Design and preparation of serine-threonine protein phosphatase inhibitors based upon the nodularin and microcystin toxin structures: Part 2. Synthesis of a functionalised nodularin macrocycle and a stripped-down microcystin macrocycle

Article abstract of DOI:10.1039/a702410j

Nodularins and microcystins are complex natural isopeptidic hepatotoxins that serve as subnanomolar inhibitors of the eukaryotic serine-threonine protein phosphatases, PP1 and PP2A. In Part 1 (A. P. Mehrotra, K. L. Webster and D. Gani, J. Chem. Soc., Perkin Trans. 1, 1997, preceding paper) each of the key structural or potentially reactive motifs within each macrocycle type was assessed as a contributor towards phosphatase inhibitory efficacy and a stripped-down nodularin-type macrocycle was identified as a suitable precursor to potentially active synthetic inhibitors. Subsequently, synthetic routes to the 19-membered nodularin macrocyclic system were developed, using solution-phase chemistry, which demonstrated that only certain cyclisation protocols were viable. Here we describe an extension of this chemistry to provide a 19-membered nodularin macrocycle, cyclo-[(3R)-3-hydroxymethyl-β-Ala-(R)-Glu-α-OMe-γ-Sar-(R)-Asp- α-OMe-β-(S)-Phe-], appropriately functionalised with a hydroxymethyl group for the incorporation of lipophilic side-chains. We also demonstrate that the 25-membered microcystin macrocycle, cyclo-[β-Ala-(R)-Glu-α-OMe-γ-Sar-(R)-Ala-(S)-Leu-(R)-Asp- α-OMe-β-(S)-Phe-], can be prepared in good yield using similar protocols in which macrocyclisation is effected through the reaction of the amino group of the (2S)-phenylalanine residue with the β-pentafluorophenyl ester of the (2R)-aspartic acid residue.

Full text of DOI:10.1039/a702410j

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Design and preparation of serine–threonine protein phosphatase
inhibitors based upon the nodularin and microcystin toxin structures:
Part 2.1 Synthesis of a functionalised nodularin macrocycle and a
stripped-down microcystin macrocycle
Antony B. Maude, Amit P. Mehrotra and David Gani*
School of Chemistry and Centre for Biomolecular Sciences, The Purdie Building,
The University, St. Andrews, Fife, UK KY16 9ST
N odularins and microcystins are complex natural isopeptidic hepatotoxins that serve as subnanomolar
inhibitors of the eukaryotic serine–threonine protein phosphatases, PP1 and PP2A. In Part 1
(A. P. M ehrotra, K . L. Webster and D. G ani, J. Chem. Soc., Perkin Trans. 1, 1997, preceding paper) each
of the key structural or potentially reactive motifs within each macrocycle type was assessed as a
contributor towards phosphatase inhibitory efficacy and a stripped-down nodularin-type macrocycle was
identified as a suitable precursor to potentially active synthetic inhibitors. Subsequently, synthetic routes
to the 19-membered nodularin macrocyclic system were developed, using solution-phase chemistry, which
demonstrated that only certain cyclisation protocols were viable. H ere we describe an extension of this
chemistry to provide a 19-membered nodularin macrocycle, cyclo-[(3R)-3-hydroxymethyl-â-Ala-(R)-G lu-
á-OM e-ã-Sar-(R)-Asp-á-OM e-â-(S )-Phe-], appropriately functionalised with a hydroxymethyl group for
the incorporation of lipophilic side-chains. We also demonstrate that the 25-membered microcystin
macrocycle, cyclo-[â-Ala-(R)-G lu-á-OM e-ã-Sar-(R)-Ala-(S )-Leu-(R)-Asp-á-OM e-â-(S )-Phe-], can be
prepared in good yield using similar protocols in which macrocyclisation is effected through the reaction
of the amino group of the (2S )-phenylalanine residue with the â-pentafluorophenyl ester of the
(2R)-aspartic acid residue.
specific inhibitors for each enzyme type, we recently demon-
Introduction
strated that the dehydroamino acid residue present in micro-
cystin was not essential in conferring activity as an inhibi-
tor.1,12 Subsequently we proceeded to evaluate the functional
role of each of the remaining conserved structural motifs
within the two classes of toxin, using data available for nat-
ural variants, and concluded that the cyclic structure, the two
free carboxylic acid groups and elements of the large rigid
lipophilic side-chain would need to be retained in active
inhibitors. This work led to the identification of a stripped
down and simpler macrocycle (than that present in each of
the natural products 1 and 2) which should serve as a frame-
work for attaching specific functionalities.11 Since our longer
term interests were to synthesise minimal analogues to probe
the active-site binding interactions and then to identify spe-
cific inhibitors for each catalytic subunit type, PP1 and PP2A,
we decided to opt for a convergent synthetic strategy in which
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 also be suitable for preparing libraries of macrocycles
and libraries of lipophilic side-chain precursors that could
be connected to give a diverse array of synthetic toxin
analogues.
To assess the viability of this strategy and, in particular, to
determine whether or not it would be possible to prepare
stripped down nodularin analogues lacking the side-chain func-
tionalities, we tested various cyclisation protocols on a model
nodularin macrocycle target, cyclo-[β-Ala-(R)-Glu-α-OMe-γ-
Sar-(R )-Asp-α-OMe-β-(S )-Phe-], 3. 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-pentafluorophenyl ester of
the (2R)-aspartate α-methyl ester residue by the free amino
The natural cyclic isopeptide toxins microcystin 1 and nodu-
larin 2, previously established as potent hepatotoxins,2 are
now known to inhibit the catalytic subunit of the mammalian
serine/threonine protein phosphatases PP1 and PP2A (but
not PP2B or 2C) with subnanomolar inhibition constants.3
Each of these catalytic activities, together with serine/
threonine protein kinase enzymes, are involved in controlling
and maintaining the delicate balance of pools of phosphoryl-
ated and dephosphorylated proteins which effect cellular
metabolism and communication.4–6 The two toxin-sensitive
enzymes, cat-PP1 and cat-PP2A, are highly homologous and
display ~50% amino acid sequence identity.7,8 Both families
of toxin, microcystins and nodularins, differ considerably
from other cyclic peptides. Both groups are cyclic tri-
isopeptides and contain two free carboxylic acid groups, an
N-methyldehydroamino acid moiety, and
a large, rigid,
lipophilic side-chain which forms part of an Adda residue
(see structures 1 and 2). These five principal motifs are the
only structural features that are conserved between the two
families.
Other naturally occurring competitive inhibitors of cat-PP1
and cat-PP2A include the powerful tumour promoter okadaic
acid, responsible for diarrhetic shellfish poisoning, and the
calyculins, tautomycin and cantharidin.9 However, none of
these compounds shows any specificity towards either of the
two enzyme types. This lack of specificity can now be rational-
ised by an analysis of the aligned amino acid sequences for the
enzymes within the context of the published X-ray crystal
structures available for cat-PP1.10 Essentially this analysis indi-
cates that the structure and composition of the active-site cleft
is extremely highly conserved and that the inhibitors interact
only with conserved regions.11
As part of a larger programme to prepare and identify
J. Chem. Soc., Perkin Trans. 1, 1997
2513

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α-semialdehydes and Wittig or Julia chemistry has been suc-
N-Me-dehydroAla
(2R)-Glu
cessfully employed in the synthesis of the Adda residue by
other groups in the recent past.13–18 Mann and co-workers
have also very recently reported on a synthesis of the Adda
residue utilising the α-semialdehyde derived from (2R,3S )-3-
methylaspartic acid as an intermediate in which the lipophilic
side-chain was introduced using a Stille coupling reaction.19,20
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 4 was devised in which the
formyl group would be masked as a primary alcohol. It was also
reasoned, by comparison to the well established peptide chem-
istry of serine and threonine residues, that such an alcohol
group could be introduced early in the synthesis of the linear
peptide precursors and could be subjected to all of the condi-
tions required for peptide coupling (masking, unmasking and
acyl-group activation) without the need for protection.21 Thus,
CH3
O
N
H
CO2H
Adda
HN
NH
(2R)-Ala
H3C
H
O
H
H2C
H3C
OCH3
O
H
O
NH
HN
H
CH3
CH3
CH3
H
H
N
H
N
H
O
H3C
H
CH3
(2S)-Arg
HN
O
H
O
H
(2S)-Leu
CO2H
NH
1a
Microcystin-LR
(2R,3S)-β-MeAsp
C
NH2
1b Microcystin-LA [(2S)-Ala replaces (2S)-Arg in 1a]
1c Microcystin-RR [(2S)-Arg replaces (2S)-Leu in 1a]
(2R)-Glu
CO2H
N-Me-dehydroBut
access to
a (3R)-3-amino(hydroxymethyl)-β-alanine [(3R)-
3-amino-4-hydroxybutyric acid (AHB)] residue would be
required.
H
CH3
O
Adda
OCH3
H3C
H
N
CH3
H
N
H
Disconnection of cyclo-[(3R)-3-hydroxymethyl-β-Ala-(R)-
Glu-α-OMe-γ-Sar-(R)-Asp-α-OMe-β-(S )-Phe-] 4 at the peptide
bond between the Phe residue and the β-carbonyl moiety of the
Asp residue, using the same strategy1,12 as employed for the
non-functionalised macrocycle 3, gives the linear isopentapep-
tide 7. The three C-terminal residues had been incorporated
into macrolactam 3 previously as the tripeptide triester (2R)-
Glu-α-OMe-γ-Sar-(2R)-Asp-α-OMe-β-OBn 8 without incident
and, therefore, the isopentapeptide (2S )-Phe-(3R)-3-hydroxy-
methyl-β-Ala-(2R)-Glu-α-OMe-γ-Sar-(2R)-Asp-α-OMe-β-OH
7 was disconnected between the (3R)-3-hydroxymethyl-β-
alanine and Glu residues to give tripeptide triester 8 and
the dipeptide (2S )-Phe-(3R)-3-hydroxymethyl-β-Ala-OH 9,
Scheme 1.
Two routes towards an appropriately protected hydroxyme-
thyl dipeptide 9 were pursued. In the first route, the β-methyl
ester hydrochloride of (2R)-aspartic acid was prepared by the
method of Schwarz et al.22,23 Refluxing a solution of the
hydrochloride in ethanol with propylene oxide gave the enan-
tiomerically pure free-base form of the β-methyl ester 10, mp
194–195 ЊC [lit.,24 194–195 ЊC for the (2S ) antipode], which was
converted into its α-trimethylsilyl ester. Without isolation the
diester was protected on nitrogen with a phenylfluorenyl
group,24 introduced using 9-bromo-9-phenylfluorene, and the α-
silyl ester was hydrolysed during the work-up to give the
required secondary amine 11, mp 160 ЊC [lit.,24 160–161 ЊC for
(2S ) antipode], which displayed the expected properties,
Scheme 2. Preparation of the mixed α-carboxy isobutyl car-
bonic anhydride followed by reduction with sodium boro-
hydride in very dry THF gave the required alcohol 12 (R = Me)
in 91% yield, mp 135–137 ЊC. This reduction protocol was
unreliable and often yielded significant amounts of lactone 13,
mp 156–158 ЊC. While the lactone could be efficiently converted
into alcohol 12 (R = H) by using sodium hydroxide in aq.
methanol the overall yields were only moderate and we wished
to assess an alternative strategy before committing the material
to the synthesis of the macrolactam.
H
O
O
NH
CH3
H
H
N
H
O
H3C
H
NH
CH3
O
H
CO2H
H
(2S)-Arg
HN
NH
2
(2R,3S)-β-MeAsp
C
Nodularin
NH2
H
CO2Me
CH3
O
N
N
H
O
O
NH
H
N
NH
O
O
H
CO2Me
H
3
group of the (2S )-phenylalanine residue proceeded in excel-
lent yield (89%).1,12 In this article we describe the extension
of this chemistry to provide a 19-membered nodularin macro-
cycle, cyclo-[(3R)-3-hydroxymethyl-β-Ala-(R)-Glu-α-OMe-γ-
Sar-(R)-Asp-α-OMe-β-(S )-Phe-], 4, appropriately functional-
ised with a hydroxymethyl group for the incorporation of
lipophilic side-chains. We also show that it is possible to prepare
the 25-membered microcystin macrocycle, cyclo-[β-Ala-(R)-
Glu-α-OMe-γ-Sar-(R)-Ala-(S )-Leu-(R)-Asp-α-OMe-β-(S )-
Phe-], 5, in good yield, effecting macrolactamisation through
the reaction of the amino group of the (2S )-phenylalanine
residue with the β-pentafluorophenyl ester of the (2R)-aspartic
acid residue.
Accordingly, commercial (2R)-(tert-N-butoxycarbonyl)-
aspartic acid β-benzyl ester was converted into the mixed car-
bonic anhydride, which was reduced with sodium borohydride
in THF to give the required alcohol 14 in an optimised yield of
89%, Scheme 3. This compares favourably with yields reported
by Bland for the preparation of the (2S)-enantiomer.25 The
removal of the N-Boc protecting group using hydrogen chloride
gas in ethyl acetate was accompanied by extensive cleavage of
the benzyl ester moiety to give (3R)-3-amino-4-hydroxybutyric
acid hydrochloride, mp 177–178 ЊC. However, this unwanted
side-reaction could be largely avoided through treatment of
compound 14 with trifluoroacetic acid (TFA) in dichloro-
Results and discussion
Synthesis of a 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,1,12 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 functionalities to provide rigidity,
we chose to use a 3-formyl group, i.e. the aldehyde 6, Scheme 1.
A similar strategy using appropriately protected aspartic
2514
J. Chem. Soc., Perkin Trans. 1, 1997

