Stereoselective synthesis of a novel pseudopeptide hapten for the generation of hydrolytic catalytic antibodies

Article abstract of DOI:10.1016/j.tetasy.2004.05.005

The synthesis of a novel hapten containing a 1,4-diamino-2,3-diolbutane core unit is described. The molecule contains four stereogenic centres and has been synthesised via a stereoselective route. The absolute stereochemistry of each stereogenic carbon at

Full text of DOI:10.1016/j.tetasy.2004.05.005

Tetrahedron:
Asymmetry
Tetrahedron: Asymmetry 15 (2004) 1847–1855
Stereoselective synthesis of a novel pseudopeptide hapten for
the generation of hydrolytic catalytic antibodies
a
a
a
ꢀ
Ana Chiva Rodriguez, Anna Pico Ramos, Geoffrey E. Hawkes,
Federico Bertib and Marina Resminia,*
aDepartment of Chemistry, Queen Mary, University of London, Mile End Road, London E1 4NS, UK
bDipartimento di Scienze Chimiche, University of Trieste, Via Giorgieri 1, 34127 Trieste, Italy
Received 22 March 2004; accepted 7 May 2004
Abstract—The synthesis of a novel hapten containing a 1,4-diamino-2,3-diolbutane core unit is described. The molecule contains
four stereogenic centres and has been synthesised via a stereoselective route. The absolute stereochemistry of each stereogenic
carbon atom has been assigned by nuclear Overhauser effect experiments carried out on a number of derivatives.
ꢀ 2004 Elsevier Ltd. All rights reserved.
1. Introduction
and efficiency of the antibody generated. These studies
have highlighted the potential of polyclonal antibodies
as a valuable tool for the evaluation of hapten design,
since their use allows assessment of the entire immune
response.
The production of artificial receptors that can achieve
recognition at the molecular level is an important goal
of organic and bioorganic chemistry. The study of cat-
alytic antibodies is certainly at the forefront of the
search for a wide range of novel enzyme-like mimics,
and aims to assemble together the basic concept of en-
zyme catalysis with the possibility of the immune system
of generating antibodies against virtually any molecule.
This research field has undergone a rapid development
process in the last decade.1;2 From the initial ‘proof of
concept’ and demonstration of fundamental enzyme-like
characteristics, antibodies have been shown to catalyse a
remarkably broad range of organic transformations,
including difficult and unfavourable chemical reactions
and the ones that are not catalysed by enzymes. More-
over research efforts have recently focused on the in-
depth study of the structural and mechanistic basis of
antibody catalysis and on the combinatorial processes
involved in the immune response itself. This has allowed
investigators to begin to understand the important
interactions taking place within the active site and to
evaluate the relationship existing between immunogen
structure and the structure of the catalytic site formed.
As a result, more emphasis has been put on improving
hapten design, which seems to influence the structure
Our previous work3;4 used anionic phosphate and
phosphonate haptens to produce sheep polyclonal cat-
alytic antibody preparations with hydrolytic activity
towards carbonate, ester and amide substrates. These
preparations effect the catalysis of the reactions of these
substrates with hydroxide ion assisted by hydrogen
bond donation at the reaction centre by high pKa side-
chains (tyrosine and arginine).5 In an attempt to pro-
duce antibodies with an active centre carboxyl group to
supply acid/base or nucleophilic catalysis in acidic
media, we are investigating the use of cationic haptens.
O
O
OH
HN
KLH
NO2
3
H2N
OH
1
Herein we report the stereoselective synthesis of the
cationic immunogen 1 and the assignment of the abso-
lute configuration of the four stereogenic centres.
* Corresponding author. Tel.: +44-207-882-3268; fax: +44-207-882-
7794; e-mail: m.resmini@qmul.ac.uk
0957-4166/$ - see front matter ꢀ 2004 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tetasy.2004.05.005

1848
A. C. Rodriguez et al. / Tetrahedron: Asymmetry 15 (2004) 1847–1855
2. Results and discussion
used for the generation of polyclonal antibodies, we
chose to synthesise the immunogen as a single stereo-
isomer to minimise the heterogeneity of the antibody
preparations.
2.1. Design and synthesis of immunogen 1
The design of immunogen 1, incorporating the 1,4-
diamino-2,3-diolbutane core unit, was inspired by
studies on mechanism-based inhibitors for the HIV-1
protease, an enzyme belonging to the family of the
aspartic proteases and characterised by two catalytically
essential aspartate residues in the active site.6–8 A num-
ber of molecules containing this functional moiety have
been shown to be effective inhibitors showing IC50 val-
ues between 0.2 and 0.4 nM.9;10
The synthetic route, shown in Scheme 1, was broadly
inspired by work carried out on similar molecules con-
taining the same 1,4-diamino-2,3-diol core unit.14 The
presence of the 4-nitrophenyl moiety in the structure of
hapten 1, with its significant electron-inductive effect,
altered the expected reactivity and prompted a number
of changes in the original route, in order to obtain the
target molecule 1, containing four stereogenic centres, as
a single stereoisomer.
Significant structural features of the hapten included: (i)
the two amino groups, expected to be protonated in vivo
and able to recruit a basic residue, such as an aspartate,
in the antibody site6–8; (ii) the secondary hydroxyl
groups, included to mimic the tetrahedral transition
state, while attempting to recruit other hydrogen
bonding residues;11 (iii) the 4-nitrophenyl moiety, a
highly antigenic residue in the immunogen and also a
chromogenic leaving group12 (as 4-nitrophenolate) to be
introduced in the substrates; (iv) the presence of the two
aromatic rings, contributing to the formation of
hydrophobic pockets in the active site of the antibody,
that would favour the substrate–antibody binding.
Furthermore the hapten contains a C2 pseudosymmetry
axis that may elicit a symmetrical active site in the
antibody resembling the active site of the HIV prote-
ase.13 The nonleaving aromatic moiety, a benzyl group,
allows us to mimic phenylalanine, resulting in the hap-
ten being a pseudopeptide. Since the hapten would be
The first step of the synthesis was the methylation of N-
Boc-L-Phe with methyl iodide and NaHCO3 to give the
corresponding ester 3. This reacted with the carbanion
generated by reaction of dimethylmethyl phosphonate
with butyl lithium to form the b-ketophosphonate 4.
