13C Labelling and electrospray mass spectrometry reveal a de novo route for inositol biosynthesis in Leishmania donovani parasite

Article abstract of DOI:10.1039/b000262n

In this paper we provide evidence for the presence of a de now route for myo-inositol biosynthesis in the protozoan parasite Leishmania donovani, by the application of ¹³C labelling and electrospray ionisation mass spectrometry (ESMS). For this myo-[1-¹³C]inositol is prepared from D-[6-¹³C]glucose and biosynthetically incorporated in the parasite promastigote cell culture. Biosynthetic phosphatidylinositol (PI) and its hydrolysis products glycero-PI and inositol are analysed by ESMS and the isotopomeric ratio determined. The incorporation experiments show substantial isotopic dilution, indicating the presence of myo-inositol 1-phosphate synthase (MIP synthase) enzyme in the parasite; this is further confirmed by incorporation of D-[6-¹³C]glucose in the parasite phosphatidylinositol. The Royal Society of Chemistry 2000.

Full text of DOI:10.1039/b000262n

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13C Labelling and electrospray mass spectrometry reveal a de novo
route for inositol biosynthesis in Leishmania donovani parasite
Parmeshwari Sahai, Mamta Chawla and Ram A. Vishwakarma*
1
Bio-organic Chemistry Lab., National Institute of Immunology, Aruna Asaf Ali Marg,
J.N.U. Complex, New Delhi 110067, India
Received (in Cambridge, UK) 12th January 2000, Accepted 3rd March 2000
Published on the Web 31st March 2000
In this paper we provide evidence for the presence of a de novo route for myo-inositol biosynthesis in the protozoan
parasite Leishmania donovani, by the application of 13C labelling and electrospray ionisation mass spectrometry
(ESMS). For this myo-[1-13C]inositol is prepared from -[6-13C]glucose and biosynthetically incorporated in the
parasite promastigote cell culture. Biosynthetic phosphatidylinositol (PI) and its hydrolysis products glycero-PI
and inositol are analysed by ESMS and the isotopomeric ratio determined. The incorporation experiments show
substantial isotopic dilution, indicating the presence of myo-inositol 1-phosphate synthase (MIP synthase) enzyme
in the parasite; this is further confirmed by incorporation of -[6-13C]glucose in the parasite phosphatidylinositol.
Introduction
The identification of major cell surface glycosylphosphatidyl-
inositol (GPI) molecules1 from the protozoan parasites
(Leishmania, Trypanosome and malaria) and their immuno-
logical role2 in the parasite infectivity and survival has
coincided with the discovery3 of an important transmembrane
signal-tranduction pathway mediated by phosphatidylinositols
and inositol phosphates. This has provided new opportunities
to study host–parasite relationships and design strategies to
combat major tropical diseases due to these parasites. Leish-
mania donovani, causing visceral leishmaniasis (kala azar),
and related species express two major type of GPI structures,
namely lipophosphoglycan (LPG) and glycosylated inositol
phospholipids (GIPLs), in very high copy numbers (more than
107 molecules cellϪ1) on their cell surface which help the para-
site to infect and proliferate within human macrophages. This
has generated major interest in their biosynthesis1 and chemical
synthesis.4 Leishmania parasites should require huge supplies of
inositol for synthesis of phosphatidylinositols (required for sig-
nalling) and GPIs (for membrane anchoring of protein and
carbohydrate antigens, receptors and adhesion molecules).
Therefore the question of whether the parasite sequesters
inositol from its hosts or it possesses machinery for inositol
biosynthesis is of interest as it may provide new targets for
chemotherapeutic intervention.
Scheme 1 Proposed mechanism of MIP synthase.
The biosynthesis of myo-inositol in yeast and bacteria is
mediated by the enzyme myo-inositol 1-phosphate synthase
(MIP synthase) which has been isolated5 and cloned6 in yeast.
