1-Oxabicyclic β-lactams as new inhibitors of elongating MPT-a key enzyme responsible for assembly of cell-surface phosphoglycans of Leishmania parasite

Article abstract of DOI:10.1039/b418247b

New iminosugars (1-oxabicyclic β-lactam disaccharides) have been synthesized as inhibitors of elongating α-D-mannosyl phosphate transferase (eMPT), a key enzyme involved in the iterative biosynthesis of cell-surface phosphoglycans of the Leishmania parasi

Full text of DOI:10.1039/b418247b

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A R T I C L E
1-Oxabicyclic b-lactams as new inhibitors of elongating MPT–a key
enzyme responsible for assembly of cell-surface phosphoglycans of
Leishmania parasite†
Dipali Ruhela, Patrali Chatterjee and Ram A. Vishwakarma*
Bio-organic Chemistry Lab, National Institute of Immunology, JNU Complex, New Delhi,
110067, India. E-mail: ram@nii.res.in; Fax: 91-11-26162125; Tel: 91-11-26717178
Received 3rd December 2004, Accepted 1st January 2005
First published as an Advance Article on the web 8th February 2005
New iminosugars (1-oxabicyclic b-lactam disaccharides) have been synthesized as inhibitors of elongating
a-D-mannosyl phosphate transferase (eMPT), a key enzyme involved in the iterative biosynthesis of cell-surface
phosphoglycans of the Leishmania parasite. The design is based on a transition-state model for this remarkable
enzyme that transfers intact a-D-mannosyl-phosphate from GDP-Man. Since these phosphoglycans are unique to
Leishmania and are essential for its infectivity and survival, their biosynthetic pathway has emerged as a novel target
for anti-leishmanial drug and vaccine design.
The most distinct feature of the LPG/PPG structure is the
Introduction
variable PG domain composed of [6Galp-b1,4-Manp-a1-PO4]n
The protozoan parasite Leishmania, responsible for multiple
repeats linked to each other via phosphodiester bonds between
human diseases, survives in extremely hostile milieu throughout
the anomeric-OH of the mannose of one repeat and 6-OH of
its life cycle in both the sand-fly vector and the human host.
the galactose of the adjoining repeat, a structure unique to
To accomplish this, all Leishmania species synthesize1 a unique
Leishmania among all the carbohydrates known in nature. The
class of molecules termed phosphoglycans (PGs), including
PG repeats form a spring-like helical supramolecular assembly
the membrane-bound lipophosphoglycan (LPG) and secreted
around the parasite that provides resistance to host enzymes
proteophosphoglycan (PPG). Substantial evidence has been
and antibodies, and constitutes the epitopes for recognition
accumulated showing that the PGs are essential virulence
of macrophage receptors. Although the biosynthetic pathway
factors, being responsible for (a) infectivity and survival of the
of LPG has not been elucidated in detail, a cell-free system
parasite in the human host; (b) attachment and maturation in
the sand-fly mid-gut; (c) recognition of the specific carbohydrate
using Leishmania microsomal membranes has been reported4
and parasite mutants have been isolated3 that are deficient in
binding sites on macrophages; (d) inhibition of normal signalling
specific genes and produce truncated LPGs. The current data4,5
of the immune system and (e) pathological lesions caused by
suggest that the PG domain is assembled by the sequential action
subsequent disease. The role(s) of PGs in parasitic virulence has
of the a-D-mannosyl phosphate transferase (MPT) and 1,4-b-
been a topic of intense debate2 in recent years, but it has now been
galactosyltransferase (GalT) enzymes (Fig. 2). At least two types
established3 that the principal determinant of virulence consists
of the PG domain and is independent of the molecular platform.
The intriguing structure (Fig. 1) of LPG of L. donovani, the
of MPTs are present, one with specificity for the GPI-anchor
and the second with specificity for the [6Galp-b1,4-Manp-a1-
PO4] repeat. These enzymes, described as the initiating-MPT and
species causing fatal visceral leishmaniasis, consists of four dis-
elongating-MPT respectively, have not been purified or cloned.
tinct domains: an alkyl-lyso-glycosylphosphatidylinositol (GPI)
The MPTs are unique enzymes that can transfer an intact a-
anchor, a conserved glycan core with an internal Galf residue,
D-mannose-phosphate moiety from the nucleotide sugar donor
variable PG repeats and a neutral oligosaccharide cap.
GDP-Man to the glycan substrate. It should be mentioned
here that in normal biology a mannose residue is transferred
1
from GDP-Man with the release of a GDP unit, whereas in
Leishmania intact a-D-mannose-phosphate is transferred with
the release of GMP (Fig. 2). Since there are no such MPT
† Electronic supplementary information (ESI) available: Copies of H
and 13C NMR spectra for new compounds (6, 1, 7, 2 and 9–13). See
http://www.rsc.org/suppdata/ob/b4/b418247b/
Fig. 1 Structure of LPG of Leishmania donovani.