Comparison of the experimentally determined value of
the specific rotation for 4 with the literature data con-
firmed that there was no evidence of racemisation for
this substrate.15 Phosphonate 4 underwent a Horner–
Wadsworth–Emmons reaction, reacting with p-nitro-
benzaldehyde and K2CO3 in ethanol to give the a,b-
unsaturated ketone 5. The trans isomer was exclusively
obtained, as shown by the coupling constant of the
alkene protons, J ¼ 16:2 Hz.16
The reduction of the ketone in 5 represented a crucial
step in the stereoselective synthesis because it would
create the second stereogenic centre in the molecule,
O
O
O
P
R'CHO, K2CO3
EtOH, N2, r.t.
P
OCH3
OCH3
CO2H
O
BocNH
CO2CH3
BocNH
CH3
BocNH
CH3I
BocNH
OCH3
OCH3
R'
NaHCO3 / DMF
r.t., N2
BuLi, THF
-78oC, N2
R
R
5
2
4
R
3
R
L-Selectride
MeOH, -78oC, N2
OH
N3
O
N3
OH
NaN3 ,NH4Cl
acetone/H2O 55oC
OH
O
BocNH
BocNH
O
O
BocNH
BocNH
R'
R'
R'
R'
CF3COPh, KHSO5,
pTsOH, 70oC, N2
CH3CN, r.t.
R
6
7
R
R
OH
R
O
8
9
PPh3, THF
N2, r.t.
O
O
O
N=PPh3
R'
O
NH2
KLH, MES pH=4.7
DMF, EDC, r.t.
O
HN
OH
3
THF/H2O
80oC
O
O
O
BocNH
1
BocNH
BocNH
R'
HCl/H2O, r.t.
NEt3, 60oC, N2
R'
12
R
O
10
R
O
11
R
O
Scheme 1. Synthetic route to hapten 1, where R ¼ benzyl and R0 ¼ 4-nitrophenyl.

A. C. Rodriguez et al. / Tetrahedron: Asymmetry 15 (2004) 1847–1855
1849
expected to further control the stereochemistry on the
formation of the other two remaining stereogenic cen-
tres. A large number of reducing reagents in different
conditions were tried, among which L-Selectride pro-
vided the most significant results in terms of diastereo-
selectivity, summarised in Table 1. The ratio of the two
diastereoisomers was determined by 1H NMR, from the
integration values of the proton attached to the carbon
that bears the hydroxylic group for each diastereoisomer.
Cram conformation of type c, which is higher in energy
by only 0.9 Kcal/mol. This conformation is likely to be
even more disfavoured in hydrogen bond acceptor sol-
vents such as THF, or donor solvents such as methanol,
which destroy the intramolecular hydrogen bond. On
the contrary, on moving to a solvent such as dichlo-
romethane, which is not hydrogen bonding, conforma-
tions b and c are expected to be very close in energy,
thus offering an explanation for the lack of selectivity in
such solvents.
Table 1. Experimental conditions investigated for the stereoselective
reduction of the ketone moiety in 5
Although a small amount of the main diastereoisomer
of 6 was obtained pure by chromatography and used for
characterisation, in the standard procedure the mixture
of the two diastereoisomers was used for the following
step, the epoxidation of 6 to give 7. An initial attempt
using m-CPBA21 did not provide the desired product
probably due to the electron-deficient characteristics of
alkene 6. Use of H2O2/NaOH led to a mixture with by-
products.22 Epoxide 7 was successfully obtained fol-
lowing reaction with CF3COPh and oxoneꢂ. Oxoneꢂ is
a triple salt composed of 2KHSO5ÆKHSO4ÆK2SO4 and
its active ingredient is potassium peroxymonosulfate,
KHSO5.23 The in situ generated dioxirane reacted with
the double bond in 6, to give epoxide 7 as a single dia-
stereoisomer in 60% yield after purification. The isola-
tion of a single product indicates that the minor
diastereoisomer of 6 present in the reaction mixture is
lost during the purification procedure.
Hydride
Solvent
Temperature
(ꢁC)
Ratio
(S,R)/(S,S)
NaBH4
DIBAL
MeOH
THF
THF
0
1:1
No reaction
)78
)78
)78
0
L-Selectride
L-Selectride
L-Selectride
L-Selectride
L-Selectride
7:3
4:6
CH2Cl2
THF
6:4
5:5
CH2Cl2
MeOH
0
)78
8.5:1.5
Diastereoselective reductions of N-protected a-amino-
ketones have been often reported in the literature, but
the selectivity is hardly predictable in a general way.17
A
number of factors are believed to contribute to the ste-
reoselectivity: the presence of chelating metals, the
bulkiness of the reducing agent and the nature of the
solvent, which is particularly important in the L-Se-
lectride reductions.18 When the reduction of a-amino
enones is carried out by chelating reagents such as alu-
minium hydrides, or in the presence of chelating metals,
anti-reduction is often observed, resulting from the hy-
dride attack on the less hindered side of the chelate
conformation of the substrate, as shown in Figure 1. On
the contrary, in the absence of chelating metals, or if the
amino group is doubly protected by bulky alkyl groups,
the reaction most often occurs via the standard Felkin–
Ahn conformation b, thus leading to syn products.17;19
Nevertheless, anti-reductions occurring via the cyclic
Cram conformation c have also been observed.14
Reaction of 7 with NaN3/NH4Cl to open the epoxide
ring provided azide 8 in good yields. The nucleophilic
reaction is regiospecific on the benzylic carbon and also
highly stereoselective since the NMR of the isolated
product shows the presence of a single diastereoisomer.
At this stage protection of the diol in 8 was required in
order to avoid formation of aziridine derivatives when
reducing the azide group to amine,24 using PPh3 in THF
followed by hydrolysis. In the next step 11 was reacted
with glutaric anhydride to introduce the linker between
the hapten and the carrier protein. The coupling of 12 to
the carrier protein KLH was successfully carried out with
EDC in 0.1 M MES at pH ¼ 4.7 and DMF, following
standard experimental procedures.25 Finally, deprotec-
tion of the Boc and isopropylidene ketal groups was
obtained by suspending the conjugate in HCl (0.2 M) for
24 h. These experimental conditions were previously
tested using the BSA conjugate of hapten 12 and by
carrying out ELISA experiments with polyclonal anti-
bodies readily available in our laboratory. The resulting
data confirmed that both the coupling and deprotection
had taken place successfully. Immunogen 1 is currently
being used in a standard immunisation programme for
the generation of sheep polyclonal antibodies.