MIP synthase is a unique enzyme catalysing (Scheme 1) oxid-
ation, enolisation, intramolecular aldol-condensation and
carbonyl-reduction steps involved in the transformation of
glucose 6-phosphate to inositol 1-phosphate. The precise mech-
anism of MIP synthase has not been elucidated but 5-keto-
glucose 6-phosphate and myo-2-inosose 1-phosphate have been
proposed7 as intermediates, and the latter was found8 to be a
competitive inhibitor of the enzyme. Interestingly, in humans
MIP synthase is produced9 only in the brain and testes and is not
expressed in cells infected by Leishmania and malaria parasites.
In spite of a major metabolic requirement of inositol for signal-
ling and GPI biosynthesis, there has been no report of the
source of inositol or presence of MIP synthase activity in the
Leishmania, Trypanosome and malaria parasites. In continu-
ation of our work10–12 on L. donovani phospholipids and GPI
molecules, we decided to explore the biosynthesis of inositol in
this parasite. In this paper we describe the use of stable isotope
[13C] labelling and electrospray ionisation mass spectrometry
(ESMS) to address the question of myo-inositol biosynthesis in
Leishmania parasites. The approach using 13C-labelled inositol
and glucose as precursors to study inositol biosynthesis in the
parasite was adopted for the following reasons: (a) in a cell
culture system parasites take up inositol from the medium
(radiolabelling studies) to incorporate in PI/GPI molecules, (b)
if the parasite was also making its own inositol (MIP synthase)
from glucose, the 13C enrichment level would become diluted in
PI/GPI products. Negative-ion ESMS was found particularly
suitable for the analysis of parasitic phospholipids, phosphati-
dylinositol (PI) and deacylated glycero-PI, which gave promin-
DOI: 10.1039/b000262n
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This journal is © The Royal Society of Chemistry 2000
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ent [M Ϫ H]Ϫ ions. The incorporation of myo-[1-13C]inositol
and -[6-13C]glucose in the parasite, and isotope dilution/
enrichment measurement of biosynthetic PI and inositol by
ESMS, led to identification of both de novo inositol bio-
synthesis and the presence of MIP synthase in the parasite.
Results and discussion
Two routes for the enantioselective synthesis of 1-myo-[1-13C]-
inositol were devised using -[6-13C]glucose as a suitable start-
ing material available in stable-isotope-labelled forms. In the
first, enzymic, approach (Scheme 2), we designed an in-vitro
Scheme 3 Synthesis of 1-myo-[1-13C]inositol (᭹ = 13C). Reagents and
conditions: (a) (i) MeOH, Dowex Hϩ resin, (ii) trityl chloride, pyridine,
DMAP, (iii) BnBr, NaH, DMF; (b) p-TsOH, pH 4; (c) (COCl)2, DMSO,
Et3N, CH2Cl2, Ϫ78 ЊC to rt; (d) Ac2O, K2CO3, CH3CN, 80 ЊC; (e)
Hg(OAc)2, acetone–water, rt, 45 min; then saturated aq. NaCl, rt, 24 h;
(f) NaBH(OAc)3, AcOH, CH3CN; (g) 10% Pd/C, hydrogen, 10 atm; (h)
NaOMe, MeOH.
Scheme 2 Enzymic synthesis of myo-[1-13C]inositol using MIP syn-
thase from S. cerevisae opi 1δ mutant (᭹ = 13C).
synthesis using a mutant strain of yeast (Saccharomyces cerevi-
siae) over-expressing myo-inositol 1-phosphate synthase. The
second, chemical, route (Scheme 3) was based on the Ferrier
reaction13 which involves intramolecular aldol-type carbocyclis-
ation of a suitably protected glucose 5-enol acetate.14
and C2-OH axial stereochemistry was confirmed by detailed
analysis of the vicinal coupling constants and NOE difference
experiments. Compound 10 was debenzylated (10% Pd/carbon
at 10 atm hydrogen) to 1-1-O-acetyl-myo-inositol 11 which on
deacetylation (NaOMe, MeOH) and purification by Amberlite
IR-120 (Hϩ) gave the desired -myo-inositol 4.
