin-bound aspartate diester 72 (loading efficiency ca. 80%)
(Scheme 13).
In order to benchmark the Asp-β-resin ester preparative
strategy, we chose to use a prototype possessing a (3R/S)-3-
amino-3-phenylpropanoic acid residue at position 1 and a Sar
residue at position 3. Hence, resin-bound aspartate 72 was
treated with piperidine and the resulting free amine was treated
with PyBOP activated Fmoc SarOH using standard SPPS
protocols. The remaining activated Fmoc amino acids were
added to the growing polypeptide chain in reverse sequence
and the completed 5,1,2,3,4-polypeptide 73 was treated with
piperidine to remove the N-terminal protection and then
cleaved from the resin using TFA. The required pentapep-
tide (2S)-Phe-(3R/S)-3-amino-3-phenylpropanoyl-[(2R)-Glu α-
OMe]-γ-Sar-[(2R)-Asp α-OMe]-β-OH 74 was obtained after
precipitation from methanolic ether in quantitative recovery
1
and displayed the expected signals in its H- and 13C-NMR
spectra (Scheme 13). The linear pentapeptide was cyclised in
DCM–DMF (9:1) using DIPEA and BOP-Cl over a period
of 7 days to give the required macrocycle 75 in 24% overall
yield after an aqueous work-up and precipitation with ether.
NMR spectral data indicated that each diastereomer (epimeric
at C-3 of the 3-amino-3-phenylpropanoic acid residue) existed
in at least two conformational/rotoisomeric forms, as was
expected.
Scheme 11 Reagents and conditions: i) IBCF, NMM, THF, Ϫ40 ЊC,
then R1R2NH, THF, 0 ЊC→rt, 74–85%; ii) PhSiH3, (Ph3P)4Pd, CH2Cl2,
54–87%.
J. Chem. Soc., Perkin Trans. 1, 2001, 1673–1695
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of 2.9 and 2.7 mM for the systems containing a Sar and (2S)-
Pro residue at position 3, respectively. These latter values are
not significantly different and errors in the determinations
are ca. 20%. Thus, the presence of a lipophilic side chain
in the Adda surrogate confers some activity to an otherwise
completely inactive macrocycle. Moreover, the similar IC50
values obtained for systems containing Sar and (2S)-Pro allows
two important conclusions. First, that for PP1 there is sufficient
room at the site close to the N-methyldehydrobutyrine residue
in the bound nodularin–enzyme complex to accommodate a
(2S)-Pro residue. Second, that the conformational restraint
provided by the pyrrolidine ring does not cause the macrocycle
to alter to a form that cannot bind to the enzyme. Both of these
results are consistent with models for nodularin binding that
mirror those that occur in the X-ray crystal structure of PP1cat
–
microcystin complexes;10,11 except that there is no covalent bond
between the dehydro amino acid residue, a potential Michael
acceptor, and the thiol group of Cys-273. Such models indicate
that the 2-methyl group in the Adda residue plays a role in
controlling the conformational structure of the macrocycle
and in binding to the protein in a hydrophobic pocket. It also
appears that the reason that phosphothreonine containing
substrates are better than those containing phosphoserine,46,47
derives, at least in part, from the presence of a methyl group
binding pocket. Such an arrangement is consistent with recent
mechanistic proposals where it is demonstrated that water
attacks the phosphate ester directly, in a ternary complex
mechanism48 and with X-ray crystal data on the structure of an
enzyme–tungstate complex.11
None of the macrocyclic compounds described here exist in a
single rotoisomeric form. Moreover, any correlation of 3-D
structure with biological activity requires at least some analysis
of the population distributions of conformers for each roto-
isomer. The effect of the 2-methyl group at position 1 on the
conformational preferences and the macrocycle and on bio-
logical activity are described in the following article,49 using
the general synthetic methods described here. In a third paper
we describe a new, sensitive and reliable assay that is suitable
for use with both PP1 and PP2A and also both other major
Ser-Thr protein phosphatase enzyme classes.50
Scheme 13 Reagents and conditions: i) CH2N2, EtOAc, Et2O, 100%; ii)
PhSiH3, (Ph3P)4Pd, CH2Cl2, 60%; iii) Wang resin (0.6–0.84 mmol gϪ1),
2,6-dichlorobenzoyl chloride, pyridine, DMF, 80% loading; iv) SPPS,
followed by cleavage of the pentapeptide from the resin; v) CH2Cl2–
DMF (9:1), DIPEA, BOP-Cl, 7 days, 24%.
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;
31P, 121.5 MHz; 19F, 282.3 MHz), Varian Gemini 200 spec-
trometer (1H, 200 MHz; 13C, 50.3 MHz), Varian Gemini 300
spectrometer (1H, 300 MHz; 13C, 75.4 MHz) and a Varian
Unity Plus 500 spectrometer (1H, 500 MHz; 13C, 125.6 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 were referenced internally on
2HOH (δ 4.68), CHCl3 (δ 7.27), C2H3O2H (δ 3.35) or (CH3)2SO
[δ 2.47].13C-NMR were referenced on C2HCl3 (δ 77.5), CH3OH
(δ 49.15) or (CH3)2SO [δ 39.70] and 31P NMR spectra to
external H3PO4 (δ 0). 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 can 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
isomeric state of the amide bond. IR spectra were recorded
on a Perkin-Elmer 1710 or a Nicolet Avatar 360 FT-IR spec-
trometer. The samples were prepared as KBr discs, Nujol mulls,
solutions in chloroform or thin films between sodium chloride
Importantly, this new synthetic protocol gave comparatively
pure products in good overall yield and for two other reasons
was selected as the method of choice. First, the linear penta-
peptide 74 and the cyclised derivative 75 did not require
chromatographic purification in order to assess purity, and
potentially could be screened for activity after the saponifi-
cation of the two methyl esters, but before full characterisation.
Second, this linear protocol did not require any post-macro-
cyclisation elaboration. We had already demonstrated that
exocyclic elaborations did not proceed in good yield, for
example, in the case of the elaboration of the Asp α-benzyl
ester precursor 27 to give the amide 28, as described above.
Biological activity
The four macrocyclic diacids, 29, 30 (R3 = H), 31 (R3 = H) and
65 were tested for biological activity against PP1cat using the
substrate Lys-Arg-Thr(P)-Ile-Arg-Arg-OH at 25 ЊC and the
malachite green assay for inorganic phosphate release.44,45 [Full
details on the activity assay of PP1 and PP2A are given in the
third paper in this series in this issue together with a description
of a new assay procedure and a comparison with other pro-
cedures.] Neither of the two macrocycles (30 and 31; R3 = H)
possessing β-alanine residues displayed any activity but the two
4-benzylpiperidyl amide derivatives (29 and 65) gave IC50 values
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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 or by the EPSRC service at
Swansea using a VG AZB-E. Fast atom bombardment spectra
were recorded using glycerol as a matrix. Major fragments are
given as percentages of the base peak intensity (100%). Flash
chromatography was performed according to the method of
Still et al.51 using Fluka Kieselgel C60 (4–60 µm mesh) silica gel.
Analytical thin layer chromatography was carried out on
0.25 mm pre-coated silica gel plates (Whatman PE SIL G/UV)
and compounds were visualised using UV fluorescence, iodine
vapour, ethanolic phosphomolybdic acid, aqueous potassium
permanganate or ninhydrin. Melting points were taken on an
Electrothermal melting point apparatus and are uncorrected.
Optical rotations were measured at 23 ЊC on a Optical Activity
AA-1000 polarimeter using 10 or 20 cm path length cells and
7 days. The reaction mixture was concentrated under reduced
pressure, redissolved in ethyl acetate (30 cm3), and was washed
with 1 mol dmϪ3 HCl (3 × 20 cm3), 5% NaHCO3 (2 × 20 cm3),
water (2 × 20 cm3) and then brine (1 × 20 cm3). The organic
phase was dried (MgSO4) and the solvent was removed under
reduced pressure to give a colourless oil. The residue was
purified by flash silica column chromatography and then by
reverse-phase (RP) HPLC, as described below for each
macrocyclic diester.
ꢀ-Allyl (2R)-aspartate ester hydrochloride 12
Method A. Acetyl chloride (7.0 cm3, 97 mmol) was added
dropwise to ice-cold allyl alcohol (50 cm3). The resulting
solution was stirred at 0 ЊC for 15 min and then at room
temperature for 1 h. (2R)-Aspartic acid (3.33 g, 25 mmol) was
added in a single portion and the suspension stirred for 18 h
and then poured into ice-cold diethyl ether (250 cm3). After
stirring at 0 ЊC for 1 h the precipitate was collected by filtration
and was washed on the pad with diethyl ether to give the hydro-
chloride 12 as a white powder (4.66 g, 89%), mp 176–178 ЊC
(lit.,35 185–186 ЊC) (Found C, 39.85; H, 5.5; N 6.6. C7H12O4NCl
requires C, 40.1; H, 5.75; N, 6.7%); νmax(Nujol)/cmϪ1 1756 (CO
are given in units of 10Ϫ1 deg cm2 gϪ1
.
Preparative RP HPLC was carried out using a PerSeptive
BioCAD SPRINT 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).
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, diisopropyl-
amine and triethylamine were distilled over CaH2. THF and
diethyl ether were dried over sodium–benzophenone and dis-
tilled under nitrogen. Thionyl chloride was distilled over sulfur
and the initial fractions were always discarded. N-Methyl-
morpholine was distilled over ninhydrin.
2
acid) and 1727 (CO ester); δH(200 MHz; H2O) 3.13 (1 H, dd,
J 5.1 and 18.1, 1 H of β-CH2), 3.17 (1 H, dd, J 5.7 and 18.1,
1 H of β-CH2), 4.57 (1 H, t, J 5.4, α-H), 4.65 (2 H, d, J 5.7,
OCH2-vinyl), 5.24–5.38 (2 H, m, vinyl CH2) and 5.84–6.03
2
(1 H, m, vinyl CH); δC(50.3 MHz; H2O) 36.80 (β-CH2), 52.06
(α-C), 69.57 (OCH2), 121.77 (vinyl CH2), 134.19 (vinyl CH),
173.60 and 173.90 (CO); m/z (EI) 174 (10%, [M Ϫ Cl]ϩ), 132
(9, [M Ϫ HCl Ϫ C3H6]ϩ), 128 (100, [M Ϫ HCl Ϫ H2 Ϫ CO2]ϩ),
74 (46) and 41 (74, C3H5ϩ).
Method B. To a stirred suspension of (2R)-aspartic acid (5.33
g, 40 mmol) in dry allyl alcohol (30 cm3) under N2, was added
dropwise chlorotrimethylsilane (15.23 cm3, 120 mmol) and
stirring was continued for 18 h. Ice-cold diethyl ether (75 cm3)
was then added and the precipitated product was collected to
afford a white crystalline solid (7.3 g, 87%), mp 184–185 ЊC
[lit.,37 185–186 ЊC (for the (2S)-isomer)]; [α]D ϩ7.7 (c 8.0 in
AcOH) [lit.,37 Ϫ9.5 (c 8.0 in AcOH) (for the (2S)-isomer)];
νmax(CH2Cl2)/cmϪ1 3364 (NH), 2965 (CH), 1751 (CO, ester) and
General solid-phase synthesis and peptide removal from Wang
resin
Solid-phase synthesis of the pentapeptides described below
were carried out using the Rainin PPS automated peptide syn-
thesiser. The syntheses employed Fmoc chemistry and the
C-terminal amino acid residues were linked to β-allyl (2R)-
N-(fluoren-9-ylmethoxycarbonyl)aspartyl-Wang resin 45 or to
α-methyl (2R)-N-(fluoren-9-ylmethoxycarbonyl)aspartyl-Wang
resin 72. Amino acids and the activating agent PyBOP were
purchased from Novabiochem chemicals, the solvents DMF,
piperidine and N-methylmorpholine from Sigma-Aldrich. A
four-fold excess of the amino acid was used for each coupling
procedure. The N-α-Fmoc protecting group from the growing
resin-bound peptide was removed using a 20% piperidine–
DMF solution and the carboxy group was activated in the
presence of 5% NMM in DMF solution. Double couplings
were performed for (2S)-proline residues. The peptides were
cleaved from the resin using a mixture of TFA–TES–H2O–
CH2Cl2 (TES = triethylsilane). Trituration with diethyl ether,
followed by lypholisation gave the required peptides as fluffy
white powders.
2
1536 (amide); δH(300 MHz; H2O) 3.03–3.06 (2 H, m, β-CH2),
4.27 (1 H, t, J 4.8, α-H), 4.60 (2 H, d, J 6, CH2O), 4.65 (3 H, s,
NH3ϩ), 5.15–5.27 (2 H, m, CH ᎐CH) and 5.77–5.90 (1 H, m,
᎐
2
2
CH ᎐CH); δ (75.4 MHz; H O) 34.05 (β-CH ), 49.37 (α-C),
᎐
2
C
2
2
66.78 (CH O), 119.00 (CH ᎐CH), 131.47 (CH ᎐CH), 170.94
᎐
᎐
2
2
2
(CO, ester) and 171.19 (CO, acid); m/z (CI) 174 (100%,
[M ϩ H Ϫ HCl]ϩ).
ꢀ-Allyl (2R)-N-(tert-butoxycarbonyl)aspartate ester 13
Triethylamine (6.0 cm3, 4.36 g, 42 mmol) was added dropwise to
a stirred solution of di-tert-butyl dicarbonate (3.92 g, 18 mmol)
and β-allyl (2R)-aspartate ester hydrochloride 12 (3.14 g,
15 mmol) in water (25 cm3) and dioxane (25 cm3). After 18 h the
solution was extracted with petroleum ether (2 × 50 cm3) and
the aqueous phase was cooled on ice and carefully acidified to
pH 3 by slow addition of 10% citric acid solution. The urethane
was then extracted into ethyl acetate (3 × 50 cm3) and the com-
bined extracts were washed with brine (2 × 25 cm3), then dried
(MgSO4), filtered and concentrated under reduced pressure
to give the N-(tert-butoxycarbonyl)aspartate ester 13 as a pale
yellow oil (3.75 g, 91%) (HRMS: found [M ϩ H]ϩ, 274.1275.
C12H20NO6 requires 274.1291); νmax(neat)/cmϪ1 3364 br (NH
and OH) and 1737 (CO); δH(300 MHz; C2HCl3) 1.41 [9 H,
s, (CH3)3], 2.87 (1 H, dd, J 4.9 and 17.0, 1 H of β-CH2), 3.01
(1 H, dd, J 4.4 and 17.0, 1 H of β-CH2), 4.52–4.62 (3 H, m,
OCH2-vinyl and α-H), 5.19–5.32 (2 H, m, vinyl CH2), 5.59 (1 H,
d, J 8.8, NH), 5.80–5.93 (1 H, m, vinyl CH) and 10.47 (1 H, br s,
OH); δC(75.4 MHz; C2HCl3) 28.10 [(CH3)3], 36.43 (β-CH2),
General cyclisation procedure using DIPEA and PFP activation
To a stirred solution of the pentapeptide activated ester (75
mg, 0.087 mmol) in CH2Cl2 (3 cm3), was added trifluoroacetic
acid (3 cm3). The reaction was allowed to stir at room
temperature for 1 h, after which time no starting material
was detected by TLC analysis. The reaction mixture was con-
centrated under reduced pressure to yield a colourless oil. The
residue was dissolved in CH2Cl2 (200 cm3), treated with
DIPEA (210 mm3), and was stirred at room temperature for
J. Chem. Soc., Perkin Trans. 1, 2001, 1673–1695
1683

View Article Online
49.69 (α-C), 65.65 (OCH2), 80.34 (C-O), 118.66 (vinyl CH2),
131.65 (vinyl CH), 155.66 (CO, urethane), 170.94 and 175.60
(CO); m/z (FAB) 296 (7%, [M ϩ Na]ϩ), 274 (20, [M ϩ H]ϩ), 218
(90, [M ϩ H Ϫ C3H4O]ϩ) and 174 (100, [M ϩ H Ϫ C5H8O2]ϩ).
502.2401); νmax(neat)/cmϪ1 3338 br (NH), 1747 (CO ester), 1693
(CO amide) and 1645 (CO urethane); δH(300 MHz; C2HCl3)
1.36 [9 H, s, (CH3)3], 1.81–1.98 [1 H, m, 1 H of β-CH2 (Glu)],
2.22–2.36 [2 H, m, 1 H of β-CH2 (Glu) and 1 H of γ-CH2
(Glu)], 2.42–2.50 [1 H, m, 1 H of γ-CH2 (Glu)], 2.84–2.93 [2 H,
m, β-CH2 (Asp)], 3.02 (3 H, s, NCH3), 3.66 (3 H, s, OCH3), 3.68
(3 H, s, OCH3), 3.76 [1 H, d, J 15.9, 1 H of CH2 (Sar)], 4.23–
4.28 [1 H, m, α-H (Glu)], 4.31 [1 H, d, J 16.2, 1 H of CH2 (Sar)],
4.52 (2 H, d, J 5.5, OCH2-vinyl), 4.84–4.88 [1 H, m, α-H (Asp)],
5.16–5.27 (2 H, m, vinyl-CH2), 5.37 [1 H, d, J 8.2, NH (Glu)],
5.77–5.90 (1 H, m, vinyl-CH) and 7.17 [1 H, d, J 8.2, NH
(Asp)]; δC(75.4 MHz; C2HCl3) 28.51 [(CH3)3], 28.97 [β-CH2
(Glu)], 35.52 (NCH3), 36.32 [γ-CH2 (Glu)], 36.72 [β-CH2
(Asp)], 48.74 [CH2 (Sar)], 52.04 [α-C (Glu)], 52.72 (OCH3),
52.80 (OCH3), 52.99 [α-C (Asp)], 65.97 (OCH2-vinyl), 80.25
[C(CH3)3], 118.94 (vinyl-CH2), 132.18 (vinyl-CH), 156.15 (CO,
urethane) and 169.20, 170.77, 171.47, 173.09 and 173.45 (CO);
m/z (FAB) 524 (2%, [M ϩ Na]ϩ), 502 (4, [M ϩ H]ϩ), 402 (100,
[M ϩ 2H Ϫ C5H9O2]ϩ), 259 (85, [M ϩ 2H Ϫ C11H18NO5]ϩ),
215 (41, C10H17NO4ϩ), 188 (53, C8H14NO4ϩ) and 144 (47,
C6H10NO3ϩ).
ꢀ-Allyl ꢁ-methyl (2R)-N-(tert-butoxycarbonyl)aspartate diester
14
To a cooled stirred solution of β-allyl (2R)-N-(tert-butoxy-
carbonyl)aspartate 13 (2.73 g, 10 mmol) in diethyl ether (20
cm3) was added dropwise excess ethereal diazomethane. After
1 h, the solution was purged with nitrogen to remove the excess
of diazomethane and was then concentrated under reduced
pressure to give the aspartate diester 14 as a colourless oil (2.69
g, 94%) (HRMS: found [M ϩ H]ϩ, 288.1438. C13H22NO6
requires 288.1447); νmax(neat)/cmϪ1 3375 (NH), 1719 (CO ester)
and 1650 (CO urethane); δH(200 MHz; C2HCl3) 1.43 [9 H, s,
(CH3)3], 2.86 [1 H, dd, J 4.7 and 17.0, 1 H of β-CH2 (Asp)], 2.99
[1 H, dd, J 4.7 and 16.9, 1 H of β-CH2 (Asp)], 3.74 (3 H, s,
OCH3), 4.52–4.65 (3 H, br d, J 5.7, α-H and OCH2-vinyl), 5.19–
5.35 (2 H, m, vinyl CH2), 5.49 (1 H, d, J 8.5, NH) and 5.78–5.98
(1 H, m, vinyl CH); δC(50.3 MHz; C2HCl3) 28.75 [(CH3)3], 37.28
(β-CH2), 50.40 (OCH3), 53.15 (α-C), 66.09 (OCH2-vinyl),
119.09 (vinyl CH2), 132.15 (vinyl CH), 156.00 (CO, urethane),
171.50 and 172.10 (CO, ester); m/z (FAB) 288 (42%, [M ϩ H]ϩ),
232 (100, [M ϩ H Ϫ C3H4O]ϩ) and 188 (85, [M ϩ H Ϫ
C5H8O2]ϩ).