H
M
O
H-
O
H-
O
Ph
Boc
Ph
N
Boc
Ph
N
NHBoc
H-
H
H
H
R
R
R
a
c
b
-1
-1
∆Hf,rel = 0.9 Kcal mol
∆Hf,rel = 0.0 Kcal mol
Figure 1. The three possible conformations of the enone 5.
Since L-Selectride is not a chelating reagent, model a can
be ruled out in our case, and the syn selectivity observed
when the reaction is carried out at )78 ꢁC in THF, or
even better in methanol, can be explained using the
Felkin–Ahn model b. A lack of selectivity is observed
when dichloromethane is used as solvent under the same
conditions. We have performed a conformational anal-
ysis on enone 5. The calculations were carried out with
the AM1 Hamiltonian20 and the systematic search yield-
ed 13 minimum energy conformations. The absolute
minimum corresponds to the expected Felkin–Ahn
conformation b, and is immediately followed by a cyclic
2.2. Determination of the absolute configuration of the
stereogenic centres of the hapten by NMR
In this section the stereogenic carbon atoms under
investigation have been numbered as shown in the
structure below for easier understanding.

1850
A. C. Rodriguez et al. / Tetrahedron: Asymmetry 15 (2004) 1847–1855
The first stereogenic centre, C1, has a known configu-
ration, since the starting material for the synthesis is
enantiomerically pure and it has been demonstrated that
no racemisation occurs during the course of the syn-
thetic pathway. Knowing the absolute configuration for
C1 allowed us to assign the absolute configuration for
the other stereogenic centres in the molecule by per-
forming a series of nuclear Overhauser effect (NOE)
experiments on different intermediates of the synthesis.
tween the ortho aromatic protons in 13, and between
H1–H2 for both possible configurations at C2 (R, H1–H2
trans; S, H1–H2 cis). These calculated distances were
Ar ¼ 2.455 A, H –H trans ¼ 2.90 A, H –H cis ¼ 2.37 A.
Therefore the calculated ratio of the distances Ar/H1–H2
trans ¼ 0.85 is in excellent agreement with the experi-
mental data, whereas the calculated ratio Ar/H1–H2
cis ¼ 1.04 is outside the limits of the experimental
data.
1
2
1
2
ꢁ
ꢁ
ꢁ
For the determination of C2, 13 was used because of the
restricted rotation between the C1–C2 bond.
Therefore, sufficient evidence has been obtained to state
that H1 and H2 present a trans disposition in 13.
This indicates that reduction of the ketone 5 has led to
the syn product 6. The diastereoselectivity obtained is in
accordance with the Felkin–Ahn model and is supported
by literature data.30
O
N3
BocN
Ph
1
3
4
2
OH
13
In order to determine the stereochemistry of C3, the
protected diol 9 was used because it presented restricted
rotation about the C2–C3 bond.
NO2
The experiments carried out on 13 were based on the
measurement of the magnitude of the NOE between the
proton attached to C1 (called H1) and the proton at-
tached to C2 (called H2).
A set of experiments similar to the ones carried out on
13 for the determination of the configuration on C2 was
performed on 9. The intensities of the NOE cross peaks
were measured at mixing times 200, 400, 600 and 800 ms.
These longer mixing times than used for 13 above gave
better signal/noise in the spectra, but the plots of the
cross peak volumes versus mixing time extends into the
nonlinear region and required the data be fitted to an
exponential of the form:
The existence of an NOE between the two protons is
shown by a cross peak in the two-dimensional NOESY
NMR experiment. The magnitude of the Overhauser
enhancement between the two protons (here H1 and H2)
can be estimated from the initial slope of a plot of cross
peak intensity (its volume) versus mixing time sm. The
mixing time is a variable time in the NOESY pulse se-
quence, during which the two hydrogens cross relax
(exchange magnetisation). This initial slope (M12) de-
pends upon the cross relaxation rate, which in turn is
proportional to the inverse sixth power of the internu-
clear distance (r12).26;27 This is an established method-
ology that has been reviewed.28;29
V ¼ V f1 ꢀ expðꢀAsmÞg
1
where V is the cross peak volume at mixing time sm, V
1
and A are constants to be determined for each NOE
build-up curve. The initial slope (limit sm ! 0) is given
by the product AÆV .
1
The reference distance was taken this time from the
benzylic protons (Bz), and the distance to be determined
is H2–H3. The initial slopes from the fitted data were
Bz ¼ 0.0572 0.0045, and H2–H3 ¼ 0.0170 0.0035.
This gave the ratio31 of the distances Bz/H2–
H3 ¼ 0.82 0.04.
In order to be able to quantify the H1–H2 distance it is
necessary to construct a similar plot for a pair of protons
with known separation that is a reference distance. This is
provided by a pair of ortho protons on the aromatic ring,
ꢁ
separated by 2.46 A. The experiments were performed at
low temperature, since the hindered amide-type rotation
about the amide bond in the Boc group resulted in broad
lines at ambient temperature and so interfered with the
measurement of the NOE effect. At low temperature,
223 K, this rotation was sufficiently slowed to result in
As before the program Chem 3D was used for the cal-
culation of the distances H2–H3 in 9 for the configura-
tions (S,R,S) and (S,R,R) corresponding to the carbons
(C1,C2,C3). The configuration C3 (S) corresponds to H2–
H3 trans, and C3 (R) to H2–H3 cis. The calculated
1
2
3
ꢁ
ꢁ
well-resolved H NMR spectra. The spectra were mea-
distances were Bz ¼ 1.79 A, H –H trans ¼ 3.08 A and
2
3
H2–H3 cis ¼ 2.33 A. The calculated ratio Bz/ H –H
ꢁ
sured with mixing times 50, 100, 150, 200 and 250 ms. The
cross peak volumes between the ortho protons (Ar) from
either side of the diagonal were averaged at each mixing
time, as were the cross peak volumes between H1 and H2.
The two plots of volume versus mixing time were linear
and fitted to straight lines, and gave slopes
Ar ¼ 0.0322 0.0058, H1–H2 ¼ 0.0122 0.0019. This
gave the ratio of the distances Ar/H1–H2 ¼ 0.85 0.05.
trans ¼ 0.58 is very different to the experimental value,
but the calculated ratio Bz/H2–H3 cis ¼ 0.77 is very close
to the experimental limit. Therefore, it can be concluded
that the configuration for C3 in 9 is R. This establishes
that the epoxide is formed syn to the OH group, possibly
favoured by coordination of the oxirane reagent to the
OH substituent. The consequence of this is that the
configuration of the four stereogenic centres in epoxide
7 results being (S,R,R,S), respectively, for (C1,C2,C3,
C4).