T h e R o y a l S o c i e t y o f C h e m i s t r y 2 0 0 5 O r g . B i o m o l . C h e m . , 2 0 0 5 , 3 , 1 0 4 3 – 1 0 4 8
T h i s j o u r n a l i s
1 0 4 3
©

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Fig. 2 Proposed biosynthetic pathway for the assembly of phosphoglycan.
Fig. 4 A comparison of the structure of a PG repeat and the designed
inhibitors.
Fig. 3 Transition state model for the reaction of eMPT.
the PG-substrate features appeared to be a good option to access
these compounds. Such cycloadditions on monosaccharide
glycals have been reported,8 where a glycal with non-polar
protecting groups undergoes reversible cycloaddition with
electron-deficient isocyanates to give a mixture of [2 + 2]
and [4 + 2] adducts. However, many such cycloadditions
are reversible, require high pressure conditions and give
very low yields e.g. cycloaddition of tri-O-benzyl-D-glucal
with trichloroacetyl-isocyanate is reported to give 24% yield.
However, despite the low yields, this chemistry holds great
potential for the synthesis of aza-sugars. Despite the known
concerns, we attempted this chemistry with an appropriate
disaccharide-glycal, hexa-O-benzyl-D-lactal (4, Scheme 1).
To our surprise, the cycloaddition of compound 4 with
trichloroacetyl-isocyanate progressed extremely well at room
temperature and pressure, providing 87% yield of exclusively the
[2 + 2] product 6, after in situ removal of the N-trichloroacetyl
group from adduct 5. This is a novel observation, where a sugar
substituent (galactose) at the 4-position of a glycal moiety plays
a remarkable role in stabilizing [2 + 2] cycloaddition products to
a great extent (from 24 to 87% yield). The same reaction when
repeated with the monosaccharide glycal, tri-O-benzyl-D-glucal,
under identical conditions gave only 24% yield, as reported
previously.8 This efficient cycloaddition provided high yielding
access to the new 1-oxabicyclic b-lactams, designed as eMPT
inhibitors. The synthesis of 1 and 2 started from known hexa-O-
benzyl lactal (4). The construction of the b-lactam ring involved
[2 + 2] cycloaddition of trichloroacetyl isocyanate to 4 at room
temperature for 18 h, progress of the reaction monitored by
following a parallel reaction in a NMR tube and observing
the spectra at different time points. The N-chloroacetyl group
was removed in situ using benzylamine at −20 ◦C, providing
2-carboxy-2-deoxy-3,6-di-O-benzyl-4-(2,3,4,6-tetra-O-benzyl-
b-galactopyranosyl)-a-D-glucopyranosyl-b-aminolactam 6 in
87% yield. The reaction proceeded regio- and stereospecifically,
with formation of b-lactam anti to the substituent at C-3.
The gluco configuration of the b-lactam ring was confirmed
enzymes in human biology, the Leishmania MPTs present novel
opportunities6 for drug design. A transition state model has been
proposed4,5 for the eMPT reaction (Fig. 3). According to this
model, a negatively charged anomeric phosphate of the first PG
repeat is the key recognition element for eMPT. Other essential
features include (a) 6-OH and 3-OH on the Man residue, (b)
the configuration of the Gal residue and (c) divalent cations
(Mg2+ and Mn2+). The model suggests that there should be at
least three binding domains in eMPT involved in a ternary
complex, the first to recognize the mannosyl-1-a-phosphate,
the second to correctly position GDP-Man with two of its
phosphodiesters complexed with divalent metal ion and the
third to hold the acceptor galactose. Since the iminosugars, the
analogues of the monosaccharides where either the ring oxygen
or the exocyclic oxygen of the anomeric carbon is replaced by
nitrogen, are known to be excellent inhibitors of carbohydrate
enzymes (glycosyltransferases and glycosidases), we decided to
examine this approach to target the eMPT and PG biosynthesis
of the Leishmania parasite. For this, we decided to mimic the a-D-
mannose-phosphate motif, leaving the acceptor part untouched,
by placing a nitrogen at the anomeric position linked directly
=
=
to a C O, isosteric to the P O of the phosphate. Since the
orientation of the 2-OH of the Man residue is not critical5 for
recognition, a constraint was designed between positions 1 and
2. This analysis suggested that the construction of a 1-oxabicylic
b-lactam ring between positions 1 and 2 of a PG substrate
would satisfy the above design requirements. Although, in a
strict sense, the designed b-lactam moiety did not fully mimic the
anomeric phosphate, the presence of an anomeric NCO linkage
(isosteric to OPO) was considered to be interesting as a probe
of MPT inhibition. As a continuation of our investigations7
into the chemistry and biology of Leishmania phosphoglycans
and related GPI molecules, we now report the synthesis and
evaluation of compound 1 and its N-alkyl analogue 2 (Fig. 4) as
new eMPT inhibitors.