Enzymic synthesis of myo-inositol
In the first step -glucose 1 was treated with hexokinase15 (Type
VI from baker’s yeast) and ATP (Scheme 2) to give -glucose
6-phosphate 2 in 81% yield. The next step involved transform-
ation of 2 to myo-inositol 1-phosphate 3 for which partially
purified MIP synthase from a genetic mutant of yeast (S.
cerevisiae, ATCC 74033, genotype MATa, opi 1δ, with MIP
synthase subunit being constitutively over-expressed)16 grown
in YEPD medium was used. In a typical incubation, 57 units of
enzyme and β-NADϩ were used for 0.306 mM glucose
6-phosphate substrate, and purification from anion- (AG1-X8)
followed by cation-exchange (Dowex HCR W2) chromato-
graphy yielded 17.2 mg of myo-inositol 1-phosphate (21.7%).
Finally, dephosphorylation of 3 by alkaline phosphatase (E.
coli), followed by ion-exchange purification gave myo-inositol 4
in 63% yield (6.7 mg).
Synthesis of myo-[1-13C]inositol
For preparation of labelled myo-[1-13C]inositol, both the
enzymic (Scheme 2) and chemical (Scheme 3) approaches were
applied using -[6-13C]glucose 1 (99 atom-% enrichment) as the
starting material and sufficient amounts (22.5 mg) of myo-
[1-13C]inositol were obtained from 200 mg of labelled glucose.
The isotopomeric ratio was determined by negative-ion ESMS
(Fig. 1, panel A1) and 13C NMR data. For larger scale syn-
thesis, we found the chemical route more convenient as it
involved less cumbersome purification steps. The overall yield
for the glucose-to-inositol synthesis by the chemical route (10
steps) was 11.25%, and for the enzymic route (three steps) was
9.9%. This is the first reported preparation of 13C-labelled
myo-inositol which would be of potential use to phosphoino-
sitide signal-transduction studies.
Chemical synthesis of myo-inositol
In an alternative strategy (Scheme 3), -glucose was first
converted to methyl 2,3,4-tri-O-benzyl-6-O-(triphenylmethyl)-
-glucopyranoside 5 by a published method17 followed by
detritylation to give methyl 2,3,4-tri-O-benzyl--glucopyrano-
side 6. The next three steps included Swern oxidation of
primary hydroxy group of 6 to aldehyde 7, preparation of the
enol acetate 8 and Ferrier rearrangement to give the corre-
sponding inosose 9. Stereoselective hydroxy-directed hydride
reduction14,18 of 9 with sodium triacetoxyborohydride led to
1-O-acetyl-3,4,5-tri-O-benzyl-myo-inositol 10 in good yield,
Establishment of ESMS conditions for isotopomer analysis
For monitoring of the incorporation of myo-[1-13C]inositol in
the parasitic cell-surface inositides, a suitable experimental
methodology was first established. This included (a) parasite
promastigote culture, (b) establishment of biosynthetic viability
by radioactive labelling and identification of PI/GPI molecules,
(c) deacylation of fatty esters of parasite diacyl-PI to glycero-PI
by NH4OH; this was done to facilitate separation of water-
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single peak at m/z 179 [M Ϫ H]Ϫ in ESMS. This was in view of
the report19 that 10% NH4OH hydrolysis at 150 ЊC released free
inositol from PI/glycero-PI by hydrolysis of phosphodiester,
and not from the glycosylated-PI molecules, which allowed
efficient separation of PI and GPI.
Having established release and identification of glycero-PI
and inositol from the parasite and the ESMS conditions, a cali-
bration curve for quantitative measurement of the isotopic
ratio of 12C:13C in biosynthetic products was prepared. For
this, [6-13C]glucose 1 was diluted with unlabelled glucose to give
1, 2, 5, 10, 20, 30, 40, and 50 atom-% and these samples were
analyzed by ESMS and the area under the peaks at m/z 179
(unlabelled) and 180 (13C-labelled) were measured and plotted
in a calibration curve that was used throughout the study to
estimate isotope dilution or enrichment during biosynthesis.