The 3D molecular modelling program Chem 3D was
used to calculate the theoretical atomic distances be-

A. C. Rodriguez et al. / Tetrahedron: Asymmetry 15 (2004) 1847–1855
1851
Having determined the stereochemistry of the epoxide
ring this leads to the assignment of the configuration of
the last stereogenic centre in the final compound, formed
by the opening of the epoxide ring with sodium azide.
The attack of the nucleophile on C4 will be on the side
opposite to the oxygen bridge and will involve the
inversion of the configuration of the carbon to which the
nucleophile will be finally attached, therefore providing
an (R)-configuration for C4.
AcOEt (30 mL) and water (10 mL). The aqueous phase
was acidified to pH 3 with HCl 1 M and extracted with
AcOEt (3 · 40 mL). The combined organic extracts were
washed with a saturated solution of NaCl (40 mL), dried
over anhydrous MgSO4 and evaporated to dryness.
Ester 3 was obtained as a brown oil (0.480 g, 91% yield)
[Rf 0.40 AcOEt–PE (15:85)]. Although a small sample
was further purified for characterisation, the crude
product was considered pure enough to be used in the
next step without further purification.
The remaining synthetic steps leading to the target
hapten do not affect the stereochemistry of any of the
centres. Furthermore the isolation of a single stereo-
isomer product with each subsequent reaction leading
from 8 to 12 confirms that the stereochemistry is pre-
served. Any racemisation occurring at this stage would
lead to formation of diastereoisomers, easily detectable
in the reaction mixture.
½aꢁ ¼ )4.2 (c 1.43, MeOH) [lit.31 ½aꢁ ¼ )4.6 (c 1.0,
D
D
1
MeOH)]; H NMR (CDCl3, 270 MHz): d 7.0–7.3 (m,
5H), d 4.9 (br, 1H), d 4.5–4.6 (m, 1H), d 3.6 (s, 3H), d
2.8–3.1 (m, 2H), d 1.3 (s, 9H); MS (FAB, m=z): 280
([MH]þ), 243 and 180.
4.3. Dimethyl [(3S)-4-phenyl-3-[N-(tert-butyloxycarbon-
yl)amino]-2-oxobutyl]phosphonate 4
3. Conclusions
Butyl lithium solution in hexane (1.6 M, 13.4 mL,
21.5 mmol) was added dropwise to a stirred solution of
dimethyl methanephosphonate (2.3 mL, 21 mmol) in
THF (20 mL) under a nitrogen atmosphere at )78 ꢁC.
The solution was stirred for 10 min at )78 ꢁC. A solution
of ester 3 (1.000 g, 3.579 mmol) in THF (5 mL) was
added dropwise and stirred for 2 h at )78 ꢁC, whereupon
TLC monitoring (AcOEt) showed that the reaction had
gone to completion and the product formed had an
Rf ¼ 0:41. Water (10 mL) was added to the reaction
solution and the aqueous layer was neutralised with HCl
1 M. The THF and the water were removed in vacuo
and the residue was partitioned between AcOEt (30 mL)
and water (10 mL). The aqueous layer was extracted
with AcOEt (3 · 30 mL). The combined organic frac-
tions were washed with a saturated solution of NaCl
(10 mL), dried over MgSO4 and concentrated to dryness.
The desired product was afforded in 90% yield (1.200 g)
as a brown oil pure enough to be directly used in the
next step. A small sample was further purified for full
characterisation.
The stereoselective synthesis of the diamino diol hapten
1 never reported before has been established, together
with the absolute configuration of the stereogenic cen-
tres, found to be (C1,C2,C3,C4): (S,R,R,R). This hapten
is currently used for the first time in an immunisation
programme for the generation of sheep polyclonal cat-
alytic antibodies.
4. Experimental
4.1. General remarks
All the chemicals used in synthesis were purchased from
Aldrich Chemical Co. (Gillingham, Dorset, UK), Lan-
caster Chemical Co. (Morecambe, Lancashire, UK),
Sigma Chemical Co. (Poole, Dorset, UK) and Merck
Ltd (Lutterworth, Leicestershire, UK). Melting points
were measured on a Reichert microscope melting point
apparatus (Reichert, Austria). Optical rotation was
measured with a polarimeter from Optical activity Ltd
(Huntingdon, UK) and all measurements were done at
25 ꢁC Model AA-100. NMR experiments were per-
formed on a JEOL EX270, on a Bruker AM250, on a
Bruker AMX-400 and on a Bruker Avance-600. Mass
spectra were recorded on a ZAB-SE4F FAB þ mass
spectrometer.
Mp 74–76 ꢁC [lit.15 74–76 ꢁC]; ½aꢁ ¼ )10.5 (c 2.65,
D
1
CHCl3) [lit.15 ½aꢁ ¼ )5.94 (c 1.80, CHCl3)]; H NMR
D
(CDCl3, 270 MHz): d 7.1–7.3 (m, 5H), d 5.4 (d,
J ¼ 8:0 Hz, 1H),
d 4.5–4.6 (m, 1H), d 3.8 (d,
J ¼ 11:3 Hz, 3H), d 3.8 (d, J ¼ 11:3 Hz, 3H), d 2.9–3.4
(m, 4H), d 1.3 (s, 9H); 13C NMR (CDCl3, 67.5 MHz): d
201.2, 155.3, 136.5, 129.4, 128.7, 127.0, 80.2, 61.3, 53.2,
37.6, 37.0, 28.3; MS (FAB, m=z): 372 ([MH]þ), 316 and
272.