1
by H NMR analysis (doublet at 5.4, J = 4.5 Hz for H-1),
Results and discussion
and also by NOE experiments (H-1/H-2 and H-4 across the
b-face of the pyranose ring). The removal of benzyl protecting
groups from compound 6 by transfer hydrogenation provided
From a synthetic point of view, a [2 + 2] heteroatom
cycloaddition on a suitable disaccharide–glycal scaffold bearing
1 0 4 4
O r g . B i o m o l . C h e m . , 2 0 0 5 , 3 , 1 0 4 3 – 1 0 4 8

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Scheme 2 Reagents and conditions: (a) mCPBA, H2O–ether, 0 ◦C, 4 h;
(b) Ac2O, py, rt, 16 h, 90%; (c) Me2NH, CH3CN, −20 ◦C, 3 h, 92%;
(d) PCl3, imidazole, CH3CN, 0 ◦C, 2 h; TEAB workup, 86%; (e) stearyl
alcohol, adamantane carbonyl chloride, 1 h; I2 oxidation, 30 min; TEAB
workup, 85%; (f) Na2CO3, MeOH, 2 h, 93%.
Scheme 1 Reagents and conditions: (a) BnBr, TBAI, NaH, DMF, rt,
4 h, 85%; (b) CCl3CONCO, CHCl3, rt, 18 h; (c) benzylamine, −20 ◦C,
87%; (d) Pd/C, HCOOH, MeOH, 50 ◦C, 12 h, 95%; (e) butyl bromide,
NaH, 0 ◦C, 3 h, 80%; (f) Pd/C, HCOOH, MeOH, 50 ◦C, 12 h, 95%.
2-carboxy-2-deoxy-4-(b-galactopyranosyl)-a-D-glucopyranosyl
1
b aminolactam (1), H NMR showing doublets at 5.48 (J =
galactopyranosyl)-a-D-mannopyranosyl phosphate (12). Final
deprotection of the above provided 13, which proved to be a
good exogenous substrate of the eMPT enzyme in the bioassay.
The microsomal membranes equipped with the machinery
for LPG biosynthesis (MPT, Gal-T and GDP-Man transporter)
were prepared from Leishmania donovani promastigotes using
a reported method.4 For the control eMPT assay, the cell
lysate was suspended in buffer (50 mM HEPES–NaOH pH 7.4,
25 mM KCl, 5 mM MgCl2, 5 mM MnCl2, 0.1 mM TLCK,
1 lg mL−1 leupeptin, 1 mM ATP and 0.5 mM DTT). Each
control assay contained 23 lM GDP-Man with 1 lCi of
GDP-[3H]Man and 25 nmol of synthetic substrate 13 and was
incubated at 28 ◦C for 20 min. The products were purified
using Sep-Pak (C-18) cartridges by sequential elution with
100 mM NH4OAc (to remove unused GDP-Man), 5, 20, 40 and
60% n-PrOH in 100 mM NH4OAc, respectively. The required
[3H]-labelled product was eluted in 60% n-PrOH and analyzed
by scintillation counting as well as by TLC (visualized by
phosphorimaging). The biochemical control assay, in triplicate,
showed 1.4% incorporation from labelled GDP-Man. Having
a control eMPT assay in hand, inhibition experiments with
synthetic b-lactams 1 and 2 were carried out at a concentration
range from 0.05 mM to 1 mM (Fig. 5). Compound 1 showed
inhibition of eMPT with an IC50 of 0.6 mM whereas the N-
alkylated compound 2 showed better inhibition with an IC50
of 0.20 mM. The maximum inhibition of 70% was reached at
0.80 mM, beyond which saturation was observed.
4.2 Hz, H-1) and 4.37 (J = 7.8 Hz, H-1ꢀ) for the two anomeric
protons.
Since the eMPT enzyme in Leishmania is known to be
associated with the Golgi membrane, success of a potential
inhibitor critically depends on its accessibility to the membrane
environment. We reasoned that placement of a small lipid tail
in the b-lactam 1 would enhance its intercalation with the
Golgi vesicles and, hence, the inhibition of eMPT. To test this
proposition an N-alkylated analogue of 1 was synthesized by
the coupling of the compound 6 with n-butyl bromide to afford
intermediate 7, which on deprotection gave water soluble 2
showing doublets at 5.38 (J = 4.2 Hz, H-1) and 4.31 (J = 7.8 Hz,
H-1ꢀ) by 1H NMR.