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CO2Me
Me
N
CO2Me
Me
N
O
O
N
H
N
H
O
O
O
HO
O
O
NH
NH
H
N
H
N
H
NH
NH
O
O
O
O
CO2Me
CO2Me
4
6
O
OH
O
CO2Me
Me
O
CO2Me
H
N
H
N
H
N
N
H2N
N
H2N
N
H
O
O
O
CO2Me
Me
O
CO2Me
O
OBn
OH
8
7
+
H
N
OH
H2N
O
O
OH
9
Scheme 1
methane. This modification gave the required, almost pure,
deprotected amine salt 15 as judged by 1H and 13C NMR
spectroscopy, which could be used directly for the next stage.
Without further purification the amino alcohol 15 was treated
with the mixed carboxylic-carbonic anhydride of N-Boc-(2S )-
phenylalanine in the presence of NMM to give N-Boc-(2S )-
Phe-(3R)-3-hydroxymethyl-β-Ala-OBn 16 in 90% overall yield
from alcohol 14. Catalytic hydrogenolysis of the benzyl ester
protecting group, Scheme 3, gave the dipeptide acid 17 (mp 95–
97 ЊC), which displayed the expected spectral and analytical
properties. Importantly, the free acid 17 and its benzyl ester
derivative 16 possessed only one set of signals in their 13C NMR
spectra, indicating that no racemisation had occurred at the α-
centre of the Asp residue during the reduction of the mixed
anhydride. Thus, compound 17, a suitably protected form of
compound 9 (see Scheme 1) was ready for reaction with the
tripeptide triester 8.
CO2H
CO2H
i, ii
CO2H
CO2Me
H2N
H2N
10
iii
CO2H
CO2Me
N
H
11
iv, v
In order to construct the linear isopeptide precursor the car-
boxyterminalof N-Boc-(2S )-Phe-(3R)-3-hydroxymethyl-β-Ala-
OH 17 was activated as the mixed carbonic anhydride and was
treated with tripeptide triester, (2R)-Glu-α-OMe-γ-Sar-(2R)-
Asp-α-OMe-β-OBn, 8 prepared as described previously1 to give
the linear pentapeptide ester 18 in 83% yield after column
chromatography, {mp 89–91 ЊC; [α]D23 ϩ9.03† (CH2Cl2)}. The
compound gave satisfactory analytical data but, as was
observed previously for sarcosine (N-methylglycine)-containing
peptides in the synthesis of macrocycle 3,1,12 both rotameric
forms of the compound corresponding to the cis and trans
configurations at the γ-Glu᎐Sar amide bond were observable
by 1H and 13C NMR spectroscopy.
OH
+
O
N
H
N
H
CO2R
O
13
vi
Scheme
2
Reagents, conditions (and yields): i, SOCl2, MeOH,
Ϫ30 → 10 ЊC (100%); ii, propylene oxide, EtOH, reflux (90%);
iii, TMSCl, Et3N, 9-bromo-9-phenylfluorene, Pb(NO3)2, CH2Cl2,
room temp. (81%); iv, IBCF, NMM, THF, Ϫ15 ЊC; v, NaBH4,
THF, Ϫ15 ЊC → room temp. (91%); vi, aq. NaOH, MeOH (85%)
(R = H)
Removal of the benzyl ester in compound 18 by catalytic
hydrogenolysis, followed by EDCI-mediated esterification of
† Specific optical rotations [α]D are given in units of 10Ϫ1 deg cm2 gϪ1
.
J. Chem. Soc., Perkin Trans. 1, 1997
2515

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the resulting free acid 19 with pentafluorophenol, proceeded
smoothly to give the N-Boc pentapeptide pentafluorophenyl
ester 20 in 61% yield over the two steps (see Scheme 4) after
purification by flash chromatography on silica gel. The N-
terminal tert-butoxycarbonyl protecting group was removed by
treatment with TFA in dichloromethane and the resulting
amine trifluoroacetate salt 21 was thoroughly dried under high
vacuum. Treatment with DIPEA, under conditions of high
dilution in dichloromethane, allowed cyclisation to proceed and
after nine days at room temperature, when periodic TLC analy-
sis showed that the conversion of starting material was com-
plete, the reaction was terminated. The required functionalised
macrocyclic pentapeptide 4 was obtained in 41% yield after
flash column chromatography on silica and elution with
CH2Cl2–MeOH (94:6), mp 96–100 ЊC. No evidence for the
formation of significant amounts of lactone was obtained for
the crude material and the purified compound displayed all
of the expected spectral and analytical properties. Macrocycle
4 showed two sets of signals in NMR spectra obtained in
deuteriochloroform corresponding to two conformations in
exchange on the NMR time-scale. This rate ~1–103 sϪ1, would
be too fast to be associated with interconverting amide
rotamers26 in small linear proline peptides,27 but could be con-
sistent with interconverting rotamers in a macrocycle where the
attainment of amide-bond planarity in both rotamers causes
other high-energy interactions.
OH
O
O
CO2H
i
O
N
H
O
N
H
O
O
O
O
14
ii
OH
O
OH
O
H
N
TFA•H2N
iii
O
N
H
O
O
O
OR
15
16 R = Bn
17 R = H
iv
The macrolactamisation of (2S )-Phe-(3R)-3-hydroxymethyl-
β-Ala-(2R)-Glu-α-OMe-γ-Sar-(2R)-Asp-α-OMe-β-OPFP 21 to
give compound 4 was noticeably more difficult than the cyclis-
ation of (2S )-Phe-β-Ala-(2R)-Glu-α-OMe-γ-Sar-(2R)-Asp-α-
OMe-β-OPFP to give macrocycle 3. One possible reason is that
the free hydroxy group forms a hydrogen bond with an amide or
Scheme 3 Reagents, conditions (and yields): i, IBCF, NMM, THF,
Ϫ15 ЊC; then NaBH4, THF, Ϫ15 ЊC → room temp. (89%); ii, TFA,
CH2Cl2; iii, (2S )-N-Boc-PheOH, IBCF, NMM, THF, Ϫ30 ЊC; then 15,
NMM, THF, Ϫ30 ЊC → room temp. (90%); iv, H2, Pd/C, EtOH
(98%)
O
OR
CO2Me
Me
N
O
CO2Me
O
O
H
N
H
N
H
N
i
HCl•H2N
N
H
17 +
O
N
H
N
O
O
O
CO2Me
Me
O
CO2Me
O
O
OH
ii
8•HCl
18 R = Bn
19 R = H
iii
CO2Me
Me
O
N
OH
N
H
NH
O
CO2Me
Me
N
O
CO2Me
F
F
O
O
H
N
HO
O
O
F
F
F
H
N
N
H
N
H
N
H
NH
O
O
O
O
O
O
CO2Me
4
20
iv
v
OH
O
CO2Me
Me
N
O
CO2Me
O
TFA•H2N
N
H
N
H
N
H
O
O
OC6H5
21
Scheme 4 Reactions, conditions (and yields): i, IBCF, NMM, THF, Ϫ30 ЊC → room temp. (83%); ii, H2, Pd/C, EtOH (97%); iii, C6F5OH, EDCI,
CH2Cl2, 0 ЊC → room temp. (61%); iv, TFA, CH2Cl2; v, DIPEA, CH2Cl2 (41%)
2516
J. Chem. Soc., Perkin Trans. 1, 1997

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ester moiety which stabilises a conformation which is not
amenable to macrolactamisation. This possibility is discussed in
more detail below.
O
CO2Me
O
O
N
H
O
Synthesis of the stripped-down microcystin macrocycle
24
In view of our experiences with the nodularin macrocycle which
had clearly demonstrated that certain cyclisation strategies were
not viable we wished first to assess the equivalent disconnection
to that which had been successfully used to prepare the non-
functionalised macrocycle 3.1,12 Natural microcystins 1 differ
from nodularins 2 in containing two extra contiguous amino
acids, a (2R)-alanine residue and a variable (2S )-α-amino acid
(leucine in microcystin-LR), that are inserted between the car-
bonyl moiety of the N-methyldehydroamino acid and the N-
atom of the (2R,3S)-3-methylaspartate residue of nodularin.
Therefore the dipeptide (2R)-Ala-(2S )-Leu was inserted into
structure 3 to define the model microcystin macrocycle target as
structure 5. The equivalent disconnection to that utilised in the
preparation of nodularin macrocycle 31,12 applied to the syn-
thesis of cyclo-[β-Ala-(2R)-Glu-α-OMe-γ-Sar-(2R)-Ala-(2S)-
Leu-(2R)-Asp-α-OMe-β-(2S )-Phe-] 5 would require the closure
to be effected between the Phe N-atom and the β-carboxy group
of the (2R)-aspartate α-methyl ester. Note that this approach
differs from that very recently described (after the completion
of our own work) for the synthesis of microcystin-LA, in which
the cyclisation step involved connecting the Adda N-atom to
the (2S )-Ala carboxy group.17 Thus, to synthesise the stripped-
down microcystin-type macrocycle 5 we needed to prepare an
appropriately protected and activated linear peptide of the
i
CO2Me
O
HCl•H2N
O
23
+
O
H
N
OH
O
N
H
O
O
22
ii
O
O
H
N
H
N
OR
O
N
sequence
(2S )-Phe-β-Ala-(2R)-Glu-α-OMe-γ-Sar-(2R)-Ala-
H
(2S )-Leu-(2R)-Asp-α-OMe-β-OH.
O
O
CO2Me
Accordingly, α-methyl γ-benzyl N-Boc-(2R)-glutamate
diester 24 was treated with hydrogen chloride in ethyl acetate to
give the deprotected hydrochloride salt 23. This was coupled to
(2S )-N-Boc-phenylalanyl-β-alanine 22, prepared as described
previously,1,12 to give the tripeptide, N-Boc-(2S )-Phe-β-Ala-
(2R)-Glu-α-OMe-γ-OBn, 25, in 95% yield (see Scheme 5).
Removal of the benzyl ester protection through catalytic
hydrogenolysis gave the free acid 26 in 91% yield {mp 115–
117 ЊC; [α]D25 ϩ14.24 (MeOH)} which displayed the expected
spectral and analytical properties. Activation of the free acid 26
as the mixed isobutyl carbonic anhydride followed by reaction
with sarcosine benzyl ester gave the tetrapeptide N-Boc-(2S )-
Phe-β-Ala-(2R)-Glu-α-OMe-γ-Sar-OBn 27 in 80% yield. This
material {mp 110–112 ЊC; [α]D25 ϩ9.1 (MeOH)} gave satisfactory
analytical and mass data but, as is expected for sarcosine-
containing peptides which exist in similarly populated cis
and trans rotameric forms about the γ-Glu᎐Sar peptide bond,
25 R =Bn
26 R = H
iii
iv
O
O
H
N
H
N
OR
N
O
N
H
CH3
O
O
O
CO2Me
27 R = Bn
28 R = H
v
Scheme 5 Reagents, conditions (and yields): i, HCl, EtOAc, 0 ЊC (93%);
ii, IBCF, NMM, THF, Ϫ30 ЊC → room temp. (95%); iii, H2, Pd/C,
MeOH (91%); iv, IBCF, NMM, THF, Ϫ30 ЊC; then MeGlyOBnؒ
pTsOH, NMM, THF, Ϫ30 ЊC → room temp. (80%); v, H2, Pd/C,
MeOH (95%)
1
two sets of signals were observed in H and 13C NMR spectra.
The benzyl ester in tetrapeptide 27 was removed by catalytic
hydrogenolysis to give the free acid, N-Boc-(2S )-Phe-β-Ala-
(2R)-Glu-α-OMe-γ-Sar-OH, 28, in good recovery which was
sufficiently pure to use in subsequent coupling reactions, as
with β-benzyl α-methyl (2R)-aspartate diester hydrochloride,
prepared as described previously,1 to give the required, fully
protected linear heptapeptide 33 in 76% yield after purification
by column chromatography. The compound 33 {mp 137–
139 ЊC; [α]D25 ϩ8.2 (MeOH)}, which now contained all of the
required amino acid building blocks, gave a satisfactory micro-
analysis and displayed the expected NMR and mass spectral
properties. Hydrogenolysis of the benzyl ester group of pro-
tected heptapeptide 33 gave the free acid 34, which was con-
verted into its pentafluorophenyl ester derivative N-Boc-(2S )-
Phe-β-Ala-(2R)-Glu-α-OMe-γ-Sar-(2R)-Ala-(2S )-Leu-(2R)-
Asp-α-OMe-β-OPFP 35 in 91% yield by reaction with penta-
fluorophenol and EDCI, as described above for target 4 (see
Scheme 6).
1
judged by an examination of H and 13C NMR spectra.
Attention was now turned to the introduction of the C-
terminal tripeptide fragment (2R)-Ala-(2S )-Leu-(2R)-Asp-α-
OMe-β-OH in the target linear heptapeptide. Accordingly, N-
Boc-(2R)-alanine, activated as its mixed anhydride, was coupled
to benzyl (2S )-leucinate to give the fully protected dipeptide 29
in 85% yield {mp 89–91 ЊC; [α]D25 Ϫ8.91 (MeOH)} which dis-
played the expected spectral and analytical properties (see
Scheme 6). Acidolysis of the N-Boc group using hydrogen
chloride gas in ethyl acetate gave the amine hydrochloride 30
in 96% yield and this material was coupled to the mixed
anhydride-activated tetrapeptide acid 28 to afford the hexapep-
tide, N-Boc-(2S )-Phe-β-Ala-(2R)-Glu-α-OMe-γ-Sar-(2R)-Ala-
(2S )-Leu-OBn 31 in 77% yield {mp 74 ЊC (softening); [α]D25
ϩ6.83 (MeOH)} which gave satisfactory analytical data. The
benzyl ester protecting group was removed by hydrogenolysis
and the resulting free acid 32 was activated and then coupled
Removal of the N-Boc protecting group with TFA in dichlo-
romethane gave the amine salt which was thoroughly dried
under high vacuum. Treatment of this salt with DIPEA in
dichloromethane, under high-dilution conditions, allowed the
cyclisation to proceed and after 7 days the reaction was
J. Chem. Soc., Perkin Trans. 1, 1997
2517

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O
O
H
N
i
O
+
RHN
O
O
OH
N
H
TsOH•H2N
O
Me
Me
O
O
29 R = ButOCO
30 R = H•HCl
ii
iii
O
O
O
H
N
H
N
H
N
OR
N
H
N
O
N
H
O
O
Me
CH3
O
O
CO2Me
31 R = Bn
32 R = H
iv
v
O
OR
O
O
O
H
N
H
N
H
N
H
N
N
H
N
O
N
H
O
CO2Me
O
Me
O
O
CO2Me
vi
CH3
33 R = Bn
34 R = H
vii
F
O
O
F
F
O
O
O
H
N
H
N
H
N
H
N
F
N
H
N
O
N
H
O
CO2Me
F
O
Me
CH3
O
O
CO2Me
35
viii, ix
MeO
HN
O
Me
O
N
O
HN
HN
Me
O
O
NH
H
N
H
N
O
O
O
OMe
O
5
Scheme 6 Reagents, conditions (and yields): i, IBCF, NMM, THF, Ϫ30 ЊC → room temp. (85%); ii, HCl, EtOAc, 0 ЊC (96%); iii, 28, IBCF,
NMM, THF, Ϫ30 ЊC → room temp. (77%); iv, H2, Pd/C, MeOH (82%); v, IBCF, NMM, THF, Ϫ30 ЊC; then H-(2R)-Asp(OBn)OMeؒHCl, NMM,
THF, Ϫ30 ЊC → room temp (76%); vi, H2, Pd/C, 2% AcOH in MeOH (95%); vii, C6F5OH, EDCI, CH2Cl2 (91%); viii, TFA, CH2Cl2, 0 ЊC; ix,
DIPEA, CH2Cl2 (67%)
complete as judged by TLC analysis. Removal of the solvents
gave a white solid, which was triturated with diethyl ether and
collected by filtration to afford the 25-membered macrocyclic
heptapeptide 5 in 67% yield, mp 276–278 ЊC (decomp.). The
compound gave satisfactory analytical and mass data but dis-
accord with the existence of cis- and trans-γ-Glu–Sar rotamers.
As for all of the macrocyclic nodularin analogue diesters that
we have prepared, the microcystin-type macrocycle diester 5 did
not dissolve in methanol or chloroform but was quite soluble in
dimethyl sulfoxide. Thus, it was possible to prepare unfunction-
alised microcystin-type macrocycles in good yield using a
1
played two sets of signals in its H and 13C NMR spectra, in
2518
J. Chem. Soc., Perkin Trans. 1, 1997