4.2. Methyl (2S)-3-phenyl-2-[N-(tert-butyloxycarbonyl)-
amino]propanoate 3
Under a nitrogen atmosphere N-Boc-
L
-Phe (0.500 g,
4.4. (1E,4S)-4-[N-(tert-Butyloxycarbonyl)amino]-1-(4-ni-
trophenyl)-5-phenylpent-1-en-3-one 5
1.78 mmol) was added to THF (4 mL); the solution was
then stirred for 5 min at 25 ꢁC. NaHCO3 solid (0.470 g,
5.59 mmol) was added followed by the addition of CH3I
(0.350 mL, 5.62 mmol) dropwise. The reaction was stir-
red for 18 h at room temperature until the TLC moni-
toring system [AcOEt–PE (15:85)] showed no increase in
the intensity of the product spot. The solvent was re-
moved in vacuo. The residue was partitioned between
p-Nitrobenzaldehyde (0.352 g, 2.51 mmol) was added
to a stirred solution of phosphonate 4 (1.150 g,
3.096 mmol) in EtOH (42 mL) under a nitrogen atmo-
sphere at room temperature. K2CO3 (0.560 g,
4.05 mmol) was added in small portions over a period of
15 min. The reaction was stirred for 1 h whereupon TLC

1852
A. C. Rodriguez et al. / Tetrahedron: Asymmetry 15 (2004) 1847–1855
monitoring (AcOEt) showed that phosphonate 4 was
consumed and that there was still some aldehyde that
had not reacted (eluting system AcOEt–PE 1:9 running
the TLC plate twice). The reacting solution was filtered
and neutralised with glacial acetic acid if necessary. The
solvent was removed in vacuo. The residue was parti-
tioned between AcOEt (45 mL) and saturated Na2S2O5
(15 mL). The aqueous phase was extracted with AcOEt
(3 · 45 mL). The combined organic layers were washed
with saturated Na2S2O5 (30 mL) until the TLC moni-
toring system [AcOEt–PE (1:9) running the TLC plate
twice] showed that there was no p-nitrobenzaldehyde in
the organic layer. The combined organic layers were
washed with saturated NaHCO3 (50 mL) and saturated
NaCl (50 mL), dried over MgSO4, filtered and concen-
trated giving 5 as a yellow solid after recrystallisation in
di-isopropyl ether (0.560 g, 60% yield) [Rf 0.16; AcOEt–
PE (1:9) running the TLC plate twice].
124.0, 79.9, 72.3, 60.5, 56.3, 38.0, 28.3; MS (FAB, m=z):
399 ([MH]þ), 343 and 325.
4.6. (1S,2S,3R,4S)-4-[N-(tert-Butyloxycarbonyl)amino]-
1,2-epoxy-1-(4-nitrophenyl)-5-phenylpentan-3-ol 7
Trifluoroacetophenone (0.860 mL, 6.12 mmol) was
added to a stirred solution of alcohol 7 (0.500 g,
1.25 mmol) (mixture of the two diastereoisomers) in
acetonitrile (35 mL). A 4 · 10ꢀ4 M EDTA solution
(23.2 mL, 9.28 · 10ꢀ3 mmol) was added as well as a solid
mixture of NaHCO3 (1.750 g, 20.83 mmol) and oxone
(3.930 g, 6.392 mmol) in small portions over a 10 min
period. The reaction was left stirring overnight exclud-
ing the flask from light. The TLC monitoring system [Rf
0.29; AcOEt–PE (15:85), running the TLC plate three
times] showed that the reaction had gone to completion
after 30 h. The solvent was evaporated and the resulting
reaction crude was partitioned between AcOEt (30 mL)
and water (10 mL). The aqueous phase was extracted
with AcOEt (3 · 30 mL) and the combined organic ex-
tracts were washed with brine (30 mL). The organic
phase was dried over MgSO4, filtered and concentrated
under reduced pressure to provide an oil that was fur-
ther purified by flash chromatography. The purification
[eluting system AcOEt–PE (2:8)] provided product 7, as
a single diastereoisomer, as a white solid in 60% yield
(0.312 g).
1
Mp 120–122 ꢁC; ½aꢁ ¼ )3.0 (c 1.59, CHCl3); H NMR
D
(CDCl3, 270 MHz): d 8.2 (d, J ¼ 9:0 Hz, 2H), d 7.6 (d,
J ¼ 16:2 Hz, 1H), d 7.6 (d, J ¼ 9:0 Hz, 2H), d 7.1–7.4
(m, 5H), d 6.7 (d, J ¼ 16:2 Hz, 1H), d 5.3 (d, J ¼ 7:1 Hz,
1H), d 4.8–5.0 (m, 1H), d 3.1 (d, J ¼ 6:7 Hz, 2H), d 1.4
(s, 9H); 13C NMR (CDCl3, 62.5 MHz): d 197, 155, 148,
141, 140, 136, 130, 129, 128, 126, 125, 123, 81, 60, 38, 28;
MS (FAB, m=z): 397 ([MH]þ), 341 and 297.
4.5. (1E,3S,4S)-4-[N-(tert-Butyloxycarbonyl)amino]-1-(4-
nitrophenyl)-5-phenylpent-1-en-3-ol 6
Mp 119–120 ꢁC; ½aꢁ ¼ )22.8 (c 0.94, CHCl3); 1H NMR
D
(CDCl3, 270 MHz): d 8.2 (d, J ¼ 8:6 Hz, 2H), d 7.4 (d,
J ¼ 8:6 Hz, 2H), d 7.1–7.3 (m, 5H), d 4.9 (d, J ¼ 9:2 Hz,
1H), d ¼ 4:1 (br, 1H), d 3.8–4.0 (m, 1H), d 3.8–3.9 (m,
1H), d 3.0 (br, 1H), d 2.8–3.0 (m, 2H), d 2.8 (d,
J ¼ 3:2 Hz, 1H), d 1.3 (s, 9H); 13C NMR (CDCl3,
67.5 MHz): 156, 148, 144, 138, 130, 129, 127, 126, 124,
81, 69, 64, 55, 54, 39, 28; MS (FAB, m=z): 415 ([MH]þ),
359 and 315.
Under a nitrogen atmosphere, enone 6 (100.0 mg,
0.2522 mmol) was dissolved in freshly distilled MeOH
(40 mL) at )78 ꢁC. L-Selectride solution (1 M, 0.760 mL,
0.760 mmol) was added and the resulting solution was
left stirring for 1 h at )78 ꢁC, whereupon TLC moni-
toring [AcOEt–PE (3:7)] showed that the reaction had
gone to completion. The reaction was acidified to pH 5
with a 1 M HCl solution and the solvent was removed by
rotary evaporation. The residue was partitioned between
AcOEt (30 mL) and saturated NaHCO3 solution (10 mL)
and the aqueous layer was extracted with AcOEt
(3 · 30 mL). The combined organic phases were washed
with saturated NaCl solution (40 mL), dried over MgSO4
and concentrated in vacuo. Purification of the residue by
flash chromatography [eluting solvent AcOEt–PE (2:8)]
gave the mixture of diastereoisomers (S,S) and (S,R) on a
8.5:1.5 ratio (78.4 mg, 78% yield). [Rf 0.35; AcOEt–PE
(3:7)]. A small sample of the main diastereosisomer (S,S)
was obtained pure from the chromatography and used to
obtain the characterisation data described below. The
minor diastereoisomer is present in very small quantities
and has not been isolated pure.