ꢀ-Allyl (2R)-[ꢁ-methyl glutamyl]-ꢂ-sarcosyl-[ꢁ-methyl (2R)-
aspartate] triester hydrochloride 18
The deprotected hydrochloride 18 was prepared in a manner
identical with that for the hydrochloride 15, by using the Boc-
protected tripeptide 17 (2.51 g, 5 mmol) to give the salt 18 as a
hygroscopic white solid (2.18 g, 100%), mp 52–54 ЊC; δH(300
MHz; C2H3O2H) 2.10–2.33 [2 H, m, β-CH2 (Glu)], 2.60–2.76
[2 H, m, γ-CH2 (Glu)], 2.84–3.02 [2 H, m, β-CH2 (Asp)], 2.94
and 3.09 (3 H, 2 × s, NCH3), 3.73 (3 H, s, OCH3), 3.85 (3 H, s,
OCH3), 4.07–4.19 [3 H, m, α-H (Glu) and CH2 (Sar)], 4.60–4.62
(2 H, m, OCH2-vinyl), 4.82–4.87 [1 H, m, α-H (Asp)], 5.22–5.36
(2 H, m, vinyl-CH2) and 5.88–6.01 (1 H, m, vinyl-CH); δC(75.4
MHz; C2H3O2H) 26.60 [β-CH2 (Glu)], 30.07 [γ-CH2 (Glu)],
35.54 [β-CH2 (Asp)], 37.00 (NCH3), 50.40 [CH2 (Sar)], 51.72
[α-C (Glu)], 53.28 [α-C (Asp)], 53.71 (OCH3), 53.94 (OCH3),
66.76 (OCH2-vinyl), 118.85 (vinyl-CH2), 133.71 (vinyl-CH) and
171.13, 171.39, 171.89, 172.70 and 174.75 (CO); m/z (EI) 401
(3%, [M Ϫ HCl]ϩ), 342 (17, [M Ϫ HCl Ϫ CO2CH3]ϩ), 259
(19, [M Ϫ HCl Ϫ C6H8NO3]ϩ), 214 (16, C10H16NO4ϩ), 188 (18,
C8H14NO4ϩ), 170 (8, C8H10O4ϩ), 143 (10, C6H9NO3ϩ), 128 (18,
C6H10NO2ϩ), 113 (19, C6H9O2ϩ), 84 (65, C4H4O2ϩ), 56 (27,
C6H10NO2ϩ) and 44 (100).
ꢀ-Allyl ꢁ-methyl (2R)-aspartate diester hydrochloride 15
Dry ethyl acetate (100 cm3) was saturated with hydrogen
chloride gas at 0 ЊC and after stirring for 1 h, a solution of
β-allyl α-methyl (2R)-N-(tert-butoxycarbonyl)aspartate diester
14 (2.69 g, 9.4 mmol) in ethyl acetate (50 cm3) was added. The
reaction mixture was stirred for 1.5 h at room temperature and
the resulting solution was concentrated under reduced pressure
to give the required hydrochloride salt 15 as a pale yellow solid
(1.89 g, 96%), mp 113–114 ЊC; νmax(Nujol)/cmϪ1 1761 and 1722
2
(CO ester); δH(300 MHz; H2O) 3.05 (1 H, dd, J 4.9 and 18.1,
1 H of β-CH2), 3.11 (1 H, dd, J 5.8 and 18.1, 1 H of β-CH2),
3.71 (3 H, s, OCH3), 4.39 (1 H, dd, J 4.9 and 5.8, α-H), 4.55
(2 H, d, J 5.5, OCH2-vinyl), 5.15–5.26 (2 H, m, vinyl-CH2)
2
and 5.76–5.89 (1 H, m, vinyl-CH); δC(75.4 MHz; H2O) 33.73
(β-CH2), 49.06 (OCH3), 53.88 (α-C), 66.75 (OCH2-vinyl),
119.08 (vinyl-CH2), 131.73 (vinyl-CH), 169.47 (CO) and 170.95
(CO); m/z (FAB) 188 (100%, [M ϩ H Ϫ HCl]ϩ) and 128 (14,
[M Ϫ HCl Ϫ CO2CH3]ϩ).
ꢀ-Allyl ꢁ-benzyl (2R)-N-(tert-butoxycarbonyl)aspartate diester
19
ꢀ-Allyl (2R)-N-(tert-butoxycarbonyl)-[ꢁ-methyl glutamyl]-ꢂ-
sarcosyl-[ꢁ-methyl (2R)-aspartate] triester 17
To a cooled stirred solution of β-allyl (2R)-N-(tert-butoxy-
carbonyl)aspartate ester 13 (5.47 g, 20 mmol) in methanol
(50 cm3) and water (10 cm3) was added 20% aqueous solution
of caesium carbonate, dropwise, until the solution was neutral
(pH 7). The mixture was concentrated under reduced pressure
to give an oil which was dissolved in water (10 cm3) and
lyophilised to afford a sticky white solid. The residue was then
dissolved in dry DMF (60 cm3) and the cooled solution was
treated with benzyl bromide (3.2 cm3, 4.60 g, 27 mmol). The
reaction mixture was stirred at room temperature for 18 h, and
then water (50 cm3) was added. The ester was extracted into
ethyl acetate (3 × 50 cm3) and the combined extracts were
washed with brine (50 cm3), dried (MgSO4), and then con-
centrated under reduced pressure to give a colourless oil.
This was purified by flash chromatography on silica (petroleum
ether–ethyl acetate; 3:1) to give the aspartate diester 19 as a
colourless oil (6.59 g, 91%) (HRMS: found [M ϩ H]ϩ,
364.1766. C19H26NO6 requires 364.1760); νmax(neat)/cmϪ1 3384
(NH), 1722 (CO ester) and 1659 (CO urethane); δH(300 MHz;
C2HCl3), 1.42 [9 H, s, (CH3)3], 2.87 (1 H, dd, J 4.7 and 17.0, 1 H
of β-CH2), 3.02 (1 H, dd, J 4.7 and 17.0, 1 H of β-CH2), 4.49–
4.54 (2 H, m, OCH2-vinyl), 4.59–4.65 (1 H, m, α-H), 5.12–5.31
To a stirred solution of N-(tert-butoxycarbonyl)-[α-methyl
(2R)-glutamyl]-γ-sarcosine 16 (3.22 g, 10 mmol) in dry THF
(50 cm3) at Ϫ40 ЊC was added N-methylmorpholine (NMM)
(1.10 cm3, 10 mmol). Isobutyl chloroformate (IBCF) (1.375
cm3, 10 mmol) was then added and the suspension was stirred
Ϫ15 ЊC for 5 min. A mixture of β-allyl α-methyl (2R)-aspartate
diester hydrochloride 15 (2.24 g, 10 mmol) and NMM
(1.10 cm3, 10 mmol) in dry THF (50 cm3) was then added. The
reaction mixture was allowed to warm to room temperature
and then stirred for a further 3 h. The hydrochloride salts
were removed by filtration and the solution was concentrated
under reduced pressure to give a yellow oil. The residue was
re-dissolved in ethyl acetate (120 cm3), and the solution was
washed successively with water (40 cm3), 10% citric acid (40
cm3), 5% sodium bicarbonate (40 cm3) and brine (40 cm3)
and then was dried (MgSO4), and concentrated under reduced
pressure. The pale yellow oil was purified by flash chroma-
tography on silica eluting with ethyl acetate to give the fully
protected tripeptide 17 as a colourless, viscous oil (3.47 g, 69%)
(HRMS: found [M ϩ H]ϩ, 502.2426. C22H36N3O10 requires
1684
J. Chem. Soc., Perkin Trans. 1, 2001, 1673–1695

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(4 H, m, OCH2Ar and vinyl CH2), 5.52 (1 H, d, J 8.5, NH),
5.78–5.91 (1 H, m, vinyl CH) and 7.33 (5 H, s, Ar-H); δC(75.4
MHz; C2HCl3) 28.04 [(CH3)3], 36.51 (β-CH2), 49.88 (α-C), 65.42
(OCH2-vinyl), 67.22 (OCH2Ar), 79.93 [OC(CH3)3], 118.51
vinyl-CH2), 128.12, 128.30 and 128.45 (Ar-CH), 131.61 (vinyl-
CH), 135.22 (Ar-C quaternary), 155.35 (CO, urethane), 170.54
and 170.88 (CO); m/z (FAB) 364 (7%, [M ϩ H]ϩ), 308 (25,
[M ϩ 2H Ϫ C4H9]ϩ), 264 (17, [M ϩ 2H Ϫ C5H9O2]ϩ), 228 (45,
with 10% citric acid solution (40 cm3) and saturated brine
(40 cm3), and then dried (MgSO4) and the solvent removed
under reduced pressure. The residue was dried under high
vacuum to give the acid 22 as a flocculent yellow solid (4.47 g,
95%), mp 56–57 ЊC; νmax(Nujol)/cmϪ1 3392 br (NH and OH),
1722 (CO ester) and 1659 (CO urethane); δH(300 MHz; C2HCl3)
1.36 [9 H, s, (CH3)3], 2.50–2.55 [1 H, br d, 1 H of β-CH2 (Asp)],
2.98–3.10 [3 H, m, 1 H of β-CH2 (Asp) and CH2 (Phe)], 4.60
[1 H, br d, α-H (Phe)], 4.81–4.86 [1 H, m, α-H (Asp)], 5.11 [3 H,
br s, CH2Ph and NH (Phe)], 5.36 [1 H, br d, NH (Asp)] and
7.14–7.36 (10 H, m, Ar-H); δC(75.4 MHz; C2HCl3) 28.60
[(CH3)3], 35.96 [β-CH2 (Asp)], 39.55 [β-CH2 (Phe)], 48.54 [α-C
(Phe)], 55.74 [α-C (Asp)], 67.90 (CH2Ph), 81.13 [C(CH3)3],
127.50, 128.59, 128.87, 129.10 and 129.81 (Ar-CH), 132.75
and 135.77 [Ar-C (quaternary)], 156.29 (CO, urethane), 170.68
(CO, acid), 172.02 (CO, amide) and 174.50 (CO, ester);
m/z (FAB) 493 (9%, [M ϩ Na]ϩ), 471 (3, [M ϩ H]ϩ), 415 (13,
[M ϩ H Ϫ C4H9]ϩ), 371 (100, [M ϩ H Ϫ C5H9O2]ϩ), 279 (33,
C13H15N2O5ϩ), 210 (25, C10H14N2O3ϩ) and 120 (95, C8H8Oϩ).
[M ϩ H Ϫ C8H8O2]ϩ), 172 (59, [M ϩ 2H Ϫ C4H9 Ϫ C8H8O2]ϩ),
ϩ
128 (90, [M ϩ 2H Ϫ C5H9O2 Ϫ C8H8O2]ϩ), 91 (86, PhCH2
)
and 57 (100, C4H9ϩ).
ꢀ-Allyl ꢁ-benzyl (2R)-aspartate diester hydrochloride 20
The deprotected hydrochloride 20 was prepared in a manner
identical with that for the hydrochloride 15, by using β-allyl α-
benzyl (2R)-N-(tert-butoxycarbonyl)aspartate diester 19 (4.36
g, 12 mmol) as the starting material to give the salt 20 as a white
solid (3.20 g, 89%), mp 97–98 ЊC; νmax(Nujol)/cmϪ1 1731 (CO);
δH(300 MHz; C2HCl3) 3.27 (1 H, dd, J 5.2 and 17.9, 1 H of
β-CH2), 3.35 (1 H, dd, J 5.2 and 17.9, 1 H of β-CH2), 4.49 (2 H,
dd, J 1.1 and 5.8, OCH2-vinyl), 4.67 (1 H, t, J 5.2, α-H), 5.11–
5.26 (4 H, m, OCH2Ar and vinyl CH2), 5.71–5.82 (1 H, m, vinyl
CH), 7.30 (5 H, s, Ar-H) and 8.88 (3 H, br, NH3); δC(75.4 MHz;
C2HCl3) 33.88 (β-CH2), 49.62 (α-C), 66.08 (OCH2-vinyl), 68.32
(OCH2Ar), 118.70 (vinyl CH2), 128.44 and 128.50 (Ar-CH),
131.55 (vinyl CH), 134.54 (Ar-C quaternary), 168.26 and
169.71 (CO); m/z (EI) 264 (4%, [M Ϫ Cl]ϩ), 178 (10,
[M Ϫ Cl Ϫ C4H6O2]ϩ), 172 (7, [M Ϫ HCl Ϫ C7H7]ϩ), 128 (100,
C6H10NO2ϩ), 91 (64, C7H7ϩ) and 41 (65, C3H5ϩ).
ꢀ-Allyl (2S)-N-(tert-butoxycarbonyl)phenylalanyl-[ꢁ-benzyl
(2R)-aspartyl]-ꢀ-[ꢁ-methyl (2R)-glutamyl]-ꢂ-sarcosyl-[ꢁ-methyl
(2R)-aspartate] tetraester 23
To a stirred solution of α-benzyl (2S)-N-(tert-butoxycarbonyl)-
phenylalanyl-(2R)-aspartate ester 22 (1.88 g, 4.0 mmol) in dry
THF (50 cm3) and DMF (5 cm3) at Ϫ40 ЊC was added N-
methylmorpholine (440 mm3, 4.0 mmol). Isobutyl chloro-
formate (550 mm3, 4.0 mmol) was added and the suspension
was stirred at Ϫ15 ЊC for 5 min. A mixture of β-allyl (2R)-[α-
methyl glutamyl]-γ-sarcosyl-[α-methyl (2R)-aspartate] triester
hydrochloride 18 (1.75 g, 4.0 mmol) and NMM (440 mm3,
4.0 mmol) in dry THF (50 cm3) and DMF (5 cm3) was added.
The reaction mixture was allowed to warm to room temper-
ature and then stirred for a further 4 h. The hydrochloride salts
were removed by filtration and the solution was concentrated
under reduced pressure to ca. 15 cm3, and then diluted with
ethyl acetate (150 cm3). The solution was successively washed
with 10% citric acid (40 cm3), 5% sodium bicarbonate (40 cm3)
and brine again (40 cm3), then dried (MgSO4), and the solvent
removed under reduced pressure. The resulting sticky yellow
solid was purified by flash chromatography on silica (ethyl
acetate) to give the fully protected pentapeptide 23 as a
pale yellow solid (2.78 g, 81%), mp 48 ЊC (softening) (HRMS:
found [M ϩ H]ϩ 854.3847. C42H56N5O14 requires 854.3824);
νmax(Nujol)/cmϪ1 3345 (NH), 1742 (CO ester) and 1722 (CO
amide); δH[500 MHz; (C2H3)2SO, mixture of rotamers] 1.48 [9
H, s, (CH3)3], 1.99–2.03 [1 H, m, 1 H of β-CH2 (Glu)], 2.11–2.18
[1 H, m, 1 H of β-CH2 (Glu)], 2.43–2.48 [1 H, m, 1 H of γ-CH2
(Glu)], 2.55 [1 H, br, 1 H of γ-CH2 (Glu)], 2.86–3.13 [6 H, m,
β-CH2 (Phe) and 2 × β-CH2 (Asp)], 2.95 and 3.07 (3 H, 2 × s,
NCH3), 3.78, 3.80 and 3.82 (6 H, 3 × s, 2 × OCH3), 4.13 [2 H,
m, CH2 (Sar)], 4.40–4.51 [2 H, m, α-H (Phe) and α-H (Glu)],
4.76 [2 H, s, OCH2 (allyl)], 4.88–4.90 [2 H, m, 2 × α-H (Asp)],
5.32 (2 H, s, CH2Ph), 5.40 (cis, 1 H, d, J 11.0, vinyl CH2), 5.49
(trans, 1 H, d, J 17.0, vinyl CH2), 6.06–6.12 (1 H, m, vinyl CH),
7.09 [0.6 H, d, J 8.0, 0.6 × NH (Phe)], 7.37–7.56 (10 H, m,
Ar-H), 7.75 [0.4 H, d, J 8.0, 0.4 × NH (Phe)], 7.82 [0.6 H, m,
0.6 × NH (Glu)], 8.53–8.59 [1.7 H, m, 1.7 × NH (Asp)], 8.62
[0.4 H, d, J 7.5, NH (Glu)] and 8.77 [0.3 H, d, J 8.1, NH (Asp)];
δC(75.4 MHz; C2HCl3, mixture of rotamers) 26.84 [β-CH2
(Glu)], 28.06 [(CH3)3], 28.70 [γ-CH2 (Glu)], 35.98 [β-CH2
(Asp)], 36.42 [β-CH2 (Asp)], 36.99 [CH2 (Phe)], 38.54 (NCH3),
48.47 [CH2 (Sar)], 48.99 (α-C), 51.72 (α-C), 52.40 (OCH3), 52.69
(OCH3), 55.40 [α-C (Phe)], 65.60 [CH2 (allyl)], 67.07 (CH2Ph),
79.73 [C(CH3)3], 118.60 (vinyl CH2), 126.73, 127.89, 128.28,
128.47, 128.55 and 129.37 (Ar-CH), 131.70 (vinyl CH), 132.01
(vinyl CH), 135.45 and 136.77 (Ar-C quaternary), 155.22
(CO, urethane), 168.70, 169.98, 170.55, 170.81, 171.26, 172.39
and 173.04 (CO); m/z (FAB) 876 (20%, [M ϩ Na]ϩ), 854
ꢀ-Allyl ꢁ-benzyl (2S)-N-(tert-butoxycarbonyl)phenylalanyl-(2R)-
aspartate diester 21
This compound was prepared in a manner identical with that
described for the glutamyl-sarcosyl-aspartate tripeptide 17,
using (2S)-N-(tert-butoxycarbonyl)phenylalanine (3.18 g, 12.0
mmol) and β-allyl α-benzyl (2R)-aspartate diester hydro-
chloride 20 (3.60 g, 12.0 mmol) to give a colourless viscous oil.
This was purified by flash chromatography on silica (petroleum
ether–ethyl acetate; 3:1) to give the phenylalanyl-aspartate
diester 21 as a colourless viscous oil which solidified slowly on
standing (5.09 g, 83%), mp 60–61 ЊC (Found C, 65.95; H,
6.9; N, 5.4. C28H34N2O7 requires C, 65.85; H, 6.7; N 5.5%);
νmax(Nujol)/cmϪ1 3345 (NH), 1746 (CO ester), 1703 (CO amide)
and 1669 (CO urethane); δH(300 MHz; C2HCl3) 1.38 [9 H, s,
(CH3)3], 2.66 [1 H, dd, J 4.6 and 17.5, 1 H of β-CH2 (Asp)],
2.93–3.10 [3 H, m, 1 H of β-CH2 (Asp) and β-CH2 (Phe)], 4.39–
4.46 [3 H, m, α-H (Phe) and OCH2-vinyl], 4.85–4.91 [1 H, m,
α-H (Asp)], 5.07–5.29 [5 H, m, vinyl CH2, OCH2Ar and NH
(Phe)], 5.74–5.87 (1 H, m, vinyl CH), 6.94 [1 H, d, J 8.0, NH
(Asp)] and 7.15–7.37 (10 H, m, Ar-H); δC(50.3 MHz; C2HCl3)
28.73 [(CH3)3], 36.52 [β-CH2 (Asp)], 39.14 [β-CH2 (Phe)], 48.77
[α-C (Phe)], 56.31 [α-C (Asp)], 66.12 (OCH2-vinyl), 67.98
(OCH2Ar), 119.26 (vinyl CH2), 127.37, 128.70, 128.92, 129.05,
129.11 and 129.72 (Ar-CH), 132.04 (vinyl CH), 135.64 and
137.04 (Ar-C quaternary), 170.61, 170.76 and 171.51 (CO);
m/z (FAB) 533 (9%, [M ϩ Na]ϩ), 511 (13, [M ϩ H]ϩ), 455 (15,
[M ϩ H Ϫ C3H4O]ϩ), 411 (100, [M ϩ H Ϫ C5H8O2]ϩ), 264 (14,
[M ϩ H Ϫ C14H17NO3]ϩ) and 120 (82).