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similar macrolactamisation strategy to that used for the nodu-
larin macrocycles.1,12
and 173.88 (CO, acid); m/z (EI) 149 (9%, [M ϩ H]ϩ), 148 (100,
Mϩ) and 102 (19, [M Ϫ CO2H ϩ H]ϩ).
The yield obtained for the formation of macrocycle 5 com-
pares well with the 56% yield reported by Chamberlain and co-
workers for the preparation of microcystin-LA dimethyl ester.17
The lengthy reaction times and relatively low yield of 41%
obtained for the preparation of the hydroxymethyl nodularin-
type macrocycle 4 compared with the model system 3 (obtained
in 89% yield), several other variants containing (2S )- and (2R)-
Pro in place of the sarcosine residue, and/or an α-benzyl ester of
(2R)-Asp in place of the Adda residue (each obtained in 50–90%
yield),28,29 and for motuporin,15 all of which were synthesised
using the same approach, suggests that the potentially H-
bonding hydroxy group retards the rate of macrolactamisation.
Nevertheless, the potential advantages offered by preforming a
functionalised macrocycle for post-cyclisation elaboration more
than outweigh the yield differences.This strategy is of particular
significance in the synthesis of libraries of macrocycles where it
is intended to modify each macrocycle with a range of exocyclic
appendages. Such synthetic studies are presently underway in
the construction of inhibitor structure–activity relationships for
PP1 and PP2A.
â-Methyl (2R)-N-(9Ј-phenylfluoren-9Ј-yl)aspartate ester 11
To a stirred solution of β-methyl (2R)-aspartate ester 10 (2.2 g,
15 mmol) in dry CH2Cl2 (30 cm3) was added chlorotrimethyl-
silane (TMSCl) (2.03 cm3, 16 mmol). Triethylamine (4.46 cm3,
32 mmol) was added after 2 h and, after another 15 min,
Pb(NO3)2 (3.3 g, 10 mmol) and a solution of 9-bromo-9-
phenylfluorene (6.42 g, 20 mmol) in dry CH2Cl2 (30 cm3) were
added. The mixture was stirred vigorously for 3 days after
which time methanol (7 cm3) was added and the reaction mix-
ture was filtered and concentrated under reduced pressure to
give a viscous residue. The residue was partioned between aq.
5% citric acid (80 cm3) and diethyl ether (80 cm3). The aqueous
phase was separated, and then was extracted with diethyl ether
(3 × 50 cm3). The combined ethereal extracts were washed with
brine (30 cm3), dried (MgSO4), and then concentrated under
reduced pressure to give a pale yellow foam. Recrystallisation
from CH2Cl2–hexane gave the required product as a crystalline
solid (4.70 g, 81%), mp 160 ЊC (lit.,24 160–161 ЊC for the 2S-
isomer) (Found: C, 74.6; H, 5.4; N, 3.5. C24H21NO4 requires C,
74.4; H, 5.4; N, 3.6%) (HRMS: Found: [M ϩ H]ϩ, 388.1549.
C24H22NO4 requires m/z, 388.1549); [α]D23 ϩ149.5 (c 0.75,
MeOH); νmax(Nujol)/cmϪ1 3291 (NH), 1749 (CO, acid) and
1696 (CO, ester); δH (200 MHz; C2HCl3) 1.93 (1 H, dd, J 4.4 and
16.9, 1 H of β-H2), 2.70 (1 H, dd, J 4.3 and 16.7, 1 H of β-H2),
2.87 (1 H, t, J 3.7, α-H), 3.66 (3 H, s, CH3) and 7.21–7.78 (13 H,
m, ArH); δC(50.3 MHz; C2HCl3), 36.43 (β-CH2), 52.55 (CH3),
53.09 (α-C), 72.98 (quaternary aliphatic), 120.88, 121.09,
125.21, 125.71, 126.28, 128.23, 128.74, 129.07, 129.14, 129.62
and 129.71 (ArCH), 140.97, 143.50, 147.47 and 149.15 (ArC
quaternary), 172.53 (CO, ester) and 175.58 (CO, acid); m/z (CI)
388 (3%, [M ϩ H]ϩ), 314 (4, [M ϩ CH2CO2CH3]ϩ) and 241
(100, PhFlϩ).
Experimental
NMR spectra were recorded on a Bruker AM-300 spectrometer
(1H, 300 MHz; 13C, 75 MHz; 19F, 282.3 MHz), a Varian Gemini
spectrometer (1H , 200 MHz; 13C, 50.3 MHz), a Varian Gemini
spectrometer (1H, 300 MHz; 13C, 75.4 MHz) and a Varian Unity
Plus 500 spectrometer (1H, 500 MHz; 13C, 125.6 MHz). 1H
NMR were referenced internally on 2HOH (δH 4.68), (C2H3)2SO
(δH 2.52), C2H3O2H (δH 3.35) and C2HCl3 (δH 7.27). 13C NMR
were referenced on C2HCl3 (δC 77.5), (C2H3)2SO (δC 35.60),
C2H3O2H (δC 49.15). J-values are given in Hz. IR spectra were
recorded using a Perkin-Elmer 1710 FT-IR spectrometer. The
samples were prepared as Nujol mulls or thin films between
sodium chloride discs. Absorption maxima are given in wave-
numbers (cmϪ1) relative to a polystyrene standard. Mps were
measured using an Electrothermal melting point apparatus and
are uncorrected. Optical rotations were measured on an Optical
Activity Ltd. AA-1000 polarimeter using 10 cm path length cells
at room temperature. Mass spectra were recorded on a VG
AutoSpec. Major fragments are given as percentages of the base
peak intensity. Solvents and common reagents were purified
according to the methods of Perrin, Armarego and Perrin.30
Analytical TLC was carried out on 0.25 mm precoated silica gel
plates (MN SIL G/UV254), and compounds were visualised by
UV fluorescence, iodine vapour, ethanolic phosphomolybdic
acid or aq. potassium permanganate. Light petroleum refers to
the fraction boiling at 40–60 ЊC.
Methyl (3R)-4-hydroxy-3-(9Ј-phenylfluoren-9Ј-ylamino)butyrate
12 (R ؍ Me)
To a stirred solution of the methyl ester 11 (2.00 g, 5.17 mmol) in
dry THF (25 cm3) at Ϫ15 ЊC was added NMM (568 mm3, 5.17
mmol). Isobutyl chloroformate (IBCF) (703 mm3, 5.17 mmol)
was added and the suspension was stirred at Ϫ15 ЊC for 5 min
and then was filtered directly into a solution of sodium borohy-
dride (118 mg, 3.10 mmol) in dry THF (15 cm3). The reaction
mixture was stirred at Ϫ15 ЊC for 15 min and was then allowed to
warm up to room temperature. After 3 h, water (10 cm3) was
carefully added and the resulting solution was concentrated
under reduced pressure until the THF had been removed. The
remaining aqueous solution was extracted with ethyl acetate
(3 × 25 cm3) and the combined organic extracts were washed
with brine (20 cm3), dried (MgSO4), and then concentrated
under reduced pressure to give the crude alcohol. Trituration
with diethyl ether gave the required alcohol as a solid which was
collected by filtration (1.75 g, 91%), mp 135–137 ЊC (Found: C,
76.9; H, 6.1; N, 3.7. C24H23NO3 requires C, 77.2; H, 6.2; N,
3.75%) (HRMS: Found: [M ϩ H]ϩ, 374.1752. C24H24NO3
requires m/z 374.1756); [α]D23 ϩ27.81 (c 0.52, MeOH);
νmax(Nujol)/cmϪ1 3382 (NH), 3300–2800br (OH) and 1706 (CO,
ester); δH (200 MHz; C2HCl3) 2.08 (1 H, dd, J 5.9 and 15.6, 1 H of
α-H2), 2.20 (1 H, dd, J 5.5 and 15.5, 1 H of α-H2), 2.59 (1 H, quin,
J 5.2, β-H), 2.71 (1 H, br s, OH), 2.99 (1 H, dd, J 4.6 and 10.8, 1 H
of CH2OH), 3.16 (1 H, dd, J 5.0 and 10.9, 1 H of CH2OH), 3.63
(3 H, s, CH3) and 7.71–7.74 (13 H, m, ArH); δC(50.3 MHz;
C2HCl3) 37.98 (α-CH2), 51.62 (β-CH), 53.00 (CH3), 64.91
(CH2OH), 72.91 (quaternary aliphatic), 120.62, 125.46, 125.66,
126.41, 127.75, 128.39, 128.71, 128.87, 128.98 and 129.04
(ArCH), 140.59, 141.04, 145.38, 150.14 and 150.34 (ArC qua-
ternary) and 173.42 (CO, ester); m/z (CI) 374 (15%, [M ϩ H]ϩ),
342 (30, [M Ϫ OCH3]ϩ), 241 (100, PhFlϩ), 134 (49, [M ϩ H Ϫ
PhFl ϩ H]ϩ) and 102 (22, [M ϩ H Ϫ PhFl Ϫ CH2OH]ϩ).
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.
â-Methyl (2R)-aspartate ester 10
β-Methyl (2R)-aspartate ester hydrochloride (7.34 g, 40 mmol)
(prepared using the procedure of Schwarz, Bumpus and Page22)
and propylene oxide (50 cm3) were refluxed in dry ethanol (150
cm3) for 3 h. On cooling the resulting precipitate was filtered off,
and washed with diethyl ether (25 cm3) to yield the title ester as
a solid (5.29 g, 90%), mp 194–195 ЊC (lit.,24 194–195 ЊC for the
2S-isomer) (Found: C, 41.0; H, 6.3; N, 9.3. C5H9NO4 requires
C, 40.8; H, 6.2; N, 9.5%) (HRMS: Found: [M ϩ H]ϩ, 148.0610.
C5H10NO4 requires m/z, 148.0610); [α]D23 Ϫ2.2 (c 0.5, water);
νmax(Nujol)/cmϪ1 1752 (CO, acid) and 1738 (CO, ester); δH (200
MHz; C2H3O2H), 2.84 (1 H, dd, J 8.3 and 17.7, 1 H of β-H2)
3.05 (1 H, dd, J 3.9 and 17.7, 1 H of β-H2), 3.75 (3 H, s, CH3)
2
and 3.88 (1 H, dd, J 3.9 and 38.3, α-H); δC(50.3 MHz; H2O)
35.43 (β-CH2), 51.65 (CH3), 53.61 (α-C), 173.49 (CO, ester)
J. Chem. Soc., Perkin Trans. 1, 1997
2519