4.7. (1R,2R,3R,4S)-1-Azido-4-[N-(tert-butyloxycarbon-
yl)amino]-1-(4-nitrophenyl)-5-phenylpentane-2,3-diol 8
Epoxide 7 (0.350 g, 0.844 mmol) was dissolved in a bi-
phasic system composed of acetone (10.8 mL) and water
(5.4 mL). A mixture of NaN3 (0.740 g, 11.3 mmol) and
NH4Cl (0.611 g, 11.3 mmol) was added as a solid. The
reacting solution was refluxed at 55 ꢁC for 30 h. Diol 8
was detected by the TLC monitoring system [Rf 0.56;
AcOEt–PE (3:7), running the TLC plate twice] and the
solvent pair was rotary evaporated. The residue was
partitioned between AcOEt (60 mL) and water (20 mL).
The aqueous phase was extracted with AcOEt
(3 · 60 mL) and the combined organic extracts were
washed with NaCl saturated solution (40 mL), dried
over MgSO4 anhydrous, filtered and concentrated to
dryness. The resultant oily product was purified by flash
chromatography [eluting solvent AcOEt–PE (15:85)] to
give 8 in 80% (0.310 g) as off-white solid.
Compound 6 (S,S): mp 110–112 ꢁC; ½aꢁ ¼ )15.9 (c 2.08,
D
CHCl3); 1H NMR (CDCl3, 270 MHz): d 8.1 (d,
J ¼ 9:0 Hz, 2H), d 7.5 (d, J ¼ 9:0 Hz, 2H), d 7.1–7.3 (m,
5H), d 6.7 (d, J ¼ 14:5 Hz, 1H), d 6.3 (dd, J ¼ 6:2,
14.5 Hz, 1H), d 4.9 (d, J ¼ 9:8 Hz, 1H), d 4.3 (br, 1H), d
3.9 (br, 1H), d 3.1 (br, 1H), d 2.9 (d, J ¼ 6:7 Hz, 2H), d
1.3 (s, 9H); 13C NMR (CDCl3, 67.5 MHz): d 156.3,
147.0, 143.3, 138.0, 135.1, 129.3, 128.7, 127.1, 126.7,
Mp 130–132 ꢁC; ½aꢁ ¼ þ40:4 (c 0.50, CHCl3); 1H NMR
D
(CDCl3, 270 MHz): d 8.2 (d, J ¼ 8:8 Hz, 2H), d 7.6 (d,

A. C. Rodriguez et al. / Tetrahedron: Asymmetry 15 (2004) 1847–1855
1853
J ¼ 8:8 Hz, 2H), d 7.2–7.4 (m, 5H), d 4.8 (d, J ¼ 5:6 Hz,
1H), d 4.7 (d, J ¼ 8:6 Hz, 1H), d 4.4 (d, J ¼ 4:2 Hz, 1H),
d 3.9–4.1 (m, 1H), d 3.5–3.7 (m, 1H), d 3.2–3.4 (m, 1H),
d 3.0 (d, J ¼ 5:6 Hz, 1H), d 2.7–3.0 (m, 2H), d 1.3 (s,
9H); 13C NMR (CDCl3, 100 MHz): d 157.7, 147.9, 143.6,
137.4, 129.3, 128.9, 128.6, 126.7, 123.6, 80.9, 73.4, 73.1,
66.8, 52.3, 37.3, 28.1; MS (FAB, m=z): 458 ([MH]þ), 358,
164 and 119.
water (52 lL, 2.8 mmol) was added and the solution was
stirred at room temperature for 24 h. The reaction solu-
tion was concentrated to dryness and the residue was
partitioned between AcOEt (60 mL) and water (20 mL).
The aqueous layer was extracted with AcOEt (3 · 60 mL).
The combined organic extracts were dried over MgSO4,
filtered and concentrated under reduced pressure. Puri-
fication of the crude by flash chromatography [eluting
system AcOEt–PE (5:5)] provided iminophosphorane 10
in 65% yield (0.200 g) as a foamy beige solid.
4.8. (S)-1-{(4R,5R)-5-[(R)-Azido-(4-nitrophenyl)methyl]-
2,2-dimethyl-1,3-dioxolan-4-yl}-N-(tert-butyloxycarbon-
yl)-2-phenylethylamine 9 and (1R,2R)-2-azido-1-[(4S,
5R)-3-(tert-butyloxycarbonyl)amino-2,2-dimethyl-1,3-
oxazolan-5-yl]-2-(4-nitrophenyl)-ethanol 13
1H NMR (CDCl3, 270 MHz): d 7.6 (d, J ¼ 8:8 Hz, 2H),
d 7.0–7.4 (m, 20H), d 6.9 (d, J ¼ 8:7 Hz, 2H), d 5.0 (br,
1H), d 4.5–4.6 (m, 1H), d 4.4–4.5 (m, 2H), d 4.1–4.3 (m,
1H), d 2.9 (dd, J ¼ 5:9, 13.2 Hz, 1H), d 2.7 (dd, J ¼ 9:1,
13.3 Hz, 1H), d 1.3 (s, 9H), d 1.1 (s, 6H); 13C NMR
(CDCl3, 67.5 MHz): d 154.9, 153.0, 146.0, 138.5, 132.4,
132.3, 131.1, 129.8, 128.4, 128.2, 126.0, 122.8, 107.8,
83.1, 78.5, 58.0, 51.1, 39.7, 28.6, 26.6, 24.6; 31P NMR
(CDCl3, 109.3 MHz): d 9.1; MS (FAB, m=z): 733
([MH]þ), 278 and 262.
p-Toluenesulfonic acid (40.0 mg, 0.232 mmol) was added
to a stirred solution of azide 8 (1.030 g, 2.251 mmol) in
dimethoxypropane (11.2 mL, 91.5 mmol). The mixture
was refluxed at 70 ꢁC and after a period of 1 h the
reaction had reached completion, as determined by TLC
monitoring system [AcOEt–PE (2:8)], RF ¼ 0:50. The
solvent was evaporated and the resultant residue was
extracted with AcOEt (50 mL) and saturated NaHCO3
aqueous solution (20 mL). The aqueous phase was ex-
tracted with AcOEt (3 · 50 mL) and the combined or-
ganic extracts were dried over MgSO4, filtered and
concentrated under reduced pressure. The crude was
purified by flash chromatography [eluting system
AcOEt–PE (5:95)] affording 9 as a white solid in 55%
(0.610 g) yield and 13 as a white solid in 28% (0.310 g).