ꢁ-Benzyl (2S)-N-(tert-butoxycarbonyl)phenylalanyl-(2R)-
aspartate ester 22
To a stirred solution of β-allyl α-benzyl N-(tert-butoxy-
carbonyl)-(2S)-phenylalanyl-(2R)-aspartate diester 21 (5.11 g,
10 mmol) and tetrakis(triphenylphosphine) palladium(0)
(0.30 g, 0.23 mmol) in dry CH2Cl2 (50 cm3) under N2, was
added with freshly distilled pyrrolidine (1.5 cm3). The reaction
mixture was stirred at room temperature for 1 h, and then
CH2Cl2 (50 cm3) was added. The resulting mixture was washed
J. Chem. Soc., Perkin Trans. 1, 2001, 1673–1695
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(12, [M ϩ H]ϩ), 754 (100, [M ϩ 2H Ϫ C5H9O2]ϩ) and 120 (90,
29.03 [γ-CH2 (Glu)], 35.73 [β-CH2 (Asp)], 36.87 [CH2 (Phe)],
C8H10Nϩ).
37.68 [β-CH2 (Asp)], 38.96 (NCH3), 48.97 [CH2 (Sar)], 49.51
(α-C), 52.11 (OCH3), 52.97 (OCH3), 53.43 (α-C), 53.66 (α-C),
55.98 [α-C(Phe)], 67.66 (CH2Ph), 80.41 [C(CH3)3], 127.29,
128.24, 128.41, 128.87, 129.01, 129.20, 129.83, 132.53 and
132.65 (Ar-CH), 135.81, 137.16 and 139.88 (Ar-C quaternary),
155.82 (CO urethane) and 167.32, 169.59, 170.94, 171.10,
171.26, 171.83, 172.13, 172.92 and 173.55 (CO); δF(282 MHz;
C2HCl3, mixture of rotamers) Ϫ153.27 (1.2 F, d, J 17.2, o-Ar-
F), Ϫ158.74 (0.6 F, t, J 22.4, p-Ar-F), Ϫ162.83 (1.2 F, t, J 20.0,
m-Ar-F), Ϫ163.51 (0.8 F, dd, J 5.1 and 17.2, o-Ar-F), Ϫ165.67
(0.8 F, t, J 21.4, m-Ar-F) and Ϫ171.10 (0.4 F, m, p-Ar-F);
m/z (FAB) 1002 (44%, [M ϩ Na]ϩ), 980 (39, [M ϩ H]ϩ), 880
(100 [M ϩ 2HϪC5H9O2]ϩ) and 120 (65, C8H10Nϩ).
(2S)-N-(tert-Butoxycarbonyl)phenylalanyl-[ꢁ-benzyl (2R)-
aspartyl]-ꢀ-[ꢁ-methyl (2R)-glutamyl]-ꢂ-sarcosyl-[ꢁ-methyl (2R)-
aspartate] triester 24
To a stirred solution of the fully protected pentapeptide 23
(2.13 g, 2.5 mmol) and tetrakis(triphenylphosphine)pal-
ladium(0) (80 mg, 0.06 mmol) in dry CH2Cl2 (25 cm3) under
N2, was added distilled pyrrolidine (375 mm3). The reaction
mixture was stirred at room temperature for 45 min, and then
CH2Cl2 (75 cm3) was added. The solution was washed with 10%
citric acid solution (40 cm3) and saturated brine (40 cm3), and
then dried (MgSO4) and the solvent removed under reduced
pressure to give the required pentapeptide free acid 24 as a
yellow solid (2.03 g, 100%), mp 66–68 ЊC (softening) (HRMS:
found [M ϩ H]ϩ, 814.3493. C39H52N5O14 requires 814.3511);
νmax(Nujol)/cmϪ1 3345 br (NH and OH), 1741 (CO ester) and
1714 (CO amide); δH(300 MHz; C2HCl3, mixture of rotamers)
1.32 [9 H, s, (CH3)3], 1.88–1.92 [1 H, m, 1 H of β-CH2 (Glu)],
2.03–2.55 [3 H, m, 1 H of β-CH2 (Glu) and β-CH2 (Asp)], 2.62–
2.70 [1 H, m, 1 H of CH2 (Phe)], 2.81–3.08 [9 H, m, 1 H of CH2
(Phe), β-CH2 (Asp) and NCH3], 3.67, 3.71 and 3.73 (6 H, 3 × s,
2 × OCH3), 4.09–4.21 [2 H, m, CH2 (Sar)], 4.47 [2 H, br, α-H
(Phe) and α-H (Glu)], 4.80 [2 H, br, 2 × α-H (Asp)], 5.14 (2 H, s,
CH2Ph), 5.23 [1 H, d, J 8.2, NH (Phe)] and 7.14–7.71 (13 H, m,
Ar-H and 3 × NH); δC(75.4 MHz; C2HCl3, mixture of rotamers)
27.09 [(CH3)3], 27.86 [β-CH2 (Glu)], 28.54 [β-CH2 (Glu)], 36.00
[β-CH2 (Asp)], 37.11 [β-CH2 (Asp)], 37.51 [CH2 (Phe)], 38.96
(NCH3), 49.01 [CH2 (Sar)], 49.82 (α-C), 52.48 (α-C), 52.54 (α-
C), 53.00 (OCH3), 53.13 (OCH3), 55.43 [α-C (Phe)], 66.28
(CH2Ph), 67.69 (CH2Ph), 80.67 [C(CH3)3], 127.30, 128.48,
128.85, 129.03, 129.10, 129.86, 132.57 and 132.70 (Ar-CH),
135.57 and 135.87 (Ar-C quaternary), 156.06 (CO, urethane),
169.39, 170.87, 171.71, 172.60, 172.95, 173.64 and 173.89 (CO);
m/z (FAB) 836 (10%, [M ϩ Na]ϩ), 814 (13, [M ϩ H]ϩ), 714 (86,
[M ϩ 2H Ϫ C5H9O2]ϩ) and 120 (100, C8H10Nϩ).
Cyclo{-[(R)-Asp ꢁ-OBn]-ꢀ-[(R)-Glu ꢁ-OMe]-ꢂ-Sar-[(R)-Asp
ꢁ-OMe]-ꢀ-(S)-Phe-} 27
To a stirred solution of pentapeptide pentafluorophenyl ester
25 (490 mg, 0.5 mmol) in dry CH2Cl2 (25 cm3) was added tri-
fluoroacetic acid (TFA) (25 cm3). The mixture was stirred for
1 h and then concentrated under reduced pressure. Toluene
(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
dry CH2Cl2 (500 cm3), then reacted with N,N-diisopropyl-
ethylamine (DIPEA) (1.5 cm3, 8.5 mmol), and the mixture
stirred at room temperature for 7 days, when TLC analysis indi-
cated that reaction was complete. The reaction mixture was
concentrated under reduced pressure and then triturated with
diethyl ether to give a solid, which was filtered off, and washed
on the pad with diethyl ether to give the cyclic product 27 as a
white powder (260 mg, 75%), mp 254–257 ЊC (decomp.)
(HRMS: found [M ϩ H]ϩ, 696.2897. C34H42N5O11 requires
696.2881); νmax(Nujol)/cmϪ1 3300 (NH), 1737 (CO ester), 1661
and 1639 (CO amide); δH[500 MHz; (C2H3)2SO, mixture of
rotamers, only selected peaks given] 1.71–1.85 [1 H, m, 1 H
of β-CH2 (Glu)], 1.96–2.01 [1 H, m, 1 H of β-CH2 (Glu)], 2.08–
2.14 [1 H, m,1 H of γ-CH2 (Glu)], 2.23–2.28 [1 H, m, 1 H of
γ-CH2 (Glu)]; m/z (FAB) 696 (15%, [M ϩ H]ϩ) and 130 (100).
ꢀ-Pentafluorophenyl (2S)-N-(tert-butoxycarbonyl)phenylalanyl-
[ꢁ-benzyl (2R)-aspartyl]-ꢀ-[ꢁ-methyl (2R)-glutamyl]-ꢂ-sarcosyl-
[ꢁ-methyl (2R)-aspartate] tetraester 25
Cyclo{-[(R)-Asp]-ꢀ-[(R)-Glu ꢁ-OMe]-ꢂ-Sar-[(R)-Asp ꢁ-OMe]-
ꢀ-(S)-Phe-} 7
To a stirred solution of pentapeptide free acid 24 (1.63 g,
2.0 mmol) in dry CH2Cl2 (50 cm3) at 0 ЊC was added penta-
fluorophenol (1.10 g, 6.0 mmol) followed by 1-(3-dimethyl-
aminopropyl)-3-ethylcarbodiimide methiodide (EDCI) (0.90 g,
3.0 mmol). The reaction mixture was allowed to warm to room
temperature and then stirred for a further 15 h. The solution
was concentrated under reduced pressure to give a yellow oil
which was purified by flash chromatography on silica (ethyl
acetate) to give the required pentafluorophenyl ester 25 as a
white waxy solid (1.69 g, 86%), mp 162–164 ЊC (HRMS:
found [M ϩ H]ϩ, 980.3333. C45H51N5O14F5 requires 980.3353);
νmax(Nujol)/cmϪ1 3313 (NH), 1800 (CO ester), 1733 (CO ester),
1697 (CO amide) and 1645 (CO urethane); δH[500 MHz;
(C2H3)2SO, mixture of rotamers] 1.30 [9 H, s, (CH3)3], 1.80–1.84
[1 H, m, 1 H of β-CH2 (Glu)], 1.92–1.99 [1 H, m, 1 H of γ-CH2
(Glu)], 2.27–2.40 [2 H, m, γ-CH2 (Glu)], 2.68–2.81 [3 H, m, 1 H
of β-CH2 (Asp) and CH2 (Phe)], 2.79 and 2.90 (3 H, 2 × s,
NCH3), 2.89–2.95 [1 H, m, 1 H of β-CH2 (Asp)], 3.17–3.26 [1 H,
m, 1 H of β-CH2 (Asp)], 3.30–3.37 [1 H, m, 1 H of β-CH2
(Asp)], 3.60, 3.62 and 3.68 (6 H, 3 × s, 2 × OCH3), 3.95–4.02
[2 H, m, CH2 (Sar)], 4.22–4.28 [1 H, m, α-H (Phe)], 4.30–4.35
[1 H, m, α-H (Glu)], 4.70–4.74 [1 H, m, α-H (Asp)], 4.82–4.88
[1 H, m, α-H (Asp)], 5.14 (2 H, s, CH2Ph), 6.92 [0.6 H, d, J 8.5,
0.6 × NH (Phe)], 7.19–7.38 (10 H, m, Ar-H), 7.56 [0.4 H,
m, 0.4 × NH (Phe)], 7.62–7.66 [0.4 H, m, 0.4 × NH (Glu)],
8.35–8.53 [2.2 H, m, 0.6 × NH (Glu) and 1.6 × NH (Asp)] and
8.76 [0.4 H, d, J 8.5, 0.4 × NH (Asp)]; δC(75.4 MHz; C2HCl3,
mixture of rotamers) 27.58 [β-CH2 (Glu)], 28.49 [(CH3)3)],
To a stirred solution of the macrocyclic benzyl ester 27 (209 mg,
0.30 mmol) in glacial acetic acid–methanol (1:1, 30 cm3) was
added 5% palladium on activated carbon (5 mg). The sus-
pension was then vigorously stirred under an atmosphere of
hydrogen for 18 h. The catalyst was removed by filtration on a
pad of pre-washed Celite and the solvent was removed under
reduced pressure to give an oily residue which was azeo-
tropically dried using methanol and toluene. The acid 7 was
obtained as a sticky white solid (154 mg, 85%) in greater than
90% purity, and was used directly, without purification, in
subsequent steps to form the exocyclic amide derivatives.
δH[500 MHz; (C2H3)2SO, one major rotamer] 1.62 and 1.87
[2 H, 2 × m, AB coupling of β-CH2 (Glu)], 1.95 and 2.30 [2 H,
2 × m, AB coupling of γ-CH2 (Glu)], 2.51–2.80 [4 H, 4 × m,
2 × AB multiplets of β-CH2 (Asp)], 2.81 [3 H, s, N-Me (Sar)],
3.13 and 3.34 [2 H, 2 × m, AB coupling of β-CH2 (Phe)], 3.62
(6 H, 2 × s, 2 × OCH3), 4.10, 4.29 and 4.38 [5 H, 3 × m, α-CH
of Glu, Phe and Asp, and α-CH2 (Sar)], 4.93 [1 H, br d, α-CH
(Asp)], 7.21 (5 H, m, Ar-H) and 7.60, 7.94, 8.49 and 8.63 (4 H,
1
4 × d, NH of Glu, Phe, Asp, Asp). The H-NMR spectrum in
d4-methanol showed two major conformers in a ratio of 2:1.
Cyclo{-[(2R)-ꢁ-4-benzylpiperidinyl-Asp]-ꢀ-[(R)-Glu]-ꢂ-Sar-
[(R)-Asp]-ꢀ-(S)-Phe-} 29
To a stirred solution of the macrocycle 7 (60 mg, 0.10 mmol) in
dry THF (10 cm3) and DMF (2 cm3) at Ϫ40 ЊC was added N-
1686
J. Chem. Soc., Perkin Trans. 1, 2001, 1673–1695

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methylmorpholine (11 mm3, 0.1 mmol). Isobutyl chloroformate
(13.75 mm3, 0.1 mmol) was added and the suspension was
stirred at Ϫ15 ЊC for 5 min. A mixture of 4-benzylpiperidine
(17.6 mm3, 0.1 mmol) and NMM (11 mm3, 0.1 mmol) in dry
THF (5 cm3) was added. The reaction mixture was allowed
to warm to room temperature and then stirred for a further
4 h. The hydrochloride salts were removed by filtration and the
solution was concentrated under reduced pressure to ca. 2 cm3,
and then diluted with ethyl acetate (10 cm3). The solution was
successively washed with 10% citric acid (5 cm3), 5% sodium
bicarbonate (5 cm3) and brine again (5 cm3), and then dried
(MgSO4). The solvents were removed under reduced pressure to
give the required amide diester 28 as a pale brown solid (33.6
mg, 44%). δH(500 MHz; C2HCl3) 1.16 and 1.87 [2 H, 2 × m, AB
coupling of β-CH2 (Glu)], 1.42 and 1.58 (4 H, 2 × d, J 8,
2 × NCH2CH2), 1.67 [2 H, m, γ-CH2 (Glu)], 2.43 (2 H, m,
NCH2CH2), 2.52 [2 H, m, β-CH2 (Asp)], 2.57 [3 H, 2 × s, N-Me
(Sar)], 2.74–3.09 [9 H, m, PhCH2, NCH2CH2, PhCH2CH,
β-CH2 (Asp) and β-CH2 (Phe)], 3.62 (6 H, 4 × s, 2 × OCH3),
4.32–4.65 [6 H, 3 × m, α-CH of Glu, Phe, Asp, Asp amide
and α-CH2 (Sar)], 7.03–7.38 (10 H, m, Ar-H); NH signals not
assigned. The 1H-NMR spectrum in d6-DMSO solution
showed at least 3 conformations. The reaction was repeated
several times; 44% was the best yield.
The crude diester 28 (15 mg, 19.7 µmol) was dissolved in
methanol (2 cm3) and water (2 cm3). Aqueous 1 mol dmϪ3
NaOH solution (50 µmol) was then added. The reaction was
allowed to stir at room temperature for 2 h, after which time the
methanol was removed under reduced pressure. The aqueous
layer was acidified using trifluoroacetic acid and the cyclic
peptide extracted using CH2Cl2 (2 cm3). The organic phase
was dried (MgSO4) and the solvent removed under reduced
pressure. The crude material was then purified by HPLC on a
C-18 reverse phase column [using isocratic reverse-phased con-
ditions eluting with acetonitrile–water (51:49) as eluent at a
flow rate of 1 cm3 minϪ1]. The eluent was monitored by UV
spectroscopy at 220 nm. The fractions corresponding to peak
1 (retention time 8.5 min) were collected and pooled together,
and the solvent was removed under reduced pressure and by
lyophilisation to give the desired compound in approximately
50% yield, and >95% purity. δH(500 MHz; C2HCl3; 2 major
conformers) 1.15 and 1.43 [4 H, 2 × m, β-CH2 (Glu) and
NCH2CH2], 1.59–1.96 [4 H, 2 × m, γ-CH2 (Glu), and NCH2-
CH2], 2.22–2.81 [10 H, 3 × m, 2 × β-CH2 (Asp) and 2 ×
NCH2CH2, PhCH2CH], 2.59 [3 H, m, N-Me (Sar)], 2.95–3.30
[4 H, m, PhCH2 and β-CH2 (Phe)], 4.18 [4 H, m, α-CH of Glu,
Phe and α-CH2 (Sar)], 4.60 [1 H, m, α-CH (Asp)], 5.11 [1 H, m,
α-CH (Asp amide)], 7.05–7.20 (10 H, m, Ar-H); NH signals
not assigned; m/z (ESϩ) 773 (1%, [M ϩ K]ϩ) and 735 (1.5,
[M ϩ H]ϩ).
C2HCl3) 24.14 [c, γ-CH2 (Pro)], 24.27 [t, γ-CH2 (Pro)], 28.40 [t,
(CH3)3], 28.75 [c, (CH3)3], 28.97 [t, β-CH2 (Pro)], 29.08 [c,
β-CH2 (Pro)], 36.78 [β-CH2 (Asp)], 47.25 [t, δ-CH2 (Pro)], 48.49
[c, δ-CH2 (Pro)], 48.68 [α-C (Asp)], 53.13 (CH3), 60.65 [c, α-C
(Pro)], 60.74 [t, α-C (Pro)], 67.29 (PhCH2), 80.81 [C(CH3)3],
128.61, 128.81 and 129.04 (Ar-CH), 136.20 (Ar-C quaternary),
156.04 (CO, urethane) and 171.06, 171.11 and 171.38 (CO,
esters); m/z (CI) 435 (100%, [M ϩ H]ϩ), 379 (55, [M ϩ H Ϫ
C4H9 ϩ H]ϩ) and 335 (97, [M ϩ H Ϫ C5H9O2ϩ H]ϩ).