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(3R)-3-(9Ј-Phenylfluoren-9Ј-ylamino)butyrolactone 13
H, br d, NH) and 7.34 (5 H, s, ArH); δC(75.4 MHz; C2HCl3)
This compound could be obtained by concentration of the
filtrate obtained from trituration of the hydroxy ester 12
(R = Me) (see above). The lactone 13 was obtained as a yellow
foam and was then recrystallised from diethyl ether–light pet-
roleum to give a solid. It was noted that addition of an excess
of sodium borohydride in the synthesis of hydroxy ester 12
(R = Me) (see preceding experiment) resulted in reduced yields
of the hydroxy ester 12 (R = Me) and the formation of larger
amounts of the lactone 13 (5–63%), mp 156–158 ЊC (Found: C,
80.6; H, 5.4; N, 4.0. C23H19NO2 requires C, 80.9; H, 5.6; N,
4.1%) (HRMS: Found: [M ϩ H]ϩ, 342.149 021. C23H20NO2
requires m/z 342.1494); [α]D23 ϩ20.0 (c 1.0, CH2Cl2); νmax(Nujol)/
cmϪ1 3322 (NH) and 1784 (CO, lactone); δH (200 MHz; C2HCl3)
2.14 (2 H, d, J 7.8, α-H2), 3.18 (1 H, quin, J 8.0, β-H), 3.77 (2 H,
quin, J 6.8, CH2OH) and 7.15–7.75 (13 H, m, ArH); δC(50.3
MHz; C2HCl3) 37.48 (α-CH2), 50.50 (β-CH), 73.21 (quaternary
aliphatic), 74.72 (CH2OH), 120.84, 120.92, 125.49, 126.41,
126.52, 128.05, 128.69, 128.80, 128.96, 129.06 and 129.36
(ArCH), 140.65, 140.90, 144.50, 149.60 and 149.76 (ArC qua-
ternary) and 176.13 (CO, lactone); m/z (CI) 342 (21%, [M ϩ
H]ϩ), 241 (17, PhFlϩ) and 102 (29, [M ϩ H Ϫ PhFl ϩ H]ϩ).
28.34 [(CH3)3], 36.08 (α-CH2), 49.48 (β-CH), 64.54 (CH2OH),
66.69 (PhCH2), 79.95 [C(CH3)3], 128.31, 128.50 and 128.75
(ArCH), 135.73 (ArC quaternary), 155.99 (CO, urethane) and
171.82 (CO, ester); m/z (CI) 310 (100%, [M ϩ H]ϩ), 291 (7,
[M ϩ H Ϫ H2O ϩ H]ϩ), 254 (95, [M ϩ H Ϫ C4H9 ϩ H]ϩ) and
210 (37, [M ϩ H Ϫ C5H9O2 ϩ H]ϩ).
Benzyl (3R)-3-amino-4-hydroxybutyrate trifluoroacetate salt 15
To a solution of benzyl (R)-3-(tert-butoxycarbonylamino)-4-
hydroxybutyrate 14 (1.10 g, 3.56 mmol) in CH2Cl2 (10 cm3) was
added TFA (10 cm3). The reaction mixture was stirred at room
temperature for 45 min, after which time the reaction was com-
plete as judged by TLC. The solution was diluted with toluene
(10 cm3) and was then concentrated under reduced pressure.
The residue was dissolved in water (20 cm3) and the solution
was washed with ethyl acetate (15 cm3). The aqueous phase was
concentrated under reduced pressure to give an oil, refractory
to crystallisation, containing up to 10% of the lactone. This
compound was used directly for the next step without further
purification (0.85 g, 74%); δH (200 MHz; 2H2O) 2.67 (2 H, br, α-
H2), 3.45–3.73 (3 H, m, CH2OH and β-H), 5.08 (2 H, s, PhCH2)
and 7.22 (5 H, s, ArH); δC(75.4 MHz; 2H2O) 33.30 (α-CH2), 49.39
(β-CH), 60.68 (CH2OH), 67.63 (PhCH2), 128.56, 128.90 and
128.99 (ArCH), 135.32 (ArC quaternary) and 171.78 (CO).
When the Boc deprotection was attempted using dry HCl(g)
in ethyl acetate, hydrolysis of the benzyl ester also occurred to
give the hydroxyamino acid salt, (3R)-3-amino-4-hydroxy-
butyric acid hydrochloride, mp 177–178 ЊC; νmax(Nujol)/cmϪ1
3250–2400br (NH and OH) and 1771 (CO); δH (200 MHz;
2H2O) 2.71–2.81 (1 H, m, 1 H of α-H2), 3.12–3.31 (1 H, m, 1 H
of α-H2), 4.31–4.40 (1 H, m, β-H) and 4.48–4.73 (2 H, m,
CH2OH); δC(75.4 MHz; 2H2O) 33.14 (α-CH2), 47.66 (β-
CH), 71.65 (CH2OH) and 177.84 (CO); m/z (CI) 102 (100%,
[M ϩ H Ϫ HCl Ϫ H2O]).
(3R)-4-Hydroxy-3-(9Ј-phenylfluoren-9Ј-ylamino)butyric acid 12
(R ؍ H)
To a stirred solution of the lactone 13 (1.00 g, 2.68 mmol) in
methanol (30 cm3) was added 1 mol dmϪ3 aq. NaOH (5 cm3).
The reaction mixture was stirred at room temperature for 2 h
and then the solvent was evaporated off under reduced pres-
sure. The resulting Naϩ salt was dissolved in water (25 cm3) and
the solution was washed with ethyl acetate (20 cm3). The aque-
ous phase was carefully partitioned between cold 10% aq. citric
acid and ethyl acetate (1:1; 40 cm3) at 0 ЊC. The aqueous phase
was extracted with ethyl acetate (3 × 25 cm3), and the combined
organic extracts were washed with brine (25 cm3), dried
(MgSO4), and concentrated under reduced pressure to yield
the crude hydroxy acid. Recrystallisation from aq. methanol
afforded the hydroxy acid 12 (R = H) as a solid (0.82 g, 85%),
mp 119–122 ЊC (Found: C, 73.1; H, 6.1; N, 3.7. C23H21NO3ؒH2O
requires C, 73.2; H, 6.1; N, 3.7%) (HRMS: Found: [M ϩ
H Ϫ H2O]ϩ, 342.1496. C23H20NO2 requires m/z 342.1494);
[α]D23 ϩ151 (c 0.6, MeOH); νmax(Nujol)/cmϪ1 3395 (NH), 3350–
2800br (OH) and 1706 (CO, acid); δH (200 MHz; C2H3O2H) 1.98
(1 H, dd, J 5.7 and 16.3, 1 H of α-H2), 2.28 (1 H, dd, J 6.5 and
16.3, 1 H of α-H2), 2.69 (1 H, quin, J 5.9, β-H), 2.99 (1 H, dd,
J 5.4 and 11.3, 1 H of CH2OH), 3.13 (1 H, dd, J 4.8 and 11.3,
1 H of CH2OH) and 7.15–7.90 (13 H, m, ArH); δC(50.3 MHz;
C2H3O2H) 38.25 (α-CH2), 54.79 (β-CH), 63.50 (CH2OH),
74.24 (quaternary aliphatic), 121.92, 122.10, 127.21, 127.31,
127.36, 129.49, 129.68, 129.81, 129.93 130.22, 131.11 and
131.31 (ArCH), 141.94, 142.70, 143.36, 147.18 and 147.59
(ArC quaternary) and 177.93 (CO, acid); m/z (CI) 342 (100%,
[M ϩ H Ϫ H2O]ϩ), 241 (93, PhFlϩ) and 102 (37, [M ϩ H Ϫ
H2O Ϫ PhFl ϩ H]ϩ).
Benzyl (3R)-3-[(tert-butoxycarbonyl)-(2S )-phenylalanylamino]-
4-hydroxybutyrate 16
To a stirred solution of (2S )-N-Boc-phenylalanine (0.58 g,
2.19 mmol) in dry THF (25 cm3) at Ϫ15 ЊC was added NMM
(240 mm3, 2.19 mmol). IBCF (300 mm3, 2.19 mmol) was added
and the suspension was stirred at Ϫ15 ЊC for 10 min. A mixture
of benzyl (3R)-3-amino-4-hydroxybutyrate trifluoroacetate salt
15 (0.71 g, 2.19 mmol) and NMM (240 mm3, 2.19 mmol) in dry
THF (20 cm3) was then added. The reaction mixture was
allowed to warm up to room temperature and was then stirred
for a further 2 h. The trifluoroacetate salts were removed by
filtration and the solution was concentrated under reduced
pressure to yield an oil. The residue was re-dissolved in ethyl
acetate (30 cm3), and the solution was washed successively with
water (20 cm3), 5% aq. NaHCO3 (20 cm3), 10% aq. citric acid
(20 cm3) and brine (20 cm3), dried (MgSO4), and concentrated
under reduced pressure to give a solid. Recrystallisation from
ethyl acetate–hexane afforded the pure dipeptide as a crystalline
solid (0.90 g, 90%), mp 72–73 ЊC; νmax(Nujol)/cmϪ1 3490–3220
(NH and OH), 1757 (CO, urethane), 1694 (CO, ester) and 1656
(CO, amide); δH [300 MHz; (C2H3)2SO, mixture of rotamers]
1.27 and 1.29 [9 H, 2 × s, (CH3)3], 2.58–2.91 [3 H, m, 1 H of
CH2 (Phe) and α-H2(Ahb)], 3.24–3.54 [3 H, m, 1 H of CH2
(Phe) and CH2OH], 4.00–4.11 [1 H, m, β-H (Ahb)], 4.38–4.48 [1
H, m, α-H (Phe)], 5.04 and 5.06 (2 H, 2 × s, OCH2Ph), 6.80 and
6.91 (1 H, 2 × d, J 8.8, NH urethane), 7.17–7.36 (10 H, m, ArH)
and 7.87 and 8.49 (1 H, 2 × d, J 5.8, NH amide); δC[75.4 MHz;
(C2H3)2SO, mixture of rotamers] 28.09 [(CH3)3], 33.82 [α-CH2
(Ahb)], 35.78 and 37.73 [CH2 (Phe)], 45.94 [β-CH (Ahb)
minor), 48.07 [β-CH (Ahb) major], 55.71 [α-C (Phe)], 62.05
(PhCH2 minor), 65.55 (PhCH2 major), 73.16 (CH2OH),
77.98 [C(CH3)3], 126.24, 126.38, 128.02, 128.07, 128.16, 128.52
and 129.35 (ArCH), 136.26 and 138.34 (ArC quaternary),
155.36 (CO, urethane) and 171.13, 171.51, 171.97 and 176.07
Benzyl (3R)-3-(tert-butoxycarbonylamino)-4-hydroxybutyrate 14
The (tert-butoxycarbonylamino)hydroxybutyrate ester 14 was
prepared in a manner identical with that described for the
hydroxy-(9Ј-phenylfluoren-9Ј-yl)butyrate 12 (R = Me), using β-
benzyl (2R)-N-(tert-butoxycarbonyl)aspartate (3.00 g, 9.29
mmol) as the starting material. The crude alcohol was recrystal-
lised from diethyl ether–hexane to give the required alcohol as a
crystalline solid (2.55 g, 89%), mp 60 ЊC (lit.,25 59–62 ЊC for 2S-
isomer) (Found: C, 62.1; H, 7.5; N, 4.5. C16H23NO5 requires: C,
62.1; H, 7.5; N, 4.5%); [α]D23 ϩ4.60 (c 0.38, in MeOH) {lit.,25 [α]D
Ϫ3.4 (c 1, MeOH (for 2S-isomer)}; νmax(Nujol)/cmϪ1 3555–
3100br (OH), 3386 (NH), 1727 (CO, urethane) and 1697 (CO,
ester); δH (300 MHz; C2HCl3) 1.42 [9 H, s, (CH3)3], 2.58–2.79 (1
H, br s, OH), 2.66 (2 H, d, J 6.0, α-H2), 3.67 (2 H, d, J 4.8,
CH2OH), 3.94–4.04 (1 H, m, β-H), 5.12 (2 H, s, PhCH2), 5.21 (1
2520
J. Chem. Soc., Perkin Trans. 1, 1997

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(CO); m/z (CI) 457 (5%, [M ϩ H]ϩ), 381 (11, [M ϩ H Ϫ
C6H5 ϩ H]ϩ), 91 (84, PhCH2ϩ) and 57 (100, C4H9ϩ).
[γ-CH2 (Glu)], 34.26 [α-CH2 (Ahb)], 34.63 (NCH3), 36.27
[β-CH2 (Asp)], 36.42 (NCH3), 37.93 [CH2 (Phe)], 46.37 [β-CH
(Ahb)], 48.88 [α-C (Asp)], 48.99 [α-C (Asp)], 50.12 [CH2 (Sar)],
52.23 (OCH3), 52.29 (OCH3), 52.65 (OCH3), 53.74 [α-C (Glu)],
53.84 [α-C (Glu)], 56.18 [α-C (Phe)], 66.36 (PhCH2), 73.62
(CH2OH), 78.53 [C(CH3)3], 126.82, 128.53, 128.62, 129.01
and 129.78 (ArCH), 136.48 and 138.53 (ArC quaternary),
155.83 (CO, urethane), 168.95, 169.07, 170.49, 171.52,
171.65, 172.37, 172.43, 172.61 and 173.47 (CO); m/z (FAB)
822 (2%, [M ϩ Na]ϩ), 800 (12, [M ϩ H]ϩ), 742 (6, [M ϩ H Ϫ
C4H9 ϩ H]ϩ), 700 (11, [M ϩ H Ϫ C5H9O2 ϩ H]ϩ) and 120 (100,
[C8H9N ϩ H]ϩ).
(3R)-3-[(tert-Butoxycarbonyl)-(2S )-phenylalanylamino]-4-
hydroxybutyric acid 17
To a solution of dipeptide benzyl ester 16 (0.90 g, 1.97 mmol) in
ethanol (25 cm3) was added 10% palladium on carbon (80 mg)
and the mixture was stirred under hydrogen for 4 h. The catalyst
was removed by filtration through a pre-washed Celite pad and
the filtrate was concentrated under reduced pressure to give the
title acid as a solid (0.71 g, 98%), mp 95–97 ЊC (Found: C, 56.35;
H, 6.95; N, 7.3. C18H26N2O6ؒH2O requires C, 56.25; H, 7.35; N,
7.3%); [α]D23 ϩ23.43 (c 0.51, MeOH); νmax(Nujol)/cmϪ1 3510–
3200 (NH and OH), 1762 (CO, urethane), 1690 (CO) and 1665
(CO, amide); δH [200 MHz; C2HCl3, mixture of rotamers] 1.35
and 1.38 [9 H, 2 × s, (CH3)3], 2.12–2.18 [1 H, m, 1 H of α-H2
(Ahb)], 2.72 [1 H, dd, J 8.0 and 17.9, 1 H of α-H2 (Ahb)], 3.00 [2
H, br, CH2 (Phe)], 4.08 (1 H, dd, J 2.8 and 9.9, 1 H of CH2OH),
4.23–4.32 [1 H, m, β-H (Ahb)], 4.43 (1 H, dd, J 6.0 and 9.9, 1 H
of CH2OH), 4.58–4.68 [1 H, m, α-H (Phe)], 5.26 [1 H, d, J 8.0,
NH (Phe)], 6.93 [1 H, d, J 6.3, NH (Ahb)] and 7.16–7.37 (5 H,
m, ArH); δH [75.4 MHz; C2HCl3, mixture of rotamers] 28.12
[(CH3)3], 34.27 [α-CH2 (Ahb) major], 35.01 [α-CH2 (Ahb)
minor], 38.63 [CH2(Phe)], 46.12 [β-CH (Ahb) major], 48.11 [β-
CH (Ahb) minor], 55.73 [α-C (Phe)], 73.62 (CH2OH), 80.40
[C(CH3)3], 126.92, 127.12, 128.30, 128.58, 128.67 and 129.27
(ArCH), 136.41 (ArC quaternary), 155.66 (CO, urethane
major), 155.91 (CO, urethane minor), 171.98, 172.12, 174.37
and 175.73 (CO); m/z (CI) 349 (13%, [M ϩ H Ϫ H2O]ϩ), 293
(100, [M ϩ H Ϫ H2O Ϫ C4H9 ϩ H]ϩ) and 249 (25, [M ϩ H Ϫ
H2O Ϫ C5H9O2 ϩ H]ϩ).
(3R)-3-[(tert-Butoxycarbonyl)-(2S )-phenylalanylamino]-4-
hydroxybutyryl-[á-methyl (2R)-glutamyl]-ã-sarcosyl-[á-methyl
(2R)-aspartate] diester 19
To a solution of pentapeptide benzyl ester 18 (0.50 g, 0.626
mmol) in ethanol (20 cm3) was added 10% palladium on carbon
(75 mg) and the mixture was stirred under hydrogen for 6 h. The
catalyst was removed by filtration through a pre-washed Celite
pad and the filtrate was concentrated under reduced pressure to
give the title compound 19 as a solid (0.43 g, 97%), mp 97–98 ЊC
(HRMS: Found: [M ϩ H]ϩ, 710.3232. C32H48N5O13 requires
m/z 710.3249); νmax(Nujol)/cmϪ1 3555br (OH and NH), 1746
(CO) and 1717 (CO); δH (300 MHz; C2HCl3, mixture of rotam-
ers) 1.41 [9 H, s, (CH3)3], 1.83–1.97 [2 H, m, β-H2 (Glu)], 2.14–
2.59 [4 H, m, γ-H2 (Glu) OH and 1 H of α-H2 (Ahb)], 2.74 [1 H,
dd, J 8.0 and 17.9, 1 H of α-H2 (Ahb)], 2.83–3.15 [4 H, m, CH2
(Phe) and β-H2 (Asp)], 3.20 and 3.11 (3 H, 2 × s, NCH3), 3.73,
3.74 and 3.76 (6 H, 3 × s, 2 × OCH3), 3.74–3.85 [2 H, br, CH2
(Sar)], 4.07 (1 H, dd, J 3.3 and 9.0, 1 H of CH2OH), 4.24–4.31
[2 H, m, α-H (Glu) and β-H (Ahb)], 4.44 (1 H, dd, J 6.0 and 9.9,
1 H of CH2OH), 4.56–4.60 [1 H, br, α-H (Phe)], 4.71–4.84 [1 H,
m, α-H (Asp)], 5.03 [1 H, d, J 8.0, NH (Phe)], 6.31 (1 H, br,
NH) and 7.17–7.43 (7 H, m, ArH and 2 × NH); δC(75.4 MHz;
C2HCl3, mixture of rotamers) 27.40 [β-CH2 (Glu)], 27.80
[β-CH2 (Glu)], 28.13 [(CH3)3], 28.47 [γ-CH2 (Glu)], 28.54
[γ-CH2 (Glu)], 34.33 [α-CH2 (Ahb)], 34.99 (NCH3), 35.30
[β-CH2 (Asp)], 35.53 (NCH3), 38.22 [CH2 (Phe)], 46.29 [β-CH
(Ahb)], 48.15 [α-C (Asp)], 49.94 [CH2 (Sar)], 52.34 (OCH3),
52.61 (OCH3), 52.98 (OCH3), 54.13 [α-C (Glu)], 55.88 [α-C,
(Phe)], 73.00 (CH2OH), 79.27 [C(CH3)3], 127.19, 128.51,
128.77, 129.08 and 129.19 (ArCH), 136.22 (ArC quaternary),
155.36 (CO, urethane), 168.87, 171.11, 171.46, 172.05, 172.63,
172.82 and 174.55 (CO); m/z (FAB) 732 ([M ϩ Na]ϩ, 12%), 710
(12, [M ϩ H]ϩ), 610 (14, [M ϩ H Ϫ C5H9O2 ϩ H]ϩ), 91 (100,
PhCH2ϩ) and 120 (100, [C8H9N ϩ H]ϩ).
â-Benzyl (3R)-3-[(tert-butoxycarbonyl)-(2S )-phenylalanyl-
amino]-4-hydroxybutyryl-[á-methyl (2R)-glutamyl]-ã-sarcosyl-
[á-methyl (2R)-aspartate] triester 18
To a stirred solution of (3R)-3-[Boc-(2S )-phenylalanylamino]-
4-hydroxybutyric acid 17 (468 mg, 1.28 mmol) in dry THF (15
cm3) at Ϫ15 ЊC was added NMM (141 mm3, 1.28 mmol). IBCF
(174 mm3, 1.28 mmol) was added and the suspension was
stirred at Ϫ15 ЊC for 10 min. A mixture of the tripeptide tri-
ester hydrochloride 8ؒHCl (623 mg, 1.28 mmol) and NMM
(141 mm3, 1.28 mmol) in a dry mixture of DMF (2 cm3) and
THF (8 cm3) was then added. The reaction mixture was
allowed to warm up to room temperature and was stirred for a
further 3 h. The hydrochloride salts were removed by filtration
and the solution was concentrated under reduced pressure. The
residue was re-dissolved in ethyl acetate (25 cm3), then washed
successively with water (15 cm3), 5% aq. NaHCO3 (20 cm3),
10% aq. citric acid (20 cm3) and brine (20 cm3), dried (MgSO4),
and concentrated under reduced pressure. The crude yellow
solid was chromatographed (CH2Cl2–MeOH, 94:6) to give the
pure pentapeptide as a crystalline solid (849 mg, 83%), mp 89–
91 ЊC (Found: C, 58.2; H, 6.9; N, 8.6. C39H53N5O13 requires C,
58.55; H, 6.7; N, 8.75%) (HRMS: Found: [M ϩ H]ϩ, 800.3700.
C39H54N5O13 requires m/z 800.3718); [α]D23 ϩ9.03 (c 0.31,
CH2Cl2); νmax(Nujol)/cmϪ1 3375br (NH and OH), 1720br (CO)
and 1684 (CO, amide); δH (300 MHz; C2HCl3, mixture of
rotamers) 1.28 [9 H, s, (CH3)3], 1.77–1.95 [2 H, m, β-H2 (Glu)],
2.17–2.45 [3 H, m, γ-H2 (Glu) and 1 H of β-H2 (Ahb)], 2.70–
2.91 [5 H, m, β-H2 (Asp), CH2 (Phe) and 1 H of β-H2 (Ahb)],
2.73 and 2.88 (3 H, 2 × s, NCH3), 3.58, 3.59 and 3.60 (6 H,
3 × s, 2 × OCH3), 3.72 [1 H, d, J 6.9, 1 H of CH2 (Sar)], 3.93–
4.18 [4 H, m, β-H (Ahb), α-H (Glu), α-H (Phe) and 1 H of CH2
(Sar)], 4.41 (2 H, d, J 5.2, CH2OH), 4.65–4.72 [1 H, m, α-H
(Asp)], 5.09 (2 H, s, PhCH2), 6.92 (1.4 H, d, J 8.2, 1.4 NH),
7.17–7.39 (10 H, m, ArH),7.52 and 7.58 (0.6 H, 2 × d, J 7.7 and
8.0, 0.6 NH), 8.37 (0.6 H, d, J 7.7, 0.6 NH), 8.50 (1 H, d, J 5.5,
NH) and 8.61 (0.4 H, d, J 8.0, 0.4 NH); δC[75.4 MHz;
(C2H3)2SO, mixture of rotamers] 28.05 [β-CH2 (Glu)], 28.28
[β-CH2 (Glu)], 28.52 [(CH3)3], 28.88 [γ-CH2 (Glu)], 29.05
â-Pentafluorophenyl (3R)-3-[(tert-butoxycarbonyl)-(2S )-phenyl-
alanylamino]-4-hydroxybutyryl-[á-methyl (2R)-glutamyl]-ã-
sarcosyl-[á-methyl (2R)-aspartate] triester 20
To a stirred solution of the pentapeptide carboxylic acid 19
(256 mg, 0.36 mmol) in CH2Cl2 (10 cm3) at 0 ЊC was added pen-
tafluorophenol (200 mg, 1.08 mmol) followed by EDCI (161
mg, 0.54 mmol). The reaction mixture was allowed to warm to
room temperature and was then stirred for a further 12 h. The
solution was concentrated under reduced pressure to give a yel-
low foam, which was purified by flash chromatography on silica
and elution with CH2Cl2 to remove pentafluorophenol, then
with CH2Cl2–MeOH (96:4) to give the required triester as an
off-white solid (193 mg, 61%), mp 61–65 ЊC (HRMS: Found:
[M ϩ Na]ϩ, 898.2932. C38H46F5N5NaO13 requires m/z
898.2910); νmax(thin film)/cmϪ1 3310br (NH and OH), 1752
(CO), 1720br (CO) and 1675br (CO, amide); δH (300 MHz;
C2HCl3, mixture of rotamers) 1.34 [9 H, s, (CH3)3], 1.86–1.95 [1
H, m, 1 H of β-H2 (Glu)], 2.24–2.56 [6 H, m, α-H2 (Ahb), γ-H2
(Glu), OH, and 1 H of β-H2 (Glu)], 2.90–3.10 [2 H, m, CH2
(Phe)], 2.95 and 3.03 (3 H, 2 × s, NCH3), 3.24–3.27 [2 H, m,
β-H2 (Asp)], 3.58 (2 H, br, CH2OH), 3.69, 3.72, 3.75 and 3.78
(6 H, 4 × s, 2 × OCH3), 3.84 [1 H, d, J 16.2, 1 H of CH2 (Sar)],
J. Chem. Soc., Perkin Trans. 1, 1997
2521