4.10. (S)-1-{(4R,5R)-5-[(R)-Amino-(4-nitrophenyl)methyl]-
2,2-dimethyl-1,3-dioxolan-4-yl}-N-(tert-butyloxycarbonyl)-
2-phenylethylamine 11
To a stirred solution of azide 9 (0.360 g, 0.723 mmol) in
anhydrous THF (66 mL) under a nitrogen atmosphere,
PPh3 (0.210 g, 0.801 mmol) was added. The solution was
left stirring over a period of 24 h. Upon disappearance of
the starting material monitored by TLC {[AcOEt–PE
(3:7)], Rf ¼ 0:05}, water (68 mL) was added and the
solution was refluxed at 80 ꢁC for 24 h. After this period,
the TLC monitoring system [AcOEt–PE (3:7) running the
TLC plate twice] showed a spot at Rf ¼ 0:39 corre-
sponding to amine 11. The solvent pair was evaporated
and the residue was partitioned between AcOEt (60 mL)
and water (20 mL). The aqueous layer was extracted with
AcOEt (3 · 60 mL). The combined organic extracts were
dried over MgSO4, filtered and concentrated under re-
duced pressure. Purification of the crude product by flash
chromatography [eluting system AcOEt–PE (3:7)] pro-
vided amine 11 (0.205 g, 60% yield) as a foamy beige solid.
Compound 9: mp 72–74 ꢁC; ½aꢁ ¼ þ15:7 (c 1.03,
D
CHCl3); 1H NMR (CDCl3, 270 MHz): d 8.2 (d,
J ¼ 8:6 Hz, 2H), d 7.4 (d, J ¼ 8:6 Hz, 2H), d 7.2–7.4 (m,
5H), d 4.9 (d, J ¼ 7:8 Hz, 1H), d 4.9 (d, J ¼ 10:2 Hz, 1H),
d 4.2–4.4 (m, 1H), d 4.1 (d, J ¼ 7:0 Hz, 1H), d 4.0 (dd,
J ¼ 6:7, 10.2 Hz, 1H), d 2.7–3.0 (m, 2H), d 1.4 (s, 9H), d
1.3 (s, 3H), d 1.2 (s, 3H); 13C NMR (CDCl3, 67.5 MHz): d
161.5, 151.4, 144.2, 136.0, 135.7, 135.4, 133.1, 132.1,
130.6, 115.2, 86.3, 69.7, 56.6, 47.7, 36.9, 35.0, 34.8, 33.2,
30.8; MS (FAB, m=z): 498 ([MH]þ), 398, 278 and 220.
Compound 13: mp 68–71 ꢁC; ½aꢁ ¼ þ64:5 (c 1.14,
D
CHCl3); 1H NMR (CDCl3, 270 MHz): d 8.2 (d,
J ¼ 8:3 Hz, 2H), d 7.4 (d, J ¼ 8:4 Hz, 2H), d 7.1 (br, 3H),
d 6.9 (br, 2H), d 4.7 (d, J ¼ 3:8 Hz, 1H), d 4.2 (br, 1H), d
3.8–3.9 (m, 1H), d 3.3–3.4 (m, 1H), d 3.0 (br, 2H), d 2.7
(br, 1H), d 1.6 (s, 6H), d 1.5 (s, 9H); 13C NMR (CDCl3,
100 MHz): d 151.0, 150.0, 146.9, 141.2, 136.1, 128.8,
127.2, 125.6, 122.4, 93.7, 79.5, 73.1, 64.9, 59.8, 38.4, 35.9,
27.5, 25.7; MS (FAB, m=z): 498 ([MHþ]), 406 and 306.
Mp 112–115 ꢁC; ½aꢁ ¼ )25.3 (c 4.14, CHCl3); 1H NMR
D
(CDCl3, 270 MHz): d 8.1 (d, J ¼ 8:6 Hz, 2H), d 7.4 (d,
J ¼ 8:6 Hz, 2H), d 7.2–7.4 (m, 5H), d 4.9 (d, J ¼ 9:8 Hz,
1H), d 4.4–4.6 (m, 1H), d 4.0–4.3 (m, 3H), d 2.8–3.0 (m,
2H), d 1.5 (br, 2H), d 1.4 (s, 9H), d 1.2 (s, 6H); 13C NMR
(CDCl3, 67.5 MHz): d 155.4, 152.3, 147.3, 138.2, 129.7,
128.6, 127.9, 126.6, 124.0, 108.3, 80.4, 79.7, 54.2, 50.3,
41.3, 28.6, 26.9, 24.4; MS (FAB, m=z): 472 ([MH]þ), 416
and 372. Anal. Calcd for C25H33O6N3: C, 63.8; H, 7.0;
N, 8.9 required; C, 59.9; H, 6.3; N, 6.8 found.
4.9. (S)-1-{(4R,5R)-5-[(R)-Triphenyliminophosphorano-
(4-nitrophenyl)methyl]-2,2-dimethyl-1,3-dioxolan-4-yl}-
N-(tert-butyloxycarbonyl)-2-phenylethylamine 10
4.11. 5-{(R)[(4R,5R)-5-(1S)-1-[N-(tert-butyloxycarbonyl)-
amino]-2-phenylethyl-2,2-dimethyl-1,3-dioxolan-4-yl]-(4-
nitrophenyl)methylamino}-5-oxopentanoic acid 12
To a solution of azide 9 (0.210 g, 0.422 mmol) in freshly
distilled THF (38 mL) under a nitrogen atmosphere, PPh3
(0.120 g, 0.457 mmol) was added. The solution was left
stirring over a period of 24 h. Upon disappearance of the
starting material monitored by TLC (AcOEt, Rf ¼ 0:27),
To a solution of amine 11 (100.0 mg, 0.2120 mmol)
in freshly distilled THF (20 mL) under a nitrogen

1854
A. C. Rodriguez et al. / Tetrahedron: Asymmetry 15 (2004) 1847–1855
atmosphere, glutaric anhydride (40.0 mg, 0.351 mmol)
and NEt3 (60 lL, 0.43 mmol) were added. The mixture
was refluxed at 60 ꢁC for a period of 22 h, after which the
reaction had gone to completion, TLC monitoring sys-
tem [AcOEt–PE (6:4)], Rf ¼ 0:24. After evaporation of
the solvent, the residue was partitioned between AcOEt
(50 mL) and water at pH ¼ 5 (15 mL). The aqueous
phase was extracted with AcOEt (3 · 50 mL) keeping the
pH of the aqueous phase at 5. The combined organic
extracts were dried over MgSO4, filtered and concen-
trated to dryness. The crude product was purified by
flash chromatography using a gradient AcOEt–cyclo-
hexane (2:8) to AcOEt–cyclohexane (5:5) to afford a
dark green oil (77.0 mg, 62% yield).