For biological evaluation of the above designed inhibitors 1
and 2, an efficient eMPT assay with the microsomal membranes
of Leishmania was required. For this we synthesized a new
lipid-linked phosphoglycan 13 (Scheme 2) as an exogenous
substrate for membrane-bound eMPT. The rationale behind
attachment of a lipid anchor was that it should help intercalation
of the substrate to Leishmania membranes during the assay
and facilitate isolation of biosynthetic products by solid-phase
extraction on reversed-phase Sep-Pak cartridges, thus enabling
a clean and high throughput eMPT assay. The synthesis of
the substrate mimic 13 started from a known7a disaccharide
Gal1,4bMan (8), which, on acetylation to the octa-acetate 9
followed by selective deprotection of the anomeric acetyl group
(Me2NH, −20 ◦C), provided the hepta-O-acetyl compound 10.
This, on reaction with anhyd PCl3 and imidazole, led to the H-
phosphonate 11 (characteristic doublet at 6.95 for JHP = 637 Hz).
Compound 11 was coupled with stearyl alcohol by adaman-
tane carbonyl chloride, followed by in situ oxidation to give
1-O-stearyl-2,3, 6-tri-O-acetyl-4-O-(2,3,4,6-tetra-O-acetyl-b-D-
In conclusion, novel substrate mimics of eMPT enzymes of the
Leishmania parasite have been designed and synthesized using
high yielding [2 + 2] cycloaddition, which showed good (lM
range) inhibition. These are the first functional inhibitors of
this remarkable enzyme and provide lead structures for further
optimization of activity for use in drug development.
O r g . B i o m o l . C h e m . , 2 0 0 5 , 3 , 1 0 4 3 – 1 0 4 8
1 0 4 5

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d, J 7.8, H-1ꢀ), 5.48 (1 H, d, J 4.2, H-1), 8.02 (1 H, br s, NH);
dC (75 MHz, CD3OD) 55.36, 61.0, 68.47, 68.87, 69.47, 71.15,
73.36, 75.52, 75.95, 76.50, 79.91, 104.03, 169.37; m/z (ES) 374.33
[M + Na]+; (HRMS found: [M + Na]+ 374.1095. C13H21O10NNa
requires m/z 374.1063).
2-Carboxy-2-deoxy-3,6-di-O-benzyl-4-(2,3,4,6-tetra-O-benzyl-b-
galactopyranosyl)-a-D-glucopyranosyl n-butyl b-aminolactam 7
Compound 6 (200 mg, 0.22 mmol) was dissolved in DMF
(4 cm3) at 0 ◦C and sodium hydride (60% dispersion, 12 mg,
0.33 mmol) was added followed by n-butyl bromide (48 mm3,
60 mg, 0.44 mmol). The mixture was stirred at 0 ◦C for 3 h,
quenched with CH3OH (1 cm3) to destroy excess NaH, diluted
with CH2Cl2 and washed with water. The organic phase was
dried (Na2SO4), concentrated and purified by silica column
chromatography (15% EtOAc in hexane) to afford compound 7
(172 mg, 80%); Rf = 0.57 in 50% EtOAc in hexane; dH (300 MHz,
CDCl3) 0.93–1.03 (3 H, t, J 7.2, CH3), 1.36–1.45 (4 H, m,
Fig. 5 Inhibition of Leishmania eMPT activity by 1 and 2.
Experimental
Solvents were purified according to standard procedures and
all reagents used were of highest purity available. The NMR
spectra (1H, 13C, 31P, 2D 1H–1H COSY and 1H–13C HET-
COR, HMQC and HMBC) were recorded on a 300 MHz
spectrometer (Bruker, Avance series) fitted with a pulse-field
gradient probe. Trimethylsilane (TMS) or residual resonance
of deuterated solvent were used as internal reference. For 31P
NMR spectra, phosphoric acid was used as external reference.
13C NMR spectra were broadband 1H decoupled or inverse
HMQC experiments. Chemical shifts are expressed in ppm
and coupling constants (J) in Hz. Where appropriate, signal
assignments were made by DEPT, 1H–1H COSY and 1H–
13C HETCOR experiments. Low and high resolution mass
spectra were recorded on Platform-II or LCT spectrometer
(Micromass-Waters) respectively using an acetonitrile–water (1 :
1) mobile phase. Optical rotations were measured at ambient
temperature with a digital Perkin-Elmer 141 polarimeter. Thin
layer chromatography was performed on Merck Kieselgel 60
F254 plates, compounds visualized either by viewing with a UV
lamp (254 nm) or by dipping into ammonium-molybdate–ceric-
sulfate developing reagent, followed by heating. Silica column
chromatography was carried out with silica gel 60 (60–120 mesh).