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4.11–4.20 [1 H, m, α-H (Glu)], 4.25–4.33 [1 H, m, β-H (Ahb)],
4.38 [1 H, d, J 16.2, 1 H of CH2 (Sar)], 4.46–4.60 [1 H, m,
α-H (Phe)], 4.98–5.05 [1 H, m, α-H (Asp)], 5.29 (1 H, br, NH
urethane), 7.16–7.29 (6 H, m, ArH and 1 NH), 7.32 (1 H, 2 × d,
J 6.9, NH) and 7.55 (1 H, s × d, J 7.7, NH); δC[75.4 MHz;
(C2H3)2SO, mixture of rotamers] 28.05 [β-CH2 (Glu)], 28.28 [β-
CH2 (Glu)], 28.54 [(CH3)3], 28.91 [γ-CH2 (Glu)], 29.07 [γ-CH2
(Glu)], 34.28 [α-CH2 (Ahb)], 34.62 (NCH3), 36.18 [β-CH2
(Asp)], 36.30 (NCH3), 37.92 [CH2 (Phe)], 46.36 [β-CH (Ahb)],
48.91 [α-C (Asp)], 49.02 [α-C (Asp)], 50.08 [CH2 (Sar)], 52.31
(OCH3), 52.59 (OCH3), 53.74 [α-C (Glu)], 56.18 [α-C (Phe)],
77.77 (CH2OH), 78.53 [C(CH3)3], 126.82, 128.62 and 129.77
(ArCH), 138.54 (ArC quaternary), 155.82 (CO, urethane) and
168.95, 171.94, 172.11, 172.33, 172.42, 172.61 and 173.50 (CO);
m/z (FAB) 898 (4%, [M ϩ Na]ϩ), 876 (3, [M ϩ H]ϩ), 776 (5,
[M ϩ H Ϫ C5H9O2 ϩ H]ϩ) and 147 (100, [C9H9NO]ϩ).
(Glu)] and 172.7 (CO); m/z (FAB) 614 (3%, [M ϩ Na]ϩ), 592
(2, [M ϩ H]ϩ), 582 (9, [M ϩ Na Ϫ CH3OH]ϩ), 560 (4,
1
[M ϩ H Ϫ CH3OH]ϩ) and 322 (100). The H and 13C NMR
spectra recorded above in (C2H3)2SO were assigned using
COSY, TOCSY, HMBC and HSQC techniques and showed the
existence of two other minor conformers (~5% of the total for
1
each). H and 13C NMR spectra were also obtained in C2HCl3
and these showed the presence of two conformations in
exchange on the NMR time-scale (see Results and discussion
section). Full details and conformational structural assign-
ments will be described for this compound together with those
for several other functionalised nodularin macrocycles in due
course.28
á-Methyl ã-benzyl (2R)-glutamate diester hydrochloride 23
Hydrogen chloride gas was bubbled into dry ethyl acetate (50
cm3) at 0 ЊC for 1 h. To this was added γ-benzyl N-(tert-
butoxycarbonyl)-α-methyl-(2R)-glutamate 24 (5.27 g, 15 mmol)
as a solution in ethyl acetate (20 cm3) and the solution was
stirred at 0 ЊC for a further 1 h before being concentrated under
reduced pressure to give the required product as a solid (4.02 g,
93%), mp 125–127 ЊC [lit.,31 129–135 ЊC for (2S )-isomer];
cyclo-[(R)-Ahb-á-OMe-(R)-Glu-ã-Sar-á-OMe-(R)-Asp-â-(S )-
Phe] diester 4
To a solution of the N-tert-butoxycarbonyl-protected penta-
fluorophenyl ester 20 (165 mg, 0.189 mmol) in CH2Cl2 (5 cm3)
was added TFA (5 cm3).The reaction mixture was stirred at
room temperature for 45 min, after which time reaction was
complete as judged by TLC. The solution containing the tri-
fluoroacetate salt 21 was diluted with toluene (10 cm3) and was
then concentrated under reduced pressure to give an oil, which
was thoroughly dried in vacuo for 6 h. The residue was dissolved
in CH2Cl2 (175 cm3), then treated with DIPEA (988 mm3, 5.67
mmol), and the mixture was stirred at room temperature for
nine days, when TLC analysis indicated that reaction was com-
plete. The reaction mixture was concentrated under reduced
pressure to give a pale brown solid. Purification by flash
chromatography on silica and elution first with CH2Cl2, then
with CH2Cl2–MeOH (94:6), gave an oil. This was triturated
with diethyl ether to give an off-white solid, which was collected
by filtration and washed on the pad with diethyl ether to give
the required functionalised macrocycle 4 as an off-white powder
(46 mg, 41%), mp 96–100 ЊC (HRMS: Found: [M ϩ Na Ϫ
CH3OH]ϩ, 582.2150. C26H33N5NaO9 requires m/z, 582.2176);
[α]D23 Ϫ64.6 (c 0.08, CH2Cl2); νmax(thin film)/cmϪ1 3307br (NH
and OH), 1760 (CO), 1742 (CO) and 1652 (CO, amide); δH [500
MHz; (C2H3)2SO, mixture of rotamers] 1.78 and 1.90 [0.76 H,
2 × m, β-H2 (Glu) minor], 1.79 and 1.94 [1.24 H, 2 × m, β-H2
(Glu) major], 2.14 and 2.78 [2 H, m, α-H2 (Ahb)], 2.27 [0.76 H,
m, γ-H2 (Glu) minor], 2.38 [1.24 H, m, γ-H2 (Glu) major], 2.51
and 2.60 [0.76 H, 2 × m, β-H2 (Asp) minor], 2.56 [1.24 H, m, β-
H2 (Asp) major], 2.74 (1.14 H, s, NCH3 minor), 2.79 and 2.90 [2
H, 2 × m, CH2 (Phe)], 2.88 (1.86 H, s, NCH3 major), 3.52, 3.55,
3.59 and 3.62 (6 H, 4 × s, 2 × OCH3), 3.87–3.99 and 3.90–4.01
[2 H, 2 × m, CH2 (Sar)], 4.02 [0.38 H, m, α-H (Glu) minor], 4.05
[0.62 H, m, α-H (Glu) major], 4.39 [1 H, m, β-H (Ahb)], 4.40 [1
H, m, α-H (Phe)], 4.54 [0.38 H, m, α-H (Asp) minor], 4.56 [0.62
H, m, α-H (Asp) major], 7.14–7.30 (5 H, m, ArH), 7.47 [0.38 H,
d, J 8.1, 0.38 NH (Glu) minor], 7.53 [0.62 H, d, J 6.7, 0.62 NH
(Glu) major], 8.12 [0.38 H, d, J 8.1, 0.38 NH (Asp) minor], 8.21
[1 H, 2 × d (overlapping), J 5.4 and 6.7, NH (Phe)], 8.33 [0.62
H, d, J 8.1, 0.62 NH (Asp) major] and 8.47 [1 H, 2 × d (over-
lapping), J 5.4 and 9.4, NH (Ahb)]; δC(125.7 MHz; (C2H3)2SO,
mixture of rotamers] 25.7 [β-CH2 (Glu) major], 25.9 [β-CH2
(Glu) minor], 28.0 [γ-CH2 (Glu) minor], 28.2 [γ-CH2 (Glu)
major], 33.4 [α-CH2 (Ahb)], 34.9 (NCH3 minor), 35.6 (NCH3
major), 36.3 [β-CH2 (Asp) major], 36.4 [β-CH2 (Asp) minor],
37.4 [CH2 (Phe)], 45.5 [β-CH (Ahb)], 48.3 [α-C (Asp) minor],
48.4 [α-C (Asp) major], 49.4 [CH2 (Sar) major], 51.3 [CH2 (Sar)
minor], 51.4 [OCH3 (Glu)], 51.5 [OCH3 (Asp)], 52.9 [α-C (Glu)
major], 53.0 [α-C (Glu) minor], 53.6 [α-C (Phe)], 76.0
(CH2OH), 126.19, 128.01 and 129.24 (ArCH), 137.88 (ArC
quaternary), 167.8 [CO (Sar) major], 167.9 [CO (Sar) minor],
168.4 [β-CO (Asp) major], 169.2 (CO), 170.7 [CO (Phe)], 171.2
[α-CO (Asp)], 171.4 (CO), 171.9 (CO), 172.2 (CO), 172.5 [α-CO
2
νmax(Nujol)/cmϪ1 1740 (CO ester); δH (200 MHz; H2O) 2.07–
2.20 (2 H, m, β-H2), 2.56 (2 H, t, J 6.9, γ-H2), 3.68 (3 H, s,
OCH3), 4.07 (1 H, t, J 6.7, α-H), 4.73 (2 H, s, PhCH2) and 7.34
2
(5 H, s, ArH); δC(75.3 MHz; H2O) 24.68 (β-CH2), 29.43 (γ-
CH2), 51.93 (OCH3), 53.53 (α-C), 67.31 (PhCH2), 128.49,
128.80 and 128.93 (ArCH), 135.52 (ArC quaternary) and
170.27 and 174.13 (CO); m/z (EI); 252 (13%, Mϩ), 192 (68,
[M Ϫ CH4 Ϫ CO2]ϩ), 160 (23, [M Ϫ PhCH2]ϩ) and 91 (100,
[PhCH2]ϩ).
ã-Benzyl (2S )-N-(tert-butoxycarbonyl)phenylalanyl-â-alanyl-
[á-methyl (2R)-glutamate] diester 25
This compound was prepared in a manner identical with that
described for the phenylalanyl(hydroxy)butyrate dipeptide 16,
using N-(tert-butoxycarbonyl)-(2S )-phenylalanyl-β-alanine 22
(2.70 g, 8.0 mol) and diester hydrochloride 23 (2.30 g, 8.0 mmol)
to give the required product as a viscous gel.This was dissolved
in warm ethyl acetate (200 cm3), and the solution was washed
and dried as described above to afford compound 25 as a solid
(4.32 g, 95%), mp 127–128 ЊC (Found: C, 63.25; H, 7.35; N, 7.0.
C30H39N3O8 requires C, 63.25; H, 6.9; N, 7.4%); νmax(Nujol)/
cmϪ1 3310 (NH), 1770 (CO, urethane), 1740 (CO, ester), 1695
(CO, ester) and 1655br (CO, amide); δH (300 MHz; C2HCl3) 1.35
[9 H, s, (CH3)3], 1.95–2.05 [1 H, m, 1 H of β-H2 (Glu)], 2.14–
2.28 [3 H, m, 1 H of β-H2 (Glu) and CH2 (β-Ala)], 2.25–2.49 [2
H, m, γ-H2 (Glu)], 2.93–3.08 [2 H, m, CH2 (Phe)], 3.43 (2 H, m,
CH2N), 3.71 (3 H, s, OCH3), 4.28 [1 H, br q, α-H (Phe)], 4.49–
4.56 [1 H, m, α-H (Glu)], 5.10 (2 H, s, PhCH2), 5.22 [1 H, d, J
8.2, NH (Phe)], 6.39 [1 H, d, J 7.7, NH (Glu)], 6.65 [1 H, t, J 5.8,
NH (β-Ala)] and 7.16–7.41 (10 H, m, ArH); δC(75.7 MHz;
C2HCl3) 26.68 [β-CH2 (Glu)], 28.10 [(CH3)3], 30.32 [γ-CH2
(Glu)], 35.43 [CH2 (β-Ala) and CH2 (Phe)], 38.60 (CH2N),
51.65 (OCH3), 52.49 [α-C (Glu)], 55.89 [α-C (Phe)], 66.57
(PhCH2), 79.97 [C(CH3)3], 126.80, 128.31, 128.38, 128.53,
128.61 and 129.37 (ArCH), 135.64 and 136.89 (ArC quater-
nary), 155.38 (CO, urethane) and 171.64, 172.39 and 172.73
(CO); m/z (FAB) 592 (14%, [M ϩ Na]ϩ), 570 (45, [M ϩ H]ϩ),
470 (100, [M ϩ H Ϫ Boc]ϩ) and 120 (62, [C8H9N ϩ H]ϩ).
(2S )-N-(tert-Butoxycarbonyl)phenylalanyl-â-alanyl-[á-methyl
(2R)-glutamate] ester 26
To a solution of tripeptide 25 (4.56 g, 8.0 mmol) in methanol
(120 cm3) was added 5% palladium on charcoal (10 mg) and the
mixture was stirred under hydrogen for 1 h, after which time a
precipitate formed. This was dissolved by the addition of gla-
cial acetic acid (20 cm3). The reaction mixture was stirred under
hydrogen for a further 20 h, and the catalyst was then removed
by filtration through a pre-washed Celite pad. The filtrate was
2522
J. Chem. Soc., Perkin Trans. 1, 1997