Biosynthetic incorporation of myo-[1-13C]inositol in Leishmania
parasite
L. donovani parasite promastigotes (DD8 strain) were first
starved in inositol-free Medium-199 with fat-free BSA for 24 h;
this was done to deplete free inositol in the cytosolic pool of the
parasite, and to increase the level of expression of putative MIP
synthase. After starving, parasites were harvested, washed, and
used for incubation in normal unlabelled (control) and myo-
[1-13C]inositol-rich (labelling) media. Initially [1-13C]inositol
(1.4 mg, 99 atom-%) was diluted with unlabelled inositol (0.6
mg) to bring the 13C level to 70 atom-%; this labelled precursor
(2 mg) was added to the biosynthetic incubation. At the begin-
ning (time t = 0) of the incubation, small aliquots from the
parasite cultures (labelling as well as control ) were taken for the
determination of 13C atom-% of the precursor. From these cul-
ture aliquots, inositol was isolated by extraction of inositides,
hydrolysis and preparative TLC (PLC). The ESMS of inositol
from the labelling sample showed that the 13C atom-% of pre-
cursor inositol was reduced to 50% (Fig. 1, panel A2) due to
minor amounts of unlabelled inositol present in the commercial
medium used for the parasite culture. It is important to mention
here that instead of analyzing the culture directly by ESMS to
determine the initial 13C label of precursor inositol, TLC purifi-
cation was necessary to eliminate the contribution of glucose
which was present in the medium and also gave an [M Ϫ H]Ϫ
peak at m/z 179. Inositol and glucose were clearly resolved (Rf
of inositol 0.15, and glucose 0.34).
After incubation of promastigotes for 24 h for both the con-
trol and biosynthetic labelling experiments, the parasites were
processed to isolate PI, glycero-PI and inositol as described in
the earlier section. The ESMS data of the control and labelling
experiments are listed in Table 1. The ratios of the abundances
of the pseudomolecular ions, i.e. [M Ϫ H]Ϫ or P at m/z 333 to
P ϩ 1 at 334 and P ϩ 2 at 335 for control glycero-PI, and
[M Ϫ H]Ϫ or P at m/z 179 to P ϩ 1 at 180 and P ϩ 2 at 181 for
control myo-inositol, were very close to the theoretical values.
ESMS of the glycero-PI from labelling experiment showed
(Fig. 1) that the 13C label from precursor myo-[1-13C]inositol
was incorporated as expected into glycero-PI and the relative
abundance of peak at m/z 334 (P ϩ 1) was found to be 21%
more in glycero-PI from the labelling experiment (Fig. 1, panel
B2) than that from the control experiment (Fig. 1, panel B1).
More interestingly when this 13C ratio in glycero-PI from the
labelling experiment (Fig. 1, panel B2) was compared with the
13C atoms (50%) present at the beginning of incubation (Fig. 1,
panel A2), it was clear that there was substantial dilution of the
label (from 50 to 21%) during incorporation of myo-[1-13C]-
inositol. This could have happened only when a de novo bio-
synthesis of inositol was also in operation in the parasite,
converting glucose to inositol (MIP synthase activity) leading
to dilution of the isotope atom-%.
Fig. 1 ESMS (negative-ion) data from experiment on biosynthetic
incorporation of myo-[1-13C]inositol in Leishmania donovani: (A1)
ESMS of synthetic myo-[1-13C]inositol prepared from -[6-13C]glucose
(99 atom-% 13C); (A2) 13C enrichment level of inositol present in the
culture medium at the start of incubation; (B1) glycero-PI from control
experiment; (B2) glycero-PI from labelling experiment; (C1) inositol
obtained by hydrolysis of glycero-PI from control experiment; (C2)
inositol obtained by hydrolysis of glycero-PI from labelling experiment.
soluble glycero-PI from other phospholipids, and GPIs which
in Leishmania parasites contain
a 1-O-alkyl-2-O-acyl-PI
moiety, (d) hydrolysis of the phosphodiester group of glycero-
PI to obtain biosynthetic inositol. In our recent study on
Leishmania cell-surface phospholipids, it was observed that
glycero-PI derived from the parasite gave a clean and prominent
molecular ion m/z 333 [M Ϫ H]Ϫ under negative-ion ESMS
conditions, suitable for determination of the isotopomeric
ratios of the biosynthetic product. Therefore it was decided to
exploit this observation for the present 13C-labelling study.