ꢀ-Benzyl [(2S)-prolyl][ꢁ-methyl (2R)-aspartate] diester hydro-
chloride 37
This compound was prepared in a manner identical to that
described for the hydrochloride 15 using the prolyl-aspartyl
diester 36, (750 mg, 1.73 mmol), to yield a white hygroscopic
solid which was not purified any further (590 mg, 93%),
mp 101–102 ЊC (Found C, 55.0; H, 6.3; N 7.4. C17H23ClN2O5
requires C, 55.1; H, 6.25; N, 7.55%) (HRMS: found
[M ϩ H Ϫ HCl]ϩ, 335.1600. C17H23N2O5 requires 335.1607);
[α]D Ϫ28.3 (c 1.32 in MeOH); νmax(CH2Cl2)/cmϪ1 3189 (NH),
2959 (CH), 1739 (CO, ester) and 1684 (CO, amide); δH(300
MHz; C2HCl3) 1.89–1.99 [3 H, m, γ-CH2, 1 H of β-CH2 (Pro)],
2.38–2.58 [1 H, m, one of β-CH2 (Pro)], 2.94 [2 H, d, J 5.7,
β-CH2 (Asp)], 3.43 [2 H, br, δ-CH2 (Pro)], 3.6 (3 H, s, CH3),
4.62–4.86 [2 H, m, α-H (Pro) and α-H, (Asp)], 5.07 [2 H, q,
J 12.3, PhCH2], 7.27 (5 H, s, Ph), 7.84 (1 H, br, NH) and 9.10
(1 H, d, J 7.5, NHϩ); δC(75.4 MHz; C2HCl3) 24.26 [γ-CH2
(Pro)], 30.39 [β-CH2 (Pro)], 36.02 [β-CH2 (Asp)], 46.74 [δ-CH2
(Pro)], 49.45 [α-CH (Asp)], 52.84 (CH3), 59.70 [α-C (Pro)],
66.86 (PhCH2), 128.17, 128.3, 128.35 and 128.49 (Ar-CH),
135.45 (Ar-C quaternary) and 169.04, 170.11 and 170.86 (3 ×
CO, esters and amides); m/z (CI) 335 (39%, [M ϩ H Ϫ HCl]ϩ),
303 (82, [M Ϫ HCl Ϫ OCH3]ϩ) and 213 (100, C10H15NO4ϩ).
ꢀ-Benzyl [ꢁ-methyl (2R)-N-(tert-butoxycarbonyl)glutamyl]-ꢂ-
(2S)-prolyl-[ꢁ-methyl (2R)-aspartate] triester 38
This compound was prepared in a manner identical to that
described for the pentapeptide 23, using α-methyl (2R)-N-(tert-
butoxycarbonyl) glutamate (277 mg, 1.19 mmol) and the hydro-
chloride triester 37 (440 mg, 1.19 mmol) to give the required
compound as a colourless oil, which was recrystallised from
ethyl acetate–hexane to give a white solid (566 mg, 82%), mp
92–93 ЊC (Found C, 57.9; H, 6.8; N 7.2. C28H39N3O10 requires
C, 58.2; H, 6.8; N, 7.3%) (HRMS: found [M ϩ H]ϩ, 578.2702.
C28H40N3O10 requires 578.2714); [α]D Ϫ19.2 (c 1.25 in MeOH);
νmax(CH2Cl2)/cmϪ1 3354 (NH), 2956 (CH) and 1742 (CO,
esters); δH(300 MHz; C2HCl3) 1.46 [9 H, s, (CH3)3], 1.64–2.69
[8 H, m, γ-CH2 and β-CH2 (Pro and Glu)], 2.87–2.98 [2 H, m,
β-CH2 (Asp)], 3.37–3.40 [δ-CH2 (Pro)], 3.52 and 3.58 [6 H, s,
2 × CH3], 4.08 [1 H, br, α-H (Pro)], 4.28–4.38 [1 H, br, α-H
(Glu)], 4.83 [1 H, q, J 4.5, α-H, (Asp)], 5.10 [1 H, d, J 5.7, NH
(urethane)], 5.13 (2 H, s, PhCH2), 7.29 (5 H, s, Ph) and 7.68
(1 H, d, J 5.7, NH); δC(75.4 MHz; C2HCl3) 22.60 [c, γ-CH2
(Pro)], 24.56 [t, γ-CH2 (Pro)], 28.03 [β-CH2 (Glu)], 28.35
[(CH3)3], 28.97 [γ-CH2 (Glu)], 30.17 [t, β-CH2 (Pro)], 32.13 [c, β-
CH2 (Pro)], 36.87 [β-CH2 (Asp)], 46.94 [t, δ-CH2 (Pro)], 47.39 [c,
δ-CH2 (Pro)], 48.71 [α-C (Asp)], 52.56 [α-C (Glu)], 2.76 [CH3
(Asp)], 52.77 [CH3 (Glu)], 60.23 [α-C (Pro)], 66.74 (PhCH2),
80.8 [C(CH3)3], 128.31, 128.38, 128.59 and 128.69 (Ar-CH),
135.80 (Ar-C quaternary), 135.91 (CO, urethane) and 170.31,
170.66, 171.27, 171.51 and 171.62 (CO, esters and amides);
m/z (CI) 578 (76%, [M ϩ H]ϩ) and 478 (100, [M ϩ H Ϫ
C5H9O2 ϩ H]ϩ).
ꢀ-Benzyl [(2S)-N-(tert-butoxycarbonyl)prolyl][ꢁ-methyl (2R)-
aspartate] diester 36
This compound was prepared in a manner identical to that
described for pentapeptide 23, using N-(tert-butoxycarbonyl)-
(2S)-proline (215 mg, 1 mmol) and α-methyl β-benzyl (2R)-
aspartate diester hydrochloride 35 (273 mg, 1 mmol), to give
the required compound as a colourless oil which was refractory
to crystallisation (343 mg, 78%) (Found C, 61.1; H, 7.3; N 6.2.
C22H30N2O7 requires C, 60.8; H, 7.0; N, 6.4%) (HRMS: found
[M ϩ H]ϩ, 435.2124. C22H31N2O7 requires 435.2131); [α]D
Ϫ5.41 (c 1.21 in MeOH); νmax(CH2Cl2)/cmϪ1 3323 (NH), 2976
(CH), 1739 (CO, urethane) and 1699 (CO, esters); δH(300 MHz;
C2HCl3) 1.43 [9 H, s, (CH3)3], 1.82–2.22 [4 H, m, γ-CH2 and β-
CH2 (Pro)], 2.85 [1 H, dd, J 17.1 and 4.8, 1 H of β-CH2 (Asp)],
3.06 [1 H, dd, J 14.1 and 4.7, 1 H of β-CH2 (Asp)], 3.09–3.45 [2
H, m, δ-CH2 (Pro)], 3.66 (3 H, s, CH3), 4.18–4.37 [1 H, m, α-H
(Pro)], 4.87 [1 H, q, J 4.5, α-H, (Asp)], 5.09 (2 H, s, PhCH2),
7.28–7.34 (5 H, m, Ar-H) and 7.37 (1 H, br, NH); δC(75.4 MHz;
ꢀ-Benzyl [ꢁ-methyl (2R)-glutamyl]-ꢂ-(2S)-prolyl-[ꢁ-methyl
(2R)-aspartate] triester hydrochloride 39
The deprotected hydrochloride was prepared in a manner iden-
tical to that described for the hydrochloride 15, using the
J. Chem. Soc., Perkin Trans. 1, 2001, 1673–1695
1687

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glutamylprolylaspartyl triester 38, (310 mg, 0.54 mmol) as a
starting material, to afford a white hygroscopic solid which was
not further purified (262 mg, 95%), mp 84–85 ЊC (HRMS:
found [M ϩ H Ϫ HCl]ϩ, 478.2182. C23H32N3O8 requires
478.2189); [α]D Ϫ41.1 (c 1.34 in MeOH); νmax(CH2Cl2)/cmϪ1
3316 (NH), 2977 (CH) and 1685 (CO, amides); δH(300 MHz;
C2HCl3) 1.80–2.80 [8 H, m, γ-CH2 and β-CH2 (Pro and Glu)],
2.86–3.04 [2 H, m, β-CH2 (Asp)], 3.4–3.59 [δ-CH2 (Pro)], 3.64,
3.67 and 3.78 [c and t, 6 H, s, (2 × CH3)], 4.26 [1 H, br, α-H
(Pro)], 4.7 [1 H, br, α-H (Glu)], 4.86 [1 H, q, J 6, α-H (Pro)], 5.11
(2 H, s, PhCH2), 7.33 (5 H, s, Ar-H), 8.15 (1 H, d, J 7.8, NH)
and 8.65 (1 H, br, NH3); δC(75.4 MHz; C2HCl3) 24.76 [γ-CH2
(Pro)], 24.91 [β-CH2 (Glu)], 29.29 [γ-CH2 (Glu)], 30.53 [β-CH2
(Pro)], 36.37 [β-CH2 (Asp)], 47.89 [δ-CH2 (Pro)], 48.94 [α-C
(Asp)], 52.71 [α-C (Glu)], 52.92 [CH3 (Asp)], 53.53 [CH3 (Glu)],
60.36 [α-C (Pro)], 67.02 (PhCH2), 128.43, 128.49 and 128.66
(Ar-CH), 135.67 (Ar-C quaternary) and 169.91, 170.67, 171.61,
172.01 and 172.06 (CO, esters, amides and acid); m/z (CI) 478
(42%, [M ϩ H Ϫ HCl]ϩ), 460 (88, [M Ϫ 2H Ϫ Me Ϫ HCl]ϩ)
and 144 (100, C6H10NO3ϩ).
ϩ31.67 (c 1.2 in MeOH); νmax(CH2Cl2)/cmϪ1 3324 (NH), 2958
(CH), 1736 (CO, urethane) and 1653 (CO, esters and amides);
δH(300 MHz; C2HCl3) 1.29 [9 H, s, (CH3)3], 1.78–2.48 [10 H, m,
γ-CH2 and β-CH2 (Pro and Glu) and α-CH2 (β-Ala)], 2.65–3.17
[4 H, m, δ-CH2 (Asp) and β-CH2 (Phe)], 3.32–3.58 [4 H, m,
δ-CH2 (Pro) and β-CH2 (β-Ala)], 3.63, 3.65 and 3.68 (c and t,
6 H, s, 2 × CH3), 4.21–4.4 [2 H, m, α-H (Phe and Pro)], 4.42–
4.58 [1 H, m, α-H, (Glu)], 4.78 [1 H, br, α-H (Asp)], 5.22 [2 H,
br, NH (urethane) and OH], 7.08–7.28 (5 H, m, Ar-H) and 7.35–
7.79 (2 H, m, NH); δC(75.4 MHz; C2HCl3) 22.66 [c, γ-CH2
(Pro)], 24.65 [t, γ-CH2 (Pro)], 26.64 [β-CH2 (Glu)], 28.30
[(CH3)3], 28.99 [γ-CH2 (Glu)], 30.06 [t, β-CH2 (Pro)], 31.41 [c,
β-CH2 (Pro)], 32.16 [α-CH2 (β-Ala)], 35.65 [β-CH2 (Asp)], 35.97
[β-CH2 (β-Ala)], 38.85 [β-CH2 (Phe)], 47.18 [t, δ-CH2 (Pro)],
47.50 [c, δ-CH2 (Pro)], 48.69 [c, α-C (Asp)], 49.10 [t, α-C (Asp)],
52.06 [α-C (Glu)], 52.62 [CH3 (Asp)], 52.74 [CH3 (Glu)], 55.91
[α-C (Phe)], 60.41 [t, α-C (Pro)], 61.39 [c, α-C (Pro)], 80.04
[C(CH3)3], 126.82, 128.54 and 129.44 (Ar-CH), 137.01 (Ar-C
quaternary), 155.78 (CO, urethane) and 171.19, 171.58, 171.67,
171.95, 172.17, 172.34, 172.56, 172.72, 173.17 and 173.55 (c and
t, CO); m/z (ES) 728 (5%, [M ϩ Na]ϩ), 706 (6, [M ϩ H]ϩ), 157
(95, C7H11NO3ϩ) and 140 (100, C7H10NO2ϩ).
ꢀ-Benzyl (2S)-N-(tert-butoxycarbonyl)phenylalanyl-ꢀ-alanyl-[ꢁ-
methyl (2R)-glutamyl]-ꢂ-(2S)-prolyl-[ꢁ-methyl (2R)-aspartate]
triester 32
ꢀ-Pentafluorophenyl (2S)-N-(tert-butoxycarbonyl)phenylalanyl-
ꢀ-alanyl-[ꢁ-methyl (2R)-glutamyl]-ꢂ-(2S)-prolyl-[ꢁ-methyl (2R)-
aspartate] triester 41
This compound was prepared in a manner identical to that
described for the pentapeptide 23, using the phenylalanyl-β-
alanyl dipeptide 34 (246 mg, 0.73 mmol) and tripeptide 39 (376
mg, 0.73 mmol) to give the required compound as a colourless
oil which was recrystallised using ethyl acetate–hexane (390 mg,
62%), mp 118–119 ЊC (Found C, 59.7; H, 6.7; N, 8.4.
C40H53N5O12ؒ0.5 H2O requires C, 59.7; H, 6.8; N, 8.7%); [α]D
ϩ31.67 (c 1.2 in MeOH); νmax(CH2Cl2)/cmϪ1 3307 (NH), 2955
(CH), 1740 (CO, urethane) and 1655 (CO, esters and amides);
δH(500 MHz; C2HCl3) 1.34 [9 H, s, (CH3)3], 1.81–2.42 [10 H, m,
γ-CH2 and β-CH2 (Pro and Glu) and α-CH2 (β-Ala)], 2.92–3.13
[4 H, m, β-CH2 (Asp) and β-CH2 (Phe)], 3.37–3.43 [4 H, m,
δ-CH2 (Pro) and β-CH2 (β-Ala)], 3.64, 3.69 and 3.71 (c and t,
6 H, s, 2 × CH3), 4.28–4.41 [2 H, m, α-H (Phe and Pro)], 4.59–
4.63 [1 H, m, α-H, (Glu)], 4.86 [1 H, q, J 5.4, α-H (Asp)], 5.14
(2 H, s, PhCH2), 5.22 [1 H, d J 5.6, NH (urethane)], 6.88 [2 H,
br, 2 × NH (amide)], 7.18–7.38 (10 H, m, Ar-H) and 7.76 (1 H,
d, J 5.4, NH); δC(75.4 MHz; C2HCl3) 22.76 [c, γ-CH2 (Pro)],
24.71 [t, γ-CH2 (Pro)], 27.08 [β-CH2 (Glu)], 28.36 [(CH3)3],
28.87 [γ-CH2 (Glu)], 30.48 [t, β-CH2 (Pro)], 31.63 [c, β-CH2
(Pro)], 32.18 [α-CH2 (β-Ala)], 35.48 [c, β-CH2 (Asp)], 35.88 [c,
β-CH2 (Asp)], 36.49 [β-CH2 (β-Ala)], 39.18 [β-CH2 (Phe)], 46.97
[t, δ-CH2 (Pro)], 47.51 [c, δ-CH2 (Pro)], 48.84 [c, α-C (Asp)],
49.31 [t, α-C (Asp)], 52.02 [α-C (Glu)], 52.63 [CH3 (Asp)], 52.82
[CH3 (Glu)], 55.82 [α-C (Phe)], 60.11 [t, α-C (Pro)], 61.37 [c, α-C
(Pro)], 66.89 (PhCH2), 80.03 [C(CH3)3], 126.83, 128.44, 128.55,
128.67, 128.73 and 129.52 (Ar-C), 135.46, 135.63 and 135.75
(Ar-C quaternary), 155.45 (CO, urethane) and 170.53, 170.82,
171.02, 171.51, 171.65, 171.78, 171.85, 172.15 and 172.62 (c and
t, CO esters and amides); m/z (FAB) 818 (72%, [M ϩ Na]ϩ), 718
(54, [M Ϫ C6H5]ϩ), 696 (73, [M ϩ H Ϫ C5H9O2 ϩ H]ϩ) and 133
(100).
To a stirred solution of the pentapeptide carboxylic acid diester
40 (130 mg, 0.18 mmol) in CH2Cl2 (20 cm3) at 0 ЊC was added
pentafluorophenol (102 mg, 0.55 mmol) followed by EDCI
(82 mg, 0.28 mmol). The reaction mixture was allowed to warm
to room temperature with stirring overnight. The solution was
concentrated under reduced pressure to yield a colourless oil
which was purified by flash chromatography on silica using
CH2Cl2–MeOH (95:5) as the eluent to give triester 41 as a white
crystalline solid (100 mg, 62%), mp 79–81 ЊC (HRMS: found
[M ϩ Na]ϩ, 894.2978. C39H46F5N5O12Na requires 894.2961);
νmax(CH2Cl2)/cmϪ1 3311 (NH), 2957 (CH), 1742 (CO, urethane)
and 1655 (CO, esters and amides); δH(300 MHz; C2HCl3) 1.33
[9 H, s, (CH3)3], 1.80–2.44 [10 H, m, γ-CH2 and β-CH2 (Pro
and Glu) and α-CH2 (β-Ala)], 2.84–3.57 [8 H, m, β-CH2 (Asp),
β-CH2 (Phe), β-CH2 (Pro) and β-CH2 (β-Ala)], 3.69 and 3.72
(6 H, s, 2 × CH3), 4.27–4.38 [2 H, m, α-H (Phe and Pro)], 4.62
[1 H, br, α-H (Glu)], 4.99 [1 H, q, J 5.7, α-CH (Asp)], 5.27 [1 H,
d, J 8.1, NH (urethane)], 6.82–6.96 (2 H, m, 2 × NH), 7.12–7.26
(5 H, m, Ar-H) and 7.88 (1 H, d, J 7.8, NH); δC(75.4 MHz;
C2HCl3) 22.73 [c, γ-CH2 (Pro)], 24.71 [t, β-CH2 (Pro)], 27.21 [β-
CH2 (Glu)], 28.30 [(CH3)3], 28.97 [γ-CH2 (Glu)], 30.36 [t, β-CH2
(Pro)], 31.67 [c, β-CH2 (Pro)], 32.17 [α-CH2 (β-Ala)], 35.50
[β-CH2 (Asp)], 35.59 [β-CH2 (β-Ala)], 39.04 [β-CH2 (Phe)],
46.90 [c, δ-CH2 (Pro)], 47.54 [t, δ-CH2 (Pro)], 48.82 [t, α-C
(Asp)], 49.48 [c, α-C (Asp)], 51.82 [α-C (Glu)], 52.63 [CH3
(Asp)], 53.08 [CH3 (Glu)], 55.89 [α-C (Phe)], 60.10 [t, α-C
(Pro)], 61.35 [c, α-C (Pro)], 79.95 [C(CH3)3], 126.84, 128.54,
128.90 and 129.44 (Ar-CH), 137.04 (Ar-C quaternary), 136.40,
138.10, 139.57, 141.60 and 142.8 (C-F, PFP), 155.51 (CO,
urethane), 166.69 (CO, PFP) and 170.78, 171.74, 171.79, 172.88
and 172.60 (c and t, CO, esters and amides); m/z (ES) 895 (34%,
[M ϩ Na ϩ H]ϩ), 873 (41, [M ϩ 2H]ϩ), 196 (27, C9H10NO4ϩ),
158 (36, C6H8NO4ϩ) and 101 (100, C5H9O2ϩ).
(2S)-N-(tert-Butoxycarbonyl)phenylalanyl-ꢀ-alanyl-[ꢁ-methyl
(2R)-glutamyl]-ꢂ-(2S)-prolyl-[ꢁ-methyl (2R)-aspartate]
diester 40
Cyclo[-ꢀ-Ala-(2R)-Glu-ꢁ-OMe-ꢂ-(2S)-Pro-(2R)-Asp-ꢁ-OMe-ꢀ-
(2S)-Phe-] 42 (R ؍ Me)
To a stirred solution of the benzyl ester 32 (130 mg, 0.165
mmol) in ethanol (20 cm3) was added 10% palladium on carbon
(20 mg) and the mixture stirred under an atmosphere of hydro-
gen for 3 h. The catalyst was removed by filtration through a
pre-washed Celite pad and the filtrate was concentrated under
reduced pressure to give the required compound as a white
crystalline solid in quantitative recovery, which was not purified
further, mp 86–87 ЊC (Found C, 54.7; H, 6.5; N, 9.3.