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concentrated under reduced pressure, using toluene to remove
the residual acetic acid azeotropically, to give a yellow oil. Trit-
uration with ethyl acetate–light petroleum then gave the
required product as an off-white solid (3.71 g, 91%), mp 115–
117 ЊC (Found: C, 57.35; H, 6.95; N, 8.6. C23H33N3O8 requires
C, 57.6; H, 6.95; N, 8.75%); [α]D25 ϩ14.24 (c 1.004, MeOH);
νmax(Nujol)/cmϪ1 3325 (NH), 1755 (CO, urethane), 1723 (CO),
1687 (CO) and 1670 (CO); δH (300 MHz; C2HCl3) 1.34 [9 H, s,
(CH3)3], 1.95–2.07 [1 H, m, 1 H of γ-H2 (Glu)], 2.32–2.39 [3 H,
m, 1 H of γ-H2 (Glu) and CH2 (β-Ala)], 2.87–3.01 [2 H, m,
CH2 (Phe)], 3.32–3.38 [1 H, m, 1 H of CH2N (β-Ala)], 3.61–3.68
[1 H, m, 1 H of CH2N (β-Ala)], 3.74 (3 H, s, OCH3), 4.45–4.50
(1 H, m, α-H), 4.64 (1 H, br m, α-H), 5.43 [1 H, d, J 8.2, NH
(Phe)], 6.64 [1 H, d, J 7.4, NH (Glu)], 6.88 [1 H, br t, NH (β-
Ala)] and 7.17–7.30 (5 H, m, ArH); δC(75.4 MHz; C2HCl3) 26.47
[β-CH2 (Glu)], 28.19 [(CH3)3], 30.22 [γ-CH2 (Glu)], 35.31
[CH2 (β-Ala)], 36.05 (CH2Ph), 38.89 (CH2N), 52.25
(OCH3), 52.66 [α-C (Glu)], 55.75 [α-C (Phe)], 80.61 [C(CH3)3],
126.97, 128.66 and 129.39 (ArCH), 136.77 (ArC quaternary),
156.22 (CO, urethane) and 171.75, 172.15 and 175.70 (CO); m/z
(FAB) 502 (30%, [M ϩ Na]ϩ), 480 (29, [M ϩ H]ϩ), 380 (100,
[M ϩ H Ϫ Boc]ϩ), 233 (35, [M ϩ 2 H Ϫ BocPhe]ϩ and 120 (57,
[C8H9N ϩ H]ϩ).
3.05–3.12 [1 H, m, 1 H of CH2Ph (Phe)], 2.93 and 3.07 (3 H,
2 × s, NCH3), 3.39–3.45 [2 H, m, CH2N (β-Ala)], 3.70 and 3.72
(3 H, 2 × s, OCH3), 4.05 [1 H, d, J 17.6, 1 H of CH2 (Sar)], 4.12
[1 H, d, J 17.6, 1 H of CH2 (Sar)], 4.23 [1 H, m, α-H (Phe)], 4.43
[1 H, m, α-H (Glu)] and 7.23 (5 H, m, ArH); δC(75.4 MHz;
C2H3O2H, mixture of rotamers) 26.13 [β-CH2 (Glu)], 27.20
[(CH3)3], 28.82 [γ-CH2 (Glu)], 33.99 and 34.76 (NCH3), 35.45
[CH2 (β-Ala)], 35.58 [CH2 (Phe)], 37.96 [CH2N (β-Ala)], 49.02
[CH2N (Sar)], 51.43 (OCH3 minor), 51.92 (OCH3 major), 52.03
[α-C (Glu)], 56.18 [α-C (Phe)], 79.27 [C(CH3)3], 126.41, 128.14
and 129.11 (ArCH), 137.44 (ArC quaternary), 156.33 (CO,
urethane) and 171.51, 172.65, 173.19 and 173.70 (CO); m/z
(FAB) 573 (57%, [M ϩ Na]ϩ), 551 (43, [M ϩ H]ϩ), 465 (17,
[M ϩ H Ϫ N-Me-Gly]ϩ), 451 (100, [M ϩ H Ϫ Boc]ϩ), 304 (14,
[M ϩ H Ϫ BocPhe]ϩ), 233 (19, [M ϩ H Ϫ BocPhe-β-Ala]ϩ)
and 120 (51, [C8H9N ϩ H]ϩ).
Benzyl (2R)-N-(tert-butoxycarbonyl)alanyl-(2S )-leucinate 29
This compound was prepared in a manner identical with that
described for the phenylalanyl(hydroxy)butyrate dipeptide 16,
using (2R)-N-(tert-butoxycarbonyl)alanine (1.89 g, 10 mmol)
and benzyl (2S )-leucinate toluene-p-sulfonate (3.94 g, 10 mmol)
to give the required product as a solid. Recrystallisation from
diethyl ether–light petroleum gave the desired product as fine
needles (3.32 g, 85%), mp 89–91 ЊC (Found: C, 64.45; H, 8.5;
N, 7.0. C21H32N2O5 requires C, 64.25; H, 8.2; N, 7.15%); [α]D25
Ϫ8.91 (c 1.01, MeOH); νmax(Nujol)/cmϪ1 3345 and 3248 (NH),
1756 (CO, urethane), 1693 (CO, ester) and 1664 (CO, amide);
δH (300 MHz; C2HCl3) 0.91 [3 H, d, J 6.0, CH3 (Leu)], 0.92 [3 H,
d, J 6.0, CH3 (Leu)], 1.35 [3 H, d, J 7.1, CH3 (Ala)], 1.45 [9 H, s,
(CH3)3], 1.51–1.69 (3 H, m, CHCH2), 4.20 [1 H, br q, α-H (Leu)],
4.65 [1 H, m, α-H (Ala)], 4.99 (1 H, br s, NH urethane), 5.13
(1 H, d, J 17.6, 1 H of PhCH2), 5.17 (1 H, d, J 17.6, 1 H of
PhCH2), 6.67 (1 H, br s, NH amide) and 7.37 (5 H, m, ArH);
δC(75.7 MHz; C2HCl3) 18.05 [CH3 (Ala)], 21.70 [CH3 (Leu)],
22.70 [CH3 (Leu)], 24.70 (CH), 28.16 [(CH3)3], 41.26 (CH2),
50.05 [α-C (Leu)], 50.69 [α-C (Ala)], 66.96 (PhCH2), 128.23,
128.41 and 128.61 (ArCH), 135.44 (ArC quaternary), 155.54
(CO urethane) and 172.76 (CO); m/z (EI) 392 (17%, Mϩ), 336
(48, [M Ϫ C4H8]ϩ), 319 (32, [M Ϫ C4H9O]ϩ), 257 (62,
[M Ϫ C8H7O2]ϩ), 91 (100, PhCH2ϩ) and 57 (80, C4H9ϩ).
Benzyl (2S )-N-(tert-butoxycarbonyl)phenylalanyl-â-alanyl-
[á-methyl (2R)-glutamyl]-ã-sarcosinate diester 27
This compound was prepared in a manner identical with that
described for the phenylalanyl(hydroxy)butyrate dipeptide 16,
using N-(tert-butoxycarbonyl)-(2S )-phenylalanyl-β-alanyl-α-
methyl-(2R)-glutamate ester 26 (3.36 g,7.0 mmol) and benzyl
sarcosinate toluene-p-sulfonate (2.46 g, 7.0 mmol) to give an oil,
which was triturated with ethyl acetate–light petroleum to
afford the desired product as a solid (3.59 g, 80%), mp 110–
112 ЊC (Found: C, 61.7; H, 7.15; N, 8.55. C33H44N4O9 requires
C, 61.85; H, 6.9; N, 8.75%); [α]D25 ϩ9.1 (c 1, MeOH);
νmax(Nujol)/cmϪ1 3340 (NH), 3310 (NH), 1755 (CO, urethane),
1690 (CO, ester), 1667 (CO, ester) and 1652 (CO, ester); δH (300
MHz; C2HCl3, mixture of rotamers) 1.35 [9 H, s, (CH3)3], 2.10–
2.55 [ 6 H, m, β- and γ-H2 (Glu) and CH2 (β-Ala)], 2.83–2.89 (2
H, m, CH2Ph), 2.95 and 3.03 (3 H, 2 × s, NCH3), 3.47 [2 H, br s,
CH2N (β-Ala)], 3.71 and 3.74 (3 H, 2 × s, OCH3), 4.14 [2 H,
2 × d, CH2 (Sar)], 4.32 [1 H, m, α-H (Phe)], 4.49 [1 H, m, α-H
(Glu)], 5.15 and 5.19 (2 H, 2 × s, PhCH2), 5.30 [1 H, br s, NH
(Phe)], 6.83 (1 H, br s, NH), 6.99 (1 H, br s, NH) and 7.19–7.34
(10 H, m, ArH); δC(75.4 MHz; C2HCl3, mixture of rotamers)
25.98 [β-CH2 (Glu)], 28.13 [(CH3)3], 29.36 [γ-CH2 (Glu)], 35.40
[CH2 (β-Ala)], 35.53 [CH2 (Phe)], 36.61 (NCH3), 38.83 [CH2N
(β-Ala)], 49.73 [CH2 (Sar)], 52.47 (OCH3), 52.50 [α-C (Glu)],
55.79 [α-C (Phe)], 67.07 (PhCH2), 79.71 [C(CH3)3], 126.79,
128.34, 128.54, 128.68, 128.81 and 129.44 (ArCH), 135.27 and
136.99 (ArC quaternary), 155.48 (CO, urethane) and 169.12,
171.56, 172.02 and 172.46 (CO); m/z (FAB) 663 (8%,
[M ϩ Na]ϩ), 641 (26, [M ϩ H]ϩ), 541 (78, [M ϩ H Ϫ Boc]ϩ),
394 (10, [M ϩ H Ϫ BocPhe]ϩ), 323 (12, [M ϩ H Ϫ BocPhe-β-
Ala]ϩ) and 120 (100, [C8H9N ϩ H]ϩ).
Benzyl (2R)-alanyl-(2S )-leucinate hydrochloride 30
Hydrogen chloride gas was bubbled into dry ethyl acetate (50
cm3) at 0 ЊC and the solution was stirred for 1 h. To this was
added the benzyl (2R)-N-(tert-butoxycarbonyl)alanyl-(2S )-
leucinate 29 (5.89 g, 15 mmol) and the solution was stirred for 1
h at room temperature. The solution was then concentrated
under reduced pressure to give a sticky solid, which was dis-
solved in the minimum volume of diethyl ether and precipitated
by addition of light petroleum. The solid was collected by filtra-
tion, and was then washed with light petroleum and dried in air
to give the desired product as a powder (4.72 g, 96%), mp 139–
140 ЊC; νmax(Nujol)/cmϪ1 3189 (NH), 1742, 1708 and 1678
(CO); δH (300 MHz; C2H3O2H) 0.90 [3 H, m, CH3 (Leu)], 0.95 [3
H, m, CH3 (Leu)], 1.51 [3 H, d, J 6.9, CH3 (Ala)], 1.65 [3 H, m,
CHCH2 (Leu)], 4.00 [1 H, q, J 6.9, α-H (Ala)], 4.51 [1 H, t, α-H
(Leu)], 5.16 (2 H, s, PhCH2) and 7.35 (5 H, s, Ph); δC(75.4 MHz;
C2H3O2H) 17.98 [CH3 (Ala)], 21.77 [CH3 (Leu)], 23.30 [CH3
(Leu)], 26.17 (CH), 41.42 (CH2), 50.34 [α-C (Ala)], 52.51 [α-C
(Leu)], 68.20 (PhCH2), 129.56, 129.65 and 129.84 (ArCH),
137.37 (ArC quaternary) and 171.46 and 173.89 (CO); m/z
(FAB) 293 (100%, Mϩ).
(2S )-N-(tert-Butoxycarbonyl)phenylalanyl-â-alanyl-[á-methyl
(2R)-glutamyl]-ã-sarcosine ester 28
To a solution of tetrapeptide diester 27 (3.20 g, 5.0 mmol) in
methanol (100 cm3) was added 5% palladium on carbon (320
mg) and the mixture was stirred under hydrogen for 12 h. The
catalyst was then removed by filtration through a pre-washed
Celite pad and the filtrate was concentrated under reduced pres-
sure to give the acid 28 as a solid (2.61 g, 95%), mp 103–105 ЊC;
νmax(Nujol)/cmϪ1 3300br (NH and OH), 1735 (CO), 1720 (CO),
1665 (CO) and 1640 (CO); δH (300 MHz; C2H3O2H, mixture of
rotamers) 1.34 [9 H, s, (CH3)3], 1.94–2.00 [1 H, m, 1 H of β-H2
(Glu)], 2.12–2.19 [1 H, m, 1 H of β-H2 (Glu)], 2.39–2.45 [3 H,
m, 1 H of CH2 (β-Ala) and γ-CH2 (Glu)], 2.50–2.56 [1 H, m, 1
H of CH2 (β-Ala)], 2.76–2.83 [1 H, m, 1 H of CH2Ph (Phe)],
Benzyl (2S )-N-(tert-butoxycarbonyl)phenylalanyl-â-alanyl-[á-
methyl (2R)-glutamyl]-ã-sarcosyl-(2R)-alanyl-(2S )-leucinate
diester 31
This compound was prepared in a manner identical with that
described for the phenylalanyl(hydroxy)butyrate dipeptide
16, using tetrapeptide 28 (1.65 g, 3.0 mmol) and dipeptide
J. Chem. Soc., Perkin Trans. 1, 1997
2523