NCH2CH CH2CH3), 3.25 (2 H, m, NCH2), 3.36–3.56 (5 H,
2
m, H-2, 6, 6ꢀ), 3.60–3.75 (2 H, m, H-3, 5), 3.80 (2 H, m, H-5ꢀ, 4ꢀ),
3.82 (2 H, m, H-2ꢀ, 3ꢀ), 4.43 (1 H, m, H-4ꢀ), 4.55 (1 H, d, J 7.5,
H-1ꢀ), 4.68–5.15 (12 H, m), 5.46 (1 H, d, J 4.2), 7.33–7.56 (30 H,
m); dC (75 MHz, CDCl3) 13.45, 20.22, 29.65, 40.29, 53.82, 68.37,
69.05, 69.40, 71.50, 72.60, 72.99, 73.04, 73.01, 73.35, 73.36,
74.54, 75.07, 75.50, 75.58, 79.44, 75.55, 82.32, 127.38–128.30
(multiple peaks), 138.18–138.77 (multiple peaks), 166.67; m/z
(ES) 970.90 [M + Na]+; (HRMS found: [M + Na]+ 970.4520.
C59H65O10NNa requires 970.4506).
2-Carboxy-2-deoxy-4-(b-galactopyranosyl)-a-D-glucopyranosyl
n-butyl b-amino lactam 2
To a solution of compound 7 (48 mg) in CH3OH (4 cm3)
was added 10% Pd/C (0.14 g) and formic acid (0.4 cm3) and
was stirred at 50 ◦C overnight, catalyst filtered and solvent
evaporated to give compound 2 (19 mg, 95%); dH (300 MHz,
CD3OD) 0.72–0.77 (3 H, t, J 7.2, CH3), 1.35–1.42 (4 H, m,
NCH2CH CH CH3), 3.05–3.09 (2 H, m, NCH2), 3.30–3.37
2
2
2-Carboxy-2-deoxy-3,6-di-O-benzyl-4-(2,3,4,6-tetra-O-benzyl-
b-galactopyranosyl)-a-D-glucopyranosyl b-aminolactam 6
(3 H, m), 3.48–3.76 (11 H, m), 4,16 (1H, m, H-4ꢀ), 4.31 (1 H, d,
J 7.8, H-1ꢀ), 5.38 (1 H, d, J 4.2, H-1); dC (75 MHz, CD3OD)
12.72, 19.69, 28.77, 40.65, 52.84, 60.95, 61.19, 67.48, 68.50,
70.88, 70.92, 72.56, 75.17, 77.66, 79.12, 103.19, 169.83; m/z
(ES) 430.37 [M + Na]+; (HRMS found: [M + Na]+ 430.1710.
C17H29O10NNa requires 430.1689).
To a solution of hexa-O-benzyl lactal7b (4, 300 mg, 0.36 mmol) in
CHCl3 (0.36 cm3) was added trichloroacetyl isocyanate (90 mm3,
0.74 mmol). The mixture was stirred at rt for 18 h to afford 5
which was characterized by 1H NMR: d 6.04 (1H, d, J = 5.4 Hz,
H-1, gluco-isomer, [2 + 2] adduct). The reaction was cooled to
1,2,3,6-Tetra-O-acetyl-4-O-(2,3,4,6-tetra-O-acetyl-b-D-
galactopyranosyl)-a-D-manno pyranose 9
◦
−20 C, treated with benzylamine (0.13 cm3, 1.17 mmol) and
the flask was warmed to rt. The organic phase was washed with
water, dried (Na2SO4), concentrated and the residue purified
by silica column chromatography (30% EtOAc in hexane) to
give the product 6 (275 mg, 87%); Rf = 0.33 in 50% EtOAc in
hexane; dH (300 MHz, CDCl3) 3.37–3.46 (5 H, m, H-2, H-6,
H-6ꢀ), 3.58–3.70 (3 H, m, H-3, 4, 5), 3.77–3.89 (3 H. m, H-2ꢀ,
3ꢀ, 5ꢀ), 4.34 (1 H, m, H-4ꢀ), 4.47 (1 H, d, J 7, H-1ꢀ), 4.58–4.97
(12 H, m, 6 × OCH2Ph), 5.40 (1 H, d, J 4.5, H-1), 6.24 (1 H, s,
NH), 7.22–7.36 (30 H, m, 6 × Ph); dC (75 MHz, CDCl3) 54.27,
68.43, 69.39, 71.48, 72.65, 73.06, 73.12, 73.37, 74.56, 75.05,
75.08, 75.35, 75.95, 76.58, 79.47, 82.31, 102.98, 127.42–128.33
(multiple peaks), 138.07–138.83 (multiple peaks), 166.90; m/z
(ES) 914.5 [M + Na]+; (HRMS, found: [M + Na]+ 914.3921.