C33H47N5O12ؒH2O requires C, 54.8; H, 6.8; N, 9.7%); [α]D
To a stirred solution of the N-(tert-butoxycarbonyl)-protected
pentafluorophenyl ester 41 (78 mg, 0.09 mmol) in CH2Cl2
(10 cm3) was added trifluoroacetic acid (10 cm3). The reaction
mixture was stirred for 1 h when the reaction was judged to be
complete by TLC. The solution was concentrated under
reduced pressure, triturated with diethyl ether (10 cm3), and
the precipitate was collected and thoroughly dried under high
1688
J. Chem. Soc., Perkin Trans. 1, 2001, 1673–1695

View Article Online
vacuum for 6 h. The residue was dissolved in CH2Cl2 (500 cm3),
treated with DIPEA (316 mm3) and then left to stir under Ar
for eight days. The reaction mixture was concentrated under
reduced pressure and immediately purified by flash chrom-
atography on silica using CH2Cl2–MeOH (94:6) as the eluant to
give a white crystalline solid which was recrystallised from
acetone–diethyl ether (28 mg, 52%), mp >220 ЊC (decomp.)
(Found C, 55.8; H, 6.4; N, 11.3. C28H37N5O9ؒH2O requires C,
55.5; H, 6.5; N, 11.55%) (HRMS: found [M ϩ H]ϩ, 588.2685.
C28H38N5O9 requires 588.2683); νmax(Nujol)/cmϪ1 3360 (NH)
and 1719 (CO, esters and amides); δH[500 MHz; (C2H3)2SO]
1.79–1.90 [5 H, m, 1 H of β-CH2 (c, Glu), β-CH2 (t, Glu) and γ-
CH2 (t, Pro)], 1.92–2.07 [6 H, m, 1 H of β-CH2 (c, Glu), γ-CH2
(c, Pro), β-CH2 (t, Pro) and 1 H of β-CH2 (c, Pro)], 2.11–2.27
[6 H, m, 1 H of β-CH2 (c, Pro), 1 H of γ-CH2 (c, Glu), γ-CH2
(t, Pro) and 1 H of α-CH2 (c and t, α-Ala)], 2.34–2.61 [5 H, m,
1 H of γ-CH2 (c, Glu), 1 H of β-CH2 (c and t, β-Ala) and 1 H
of β-CH2 (c and t, Asp)], 2.68–2.82 [3 H, m, 1 H of β-CH2 (c
and t, Asp), 1 H of β-CH2 (t, Phe)], 2.90–3.03 [2 H, m, 1 H of
β-CH2 (c and t, Phe)], 3.08–3.25 [3 H, m, 1 H of β-CH2 (c, Phe)
and 1 H of α-CH2 (c and t, β-Ala)], 3.40–3.59 [6 H, m, δ-CH2
(c and t, Pro) and 1 H of β-CH2 (c and t, β-Ala)], 3.62, 3.64,
3.66, and 3.71 (12 H, s, 2 × CH3, c and t), 4.13–4.25 [3 H, m,
α-H (c, Phe), α-H (t, Glu) and α-H (c, Asp)], 4.28 [1 H, dd, J 8.3
and 2.7, α-H (c, Pro)], 4.38–4.44 [2 H, m, α-H (c, Glu) and α-H
(t, Pro)], 4.47 [1 H, sep, J 3.1, α-H, (t, Asp)], 4.56 [1 H, q, J 5.5,
α-H, (t, Phe)], 7.21–7.34 (10 H, m, c and t, Ph), 7.78 [1 H, t,
J 5.5, NH (c, β-Ala)], 7.88 [1 H, t, J 5.5, NH (t, β-Ala)], 8.11
[1 H, d, J 6.7, NH (c, Asp)], 8.20 [1 H, d, J 9.2, NH (t, Phe)],
8.21 [1 H, d, J 7.9, NH (t, Glu)], 8.30 [1 H, d, J 7.3, NH (c,
Phe)], 8.32 [1 H, d, J 7.9, NH (c, Glu)] and 8.38 [1 H, d, J 7.3,
NH (t, Asp)]; δC[125.8 MHz; (C2H3)2SO] 21.88 [c, γ-CH2 (Pro)],
23.92 [t, γ-CH2 (Pro)], 26.02 [c, β-CH2 (Glu)], 26.66 [t, β-CH2
(Glu)], 28.87 [t, γ-CH2 (Glu)], 29.31 [c, γ-CH2 (Glu)], 30.53 [t,
β-CH2 (Pro)], 31.46 [c, β-CH2 (Pro)], 34.49 [α-CH2 (β-Ala)],
34.78 [c, β-CH2 (Asp)], 34.93 [c, β-CH2 (Asp)], 35.57 [β-CH2
(β-Ala)], 37.77 [c, β-CH2 (Phe)], 39.18 [t, β-CH2 (Phe)], 46.31
[t, δ-CH2 (Pro)], 46.84 [c, δ-CH2 (Pro)], 49.4 [c, α-C (Asp)], 50.1
[t, α-C (Asp)], 50.8 [α-C (Glu)], 51.79, 51.9, 52.05 and 52.14
(c ϩ t, CH3), 53.71 [t, α-C (Phe)], 55.52 [c, α-C (Phe)], 59.65
[t, α-C (Pro)], 59.71 [c, α-C (Pro)], 126.12, 127.91, 128.03,
128.92 and 129.03 (Ar-CH), 137.82 and 137.86 (Ar-C quater-
nary) and 168.88, 170.13, 170.98, 171.08, 171.14, 171.3, 171.42,
171.96, 172.18 and 172.6 (CO, ester and amides); m/z (CI)
588 (8%, [M ϩ H]ϩ), 556 (25, [M Ϫ OCH3]ϩ) and 391 (100,
[M Ϫ C10H14NO3]ϩ).
6.21 [2 H, m, CH ᎐CH and NH], 7.25–7.82 (8 H, m, Ar-H) and
᎐
2
9.01 (1 H, br, OH); δC(75.4 MHz; C2HCl3) 36.54 (β-CH2), 47.20
(fluorenyl CH), 50.51 (α-C), 66.07 and 67.60 (2 × CH2O),
119.05 (CH ᎐CH), 120.510, 125.28, 127.26, 127.53, 127.90 and
᎐
2
128.41 (Ar-CH), 131.98 (CH ᎐CH), 141.44, 143.78 and 143.92
᎐
2
(Ar-C quaternary), 156.35 (CO, urethane), 170.96 (CO, ester)
and 175.46 (CO, acid); m/z (CI) 396 (12%, [M ϩ H]ϩ) and 179
(100, C14H11ϩ).
ꢀ-Allyl (2R)-N-(fluoren-9-ylmethoxycarbonyl)aspartyl-Wang
resin 45
A suspension of Wang resin (500 mg, 0.84 mmol gϪ1 OH substi-
tution) and β-allyl (2R)-N-(fluoren-9-ylmethoxycarbonyl)-
aspartate 44 (660 mg, 1.67 mmol) were stirred in dry DMF (2.5
cm3) under N2 for 15 min. Pyridine (100 mm3, 2.77 mmol) and
2,6-dichlorobenzoyl chloride (105 mm3, 1.67 mmol) were added
and the suspension was stirred for 20 h and then filtered and
washed successively with DMF (25 cm3), CH2Cl2 (25 cm3)
and methanol (25 cm3). The percentage loading was checked
and determined to be 70%.52 The remaining hydroxy groups of
the resin were benzoylated with benzoyl chloride and pyridine
in CH2Cl2 for 2 h, washed with DMF (25 cm3), CH2Cl2 (25 cm3)
and methanol (25 cm3), and dried under vacuum and checked
once again for percentage loading (772 mg, 70% loading).
ꢂ-Allyl (2R)-glutamate hydrochloride 46
To a stirred suspension of (2R)-glutamic acid (2.21 g, 15 mmol)
in dry allyl alcohol (70 cm3) under N2 was added dropwise
chlorotrimethylsilane (4.75 cm3, 47.5 mmol). The resulting
solution was stirred at RT for 18 h, after which diethyl ether
(200 cm3) was added at 0 ЊC to give a white precipitate which
was collected by filtration, washed with diethyl ether and dried
under vacuum (2.18 g, 65%), mp 131–132 ЊC [lit.,52 130–132 ЊC
(for the (2S)-isomer)], [α]D Ϫ22.1 (c 1.1 in MeOH) {lit.,52
[α]D ϩ22.5 (c 1 in MeOH) [for the (2S) isomer]}; νmax(CH2Cl2)/
2
cmϪ1 2863 br (OH) and 1729 (CO, ester); δH(300 MHz; H2O)
2.08–2.21 (2 H, sep, J 7.2, β-CH2), 2.55 (2 H, t, J 6.0, γ-CH2),
3.98 (1 H, t, J 6.9, α-H), 4.52 (2 H, d, J 5.7, CH2O), 5.21 (2 H,
dd, J 15.9, 1.5 CH ᎐CH) and 5.84 (1 H, m, CH ᎐CH); δ (75.4
᎐
᎐
2
2
C
2
MHz; H2O) 25.00 (β-CH2), 29.75 (γ-CH2), 52.31 (α-C), 66.28
(CH2O), 171.79 (CO, ester) and 174.23 (CO, acid); m/z (CI) 188
(84%, [M Ϫ Cl]ϩ) and 130 (100, C5H8NO3ϩ).
ꢂ-Allyl (2R)-N-(fluoren-9-ylmethoxycarbonyl)glutamate 47
To an ice-cold stirred suspension of γ-allyl (2R)-glutamate
hydrochloride 46 (943 mg, 4.22 mmol) in water (30 cm3) was
added potassium carbonate (945 mg, 6.75 mmol). A pre-stirred
solution of fluoren-9-ylmethoxycarbonyl chloride (1.21 g, 4.64
mmol) in dioxane (30 cm3) was added. The resulting solution
was warmed to room temperature and stirred for 4 hours, then
poured into water (20 cm3) and the dioxane was removed under
reduced pressure. The aqueous solution was washed with
diethyl ether (2 × 50 cm3) and acidified to pH 2 at 0 ЊC with
6 mol dmϪ3 HCl and then extracted with diethyl ether (3 × 50
cm3). The ethereal solutions were combined, dried (MgSO4)
and the solvent was removed under reduced pressure to give the
required compound as a white crystalline solid in quantitative
recovery, which was not purified further, mp 113–114 ЊC
(HRMS: found [M ϩ H]ϩ, 410.1595. C23H24NO6 requires
410.1603); [α]D ϩ30.4 (c 1.3 in MeOH); νmax(CH2Cl2)/cmϪ1 3364
(NH), 2965 (CH) and 1746 (CO, urethane); δH(300 MHz;
C2HCl3) 2.01–2.58 (4 H, m, γ-CH2 and β-CH2), 4.18–4.62 (6 H,
m, 2 × CH2O, fluorenyl CH and α-H), 5.21–5.58 (2 H, m,
CH ᎐CH), 5.58 [1 H, d, J 7.8, NH (urethane)], 5.82–5.98 (1 H,
ꢀ-Allyl (2R)-N-(fluoren-9-ylmethoxycarbonyl)aspartate 44
To an ice-cold stirred suspension of γ-allyl (2R)-aspartate
hydrochloride 12 (530 mg, 2.5 mmol) in water (20 cm3) was
added potassium carbonate (560 mg, 4 mmol). A pre-stirred
solution of fluoren-9-ylmethoxycarbonyl chloride (717 mg,
2.75 mmol) in dioxane (20 cm3) was added. The resulting solu-
tion was warmed to room temperature and stirred for 4 h, then
poured into water (15 cm3) and the dioxane was removed under
reduced pressure. The aqueous solution was washed with
diethyl ether (2 × 30 cm3) and acidified to pH 2 at 0 ЊC with
6 mol dmϪ3 HCl and then extracted with diethyl ether (3 × 30
cm3). The ethereal solutions were combined, dried (MgSO4)
and the solvent was removed under reduced pressure to give the
required compound as a white crystalline solid, which was not
purified further (930 mg, 94%), mp 110–111 ЊC (Found C, 66.3;
H, 5.4; N, 3.6. C22H21NO6 requires C, 66.8; H, 5.35; N, 3.55%)
(HRMS: found [M ϩ H]ϩ, 396.1450. C22H22NO6 requires
396.1447); [α]D ϩ3.04 (c 0.46 in MeOH); νmax(CH2Cl2)/cmϪ1
3363 (NH), 2956 (CH) and 1741 (CO, urethane); δH(300 MHz;
C2HCl3) 2.94 (1 H, dd, J 18 and 3.4, 1 H of β-CH2), 3.13 (1 H,
dd, J 18 and 3.45, 1 H of β-CH2), 4.20–4.79 (6 H, m, 2 × CH2O,
᎐
2
m, CH ᎐CH) and 7.23–7.79 (8 H, m, Ar-H); δ (75.4 MHz;
᎐
2
C
C2HCl3) 27.42 (β-CH2), 30.40 (γ-CH2), 47.24 (fluorenyl CH),
53.39 (α-C), 65.67 and 67.34 (2 × CH O), 118.68 (CH ᎐CH),
᎐
2
2
fluorenyl CH and α-H), 5.21–5.38 (2 H, m, CH ᎐CH), 5.82–
120.13, 125.23, 127.25 and 127.89 (Ar-CH), 132.03 (CH ᎐CH),
᎐
2
᎐
2
J. Chem. Soc., Perkin Trans. 1, 2001, 1673–1695
1689

View Article Online
141.46, 143.76 and 143.97 (Ar-C quaternary), 156.42 (CO,
urethane) and 172.5 and 175.66 (CO, ester and acid); m/z (CI)
424 (17%, [M ϩ 2 H ϩ Na]ϩ), 410 (64, [M ϩ H]ϩ), 181 (100,
C14H13ϩ) and 130 (48, C5H8NO3ϩ).
H, m, γ-CH2 and β-CH2 (Pro and Glu) and α-CH2 (β-Ala)],
2.82–3.14 [4 H, m, β-CH2 (Asp) and β-CH2 (Phe)], 3.32–3.47 [4
H, m, δ-CH2 (Pro) and β-CH2 (β-Ala)], 3.77 (3 H, s, CH3), 4.16–
4.88 [9 H, m, 4 × α-H, 2 × CH2O and fluorenyl CH], 4.97 [1 H,
br, NH (urethane)], 5.14–5.36 (2 H, m, CH ᎐CH), 5.76–5.94
᎐
2
(1 H, m, CH ᎐CH), 6.18 (2 H, br, NH) and 7.00–7.23 (15 H, m,
᎐
2
ꢁ-Methyl ꢂ-allyl (2R)-N-(fluoren-9-ylmethoxycarbonyl)-
glutamate 48
Ar-H, 2 × NH); δC(75.4 MHz; C2HCl3) 24.53 [γ-CH2 (Pro)],
26.86 [β-CH2 (Glu)], 28.84 [γ-CH2 (Glu)], 29.77 [β-CH2 (Pro)],
34.97 [α-CH2 (β-Ala)], 35.36 [β-CH2 (Asp)], 36.34 [β-CH2
(β-Ala)], 38.71 [β-CH2 (Phe)], 46.90 [δ-CH2 (Pro)], 47.42
[α-C (Asp)], 51.55 [α-C (Glu)], 52.51 [CH3 (Asp)], 56.02 [α-C
(Phe)], 60.00 [α-C (Pro)], 65.56 and 67.31 (2 × CH2O), 118.45
This compound was prepared in a manner identical to diester
14, using γ-allyl ester 47 (1.04 g, 2.54 mmol) to afford the
required compound as a white crystalline solid in quantitative
recovery, mp 84–85 ЊC (Found C, 67.9; H, 5.8; N, 3.5.
C24H25NO6 requires: C, 68.1; H, 5.95; N, 3.3%) (HRMS:
found [M ϩ H]ϩ, 424.1766. C24H26NO6 requires 424.1760); [α]D
ϩ22.14 (c 0.56 in MeOH); νmax(CH2Cl2)/cmϪ1 3364 (NH), 2965
(CH) and 1751 (CO, urethane); δH(300 MHz; C2HCl3) 1.95–
2.53 (4 H, m, γ-CH2 and β-CH2), 3.75 (3 H, s, CH3), 4.09–4.61
(6 H, m, 2 × CH2O, fluorenyl CH and α-H), 5.21–5.38 (2 H, m,
(CH ᎐CH), 119.96, 125.14, 126.87, 127.1, 127.75, 128.49,
᎐
2
128.64, 129.41, 130.13, 131.83 and 133.37 (Ar-CH), 136.67
(CH ᎐CH), 141.29 and 141.74 (Ar-C quaternary), 156.55 (CO,
᎐
2
urethane) and 170.61, 171.94 and 172.7 (CO esters and amides);
m/z (ES) 892 (20%, [M ϩ K]ϩ), 876 (42, [M ϩ Na]ϩ), 855 (28,
[M ϩ 2H]ϩ), 718 (49) and 102 (100).
CH ᎐CH), 5.56 [1 H, d, J 8.1, NH (urethane)], 5.84–5.97 (1 H,
᎐
2
m, CH ᎐CH) and 7.26–7.77 (8 H, m, Ar-H); δ (75.4 MHz;
᎐
2
C
C2HCl3) 27.04 (β-CH2), 30.29 (γ-CH2), 47.29 (fluorenyl CH),
52.70 (CH3), 53.48 (α-C), 65.54 and 67.21 (2 × CH2O), 118.61
(2S)-Phenylalanyl-ꢀ-alanyl-[ꢁ-methyl (2R)-glutamyl]-ꢂ-(2S)-
prolyl-[(2R)-aspartate] ester 55
(CH ᎐CH), 120.14, 125.22, 127.23 and 127.87 (Ar-CH), 132.14
᎐
2
To a stirred suspension of resin 53 (495 mg, 0.3 mmol) in
DMSO–THF–0.5 mol dmϪ3 HCl–NMM (2:2:1:0.1), (25 cm3)
under Ar was added tetrakis(triphenylphosphine)Pd(0) (104
mg, 9 × 10Ϫ5 mol). Slow stirring was continued for 3 h, after
which time the resin was collected by filtration and washed with
THF (25 cm3), CH2Cl2 (25 cm3), and methanol (25 cm3). The
resin was then treated with 20% piperidine–DMF (10 cm3) for
30 min (monitoring deprotection using the Ninhydrin Test37),
after which time the resin was collected by filtration and washed
with DMF, CH2Cl2 and methanol successively. The washed
resin was then treated with cleavage mixture TFA–TES–H2O–
CH2Cl2 (40:2:5:53) for 1 h, and the resin was removed by fil-
tration and the solvents concentrated under reduced pressure
(4–5 cm3). The required peptide was then precipitated by the
addition of excess diethyl ether to give a white solid in quanti-
tative recovery (177 mg); νmax(CH2Cl2)/cmϪ1 3200 br (NH and
OH) and 1729 br (CO, esters and amides); δH(300 MHz; 2H2O)
1.44–2.39 [10 H, m, γ-CH2, β-CH2 (Pro and Glu) and α-CH2
(β-Ala)], 2.64–3.04 [4 H, m, β-CH2 (Asp) and β-CH2 (Phe)],
3.17–3.40 [4 H, m, δ-CH2 (Pro) and β-CH2 (β-Ala)], 3.57 (t, 3 H,
s, CH3), 3.59 (c, 3 H, s, CH3), 4.00 (1 H, t, J 7.4 α-H), 4.08–4.38
(2 H, m, 2 × α-H), 4.58–4.61 (1 H, m, α-H) and 7.02–7.38 (5 H,
(CH ᎐CH), 141.46, 143.85 and 144.03 (Ar-C quaternary),
᎐
2
156.13 (CO, urethane) and 172.50 (CO, ester); m/z (CI) 424
(45%, [M ϩ H]ϩ), 179 (94, C14H11ϩ), 144 (78, C6H10NO3ϩ) and
57 (100, C4H9ϩ).