View Article Online
hydrochloride 30 (0.99 g, 3.0 mmol) to give the required product
as an off-white solid (1.90 g, 77%), mp 74 ЊC (softening)
(Found: C, 61.0; H, 7.65; N, 9.9. C42H60N6O11 requires C, 61.15;
H, 7.35; N, 10.2%); [α]D25 ϩ6.83 (c 1.26, MeOH); νmax(Nujol)/
cmϪ1 3297 (NH), 1751 (CO, urethane), 1732 (CO, amide) and
1664 (CO, ester); δH [500 MHz; (C2H3)2SO, mixture of rotamers]
0.83 [3 H, d, J 5.5, CH3 (Leu)], 0.88 [3 H, d, J 6.5, CH3 (Leu)],
1.23 [3 H, 2 × d, CH3 (Ala)], 1.31 [9 H, s (CH3)3], 1.50–1.65 (3
H, m, CHCH2), 1.80–1.84 [1 H, m, 1 H of β-H2 (Glu)], 1.91–
2.03 [1 H, m, 1 H of β-H2 (Glu)], 2.28–2.32 [3 H, m, CH2 (β-
Ala) and 1 H of γ-H2 (Glu)], 2.39–2.43 [1 H, m, 1 H of γ-H2
(Glu)], 2.67–2.80 [2 H, m, CH2 (Phe)], 2.78 and 2.95 (3 H, 2 × s,
NCH3), 3.23 [2 H, m, CH2N (β-Ala)], 3.61 (3 H, 2 × s, OCH3),
3.89–4.13 [3 H, m, CH2 (Sar) and 1 × α-H], 4.24–4.42 (3 H, m,
3 × α-H), 5.14 (2 H, s, PhCH2), 6.82 (1 H, d, J 9.5, NH
urethane), 7.19–7.41 (10 H, m, ArH), 7.91 [1 H, br s, NH (β-Ala)]
and 8.02, 8.20, 8.27, 8.30 and 8.36 (5 H, 4 × d and 1 × t, NH
amide); δC[125.75 MHz; (C2H3)2SO, mixture of rotamers] 18.55
[CH3 (Ala) minor], 18.78 [CH3 (Ala) major], 21.12 [CH3 (Leu)],
21.23 [CH3 (Leu)], 22.71 [CH3 (Leu)], 24.23 [CH (Leu)], 26.43
[β-CH2 (Glu) major], 26.58 [β-CH2 (Glu) minor], 28.16 [(CH3)3],
28.68 [γ-CH2 (Glu)], 34.24 [CH2 (β-Ala)], 34.82 (NCH3
major), 35.20 [CH2 (Phe)], 36.11 (NCH3 minor), 37.71 [CH2N
(β-Ala)], 42.69 [CH2 (Leu)], 48.05 [CH2 (Sar)], 50.29 [α-C (Ala)],
51.54(OCH3),51.63[α-C (Leu)],51.74(OCH3),51.80[α-C (Glu)],
55.76 [α-C (Phe)], 65.92 (PhCH2), 77.98 [C(CH3)3], 126.14,
127.80, 128.01, 128.44 and 129.20 (ArCH), 135.96 and 138.26
(ArC quaternary), 155.17 (CO, urethane) and 167.96, 170.67,
170.75, 171.54, 172.24, 172.33 and 172.50 (CO); m/z (FAB) 725
(95%, [M ϩ Na Ϫ C5H9O2]ϩ) and 120 (100, [C8H9N ϩ H]ϩ).
using hexapeptide 32 (1.18 g, 1.6 mmol) and β-benzyl α-methyl
(2R)-aspartate hydrochloride (0.44 g, 1.6 mmol) to give a crude
solid which was then dried under reduced pressure over phos-
phorus pentoxide. Recrystallisation from ethyl acetate–light
petroleum gave the required product as an off-white solid (1.16
g, 76%), mp 137–139 ЊC (softening) (Found: C, 58.35; H, 7.15;
N, 10.0. C47H67N7O14ؒH2O requires C, 58.05; H, 7.15; N,
10.1%); [α]D25 ϩ8.2 (c 1.525, MeOH); νmax(Nujol)/cmϪ1 3306
(NH), 1747 (CO, urethane), 1698 (CO), 1664 (CO) and 1634
(CO); δH [300 MHz; (C2H3)2SO, mixture of rotamers] 0.77 [3 H,
d, J 6.6, CH3 (Leu)], 0.80 [3 H, d, J 6.6, CH3 (Leu)], 1.18 [3 H,
2 × d, CH3 (Ala)], 1.26 [9 H, s, (CH3)3], 1.38–1.60 [3 H, m,
CHCH2 (Leu)], 1.72–1.93 [2 H, m, β-H2 (Glu)], 2.25–2.36 [6 H,
m, CH2 (β-Ala), γ-H2 (Glu) and β-H2 (Asp)], 2.64–2.74 [1 H, m,
1 H of CH2Ph (Phe)], 2.81–2.92 [1 H, m, 1 H of CH2Ph (Phe)],
2.72 and 2.88 (3 H, 2 × s, NCH3), 3.24 [2 H, m, CH2N (β-Ala)],
3.57 (3 H, s, OCH3), 3.56 and 3.60 (3 H, 2 × s, OCH3), 3.83 [1
H, d, J 16.5, 1 H of CH2 (Sar)], 3.95–4.11 [3 H, m, 2 × α-H and
1 H of CH2 (Sar)], 4.23–4.36 (2 H, m, 2 × α-H), 4.66 [1 H, m, α-
H (Asp)], 5.07 and 5.09 (2 H, 2 × s, PhCH2), 6.84 and 7.17 [1 H,
2 d, J 8.8, NH (Phe)], 7.22 (5 H, s, ArH), 7.34 (5 H, s, ArH),
7.92 [1 H, br t, NH (β-Ala)], 8.05 (1 H, d, J 6.8, NH), 8.15–8.31
(2 H, m, 2 × NH) and 8.41 and 8.48 [1 H, 2 × d, J 8.0, NH
(Asp)]; δC[75.4 MHz; (C2H3)2SO, mixture of rotamers] 18.68
[CH3 (Ala)], 21.72 [CH3 (Leu)], 23.39 [CH3 (Leu)], 24.59 [CH
(Leu)], 27.96 [(β-CH2 (Glu)], 28.52 [(CH3)3], 35.21 [β-CH2
(Asp)], 35.59 [CH2 (β-Ala)], 36.31 (NCH3), 36.84 [CH2 (Phe)],
38.09 [CH2N (β-Ala)], 48.92 [CH2 (Sar)], 51.04 (α-C), 52.25
(OCH3), 52.62 (OCH3), 56.21 [α-C (Phe)], 66.32 (OCH2Ar),
78.46 [C(CH3)3], 126.70, 128.45, 128.56, 128.99 and 129.75
(ArCH), 136.46 and 138.83 (ArC quaternary), 155.79 (CO,
urethane) and 168.85, 170.40, 171.39, 171.59, 172.21, 172.58
and 173.16 (CO); m/z (FAB) 976 (36%, [M ϩ Na]ϩ), 954 (19,
[M ϩ H]ϩ), 876 (7, [M ϩ Na Ϫ C5H9O2]ϩ), 854 (100,
[M ϩ H Ϫ C5H9O2]ϩ) and 120 (95, [C8H9N ϩ H]ϩ).
(2S )-N-(tert-Butoxycarbonyl)phenylalanyl-â-alanyl-[á-methyl
(2R)-glutamyl]-ã-sarcosyl]-(2R)-alanyl-(2S )-leucine ester 32
To a solution of hexapeptide diester 31 (1.65 g, 2.0 mmol) in
methanol (50 cm3) was added 5% palladium on charcoal (10
mg) and the mixture was stirred under hydrogen for 18 h. The
catalyst was then removed by filtration through a pre-washed
Celite pad and the filtrate was concentrated under reduced pres-
sure to give the required product as a solid (1.21 g, 82%), mp
82 ЊC (softening); νmax(Nujol)/cmϪ1 3297 (NH and OH), 1747
(CO, urethane), 1722 (CO, ester) and 1664 (CO, amide); δH (300
MHz; C2H3O2H, mixture of rotamers) 0.93 [6 H, m, 2 × CH3
(Leu)], 1.34 [ 9 H, s, (CH3)3], 1.35 [3 H, d, CH3 (Ala)], 1.65 [3 H,
br s, CHCH2 (Leu)], 1.95 [1 H, m, 1 H of β-H2 (Glu)], 2.17 [1 H,
m, 1 H of β-H2 (Glu)], 2.42 [3 H, br s, CH2 (β-Ala) and 1 H of
γ-H2 (Glu)], 2.53 [1 H, br t, 1 H of γ-H2 (Glu)], 2.81 [1 H, m, 1
H of CH2Ph (Phe)], 3.06 [1 H, m, 1 H of CH2Ph (Phe)], 2.91
and 3.07 (3 H, 2 × s, NCH3), 3.42 [2 H, br s, CH2N (β-Ala)],
3.70 (3 H, s, OCH3), 4.03 and 4.11 [2 H, 2 × s, CH2 (Sar)], 4.24
(1 H, m, α-H), 4.40–4.48 (3 H, m, 3 × α-H) and 7.19–7.34 (5 H,
m, ArH); δC(75.4 MHz; C2H3O2H, mixture of rotamers) 16.65
[CH3 (Ala) major], 17.11 [CH3 (Ala) minor], 20.36 [CH3 (Leu)],
22.03 [CH3 (Leu)], 24.55 [CH (Leu)], 26.27 [β-CH2 (Glu)], 27.22
[(CH3)3], 28.72 [γ-CH2 (Glu)], 34.02 [CH2 (β-Ala)], 34.75
(NCH3 minor), 35.47 [CH2 (Phe)], 36.11 (NCH3 major), 37.97
[CH2N (β-Ala)], 40.21 [CH2 (Leu) major], 40.41 [CH2 (Leu)
minor], 48.78 [CH2 (Sar)], 50.73 [α-C (Ala)], 51.19 [α-C (Leu)],
51.46 (OCH3), 51.92 [α-C (Glu)], 56.16 [α-C (Phe)], 79.26
[C(CH3)3], 126.42, 128.14 and 129.12 (ArCH), 137.43 (ArC
quaternary), 156.32 (CO, urethane) and 169.00, 169.81, 172.65,
173.19, 173.52, 173.99 and 174.92 (CO); m/z (FAB) 757 (100%,
[M ϩ Na]ϩ), 735 (6, [M ϩ H]ϩ), 679 (8, [M ϩ H Ϫ C4H9]ϩ), 657
(31, [M ϩ Na Ϫ C5H9O2]ϩ), 635 (96, [M ϩ H Ϫ C5H9O2]ϩ) and
120 (100, [C8H9N ϩ H]ϩ).
(2S )-N-(tert-Butoxycarbonyl)phenylalanyl-â-alanyl-[á-methyl
(2R)-glutamyl]-ã-sarcosyl-(2R)-alanyl-(2S )-leucyl-[á-methyl
(2R)-aspartate] diester 34
To a solution of triester 33 (0.50 g, 0.52 mmol) in a mixture of
methanol (50 cm3) and glacial acetic acid (1 cm3) was added 5%
palladium on carbon (10 mg) and the mixture was stirred under
hydrogen for 18 h. The catalyst was removed by filtration
through a pre-washed Celite pad and the filtrate was concen-
trated under reduced pressure to give a yellow oil. Residual
acetic acid was removed azeotropically with toluene under
reduced pressure to give the required product as an off-white
solid (0.43 g, 95%), mp 114–116 ЊC (softening); νmax(Nujol)/
cmϪ1 3316br (NH and OH), 1727br (CO) and 1664br (CO);
δH (300 MHz; C2H3O2H, mixture of rotamers) 0.88–0.94 [6 H,
m, 2 × CH3 (Leu)], 1.34 [9 H, s, (CH3)3], 1.65 [3 H, br d, CHCH2
(Leu)], 1.89–1.98 [1 H, m, 1 H of β-H2 (Glu)], 2.13–2.24 [1 H,
m, 1 H of β-H2 (Glu)], 2.31–2.56 [4 H, m, γ-H2 (Glu) and
CH2 (β-Ala)], 2.78–2.84 [2 H, m, β-H2 (Asp)], 2.99–3.14 [2 H, m,
CH2Ph (Phe)], 2.92 and 3.07 (3 H, 2 × s, NCH3), 3.37–3.48 [2
H, m, CH2N (β-Ala)], 3.64, 3.68 and 3.69 (6 H, 3 × s,
2 × OCH3), 3.97–4.11 [2 H, m, CH2 (Sar)], 4.25–4.48 (4 H, m,
4 × α-H), 4.74 [1 H, t, J 5.8, α-H (Asp)] and 7.08–7.29 (5 H, m,
ArH); δC(75.4 MHz; C2H3O2H, mixture of rotamers) 17.64
[CH3 (Ala) major], 18.02 [CH3 (Ala) minor], 21.58 [CH3 (Leu)],
21.80 [CH3 (Leu)], 23.60 [CH (Leu)], 26.01 [β-CH2 (Glu)], 27.83
[(CH3)3 minor], 28.75 [(CH3)3 major], 30.27 [γ-CH2 (Glu)],
35.65 [β-CH2 (Asp)], 36.28 [CH2 (β-Ala)], 36.99 [CH2 (Phe)],
37.27 (NCH3 minor), 37.68 (NCH3 major), 39.51 [CH2N (β-
Ala)], 41.48 [CH2 (Leu)], 50.61 [CH2 (Sar)], 50.86 (α-C), 52.59
(α-C), 52.99 (OCH3), 53.14 (OCH3), 53.15 (α-C), 57.66 [α-C
(Phe)], 80.77 [C(CH3)3], 126.55, 127.94, 129.46, 129.66, 130.18
and 130.64 (ArCH), 138.96 (ArC quaternary), 157.81 (CO,
urethane) and 171.54, 173.10, 174.16, 174.68 and 175.51 (CO);
m/z (FAB) 908 (15%, [M ϩ 2Na Ϫ H]ϩ), 886 (90, [M ϩ Na]ϩ),
â-Benzyl (2S )-N-(tert-butoxycarbonyl)phenylalanyl-â-alanyl-
[á-methyl (2R)-glutamyl]-ã-sarcosyl-(2R)-alanyl-(2S)-leucyl-
[á-methyl (2R)-aspartate] triester 33
This compound was prepared in a manner identical with that
described for the phenylalanyl(hydroxy)butyrate dipeptide 16,
2524
J. Chem. Soc., Perkin Trans. 1, 1997