C55H57O10NNa requires m/z 914.3880).
Acetic anhydride (4 cm3) was added dropwise to a stirring
solution of known compound 87a (700 mg, 2.04 mmol) in
◦
anhydrous pyridine (6 cm3) at 0 C. The reaction mixture was
brought to rt and stirred for 16 h. The mixture was poured over
ice and the product crystallized to afford compound 9 (1.25 g,
90%); dH (300 MHz, CDCl3) 1.8–2.2 (24 H, m, 8 × COCH3),
3.86–3.99 (2H, m, H-6ꢀ), 3.93 (1 H, br dd, H-5ꢀ), 4.15 (1 H,
m, H-5), 4.05–4.25 (2 H, m, H-6), 4.44 (1 H, dd, J4,5 J3,4 9.6,
H-4), 4.53 (1 H, d, J1 ,2
ꢀ
ꢀ
7.8, H-1), 4.96 (1 H, dd, J3 ,4 3.4, H-
ꢀ
ꢀ
3ꢀ), 5.10 (1 H, m, H-2ꢀ), 5.40 (1 H, dd, H-2), 5.42 (1 H, d,
J3,2 3, H-3), 5.47 (1 H, br d, H-4ꢀ), 6.00 (1 H, d, J 1.9, H-1); dC
(75 MHz, CDCl3) 20.38–20.77, 60.73, 62.10, 66.50, 68.33, 69.06,
69.08, 70.43, 70.82, 70.89, 73.83, 90.31, 101.17, 169.14–169.98
(multiple peaks); m/z (ES) 701.5 [M + Na+]; (HRMS found:
[M + Na]+ 701.1985. C28H38O19Na requires 701.1905).
2-Carboxy-2-deoxy-4-(b-galactopyranosyl)-a-D-glucopyranosyl
b-aminolactam 1
2,3,6-Tri-O-acetyl-4-O-(2,3,4,6-tetra-O-acetyl-b-D-
galactopyranosyl)-a-D-manno pyranose 10
To a solution of 6 (240 mg, 0.28 mmol) in CH3OH (24 cm3)
was added Pd–C (10%, 800 mg) and formic acid (2.4 cm3). The
mixture was stirred at 50 ◦C overnight, catalyst filtered and
solvent evaporated to afford compound 1 (90 mg, 95%); Rf =
0.27 in 40% CH3OH in CH2Cl2; dH (300 MHz, CD3OD) 3.39–
3.69 (7 H, m), 3.78–3.84 (6 H, m), 4.17 (1 H, m, H-4ꢀ), 4.37 (1 H,
Compound 9 (600 mg, 0.89 mmol) was dissolved in anhydrous
CH3CN saturated with dimethylamine (40 cm3) at −20 ◦C and
stirred for 3 h, after which TLC confirmed the disappearance of
the starting material. Excess dimethylamine was removed under
1 0 4 6
O r g . B i o m o l . C h e m . , 2 0 0 5 , 3 , 1 0 4 3 – 1 0 4 8

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a reduced pressure, below 30 ◦C, and the reaction mixture was
concentrated to provide the desired product 10 (517 mg, 92%
yield); dH (300 MHz, CDCl3) 1.8–2.2 (21 H, m, 7 × COCH3),
3.80–3.90 (2H, m, H-6ꢀ), 3.91 (1 H, br dd, H-5ꢀ), 4.15 (1 H, m,
H-5), 4.2 − 4.0 (2 H, m, H-6), 4.43 (1 H, dd, J4,5 J3,4 9.69, H-4),
(1 H, m, H-2ꢀ), 5.22 (1 H, dd, J1,2 1.5, J1,OH 3, H-1), 5.38 (1 H,
m, H-2), 5.42 (1 H, d, J3,2 3, H-3), 5.45 (1 H, br d, H-4ꢀ); m/z
(ESMS) 636 [M+]; (HRMS found: [M]+ 636.1982. C26H36O18
requires 636.1902).
CH2), 3.41–3.53 (4 H, m, H-2, 5, 5ꢀ, 2ꢀ), 3.61–3.87 (12 H, m),
4.34 (1 H, br d, J 6.3, H-1ꢀ), 5.30 (1 H, dd, J 6.8 and 1.9, H-1);
dH (75 MHz, D2O) 13.87, 20.03, 29.47–30.06, 31.96, 36.24, 39.50,
60.42, 62.15, 66.28, 68.58, 69.87, 70.17, 70.66, 70.94, 71.28,
72.36, 75.26, 95.75, 104.69; dP (100 MHz, D2O) −1.72; m/z (ES)
673.6 [M − H]−; (HRMS found: [M − H]− 673.3594. C30H58O14P
requires 673.3564).