ꢁ-Methyl (2R)-N-(fluoren-9-ylmethoxycarbonyl)glutamate ester
49
To a stirred solution of diester 48 (423 mg, 1 mmol) and phenyl-
silane (247 mm3, 2 mmol) in dry CH2Cl2 (30 cm3) under Ar was
added tetrakis(triphenylphosphine)Pd(0) (23 mg, 0.02 mmol).
The reaction mixture was allowed to stir at room temperature
for 2 h (when no starting material was detected by TLC) and
was then concentrated under reduced pressure. The residue was
purified by flash chromatography on silica using ethyl acetate–
hexane (60:40) as the eluent to give an amorphous white solid
(310 mg, 81%), mp 130–131 ЊC (Found C, 64.6; H, 5.45; N, 3.7.
C21H21NO6ؒ0.5 H2O requires C, 64.3; H, 5.65; N, 3.55%)
(HRMS: found [M ϩ H]ϩ, 384.1443. C21H22NO6 requires
384.1447); [α]D ϩ19.5 (c 0.63 in MeOH); νmax(CH2Cl2)/cmϪ1
3355 (NH), 2965 (CH) and 1732 (CO, urethane); δH(300 MHz;
C2HCl3) 1.92–2.57 (4 H, m, γ-CH2 and β-CH2), 3.75 (3 H,
s, CH3), 4.18–4.56 (4 H, m, CH2O, fluorenyl CH and α-H), 5.45
[1 H, d, J 7.2, NH (urethane)] and 7.25–7.77 (8 H, m, Ar-H);
δC(75.4 MHz; C2HCl3) 27.57 (β-CH2), 29.89 (γ-CH2), 47.28
(fluorenyl CH), 52.74 (CH3), 53.29 (α-C), 67.24 (CH2O), 120.14,
125.19, 127.23 and 127.88 (Ar-CH), 141.47 and 143.81 (Ar-C
quaternary), 156.15 (CO, urethane) and 177.54 (CO, acid); m/z
(CI) 384 (7%, [M ϩ H]ϩ), 179 (31, C14H11ϩ) and 144 (100,
C6H10NO3ϩ).
2
m, Ar-H); δC(75.4 MHz; H2O) 22.13 [c, γ-CH2 (Pro)], 24.05
[t, γ-CH2 (Pro)], 25.71 [β-CH2 (Glu)], 29.66 [γ-CH2 (Glu)], 29.77
[t, β-CH2 (Pro)], 31.74 [t, β-CH2 (Pro)], 34.23 [α-CH2 (β-Ala)],
35.26 [β-CH2 (Asp)], 35.45 [β-CH2 (β-Ala)], 36.88 [β-CH2
(Phe)], 47.93 [c, δ-CH2 (Pro)], 47.86 [t, δ-CH2 (Pro)], 48.98 [t,
α-C (Asp)], 49.19 [c, α-C (Asp)], 52.15 [α-C (Glu)], 52.94 [CH3
(Asp)], 54.44 [α-C (Phe)], 60.25 [t, α-C (Pro)], 60.8 [c,
α-C (Pro)], 128.06, 129.23, 129.44, 130.63, 133.75 and 133.9
(Ar-CH), 135.89 (Ar-C quaternary) and 173.43, 173.64, 173.72,
173.89, 173.97, 174.27 and 174.4 (CO esters and amides);
m/z (ES) 620 (5%, [M ϩ K]ϩ), 614 (10, [M ϩ Na]ϩ) and 592
(100, [M ϩ H]ϩ).
ꢀ-Allyl (2S)-N-(fluoren-9-ylmethoxycarbonyl)phenylalanyl-ꢀ-
alanyl-[ꢁ-methyl (2R)-glutamyl]-ꢂ-(2S)-prolyl-[(2R)-aspartate]
diester 53
β-Allyl (2S)-N-(fluoren-9-ylmethoxycarbonyl)phenylalanyl-β-
alanyl-[(2R)-α-methyl glutamyl]-γ-(2S)-prolyl-[(2R)-aspartate-
Wang resin] diester 53 (2.1 g, 426 mg peptidyl content, 0.05
mmol) was synthesised on the peptide synthesiser and treated
with cleavage mixture TFA–TES–H2O–CH2Cl2 (40:2:5:53) at
room temperature for 1 h. The resin was collected by filtration
and the solvents were concentrated under reduced pressure
(4–5 cm3). The peptide was then precipitated with excess diethyl
ether to afford a white solid in quantitative recovery (426 mg),
mp 115–116 ЊC (Found C, 59.6; H, 6.6; N, 7.9. C45H51N5O12ؒ
3 H2O requires C, 59.5; H, 6.35; N, 7.7%); [α]D Ϫ13.0 (c 1.3 in
MeOH); νmax(CH2Cl2)/cmϪ1 3276 (NH), 2977 (CH) and 1727 br
(CO, esters and amides); δH(300 MHz; C2HCl3) 1.76–2.54 [10
Cyclo[-ꢀ-Ala-(2S)-Glu-ꢁ-OMe-ꢂ-(2R)-Pro-(2R)-Asp-ꢀ-(2S)-
Phe-] 57
To a stirred suspension of resin 55 (100 mg, 0.06 mmol)
in DMF (5 cm3) and DIPEA (102 mm3, 0.6 mmol) under Ar
was added PyBOP (157 mg, 0.3 mmol) and HOBt (41 mg,
0.3 mmol). The resin was stirred slowly for 7 days (continuous
monitoring of progress using the Ninhydrin Test), and then
removed by filtration and washed successively with THF (25
cm3), CH2Cl2 (25 cm3) and methanol (25 cm3). The resulting
resin 56 was treated with cleavage mixture TFA–TES–H2O–
CH2Cl2 (40:2:5:53) for 1 h and then removed by filtration, and
the filtrate concentrated under reduced pressure (4–5 cm3). The
1690
J. Chem. Soc., Perkin Trans. 1, 2001, 1673–1695

View Article Online
peptide was precipitated by the addition of excess diethyl ether
to give a white solid which was purified by reverse phase
HPLC (10 mg, 30%), mp >180 ЊC (decomp.) (HRMS: found
[M ϩ H Ϫ H2O]ϩ, 556.2420. C27H34N5O8 requires 556.2407);
νmax(CH2Cl2)/cmϪ1 3302 (NH) and 1735 and 1670 (CO, esters
and amides); δH[500 MHz; (C2H3)2SO] 1.72–2.02 [11 H, m, 1 H
of β-CH2 (c and t, Pro), 1 H of β-CH2 (c, Pro), γ-CH2 (c and t,
Pro) and β-CH2 (c and t, Glu)], 2.12–2.55 [15 H, m, γ-CH2 (c
and t, Glu), 1 H of γ-CH2 (c and t, Glu), 1 H of β-CH2 (t, Pro),
1 H of β-CH2 (c and t, Asp) and α-CH2 (c and t, β-Ala)], 2.66–
3.20 [8 H, m, 1 H of β-CH2 (c and t, β-Ala), β-CH2 (c and t,
Phe) and 1 H of β-CH2 (c and t, Asp)], 3.40–3.49 [6 H, m, 1 H
of β-CH2 (c and t, β-Ala) and δ-CH2 (c and t, Pro)], 3.55, 3.67,
3.78, and 3.79 [12 H, s, 2 × CH3, c and t], 4.08–4.48 [8 H, m,
α-H (c and t, Phe), α-H (c and t, Glu), α-H (c and t, Asp) and α-
H (c and t, Pro)], 7.21–7.39 (10 H, m, Ar-H, c and t), 7.75–7.97
[3 H, m, NH (c and t, β-Ala), NH (c, Asp)], 8.14–8.29 [5 H, m,
NH (t, Asp), NH (c and t, Phe) and NH (c and t, Glu)]; δC(75.4
MHz; C2H3O2H) 23.89 [γ-CH2 (Pro)], 26.18 [β-CH2 (Glu)],
29.25 [γ-CH2 (Glu)], 31.61 [β-CH2 (Pro)], 34.39 [α-CH2 (β-
Ala)], 35.11 [β-CH2 (Asp)], 37.03 [β-CH2 (β-Ala)], 38.76 [β-CH2
(Phe)], 46.68 [δ-CH2 (Pro)], 46.91 [α-C (Asp)], 51.53 [α-C
(Glu)], 51.73 (CH3), 56.53 [α-C (Phe)], 59.5 [α-C (Pro)], 126.11,
127.93, 128.45, 128.96 and 129.34 (Ar-CH), 135.25 (Ar-C
quaternary) and 169.12, 170.28, 170.71, 171.18 and 172.28 (CO
ester, amides and acid); m/z (ES) 596 (4%, [M ϩ Na]ϩ), 574 (16,
[M ϩ H]ϩ) and 212 (100).
65.73 and 67.00 (2 × CH O), 118.60 (CH ᎐CH), 120.05, 125.19,
᎐
2 2
126.90, 127.15, 127.79, 128.55, 129.48 and 131.86 (Ar-CH),
136.97 (CH ᎐CH), 141.33 and 143.93 (Ar-C quaternary),
᎐
2
156.12 (CO, urethane) and 170.41, 171.71, 171.87 and 172.66
(CO, esters and amides); m/z (ES) 891 (12%, [M ϩ H ϩ Na]ϩ),
869 (50, [M ϩ 2H]ϩ), 646 (28, [M ϩ 2H Ϫ C15H11O2]ϩ) and 111
(100).
(2S)-N-(Fluoren-9-ylmethoxycarbonyl)phenylalanyl-ꢀ-alanyl-
[ꢁ-methyl (2R)-glutamyl]-ꢂ-(2S)-prolyl-[ꢁ-methyl (2R)-aspartic
acid]
This compound was prepared through allyl group deprotection
as described for ester 22, using the triester 60 (220 mg,
0.25 mmol). The required compound was obtained as a white
crystalline solid (150 mg, 72%), mp >124 ЊC (decomp.); [α]D
Ϫ13.5 (c 1 in MeOH); νmax(CH2Cl2)/cmϪ1 3300 (NH), 3229 br
(OH), and 1801 and 1721 (CO, esters and amides); δH(300
MHz; C2H3O2H) 1.87–2.45 [8 H, m, γ-CH2, β-CH2 (Pro and
Glu) and α-CH2 (β-Ala)], 2.74–3.14 [4 H, m, β-CH2 (Asp) and
β-CH2 (Phe)], 3.28–3.52 [4 H, m, δ-CH2 (Pro) and β-CH2 (β-
Ala)], 3.58, 3.59, 3.65 and 3.66 (c and t, 6 H, 4 × s, CH3), 4.09–
4.85 [7 H, m, 4 × α-H, CH2O, fluorenyl CH], and 7.18–7.77
(13 H, m, Ar-H); δC(75.4 MHz; C2H3O2H) 24.12 [γ-CH2 (Pro)],
26.53 [β-CH2 (Glu)], 29.5 [γ-CH2 (Glu)], 31.85 [β-CH2 (Pro)],
34.84 [α-CH2 (β-Ala)], 35.53 [β-CH2 (Asp)], 37.63 [β-CH2
(β-Ala)], 37.92 [β-CH2 (Phe)], 47.23 [δ-CH2 (Pro)], 47.67 [α-C
(Asp)], 49.60 [α-C (Glu)], 51.97 [CH3 (Asp)], 52.05 [CH3 (Glu)],
56.74 [α-C (Phe)], 60.41 [α-C (Pro)], 66.72 (CH2O), 119.66,
124.92, 125.07, 126.5, 126.92, 127.54, 128.21 and 129.13
(Ar-CH), 137.39, 141.25 and 143.90 (Ar-C quaternary), 156.87
(CO, urethane) and 171.98, 172.32, 172.66, 172.94, 173.12 and
176.14 (CO, esters, amides and acid).
Cyclo[-ꢀ-Ala-(2R)-Glu-ꢂ-(2S)-Pro-(2R)-Asp-ꢀ-(2S)-Phe-]
To a stirred solution of the cyclic pentapeptide 57 (0.03 mmol)
in methanol (2 cm3) and water (2 cm3) was added NaOH (0.065
mmol). The reaction was allowed to stir at room temperature
for 2 h, after which time the methanol was removed under
reduced pressure. The aqueous layer was acidified using tri-
fluoroacetic acid and the cyclic peptide extracted using CH2Cl2
(2 cm3). The organic phase was dried (MgSO4) and the solvent
removed under reduced pressure. m/z (ES) 582 (3%, [M ϩ
Na]ϩ), 561 (7, [M ϩ 2H]ϩ); >95% HPLC purity on a Poros 10
R2/H reverse phase column [using isocratic reverse-phased
conditions eluting with acetonitrile–water (24:76) as eluent at a
flow rate of 5 cm3 minϪ1. The eluent was monitored by UV
spectroscopy at 220 nm. The fractions corresponding to peak
1 (retention time 1.7 min) were collected and pooled together,
and the solvent removed under reduced pressure and by
lyophilisation].
(2S)-Phenylalanyl-ꢀ-alanyl-[ꢁ-methyl (2R)-glutamyl]-ꢂ-(2S)-
prolyl-[ꢁ-methyl (2R)-aspartate] diester 61
To a stirred solution of the (2S)-N-(fluoren-9-ylmethoxy-
carbonyl)phenylalanyl-β-alanyl-[α-methyl
(2R)-glutamyl]-γ-
(2S)-prolyl-[α-methyl (2R)-aspartic acid] (100 mg, 0.12 mmol)
in dry DMF (3 cm3) was added piperidine (17 mm3, 0.18 mmol).
The reaction mixture was allowed to stir at room temperature
for 45 min, and then concentrated under reduced pressure
and redissolved in water (10 cm3) and acidified with TFA. The
resulting solution was washed with CH2Cl2 and the acidic layer
concentrated under reduced pressure to yield the required com-
pound as a white hygroscopic solid (60 mg, 83%), mp 67–69 ЊC;
[α]D ϩ14.5 (c 1.1 in MeOH); νmax(CH2Cl2)/cmϪ1 3286 (NH),
2955 br (OH), and 1746 and 1670 (CO, esters and amides);
δH(300 MHz; 2H2O) 1.45–2.18 [10 H, m, γ-CH2, β-CH2 (Pro and
Glu) and α-CH2 (β-Ala)], 2.64–3.04 [4 H, m, β-CH2 (Asp)
and β-CH2 (Phe)], 3.15–3.36 [4 H, m, δ-CH2 (Pro) and β-CH2
(β-Ala)], 3.57 and 3.60 (6 H, 2 × s, 2 × CH3), 3.97–4.59 [4 H,
m, 4 × α-H] and 7.07–7.38 (5 H, m, Ar-H); δC(75.4 MHz;
C2H3O2H) 24.07 [γ-CH2 (Pro)], 26.20 [β-CH2 (Glu)], 29.48
[γ-CH2 (Glu)], 31.41 [β-CH2 (Pro)], 34.88 [α-CH2 (β-Ala)],
35.34 [β-CH2 (Asp)], 36.42 [β-CH2 (β-Ala)], 40.15 [β-CH2
(Phe)], 46.84 [δ-CH2 (Pro)], 46.90 [α-C (Asp)], 49.86 [α-C
(Glu)], 51.58 and 52.40 (2 × CH3), 56.06 [α-C (Phe)], 60.53
[α-C (Pro)], 125.11, 126.73, 126.92, 128.42, 128.59 and 129.19
(Ar-CH), 136.02 (Ar-C quaternary) and 170.55 and 172.39
(CO ester, amides and acid); m/z (ESϩ) 606 (100%, [M ϩ H]ϩ),
590 (18, [MϪ CH3]ϩ) and 324 (9, C15H20N2O6ϩ).
ꢀ-Allyl (2S)-N-(fluoren-9-ylmethoxycarbonyl)phenylalanyl-ꢀ-
alanyl-[ꢁ-methyl (2R)-glutamyl]-ꢂ-(2S)-prolyl-[ꢁ-methyl (2R)-
aspartate] triester 60
The triester was prepared in a manner identical to diester 14,
using diester 53 (853 mg, 1 mmol) to give the required com-
pound as a white crystalline solid in quantitative recovery.
Mp 120–121 ЊC (Found C, 61.9; H, 6.3; N, 7.7. C46H53N5O12ؒ
1.5 H2O requires C, 61.7; H, 6.3; N, 7.8%); [α]D Ϫ17.8 (c 0.25
in MeOH); νmax(CH2Cl2)/cmϪ1 3301 (NH), 2953 (CH), and 1739
and 1652 (CO, esters and amides); δH(300 MHz; C2HCl3) 1.82–
2.52 [10 H, m, γ-CH2, β-CH2 (Pro and Glu) and α-CH2 (β-Ala)],
2.79–3.19 [4 H, m, β-CH2 (Asp) and β-CH2 (Phe)], 3.25–3.80
[4 H, m, δ-CH2 (Pro) and β-CH2 (β-Ala)], 3.51, 3.60, 3.68 and
3.72 (c and t, 6 H, 4 × s, CH3), 4.08–4.97 [9 H, m, 4 × α-H,
2 × CH2O and fluorenyl CH], 5.09–5.37 (2 H, m, CH2=CH),
5.68 [1 H, d, J 7.9, NH, (urethane)], 5.79–5.86 (1 H, m,
CH2=CH), 6.90 (1 H, d, J 7.7, NH), 7.03 (1 H, br, NH) and
7.18–7.76 (15 H, m, Ar-H, 2 × NH); δC(75.4 MHz; C2HCl3)
24.63 [γ-CH2 (Pro)], 27.07 [β-CH2 (Glu)], 29.14 [γ-CH2 (Glu)],
30.36 [β-CH2 (Pro)], 35.28 [α-CH2 (α-Ala)], 35.73 [β-CH2
(Asp)], 36.33 [β-CH2 (β-Ala)], 39.07 [β-CH2 (Phe)], 47.14
[δ-CH2 (Pro)], 47.5 [α-C (Asp)], 51.90 [α-C (Glu)], 52.59 [CH3
(Asp)], 52.86 [CH3 (Glu)], 56.10 [α-C (Phe)], 60.09 [α-C (Pro)],
Cyclo[-ꢀ-Ala-(2R)-Glu-ꢁ-OMe-ꢂ-(2S)-Pro-(2R)-Asp-ꢁ-OMe-ꢀ-
(2S)-Phe-] 42 (R ؍ Me)
To a stirred solution of the pentapeptide 61 (60 mg, 9.92 × 10Ϫ5
mol) in DMF (80 cm3) was added PyBOP (28.4 mg, 0.11 mmol)
and DIPEA (102 mm3, 0.99 mmol). The resulting solution was
stirred at room temperature for 7 days and then concentrated
J. Chem. Soc., Perkin Trans. 1, 2001, 1673–1695
1691

View Article Online
under reduced pressure and immediately purified by flash
chromatography on silica using CH2Cl2–MeOH (94:6) as the
eluent to give a white solid which contained PyBOP as a con-
taminant. Further purification by preparative HPLC on a Poros
10 R2 reverse-phase column [using isocratic reverse-phased
conditions, eluting with acetonitrile–water (18:82) as eluent at a
flow rate of 2 cm3 minϪ1, with the detector set at 220 nm] gave
the required compound which possessed identical analytical
data to the compound prepared previously from the penta-
fluorophenyl ester (see compound 42 above).