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808 (7, [M ϩ H Ϫ C4H9]ϩ), 786 (52, [M ϩ Na Ϫ C5H9O2]ϩ),
764 (56, [M ϩ H Ϫ C5H9O2]ϩ) and 120 (100, [C8H9N ϩ H]ϩ).
CHCH2 (Leu)], 1.78–1.91 [2 H, m, β-H2 (Glu)], 2.18–2.42 [6 H,
m, α-H2 (Glu), β-H2 (Asp) and CH2(β-Ala)], 2.63–2.71 [1 H, m,
1 H of CH2Ph (Phe)], 2.89–3.05 [1 H, m, 1 H of CH2Ph (Phe)],
2.75 and 2.91 (3 H, 2 × s, NCH3), 3.09–3.14 [1 H, m, 1 H of
CH2N (β-Ala)], 3.43 and 3.47 (3 H, 2 × s, OCH3), 3.51–3.56 [1
H, m, 1 H of CH2N (β-Ala)], 3.58 and 3.59 (3 H, 2 × s, OCH3),
3.80 [1 H, d, 1 H of CH2N (Sar)], 4.00–4.40 [6 H, m, 1 H of
CH2N (Sar) and 5 × α-H], 7.18–7.26 (5 H, m, ArH and 7.90–
8.27 (6 H, m, 6 × NH); δC[75.4 MHz; (C2H3)2SO, mixture of
rotamers] 16.88 [CH3 (Ala) major], 18.11 [CH3 (Ala) minor],
21.08 [CH3 (Leu) major], 21.18 [CH3 (Leu) minor], 22.97 [CH3
(Leu) major], 23.06 [CH3 (Leu) minor], 24.25 [CH (Leu)], 26.43
[β-CH2 (Glu)], 28.91 [γ-CH2 (Glu) minor], 29.30 [γ-CH2 (Glu)
major], 34.27 [β-CH2 (Asp)], 35.16 [CH2 (β-Ala)], 36.37 (NCH3
major), 36.44 (NCH3 minor), 37.39 [CH2 (Phe)], 37.59 [CH2N
(β-Ala)], 48.65 [CH2 (Sar) minor], 48.93 [CH2 (Sar) major],
49.52 [α-C (Ala)], 49.98 [α-C (Asp)], 50.90 [α-C (Leu)], 51.12 [α-
C (Glu)], 51.53 (OCH3), 51.73 (OCH3), 51.93 (OCH3), 54.07 [α-
C (Phe) minor], 54.43 [α-C (Phe) major], 126.35, 128.20, 129.23
and 129.29 (ArCH), 138.08 and 138.14 (ArC quaternary) and
168.31, 169.25, 170.95, 171.08, 171.25, 171.41, 171.54, 171.62,
171.88, 172.35, 172.67, 172.74 and 172.78 (CO); m/z (FAB) 768
(25%, [M ϩ Na]ϩ), 746 (23, [M ϩ H]ϩ) and 120 (100,
[C8H9N ϩ H]ϩ).
â-Pentafluorophenyl (2S )-N-(tert-butoxycarbonyl)phenylalanyl-
â-alanyl-[á-methyl (2R)-glutamyl]-ã-sarcosyl-(2R)-alanyl-(2S )-
leucyl-[á-methyl (2R)-aspartate] triester 35
To a stirred solution of the heptapeptide carboxylic acid 34
(0.43 g, 0.5 mmol) in CH2Cl2 (15 cm3) at 0 ЊC was added pen-
tafluorophenol (0.28 g, 1.5 mmol) followed by EDCI (0.23 g,
0.75 mmol). The reaction mixture was stirred at 0 ЊC for 1 h,
was then allowed to warm up to room temperature, and was
stirred for a further 24 h. The solution was concentrated under
reduced pressure and the residual brown solid was purified by
flash chromatography on silica (CH2Cl2 to remove excess of
pentafluorophenol, then with CH2Cl2–MeOH, 94:6) to give the
required product as a waxy solid (0.47 g, 91%), mp 142–144 ЊC;
νmax(Nujol)/cmϪ1 3316 (NH), 1795 (CO), 1747 (CO), 1717 (CO),
1659 (CO) and 1644 (CO); δH [300 MHz; (C2H3)2SO, mixture of
rotamers] 0.80 [3 H, d, J 6.6, CH3 (Leu)], 0.83 [3 H, d, J 6.8,
CH3 (Leu)], 1.19 [3 H, 2 × d, CH3 (Ala)], 1.26 [9 H, s, (CH3)3],
1.44–1.54 [3 H, m, CHCH2 (Leu)], 1.76–1.96 [2 H, m, β-H2
(Glu)], 2.20–2.40 [4 H, m, CH2 (β-Ala) and γ-H2 (Glu)], 2.68–
2.75 [1 H, m, 1 H of CH2Ph (Phe)], 2.74 and 2.91 (3 H, 2 × s,
NCH3), 3.07–3.39 [5 H, m, 1 H of CH2Ph (Phe), CH2N (β-Ala)
and β-H2 (Asp)], 3.56, 3.58 and 3.63 (6 H, 3 × s, 2 × OCH3),
3.87 [1 H, d, J 16.3, 1 H of CH2 (Sar)], 3.96–4.08 [2 H, m, 1 H
of CH2 (Sar) and 1 × α-H], 4.25–4.37 (3 H, m, 3 × α-H), 4.77 [1
H, m, α-H (Asp)], 6.85 [1 H, d, J 8.4, NH (Phe)], 7.16 (1 H, d, J
5.3, NH), 7.22 (5 H, s, ArH), 7.92 [1 H, br t, NH (β-Ala)], 8.08
(1 H, br t, NH), 8.19–8.31 (2 H, m, 2 × NH) and 8.59 and 8.65
[1 H 2 × d, J 8.1, NH (Asp)]; δC[75.4 MHz; (C2H3)2SO, mixture
of rotamers] 18.27 [CH3 (Ala) major], 18.63 [CH3 (Ala) minor],
21.19 [CH3 (Leu)], 22.94 [CH3 (Leu)], 24.13 [CH (Leu)], 28.08
[(CH3)3], 28.61 [γ-CH2 (Glu)], 34.91 [CH2 (β-Ala) and β-CH2
(Asp)], 35.15 [CH2 (Phe)], 36.02 (NCH3), 37.66 [CH2N (β-Ala)],
48.26 [CH2 (Sar)], 50.08 (α-C), 50.58 (α-C), 51.50 (OCH3),
51.77 (OCH3), 52.42 (α-C), 55.78 [α-C (Phe)], 78.00 [C(CH3)3],
126.25, 128.10 and 129.32 (ArCH), 138.41 (ArC quaternary),
155.36 (CO, urethane), 166.79, 168.02, 168.44, 170.63, 170.93,
171.77, 172.14, 172.34 and 172.73 (CO); δF [282.3 MHz;
(C2H3)2SO] Ϫ154.16 (2 F, t, J 19.8 Ar o-F), Ϫ159.13 (1 F, dt, J
5.9 and 23.8, Ar p-F), Ϫ163.22 (2 F, dd, J 5.9 and 19.8, Ar o-F),
Ϫ164.08 (2 F, t, J 21.8, Ar m-F), Ϫ166.91 (2 F, t, J 21.8, Ar m-
F) and Ϫ173.40 (1 F, dt, J 5.9 and 23.8, Ar p-F); m/z (FAB)
1052 (25%, [M ϩ Na]ϩ), 1030 (18, [M ϩ H]ϩ), 930 (100,
[M ϩ H Ϫ Boc]ϩ) and 120 (83, [C8H9N ϩ H]ϩ).
Acknowledgements
We thank the BBSRC and EPSRC for grants GR/K64068 and
B05162, Dr T. Rutherford for obtaining and interpreting NMR
data, Dr J. Wilkie for performing computational analyses of
enzyme-bound toxin conformations, Dr M. Akhtar for scien-
tific support and the Wellcome Trust for an NMR equipment
grant.
Abbreviations
COSY, 2-D homonuclear chemical-shift correlation spec-
troscopy; HMBC, 2-D heteronuclear multiple-bond correlation
spectroscopy; HSQC, proton-detected single-quantum-
coherence heteronuclear correlation spectroscopy; TOCSY,
phase-sensitive 2-D total correlation spectroscopy; AHB, 3-
amino-4-hydroxybutyric acid; DIPEA, N,N-diisopropyl-
ethylamine; DMF, N,N-dimethylformamide; EDCI, 1-(3-
dimethylaminopropyl)-3-ethylcarbodiimide methiodide; IBCF,
isobutyl chloroformate; NMM, 4-methylmorpholine; PFP,
pentafluorophenyl; PP1 serine/threonine protein phosphatase
type 1; PP2A, serine/threonine protein phosphatase type 2A;
TFA, trifluoroacetic acid; THF, tetrahydrofuran; TMSCl,
chlorotrimethylsilane.
cyclo-[â-Ala-á-OMe-(R)-Glu-ã-Sar-(R)-Ala-(S )-Leu-á-OMe-
(R)-Asp-â-(S )-Phe] 5
To a stirred solution of the N-(tert-butoxycarbonyl)-protected
pentafluorophenyl ester 35 (0.25 g, 0.24 mmol) in dry CH2Cl2
(10 cm3) was added TFA (5 cm3). The mixture was stirred for 90
min and was then diluted with toluene (5 cm3), and concen-
trated under reduced pressure to give a pale brown solid, which
was thoroughly dried in vacuo for 6 h. The resulting residue was
dissolved into dry CH2Cl2 (250 cm3), and the solution was
treated with DIPEA (0.71 cm3, 4.0 mmol) stirred at room tem-
perature while the reaction was followed by TLC (94:6
CH2Cl2–MeOH) which indicated complete reaction after seven
days. The solution was then concentrated under reduced pres-
sure to give a solid,which was triturated with diethyl ether and
then collected by filtration and washed on the pad with diethyl
ether to afford the pure macrocycle 5 as a powder (120 mg,
67%), mp 276–278 ЊC (decomp.) (Found: C, 55.7; H, 6.75; N,
12.6. C35H51N7O11ؒ1/2H2O requires C, 55.7; H, 6.95; N, 13.0%)
(HRMS: Found: [M ϩ Na]ϩ, 768.3524. C35H51N7NaO11
requires m/z 768.3544); νmax(Nujol)/cmϪ1 3306 (NH), 1751
(CO), 1708 (CO), 1664 (CO) and 1634 (CO); δH [300 MHz;
(C2H3)2SO, mixture of rotamers] 0.76–0.86 [6 H, m, 2 × CH3
(Leu)], 1.16 and 1.19 [3 H, 2 × d, CH3 (Ala)], 1.39–1.55 [3 H, m,
References
1 A. P. Mehrotra, K. L. Webster and D. Gani, J. Chem. Soc., Perkin
Trans. 1, 1997, preceding paper.
2 W. W. Carmichael, J. Appl. Bacteriol., 1992, 72, 445.
3 R. E. Honkanen, J. Zwiller, S. L. Dailly, B. S. Khatra, M. Dukelow
and A. L. Boynton, J. Biol. Chem., 1991, 266, 6614.
4 S. Shenolikar and A. C. Nairn, Adv. Sec. Mess. Phosphoprotein Res.,
1991, 23, 1.
5 M. Bollen and W. Stalmans, Crit. Rev. Biochem. Mol. Biol., 1992, 27,
227.
6 A. A. Depaoli-Roach, I.-K. Park, V. Cerovsky, C. Csortos, S. D.
Durbin, M. J. Kuntz, A. Sitikov, P. M. Tang, A. Verin and
S. Zolnierowicz, Adv. Enzyme Regul., 1994, 34, 199.
7 P. Cohen, Annu. Rev. Biochem., 1989, 58, 453.
8 P. T. W. Cohen, N. D. Brewis, V. Hughes and D. J. Mann, FEBS
Lett., 1990, 268, 355.
9 C. F. B. Holmes and M. P. Boland, Curr. Opin. Struct. Biol., 1993,
3, 934.
10 J. Goldberg, H. Huang, Y. Kwon, P. Greengard, A. C. Nairn and
J. Kuriyan, Nature, 1995, 376, 745.
11 J. Wilkie and D. Gani, unpublished results.
12 A. P. Mehrotra and D. Gani, Tetrahedron Lett., 1996, 37, 6915.
J. Chem. Soc., Perkin Trans. 1, 1997
2525

View Article Online
13 M. Namikoshi, K. L. Rinehart, A. M. Dahlem, V. R. Beasley and
W. W. Carmichael, Tetrahedron Lett., 1989, 30, 4349.
14 T. K. Chakraborty and S. P. Joshi, Tetrahedron Lett., 1990, 31, 2043.
15 M. F. Beatty, C. Jennings-White and M. A. Avery, J. Chem. Soc.,
Perkin Trans. 1, 1992, 1637.
25 J. M. Bland, Synth. Commun., 1995, 25, 467.
26 K. Wüthrich, NMR of Proteins and Nucleic Acids, Wiley
Interscience, 1986, New York.
27 M. M. Lenman, S. L. Ingham and D. Gani, J. Chem. Soc., Chem.
Commun., 1996, 85.
16 R. J. Valentokovich and S. L. Schreiber, J. Am. Chem. Soc., 1995,
117, 9069.
17 J. M. Humphrey, J. B. Aggen and R. A. Chamberlain, J. Am. Chem.
Soc., 1996, 118, 11759.
28 K. L. Webster, A. P. Mehrotra, A. B. Maude, D. Gani and
T. J. Rutherford, Parts 3 and 4 in this series, in preparation. See also
K. L. Webster, T. J. Rutherford and D. Gani, Tetrahedron Lett.,
1997, 38, 5713.
18 H. Y. Kim and P. L. Toogood, Tetrahedron Lett., 1996, 37, 2349.
19 F. D’Aniello and A. Mann, J. Org. Chem., 1996, 61, 4870.
20 F. D’Aniello, A. Mann, A. Schoenfelder and M. Taddei,
Tetrahedron, 1997, 53, 1447.
29 A. B. Maude and D. Gani, unpublished results.
30 D. D. Perrin, W. L. F. Armarego and D. R. Perrin, in Purification of
Laboratory Chemicals, Pergamon Press, Oxford, 1980.
31 G. R. Pettit and P. S. Nelson, J. Org. Chem., 1986, 51, 1282.
21 P. Hormozdiari and D. Gani, Tetrahedron Lett., 1996, 37, 8227.
22 H. Schwarz, F. M. Bumpus and I. H. Page, J. Am. Chem. Soc., 1957,
79, 5697.
23 M. Goodman and F. Boardman, J. Am. Chem. Soc., 1963, 85, 2483.
24 P. Gmeiner, P. L. Feldman, M. Y. Chu-Moyer and H. Rapoport,
J. Org. Chem., 1990, 55, 3068.
Paper 7/02410J
Received 8th April 1997
Accepted 29th April 1997
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J. Chem. Soc., Perkin Trans. 1, 1997