4.53 (1 H, d, J1 ,2
ꢀ
ꢀ
7.9, H-1), 4.95 (1 H, dd, J3 ,4
3.4, H-3ꢀ), 5.11
ꢀ
ꢀ
Parasite culture and preparation of microsomal membranes
The promastigotes of Leishmania donovani (DD8 strain) were
grown at 25 ◦C in Dulbecco’s modified Eagle’s medium
supplemented with 0.05 mM adenosine, 0.05 mM xanthine,
1 mg L−1 biotin, 40 mg L−1 Tween-80, 5 mg L−1 hemin, 0.5%
triethanolamine, 0.3% BSA, 50 mg L−1 gentamycin and 10%
heat inactivated fetal bovine serum, with the pH maintained
at 7.2. The promastigote◦s were harvested by centrifugation at
3000 g for 10 min at 20 C, the pellet resuspended in ice cold
phosphate buffered saline (20 mM, pH = 7.2) and centrifuged
once again. The promastigotes were counted using a Neubauer
chamber. For rupturing, the cell pellet (6.5 × 109 cells) was
suspended in 5 mL of hypotonic buffer (0.1 mM TLCK and
1 lg ml−1 leupeptin) and sonicated on ice (6 × 10 s pulses with
3 s intervals), breaking assessed using a light microscope. The
above suspension was centrifuged at 3000 g for 5 min to remove
debris and the supernatant was centrifuged at 100 000 g for 1 h
at 4 ◦C to obtain a membrane pellet, which was suspended in the
buffer (50 mM HEPES–NaOH, pH = 7.4, 25 mM KCl, 5 mM
MgCl2, 5 mM MnCl2, 0.1 mM TLCK, 1 lg mL−1 leupeptin,
1 mM ATP and 0.5 mM DTT). The concentration of the protein
present in this solution was 13 mg mL−1.
2,3,6-Tri-O-acetyl-4-O-[2,3,4,6-tetra-O-acetyl–b-D-
galactopyranosyl]-a-D-mannopyranosyl H-phosphonate 11
To a stirred solution of◦imidazole (1 g, 14.68 mmol) in anhydrous
CH3CN (20 cm3) at 0 C was added PCl3 (0.8 cm3, 9.14 mmol)
and triethylamine (2.4 cm3, 0.86 mmol). The mixture was stirred
for 20 min and a solution of compound 10 (500 mg, 0.786 mmol)
in anhydrous CH3CN (20 cm3) was added. The mixture was
stirred at 0 ◦C for 2 h and quenched with 1 M TEAB (10 cm3)
and further stirred for 15 min, diluted with CH2Cl2, organic layer
washed with ice cold water (2 × 10 cm3) and cold 1 M TEAB
buffer (2 × 10 cm3), dried over Na2SO4 and concentrated to yield
compound 11 (500 mg, 86%) as a triethylammonium salt. Rf =
0.35 in 20% CH3OH in CH2Cl2; dH (300 MHz, CDCl3) 1.90–2.08
(21 H, m, 7 × OCOCH3), 3.96 (2 H, m, H-5ꢀ, H-5), 3.80–3.95
(2 H, m, H-6ꢀ), 4.24–4.13 (2 H, m, H-6), 4.47 (1 H, d, J 7.8, H-4),
4.54 (1 H, d, J 7.8, H-1ꢀ), 4.96 (1 H, dd, J 3.3 and 7.8, H-3ꢀ),
5.05 (1 H, dd, J 2.1 and 7.8, H-2ꢀ), 5.20–5.40 (2 H, m, H-4ꢀ, 3),
5.28 (1 H, dd, J 2.1 and 3.6, H-2), 6.95 (1 H, dd, JHP 637, H-1);
dP (100 MHz, CDCl3) 0.129; m/z (ES) 699.2 [M − Et3N–H]−;
(HRMS found: [M − Et3N–H]− 699.1593. C26H36O20P requires
699.1538).