conditions, eluting with acetonitrile–water (35:65) at a flow rate
of 4.5 cm3 minϪ1. The eluent was monitored by UV spectroscopy
at 220 nm. The fractions corresponding to peak 1 (retention
time 2.95 min) were collected and pooled together, and the
solvent removed under reduced pressure and by lyophilisation]
(36 mg, 17%), mp > 167 ЊC (decomp.), νmax(CH2Cl2)/cmϪ1 3301
(NH), 2939 (CH), and 1732 and 1672 (CO, esters and amides)
(HRMS: found [M ϩ Na]ϩ, 797.3479. C40H50N6O10Na requires
797.3486); δH[300 MHz; (C2H3)2SO, mixture of rotamers] 1.40–
1.73 [8 H, m, β-CH2 (Glu), γ-CH2 (Pro) and 2 × CH2-CH2NH],
1.84–2.05 [3 H, br, 1 H of γ-CH2 (Glu) and β-CH2 (Pro)], 2.10–
2.57 [6 H, m, 1 H of γ-CH2 (Glu), 2 × CH2-CH2NH and 1 H of
β-CH2 (Asp)], 2.68–3.21 [10 H, m, 1 H of β-CH2 (Asp),
CHCH2Ph, CHCH2Ph, δ-CH2 (Pro), β-CH2 (Asp) and β-CH2
(Phe)], 3.63 and 3.72 (3 H, 2 × s, OCH3), 4.07–4.61 (5 H, m, α-
CH of Glu, Pro, Phe and 2 × Asp), 7.21–7.40 (10 H, m, Ar-H),
7.78–8.36 [3 H, m, NH (Phe) and 2 × NH (Asp)] and 8.58–8.62
[1 H, m, NH (Glu)]; δC[75.4 MHz; (C2H3)2SO] 23.47 [γ-CH2
(Pro)], 26.09 [β-CH2 (Glu)], 29.28 [β-CH2 (Pro)], 30.53 [γ-CH2
(Glu)], 31.42 (2 × CH2-CH2NH), 35.16 [β-CH2 (Asp)], 36.24 [β-
CH2 (Asp)], 37.97 (CHCH2Ph), 38.15 [β-CH2 (Phe)], 42.86
(2 × CH2-CH2NH), 46.31 (CH2Ph), 46.79 [δ-CH2 (Pro)], 46.91
[α-CH (Asp)], 47.23 [α-CH (Asp)], 51.03 [α-CH (Glu)], 52.05
(OCH3), 56.71 [α-CH (Phe)], 59.62 [α-CH (Pro)], 126.11,
126.68, 127.19, 127.53, 127.93, 128.31, 128.85, 128.96, 129.34
and 129.58 (Ar-CH), 135.38 and 136.96 (Ar-C quaternary) and
169.93, 170.27, 170.88, 171.34, 171.76, 172.41 and 173.22 (CO
esters, amides and acid); m/z (ES) 797 (100%, [M ϩ Na]ϩ) and
775 (32, [M ϩ H]ϩ).
N-(Fluoren-9-ylmethoxycarbonyl)-(2S)-phenylalanyl-[ꢁ-4-
benzylpiperidinyl-(2R)-aspartyl]-[ꢁ-methyl (2R)-glutamyl]-ꢂ-
(2S)-prolyl-ꢀ-allyl-(2R)-aspartyl-Wang resin 62; and N-(fluoren-
9-ylmethoxycarbonyl)-(2S)-phenylalanyl-[ꢁ-4-benzylpiperidinyl-
(2R)-aspartyl]-[ꢁ-methyl (2S)-glutamyl]-ꢂ-(2S)-prolyl-ꢀ-allyl-
(2R)-aspartic acid 63
N-(Fluoren-9-ylmethoxycarbonyl)-(2S)-phenylalanyl-[α-4-
benzylpiperidinyl-(2R)-aspartyl]-[α-methyl (2R)-glutamyl]-γ-
(2S)-prolyl-β-allyl-(2R)-aspartyl-Wang resin 62 was syn-
thesised on the peptide synthesiser [using Fmoc chemistry as
previously described (see general procedure)] starting from
β-allyl-(2R)-N-(Fmoc)-aspartyl-Wang resin 45 (512 mg, 0.3
mmol) and incorporating the isoasparagine derivative 68f in
quantitative recovery (828 mg). For characterisation, a sample
of the fully protected resin-bound pentapeptide 62 (50 mg,
18 µmol) was treated with cleavage mixture TFA–TES–H2O–
CH2Cl2 (40:2:5:53) at room temperature for 1 h. The polymer
support was collected by filtration and the solvents were con-
centrated under reduced pressure (4–5 cm3). The peptide was
then precipitated with excess diethyl ether to afford the required
pentapeptide 63 as a white solid in quantitative recovery, which
did not require further purification, mp 134–136 ЊC (HRMS:
found [M ϩ H ϩ Na]ϩ, 1077.4556. C58H66N6O13Na requires
1077.4586); [α]Dϩ30 (c 1 in MeOH); νmax(CH2Cl2)/cmϪ1 3302
(NH), 2952 (CH), and 1739 (CO, esters and amides); δH(300
MHz; C2HCl3) 1.18–1.43 (4 H, m, 2 × CH2-CH2NH), 1.70
(4 H, br, CH2-CH2NH), 2.46–3.04 [5 H, m, CHCH2Ph,
CHCH2Ph and β-CH2 (Asp)], 1.81–2.58 [10 H, m, γ-CH2,
β-CH2 (Pro and Glu) and α-CH2 (β-Ala)], 2.84–3.37 [6 H, m,
2 × β-CH2 (Asp) and β-CH2 (Phe)], 3.42–3.71 [4 H, m, δ-CH2
(Pro) and β-CH2 (β-Ala)], 3.72 (c, 3 H, s, CH3), 3.8 (t, 3 H, s,
CH3), 4.09–5.00 [10 H, m, 5 × α-H, 2 × CH2O and fluorenyl
Cyclo{-(2S)-Phe-[(2R)-ꢁ-4-benzylpiperidinyl-Asp]-(2R)-Glu-ꢂ-
(2S)-Pro-ꢀ-(2R)-Asp-} 65
This compound was prepared in a manner identical to that
described for cyclo[-β-Ala-(2R)-Glu-γ-(2S)-Pro-(2R)-Asp-β-
(2S)-Phe-] using cyclo{-(2S)-Phe-[(2R)-α-4-benzylpiperidinyl-
Asp]-(2R)-α-OMe-Glu-γ-(2S)-Pro-β-(2R)-Asp-}
64.
m/z
(MALDITOF) 784 (8%, [M ϩ H ϩ Na]ϩ), 762 (3, [M ϩ 2H]ϩ);
>95% HPLC purity on a C-18 reverse phase column [using
isocratic reverse-phased conditions eluting with acetonitrile–
water (51:49) at a flow rate of 1 cm3 minϪ1. The eluent was
monitored by UV spectroscopy at 220 nm. The fractions corre-
sponding to peak 1 (retention time 8.45 min) were collected
and pooled together, and the solvent removed under reduced
pressure and by lyophilisation].
CH], 5.01–5.37 (4 H, m, CH ᎐CH and CH Ph), 5.65 [t, 1 H, d,
᎐
2
2
J 8.8, NH (urethane)], 5.75 [c, 1 H, d, J 8.4, NH (urethane)],
5.83–5.94 (1 H, m, CH ᎐CH), 6.31 (c, 1 H, d, J 7.9, NH), 6.39
᎐
2
ꢀ-Allyl (2R)-N-(fluoren-9-ylmethoxycarbonyl)aspartic
ꢁ-benzylamide 66b
(t, 1 H, d, J 7.8, NH) and 7.11–7.77 (21 H, m, 2 × NH and Ar-
H); δC(75.4 MHz; C2HCl3) 24.51 [γ-CH2 (Pro)], 25.53 [β-CH2
(Glu)], 28.71 [γ-CH2 (Glu)], 29.66 [β-CH2 (Pro)], 35.34 [α-CH2
(β-Ala)], 35.43 and 35.67 [β-CH2 (Asp)], 36.67 [β-CH2 (β-Ala)],
39.28 [β-CH2 (Phe)], 43.48 [δ-CH2 (Pro)], 46.90 and 47.17 [α-C
(Asp)], 51.04 [α-C (Glu)], 52.81 [CH3 (Asp)], 55.69 [α-C (Phe)],
60.68 [α-C (Pro)], 65.54, 66.95 and 67.75 (3 × CH2O), 118.46
This compound was prepared in a manner identical to that
described for pentapeptide 23, using the ester 44 (790 mg,
2 mmol) and benzylamine (218 mm3, 2 mmol) to give the
required compound as a white crystalline solid (715 mg,
74%), mp 141–142 ЊC (Found C, 71.3; H, 5.9; N, 6.0.
C29H28N2O5ؒ0.25H2O requires C, 71.2; H, 5.8; N, 5.75%)
(HRMS: found [M ϩ 2H]ϩ, 486.2169. C29H30N2O5 requires
486.2155); [α]D ϩ13.84 (c 0.43 in MeOH); νmax(CH2Cl2)/cmϪ1
3296 (NH) and 1756 and 1742 (CO, esters and amide); δH(300
MHz; C2HCl3) 1.91 (2 H, s, PhCH2), 2.59 (1 H, dd, J 10.4 and
6.4, 1 H of β-CH2), 2.72 (1 H, dd, J 10.4 and 6.2, 1 H of β-CH2),
4.16–4.78 (4 H, m, CH2O, fluorenyl CH and α-H), 5.21–5.38
(CH ᎐CH), 120.13, 123.71, 125.22, 127.05, 127.26, 127.57,
᎐
2
127.93, 128.36, 128.57, 128.88, 129.62 and 132.12 (Ar-CH),
136.44 (CH ᎐CH), 135.22, 136.43, 138.24, 141.41, 141.74 and
᎐
2
142.24 (Ar-C quaternary), 156.65 (CO, urethane) and 170.45,
170.95, 171.38, 171.84 and 173.33 (CO esters, amides and acid);
m/z (ES) 1077 (1%, [M ϩ Na]ϩ), 211 (100), 195 (57, [C14H11O]ϩ)
and 157 (78, [C7H9O4]ϩ).
(2 H, m, CH ᎐CH), 5.82–5.91 [2 H, m, CH ᎐CH, NH
᎐
᎐
2
2
(urethane)], 6.81 (1 H, br, NH) and 7.22–7.81 (13 H, m, Ar-H);
δC(75.4 MHz; C2HCl3) 36.21 (β-CH2), 43.71 (PhCH2), 47.23
(fluorenyl CH), 51.19 (α-C), 65.91 and 67.28 (2 × CH2O),
Cyclo{-(2S)-Phe-[(2R)-ꢁ-4-benzylpiperidinyl-Asp]-(2R)-ꢁ-
OMe-Glu-ꢂ-(2S)-Pro-ꢀ-(2R)-Asp-} 64
This compound was prepared in a manner identical to that for
compound 57 using the resin-bound precursor 62 (750 mg,
0.27 mmol) which was selectively deprotected and cyclised to
give the required compound after purification using prepar-
ative HPLC on a C-18 column [using isocratic reverse-phased
118.97 (CH ᎐CH), 120.18, 125.08, 127.22, 127.65, 127.93 and
᎐
2
128.83 (Ar-CH), 131.66 (CH ᎐CH), 141.46 and 143.74 (Ar-C
᎐
2
quaternary), 152.89 (CO, urethane), 168.02 (CO, amide) and
170.25 (CO, ester); m/z (CI) 486 (92%, [M ϩ 2H]ϩ), 263 (100,
[M ϩ 2H Ϫ C14H11CO2]ϩ) and 179 (60, C14H11ϩ).
1692
J. Chem. Soc., Perkin Trans. 1, 2001, 1673–1695

View Article Online
(2R)-N-(Fluoren-9-ylmethoxycarbonyl)aspartic ꢁ-benzylamide
68b
3290 (NH), 2963 (CH) and 1732 and 1647 (CO, esters and
amide); δH(300 MHz; C2HCl3) 2.43 (2 H, br, PhCH2), 2.67 (1 H,
dd, J 16.2 and 5.7, 1 H of β-CH2), 2.87 (1 H, dd, J 15.9 and 6.6,
1 H of β-CH2), 3.43–3.76 [8 H, m, 4 × CH2 (Piz)], 4.17–4.64
(3 H, m, CH2O and fluorenyl CH), 5.09 (1 H, q, J 6.6, α-H),
This compound was prepared through allyl group deprotection
in a manner identical to ester 22, using the diester 66b (575 mg,
1.19 mmol) to give the required compound as a white crystal-
line solid (462 mg, 87%), mp 118–119 ЊC (HRMS: found [M ϩ
H]ϩ, 445.1769. C26H25N2O5 requires 445.1763); [α]D ϩ6.88 (c
0.32 in MeOH); νmax(Nujol)/cmϪ1 3335 (NH) and 1742 and
1712 (CO, esters and amide); δH(300 MHz; C2H3O2H) 1.98
(2 H, s, PhCH2), 2.77 (1 H, dd, J 11.1 and 6.5, 1 H of β-CH2),
2.91 (1 H, dd, J 10.7 and 6.3, 1 H of β-CH2), 4.07–4.68 (4 H, m,
CH2O, fluorenyl CH and α-H), 4.99 (1 H, br, OH) and 7.18–
7.86 (13 H, m, Ar-H); δC(75.4 MHz; C2H3O2H) 35.87 (β-CH2),
42.84 (PhCH2), 47.03 (fluorenyl CH), 51.85 (α-C), 65.62 and
66.89 (2 × CH2O), 119.69, 125.0, 126.93, 127.09, 127.55 and
128.23 (Ar-CH), 138.46, 141.28 and 143.93 (Ar-C quaternary),
157.07 (CO, urethane) and 170.15 and 172.81 (CO, amide and
acid); m/z (CI) 445 (6%, [M ϩ H]ϩ) and 179 (100, C14H11ϩ).
5.21–5.35 (2 H, m, CH ᎐CH), 5.84–5.97 (1 H, m, CH ᎐CH),
᎐
᎐
2
2
6.06 [1 H, d, J 9.6, NH (urethane)] and 7.26–7.77 (13 H, m,
Ar-H); δC(75.4 MHz; C2HCl3) 37.70 (β-CH2), 42.45 (PhCH2),
47.23 (fluorenyl CH), 47.54 (piperazyl CH2), 52.62 (α-C), 65.7
and 67.23 (2 × CH O), 118.67 (CH ᎐CH), 120.15, 125.26,
᎐
2
2
127.23, 127.53, 127.90, 128.50 and 129.31 (Ar-CH), 132.03
(CH ᎐CH), 137.43, 141.44 and 143.86 (Ar-C quaternary),
᎐
2
155.74 (CO, urethane) and 168.86 and 170.46 (CO, amide
and ester); m/z (CI) 554 (100%, [M ϩ H]ϩ), 358 (14, [M Ϫ
C14H11O]ϩ), 179 (24, C14H11ϩ) and 57 (57, C3H5Oϩ).
(2R)-N-(Fluoren-9-ylmethoxycarbonyl)aspartic ꢁ-benzyl-
piperazinylamide 68g
This compound was prepared through allyl group deprotection
in a manner identical to diester 21, using γ-allyl-(2R)-N-
(2R)-N-(Fluoren-9-ylmethoxycarbonyl)aspartic ꢁ-4-benzyl-
piperidinylamide 68f
(fluoren-9-ylmethoxycarbonyl)aspartic
α-benzylpiperazinyl-
amide 66g, (575 mg, 1.04 mmol) to give the required compound
as a white hygroscopic solid which still showed signs of impur-
ity (763 mg, 69%), mp 74–75 ЊC; νmax(CH2Cl2)/cmϪ1 3314 (NH),
3064 br (OH) and 1718 and 1635 (CO, esters and amide);
δH(300 MHz; C2H3O2H) 2.01 (2 H, s, PhCH2), 2.57–3.98 (10 H,
m, β-CH2, CH2 × 4 Piz), 4.04–4.73 (3 H, m, CH2O, fluorenyl
CH), 4.98–5.04 (1 H, m, α-H) and 6.83–7.99 (13 H, m, Ar-H);
δC(75.4 MHz; C2HCl3) 37.47 (β-CH2), 37.95 (PhCH2), 42.01
(Piz CH2), 46.94 (fluorenyl CH), 47.49 (α-C), 67.24 (2 × CH2O),
119.84, 124.59, 125.03, 125.93, 127.58, 128.16 and 128.60 (Ar-
CH), 139.59, 141.13, 143.53 and 143.59 (Ar-C quaternary),
155.68 (CO, urethane) and 168.88 and 173.66 (CO, amide and
ester); m/z (CI) 511 (1%, [M Ϫ 2H]ϩ) and 179 (100, C14H11ϩ).
To a stirred solution of ester 44 (1 g, 2.53 mmol) in dry THF
(25 cm3) at Ϫ15 ЊC was added N-methylmorpholine (276 mm3,
2.53 mmol). Isobutyl chloroformate (343 mm3, 2.53 mmol) was
added and the suspension was stirred at Ϫ15 ЊC for a further
5 min. A solution of 4-benzylpiperidine (356 mg, 2.53 mmol)
and N-methylmorpholine (276 mm3, 2.53 mmol) in THF (25
cm3) was added and the mixture left to stir overnight. The
hydrochloride salts were removed by filtration and the filtrate
concentrated under reduced pressure to give a pale yellow oil
which was redissolved in ethyl acetate (15 cm3), washed succes-
sively with water (10 cm3), 5% aqueous NaHCO3 solution (15
cm3), 10% citric acid solution (15 cm3) and then brine (15 cm3).
The organic phase was dried (MgSO4) and concentrated under
reduced pressure to give the required diester 66f which was
immediately used in the next reaction.
3-Amino-3-(4Ј-bromophenyl)propanoic acid 69 (R ؍ Br)
To a stirred solution of the above diester and phenylsilane
(624 mm3, 5.06 mmol) in dry CH2Cl2 (30 cm3) under Ar was
added tetrakis(triphenylphosphine)Pd(0) (58 mg, 0.05 mmol).
The reaction was allowed to stir at room temperature for 18 h,
and was then concentrated under reduced pressure. The residue
was immediately purified by flash chromatography on silica
using ethyl acetate–hexane (60:40) as the eluent to give a white
solid (700 mg, 54%), mp 79–81 ЊC (Found C, 71.3; H, 6.2;
N, 5.1. C31H32N2O5ؒ0.5H2O requires C, 71.4; H, 6.4; N, 5.4%);
[α]D ϩ27.3 (c 0.3 in MeOH); νmax(CH2Cl2)/cmϪ1 3279 (NH),
3100 br (OH) and 1718 (CO, ester); δH(300 MHz; C2HCl3) 1.13–
1.28 (5 H, m, CHCH2Ph and 2 × CH2-CH2NH), 1.70 (4 H, br,
2 × CH2-CH2NH), 2.46–3.04 [4 H, m, PhCH2 and β-CH2
(Asp)], 3.98–4.59 (4 H, m, CH2O, fluorenyl CH and α-H), 5.09
(1 H, br, OH), 6.18 [1 H, dd J 21 and 9, NH (urethane)] and
7.04–7.77 (13 H, m, Ar-H); δC(75.4 MHz; C2HCl3) 31.42
(2 × CH2-CH2NH), 37.47 (β-CH2), 37.95 (2 × CHCH2Ph),
42.94 (CH2-CH2NH), 46.19 (PhCH2), 46.94 (α-C), 47.49
(fluorenyl CH), 67.09 (CH2O), 119.84, 125.03, 125.93, 126.96,
127.58, 128.16 and 128.60 (Ar-CH), 139.59, 141.13, 143.53 and
143.59 (Ar-C quaternary), 155.68 (CO, urethane), 169.88 (CO,
ester) and 173.66 (CO, acid); m/z (CI) 513 (1%, [M ϩ H]ϩ) and
179 (100, C14H11ϩ).