References and notes
1. Thomas, N. R. Nat. Prod. Rep. 1996, 13, 479–511.
2. Stevenson, J. D.; Thomas, N. R. Nat. Prod. Rep. 2000, 17,
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Pavli, E. P.; Watts, A. B.; Verma, C.; Gallacher, G.;
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P. G. Nature 1989, 338, 269–271.
7. Reymond, J. L.; Janda, K. D.; Lerner, R. A. Angew.
Chem., Int. Ed. Engl. 1991, 30(12), 1711–1713.
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6573–6574.
9. Kempf, D. J.; Codacovi, L.; Wang, X. C.; Kohlbrenner,
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1H NMR (CDCl3, 250 MHz): d 8.1 (d, J ¼ 8:5 Hz, 2H),
d 7.4 (d, J ¼ 8:5 Hz, 2H), d 7.0–7.3 (m, 5H), d 4.7–5.0
(m, 3H), d 4.0–4.2 (m, 3H), d 2.6–2.9 (m, 2H), d 2.3 (t,
J ¼ 6:8 Hz, 2H), d 2.2 (t, J ¼ 7:2 Hz, 2H), d 1.7–1.9 (m,
2H), d 1.4 (s, 9H), d 1.3 (s, 6H); 13C NMR (CDCl3,
67.5 MHz): d 176.8, 172.8, 156.3, 147.7, 147.3, 137.8,
129.6, 128.8, 128.3, 127.0, 123.8, 108.7, 80.9, 76.6, 54.4,
51.0, 41.1, 35.1, 33.3, 28.6, 27.2, 24.7, 20.5; MS (FAB,
m=z): 608 ([MNa]þ), 586 ([MH]þ), 486 and 472. Anal.
Calcd for C30H39O9N3: C, 61.5; H, 6.7; N, 7.2 required;
C, 60.9; H, 6.8; N, 8.4 found.
11. Shokat, K. M.; Ko, M. K.; Scanlan, T. S.; Kochersperger,
L.; Yonkovich, S.; Thaisrivongs, S.; Schultz, P. G. Angew.
Chem., Int. Ed. Engl. 1990, 29, 1296–1303.
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372.
4.12. Coupling of 12 to KLH
To KLH (25.0 mg, 3.7 · 10ꢀ6 mmol) reconstituted in
water (2.5 mL), 0.1 M PBS pH ¼ 7.3 (5 mL) was added.
After the solution was stirred gently, acid 12 (20.0 mg,
3.4 · 10ꢀ2 mmol) previously dissolved in DMF (1.5 mL),
was added dropwise followed by the addition of 0.1 M
MES pH ¼ 4.7 (5 mL). EDC (25.0 mg, 0.13 mmol) was
added portionwise and the mixture was left stirring for a
period of 5 h. Upon completion of the reaction,
the reacting solution was dialysed against water
using dialysis tubing-Visking size 8 32/32 in. and freeze
dried.
13. Marastoni, M.; Salvadori, S.; Bortolotti, F.; Tomatis, R.
J. Pept. Res. 1997, 49, 538–544.
14. Benedetti, F.; Miertus, S.; Norbedo, S.; Tossi, A.;
Zlatoidzky, P. J. Org. Chem. 1997, 62, 9348–9353.
15. Deziel, R.; Pante, R.; Caron, V.; Grenier, L.; Llinas-
Brunet, M.; Duceppe, J.-S.; Malenfant, E.; Moss, N.
J. Org. Chem. 1996, 61, 2901–2903.
16. Pretsch, E.; Bulhmann, P.; Afforlter, C. Structure Deter-
mination of Organic Compounds; Springer: New York,
2000; p 168.
ꢁ
17. Vabenø, J.; Brisander, M.; Lejon, T.; Luthman, K. J. Org.
Chem. 2002, 67, 9186–9191.
18. Koskinen, A. M.; Koskinen, P. M. Tetrahedron Lett. 1993,
34(42), 6765–6768.
19. Reetz, M. T. Chem. Rev. 1999, 99, 1121.
20. Dear, M. J. S.; Reynold, C. H. J. Comp. Chem. 1986, 2,
140.
4.13. Deprotection of the amino and isopropylidene ketal
groups in conjugate of 12 to KLH
21. Fieser, L. F.; Fieser, M. Reagents for Organic Synthesis,
1st ed.; John Wiley and Sons: USA, 1979; pp 62–
63.
22. Zwanenburg, B.; Wiel, J. Tetrahedron Lett. 1970, 12, 935–
936.
The conjugate KLH-12 (50.0 mg, 7.4 · 10ꢀ6 mmol) was
dissolved in 0.2 M HCl (50.0 mL, 10 mmol). The solu-
tion was left stirring gently over a period of 24 h. The
mixture was dialysed against water using dialysis tubing-
Visking size 8 32/32 in. and freeze dried.
23. Grocock, E. L.; Marples, B. A.; Toon, R. C. Tetrahedron
2000, 56, 989–992.
24. Florez-Alvarez, J.; Brocklehurst, K.; Gallacher, G.; Res-
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26. Sanders, J. K. M.; Hunter, B. K. Modern NMR Spectro-
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27. Neuhaus, D.; Williamson, M. P. The Nuclear Overhouser
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New York, 1989; p 35.
We wish to thank Dr. H. Tomms (ULIRS) for per-
forming the NMR spectra. Queen Mary, University of
London, (studentship to A.C.R.), EC Erasmus Pro-
gramme (A.P.R.) the Royal Society, the London Central
Research Fund and the EPSRC (Award GR/R04102/01
towards purchase of the Avance-400NMR spectrome-
ter) are acknowledged for financial support.
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