Elongating-MPT control and inhibition assay
Stearyl-2,3,6-tri-O-acetyl-4-O-(2,3,4,6-tetra-O-acetyl-b-D-
galactopyranosyl)-a-D-mannopyranosyl phosphate 12
For the control eMPT assay, each tube was prepared by adding
12.5 mm3 of 1% Chaps, 28 mm3 of 200 lM GDP-Man, 10 mm3 of
GDP-[3H]Man (1lCi) and 17 lg (25 nmol) of synthetic substrate
13. The contents were lyophilized and then 250 mm3 of above
membrane suspension (1.4 × 108 cell equivalent) was added to
each tube. The tubes were incubated at 28 ◦C for 20 min, cooled
to 0 ◦C and the membranes pelleted at 4 ◦C for 10 minutes
by centrifugation at 3000 g. The supernatants (250 mm3) were
diluted with 500 mm3 of 100 mM NH4OAc buffer and loaded
onto C18 Sep-Pak cartridges (preactivated by equilibrating and
washing with 100 mM NH4OAc). Each cartridge was washed
with 5 cm3 of 100 mM NH4OAc followed by 1.5 cm3 each
of 5, 20, 40 and 60% n-propanol in 100 mM NH4OAc. The
desired [3H] labelled PG products were eluted in 60% n-propanol
fraction, the eluates concentrated and redissolved in 100 mm3 of
60% n-propanol and the radioactivity measured by scintillation
counting. The control assay was carried out in triplicate and
the % incorporation was calculated for eMPT activity. The
inhibition with synthetic 1 and 2 was carried using the above
assay, in which the inhibitors were added in a concentration
range from 0.05 to 1.0 mM, the experiments conducted in
triplicate and averaged. The incorporation of [3H]-Man was
compared with the cpm present in control assay (incubation
of reaction components with the substrate 13 but without 1 and
2). The IC50 values were determined (GraphPad Prism 4.01)
from response curves in which concentations ranged from 0.05
to 1 mM.
A mixture of the above disaccharide H-phosphonate 11 (25 mg,
0.031 mmol) and stearyl alcohol (11 mg, 0.04 mmol) was dried by
evaporation of pyridine (2 × 0.5 cm3). The residue was dissolved
in anhydrous pyridine (1 cm3), adamantane carbonyl chloride
(16 mg, 0.08 mmol) was added, and the mixture was stirred
at rt for 1 h after which a freshly prepared solution of iodine
(16 mg, 0.063 mmol) in 95% aq. pyridine (3 cm3) was added.
After 30 min CH2Cl2 was added, the solution washed with 1M
Na2S2O3 and 1M TEAB, dried (Na2SO4) and concentrated. The
residue was purified by silica column chromatography (2.5%
CH3OH in CH2Cl2 with 1% Et3N) to afford the coupled product
12 (29 mg, 85%); Rf = 0.46 in 20% CH3OH in CH2Cl2; dH
(300 MHz, CDCl3) 0.84 (3 H, t, CH3), 1.23–1.45 (34 H, m, lipid
CH2), 1.85–2.12 (21 H, m, 7 × OCOCH3), 3.84–4.16 (6 H, m, H-
5/5ꢀ, H-6/6ꢀ), 4.51 (1 H, d, J 7.8, H-1ꢀ), 4.85–5.01 (2 H, m, H-2ꢀ,
3ꢀ), 5.25 (3 H, m, H-4, 4ꢀ, 3), 5.52 (1 H, dd, J 2.1 and 3.6, H-2),
5.69 (1 H, dd, JHP 6.8 and J1,2 1.9, H-1); dC (75 MHz, CDCl3)
13.99, 20.48–20.77, 22.56, 27.8–29.59, 31.80, 36.44, 38.78, 52.82,
60.69, 68.99, 69.48, 70.23, 70.91, 76.52, 93.26, 100.93, 168.99–
170.42 (multiple peaks); dP (100 MHz, CDCl3) −2.90; m/z (ES)
967.5 [M − H]−; (HRMS found: [M − H]− 967.4315. C44H72O21P
requires 967.4304).
Stearyl-4-b-D-galactopyranosyl-a-D-mannopyranosyl-phosphate
13
To a solution of compound 12 (15 mg, 0.014 mmol) in anhydrous
CH3OH (2.5 cm3) was added anhydrous Na2CO3 (16 mg,
0.15 mmol). The mixture was stirred at rt for 2 h, excess Na2CO3
removed by filtration and solvent evaporated to yield the target
exogenous eMPT substrate 13 (9 mg, 93% yield); Rf = 0.55 in
10 : 10 : 3 CH3OH–CH2Cl2–0.25% KCl; dH (300 MHz, D2O)
0.76 (3H, t, CH3), 1.16–1.83 (34 H, m, lipid CH2), 3.22 (2H, m,
Acknowledgements
Authors acknowledge the financial support (SR/S5/OC-
11/2002) from the Department of Science and Technology
(Government of India), the Department of Biotechnology
(Government of India) for providing a core grant and to the
Institute for infrastructure support.
O r g . B i o m o l . C h e m . , 2 0 0 5 , 3 , 1 0 4 3 – 1 0 4 8
1 0 4 7

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