To a stirred solution of malonic acid (2.60 g, 25 mmol) and
ammonium acetate (3.85 g, 50 mmol) in methanol (35 cm3) was
added 4-bromobenzaldehyde (4.63 g, 25 mmol). The suspension
was then refluxed for 6 h. Upon cooling the reaction mixture
was filtered and then washed on the pad with cold ethanol to
give the required product as a white solid (3.05 g, 50%), mp
245–247 ЊC (softens at 225 ЊC) (lit.,41 264–265 ЊC) (Found C,
44.75; H, 4.0; N, 5.7. C9H10NO2Br requires C, 44.45; H, 4.15; N,
5.75%); νmax(KBr)/cmϪ1 3005 br (NH3ϩ), 2875 (CH’s), 2546(s),
2156(s), 1630 (amino acid I), 1593 (amino acid II, CO2Ϫ) and
1558 (NH3ϩ); δH(300 MHz; H2O) 3.63 (2 H, ABX, β-CH2),
2
4.18 (1 H, apparent dd, J 7.3, 7.4, α-CH), 7.25 (2 H, m, Ar-H)
2
and 7.50 (2 H, m, Ar-H); δC(75.5 MHz; H2O) 47.5 (β-CH2),
53.1 (α-CH), 121.0 (Ar-CH para), 129.0 (Ar-CH meta), 132.2
(Ar-CH ortho), 144.3 (Ar-C quaternary) and 180.6 (CO, acid);
m/z (FAB) 290 and 288 (47 and 49%, Br isotopes, [M Ϫ
H ϩ 2Na]ϩ), 137 (100) and 116 (100).
3-[N-(Fluoren-9-ylmethoxycarbonyl)amino]-3-(4Ј-bromophenyl)-
propanoic acid 67 (R ؍ Br)
To a stirred solution of 3-amino-3-(4Ј-bromophenyl)propanoic
acid 69 (R = Br) (1.22 g, 5 mmol) in 1 mol dmϪ3 aqueous NaOH
(20 cm3) cooled in an ice-bath was added a solution of fluoren-
9-ylmethoxycarbonyl chloride (1.42 g, 5.5 mmol) in dioxane (20
cm3). The reaction mixture was warmed to room temperature
and stirred for 4 h and then poured into water (20 cm3). The
dioxane was removed under reduced pressure and the resulting
aqueous solution was washed with diethyl ether (2 × 25 cm3),
then cooled to 0 ЊC and carefully acidified to pH 2 with a solu-
tion of 6 mol dmϪ3 HCl. The mixture was then extracted with
ethyl acetate (3 × 20 cm3), dried (MgSO4) and the solvent was
removed under reduced pressure to give a white solid (1.11 g,
ꢀ-Allyl (2R)-N-(fluoren-9-ylmethoxycarbonyl)aspartic ꢁ-benzyl-
piperazinylamide 66g
The α-amide was prepared in a manner identical to that
described for pentapeptide 23, using aspartate monoester 44
(790 mg, 2 mmol) and benzylpiperazine (348 mm3, 2 mmol) to
give the required compound as a white waxy solid (763 mg,
81%) (HRMS: found [M ϩ H]ϩ, 554.2667. C33H36N3O5 requires
554.2655); [α]D ϩ4.19 (c 0.31 in MeOH); νmax(CH2Cl2)/cmϪ1
J. Chem. Soc., Perkin Trans. 1, 2001, 1673–1695
1693

View Article Online
48%), mp 177–179 ЊC; νmax(KBr)/cm1 3334 (NH), 3400–2800 br
(OH, acid), 3042 (CHЈs) and 1695 (CO, urethane); δH[300 MHz;
(C2H3)2SO] 2.68 (2 H, ABX, β-CH2), 4.19–4.29 (3 H, m,
OCH2CH), 4.84–4.90 (1 H, m, α-CH), 7.18–7.89 (13 H, m, Ar-
H), 7.98 (1 H, d, J 8.4, NH) and 12.23 (1 H, br s, CO2H); δC[75.4
MHz; (C2H3)2SO] 40.9 (β-CH2), 46.9 (OCH2CH), 51.4 (α-CH),
65.6 (OCH2), 120.3, 125.3, 127.3, 128.9, 130.4 and 131.4 (Ar-
CH), 140.9, 142.5, 143.9 and 144.1 (Ar-C quaternary), 155.5
(CO, urethane) and 171.8 (CO, acid); m/z (ES) 488 and 490 (100
and 96%, Br isotopes, [M ϩ Na]ϩ).
g), mp 96–97 ЊC (Found C, 67.4; H, 5.7; N, 3.5. C23H23NO6
requires C, 67.5; H, 5.65; N, 3.4%) (HRMS: found [M ϩ H]ϩ,
410.1609 C23H24NO6 requires 410.1603); [α]D Ϫ15 (c 0.3 in
MeOH); νmax(CH2Cl2)/cmϪ1 3374 (NH), 2965 (CH) and 1751
(CO, urethane); δH(300 MHz; C2HCl3) 2.91 (1 H, dd, J 17.4
and 4.1, 1 H of β-CH2), 3.09 (1 H, dd, J 16.8 and 4.5, 1 H of β-
CH2), 3.77 (3 H, s, CH3), 4.20–4.74 (6 H, m, 2 × CH2O, fluor-
enyl CH and α-H), 5.21–5.38 (2 H, m, CH ᎐CH), 5.82–6.21
᎐
2
[2 H, m, CH ᎐CH and NH (urethane)] and 7.23–7.79 (8 H, m,
᎐
2
Ar-H); δC(75.4 MHz; C2HCl3) 36.75 (β-CH2), 47.23 (fluorenyl
CH), 50.53 (α-C), 53.01 (CH3), 65.89 and 67.4 (2 × CH2O),
3-Amino-3-(4Ј-methoxyphenyl)propanoic acid 69 (R ؍ OMe)
118.92 (CH ᎐CH), 120.15, 125.28, 127.23 and 127.89, 127.9
᎐
2
(Ar-CH), 131.76 (CH ᎐CH), 141.44, 143.86 and 143.98 (Ar-C
᎐
2
This compound was prepared in a manner identical to that
described for the 3-(4Ј-bromophenyl) analogue 69 (R = Br)
using 4-methoxybenzaldehyde (3.40 g, 25 mmol) to give the
required product as a white solid (2.78 g, 57%), mp 222–224 ЊC
(lit.,42 228–229 ЊC) (Found C, 65.2; H, 6.6; N, 8.55. C9H11NO2
requires C, 65.4; H, 6.7; N, 8.5%); νmax(KBr)/cmϪ1 2957 br
(NH3ϩ), 2932 (CHЈs), 2631(s), 2150(s), 1673 (amino acid I),
1610 (amino acid II, CO2Ϫ) and 1548 (–NH3ϩ); δH(300 MHz;
2H2O) 2.53 (2 H, ABX, β-CH2), 3.81 (3 H, s, OCH3), 4.19 (1 H,
apparent dd, J 7.4, 7.4, α-CH), 6.97 (2 H, m, Ar-H) and 7.32
quaternary), 156.12 (CO, urethane) and 170.76 and 171.32 (CO,
esters); m/z (CI) 410 (57%, [M ϩ H]ϩ) and 179 (100, C14H11ϩ).
ꢁ-Methyl (2R)-N-(fluoren-9-ylmethoxycarbonyl)aspartate 71
The monoester was prepared in a manner identical to α-methyl
(2R)-N-(fluoren-9-ylmethoxycarbonyl)glutamate 46, using
methyl allyl diester 70 (242 mg, 0.50 mmol) to give the required
compound as a white crystalline solid (134 mg, 60%), mp 124–
125 ЊC (Found C, 64.8; H, 5.2; N, 3.6. C20H19NO6 requires C,
65.05; H, 5.2; N, 3.8%) (HRMS: found [M ϩ H]ϩ, 370.1298.
C20H20NO6 requires 370.1291); [α]D ϩ19.05 (c 0.21 in MeOH);
νmax(CH2Cl2)/cmϪ1 3335 (NH), 3100 br (OH) and 1727 (CO,
ester); δH(300 MHz; C2H3O2H) 2.82 (1 H, m, β-CH2), 3.71 (3 H,
s, CH3), 4.08–4.62 (4 H, m, CH2O, fluorenyl CH and α-H)
and 7.24–7.82 (13 H, m, Ar-H); δC(75.4 MHz; C2HCl3) 35.76
(β-CH2), 47.03 (fluorenyl CH), 50.69 (α-C), 51.71 (CH3), 66.85
(CH2O), 119.64, 124.96, 126.88 and 127.50 (Ar-CH), 134.33,
141.28, 143.88 and 143.93 (Ar-C quaternary), 157.06 (CO,
urethane), 171.91 (CO, ester) and 172.57 (CO, acid); m/z (CI)
370 (99%, [M ϩ H]ϩ), 192 (64, [M ϩ 2H Ϫ C14H11]ϩ), 179 (100,
C14H11ϩ) and 148 (85, C5H10NO4ϩ).
2
(2 H, m, Ar-H); δC(75.5 MHz; H2O) 47.6 (β-CH2), 53.0 (α-
CH), 56.0 (OCH3), 114.7 (Ar-CH meta), 128.3 (Ar-CH ortho),
137.9 (Ar-C quaternary), 158.5 (Ar-CH para) and 180.8 (CO,
acid); m/z (FAB) 240 (36%, [M Ϫ H ϩ 2Na]ϩ), 137 (57) and
115 (100).
3-Amino-3-phenylpropanoic acid 69 (R ؍ H)
This compound was prepared in a manner identical to that
described for the 3-(4Ј-bromophenyl) analogue 69 (R = Br)
using benzaldehyde (3.85 g, 25 mmol) to give the required
product as a white solid (2.15 g, 52%), mp 216–218 ЊC (lit.,42
218–219 ЊC) (Found C, 61.4; H, 6.95; N, 7.45. C10H13NO3
requires C, 61.5; H, 6.7; N, 7.2%); νmax(KBr)/cmϪ1 3020 br
(NH3ϩ), 2940 (CH’s), 2610(s), 2206(s), 1625 (amino acid I),
1581 (amino acid II, CO2Ϫ) and 1516 (–NH3ϩ); δH(300 MHz;
2H2O) 2.57 (2 H, ABX, β-CH2), 4.23 (1 H, apparent dd, J 7.3,
7.3, α-CH) and 7.29–7.43 (5 H, m, Ar-H); δC(75.5 MHz; 2H2O)
47.6 (β-CH2), 53.6 (α-CH), 127.0 (Ar-CH meta), 127.9 (Ar-CH
para), 129.3 (Ar-CH ortho), 145.1 (Ar-C quaternary), and 180.7
(CO, acid); m/z (FAB) 210 (36%, [M Ϫ Hϩ 2Na]ϩ), 137 (54)
and 116 (100).
ꢁ-Methyl (2R)-N-(fluoren-9-ylmethoxycarbonyl)aspartyl-Wang
resin 72
The resin ester was prepared in a manner identical to that
described for β-allyl (2R)-N-(fluoren-9-ylmethoxycarbonyl)-
aspartyl-Wang resin 45, using α-methyl ester 71 (1 g, 2.71
mmol). The loading was determined to be 80%.53
(2S)-Phenylalanyl-[3-phenylpropanoyl]-[ꢁ-methyl (2R)-
glutamyl]-sarcosyl-[ꢁ-methyl (2R)-aspartate] diester 74
3-[N-(Fluoren-9-ylmethoxycarbonyl)amino]-3-phenylpropanoic
acid 67 (R ؍ H)
(2S)-Phenylalanyl-[3-phenylpropanoyl]-[α-methyl
glutamyl]-sarcosyl-[α-methyl (2R)-aspartate-Wang
(2R)-
resin]
This compound was prepared in a manner identical to that
described for the 3-(4Ј-bromophenyl) analogue 67 (R = Br)
using 3-amino-3-phenylpropanoic acid 69 (R = H) to give a
white solid (1.49 g, 77%), mp 186–188 ЊC (Found C, 74.35; H,
5.45; N, 3.54. C24H21NO4 requires C, 74.4; H, 5.45; N, 3.6%);
νmax(KBr)/cmϪ1 3365 (NH), 3400–2800 br (OH, acid), 3043
(CH’s) and 1740 (CO, urethane); δH[300 MHz; (C2H3)2SO] 2.68
(2 H, ABX, β-CH2), 4.17–4.29 (3 H, m, OCH2CH), 4.95 (1 H,
apparent q, J 8.4, α-CH), 7.22–7.90 (13 H, m, Ar-H), 7.97 (1 H,
d, J 8.6, NH) and 12.31 (1 H, s, CO2H); δC[75.4 MHz;
(C2H3)2SO] 41.2 (β-CH2), 46.9 (OCH2CH), 51.8 (α-CH), 65.5
(OCH2), 120.3, 125.3, 126.5, 127.2, 127.8 and 128.5 (Ar-CH),
140.9, 143.0, 143.9 and 144.1 (Ar-C quaternary), 155.5 (CO,
urethane) and 171.9 (CO, acid); m/z (ES) 410 (100%, [M ϩ
Na]ϩ).
diester was synthesised on the peptide synthesiser and treated
with cleavage mixture TFA–TES–H2O–CH2Cl2 (40:2:5:53)
at room temperature for 1 h. The resin was filtered off and
the solvent concentrated under reduced pressure (4–5 cm3).
Trituration with excess diethyl ether resulted in the pentapep-
tide 74 being obtained as a white solid (150 mg), mp >200 ЊC
(decomp.), νmax(KBr disc)/cmϪ1 2500–3000 br (OH), 1730 (CO,
ester) and 1670 (CO, amides); δH[300 MHz; (C2H3)2SO)] 1.60–
1.90 [2 H, m, β-CH2 (Glu)], 2.00–2.30 [2 H, m, γ-CH2 (Glu)],
2.60–2.80 [4 H, m, α-CH2 (Prop) and β-CH2 (Asp)], 2.93 and
2.96 (3 H, 2 × s, NCH3), 2.82–3.05 [2 H, m, PhCH2], 3.56–3.65
(6 H, m, 2 × CH3), 3.80–4.02 [3 H, m, α-H (Phe) and CH2
(Sar)], 4.10–4.23 [1 H, m, α-H (Glu)], 4.57–4.63 [1 H, m,
α-H (Asp)], 5.15–5.25 [1 H, m, α-H (Prop)], 7.02–7.40 (10 H, m,
Ar-H), 8.10 [2 H, br, NH2 (Phe)], 8.32 [1.5 H, br, NH (Glu) and
0.5 NH (Asp)], 8.53 [0.5 H, m, 0.5 NH (Asp)], 8.78 [1 H, m,
NH (Prop)].
ꢁ-Methyl ꢂ-allyl (2R)-N-(fluoren-9-ylmethoxycarbonyl)-
aspartate 70
Cyclo[-3-phenylpropanoyl-(2R)-Glu-ꢁ-OMe-ꢂ-Sar-(2R)-Asp-ꢁ-
OMe-ꢀ-(2S)-Phe-] 75
This compound was prepared in a manner identical to diester
14, using γ-allyl (2R)-(fluoren-9-ylmethoxycarbonyl)aspartate
monoester 44 (1.00 g, 2.54 mmol) to give the required com-
pound as a white crystalline solid in quantitative recovery (1.04
To a stirred solution of the pentapeptide 74 (350 mg,
0.6 mmol), in CH2Cl2–DMF (9:1, 50 cm3) was added DIPEA
1694
J. Chem. Soc., Perkin Trans. 1, 2001, 1673–1695

View Article Online
(150 mm3, 1.5 mmol). The mixture was cooled to 0 ЊC using an
ice bath, and then BOP-Cl (156 mg, 0.66 mmol) was added and
the mixture stirred at 0 ЊC for at 6 h. The solution was allowed
to warm up to room temperature and stirred for a further 7
days. The resulting solution was then washed with 10% citric
acid, brine, 5% sodium bicarbonate, distilled water and finally
brine again. The organic layer was separated, dried (MgSO4)
and solvent removed under reduced pressure to a volume of
1 cm3. Tituration with diethyl ether (10 cm3) caused the com-
pound to precipitate out as a white solid (80 mg, 24%),
mp >220 ЊC (decomp.) (HRMS: found [M ϩ H]ϩ, 638.2847.
C32H40N5O9 requires 638.2826); νmax(KBr disc)/cmϪ1 2500–3000
br (OH), 1730 (CO, ester) and 1670 (CO, amides); δH[500 MHz;
(C2H3)2SO, mixture of rotamers] 1.65–1.95 [2 H, m, β-CH2
(Glu)], 2.00–2.20 [2 H, m, γ-CH2 (Glu)], 2.35–2.71 [4 H, m,
α-CH2 (Prop) and β-CH2 (Asp)], 2.70–2.75 [1 H, m, 1 H of
PhCH2], 2.82 and 2.84 (3 H, 2 × s, NCH3), 3.00–3.17 [1 H, m,
1 H of PhCH2 (Phe)], 3.63, 3.65 and 3.67 (6 H, 3 × s, 2 × CH3),
4.02 [2 H, AB, J 15.5, CH2 (Sar)], 4.21–4.28 [1 H, m, α-H (Glu)],
4.48 [1 H, m, α-H (Asp)], 4.45 and 4.61 [1 H, 2 × m, α-H (Phe)],
5.26–5.44 [1 H, m, α-H (Prop)], 7.05–7.40 (10 H, m, Ar-H), 7.88
[0.75 H, m, NH (Phe)], 8.17 [0.75 H, m, NH (Asp)], 8.37 [0.25
H, m, NH (Phe)], 8.51 [0.75 H, m, 0.5 NH (Glu)], 8.64 [0.25 H,
m, NH (Prop)] and 8.89 [1 H, m, NH (Prop)]; δC[75.4 MHz;
(C2H3)2SO)] 26.76 [β-CH2 (Glu)], 29.20 [γ-CH2 (Glu)], 34.52,
35.78 (NCH3), 36.84 [β-CH2 (Asp)], 36.84 and 37.98 [PhCH2],
43.43 [β-CH2 (Prop)], 49.61 [α-C (Asp)], 50.96 [α-C (Prop)],
51.75 [α-C (Glu)], 51.95 and 52.25 [CH2 (Sar) and 2 × CH3],
53.19 and 54.30 [α-C (Phe)], 126.18, 126.34, 126.87, 127.05,
128.04, 128.24, 128.34, 128.42, 128.50, 129.05, 129.20 and
129.45 (Ar-CH and Ar-C quaternary) and 168.16, 168.52,
170.24, 170.26, 171.58, 172.40 and 172.88 (CO, amides, esters);
m/z (FAB) 660 (65%, [M ϩ Na]ϩ) and 638 (100, [M ϩ H]ϩ).
12 J. Wilkie and D. Gani, unpublished results.
13 M. Namikoshi, K. L. Rinehart, R. Sakai, R. R. Stotts, A. M.
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Acknowledgements
6319.
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20, 3157.
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Fr., 1987, 1079.
The authors would like to thank Dr R. Beri for providing
a recombinant strain of E. coli expressing PP1, P. Coffey for
preliminary experimental work on β-amino acid, and
Dr M. Akhtar for scientific support. We thank the BBSRC
Biomolecular Sciences Committee for grants B05162 and B/49/
B07992 and Astra-Zeneca for a Strategic Research Fund and
a CASE award to KLW.
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