The parasite PI/GPI fraction was digested with 4 M NH4OH
at room temp. and, after purification, ESMS showed two prom-
inent quasimolecular ions [M Ϫ H]Ϫ at m/z 333.1 and 242.1 for
glycero-PI and glycero-PC, respectively. To confirm the identity
of these compounds, three samples (PI, PC and soybean
lecithin containing both PI and PC) were digested separately
with 4 M NH4OH and analysed by ESMS which showed m/z
333.1 for glycero-PI and m/z 242.1 for the glycero-PC, and for
the lecithin sample both these ions were seen; the structures
were also confirmed by 1H and 31P NMR spectroscopy. Further
digestion of glycero-PI in 10% NH4OH at 150 ЊC for 18 h fol-
lowed by purification led to the free myo-inositol, showing a
To confirm this observation, the glycero-PI from control and
labelling experiments were further hydrolysed with 10%
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Table 1 Electrospray mass spectrometry experimental results
glycero-PI
(P/P ϩ 1/P ϩ 2)
P = 333
myo-Inositol
(P/P ϩ 1/P ϩ 2)
P = 179
Control
Biosynthetic
Control
Biosynthetic
Peak intensity proportions from labelling with [1-13C]inositol
Peak intensity proportions from labelling with [6-13C]glucose
100:8:2
100:8:2
100:35:4
100:43:10
100:11:2
100:11:2
100:26:4
100:48:11
NH4OH at 150 ЊC in sealed tubes and purified (PLC) to obtain
hydrolysed free inositol. The ESMS of the samples showed the
relative abundance of peak at m/z 180 to be 13% more in
inositol obtained by hydrolysis of glycero-PI from labelling
experiment (Fig. 1, panel C2) than that from the control
experiment (Fig. 1, panel C1). The starting ratio of peaks at
m/z, 179/180 (1:1, Fig. 1, panel A2) was considerably diluted
(37%) due to contribution from de novo biosynthesis. This
further showed the presence of MIP synthase activity in the
parasite.
Incorporation of D-[6-13C]glucose in the parasite
Keeping in view the results obtained from myo-[1-13C]inositol
incorporation and to further complement the existence of a
de novo biosynthetic route for inositol, the Leishmania pro-
mastigotes were starved in glucose-free Dulbecco’s modified
Eagle medium (DMEM) and then incubated with -[6-13C]-
glucose (99 atom-%) along with a parallel control experiment.
ESMS data of glycero-PI from labelling experiment showed
that the 13C label from precursor -[6-13C]glucose was incorpor-
ated into glycero-PI and the relative abundance of peak at m/z
334 was found to be 28% more in glycero-PI from the labelling
experiment (Fig. 2, panel D2) than the glycero-PI from the
control experiment (Fig. 2, panel D1). ESMS of the inositol
samples obtained by complete hydrolysis of glycero-PI (from
control as well as labelling experiments) showed that the relative
abundance of peak m/z 180 was 27% more in inositol obtained
by hydrolysis of glycero-PI from the labelling experiment (Fig.
1, panel E2) than the inositol from the control experiment (Fig.
1, panel E1). These glucose labellings unambiguously con-
firmed the results obtained from inositol-labelling experiments.
In summary, we have synthesised 1-myo-[1-13C]inositol by
two independent routes and incorporated this into Leishmania
donovani parasites. The isotopomeric analysis by negative-ion
ESMS showed substantial dilution of 13C atom-% in the bio-
synthetic glycero-PI and inositol products showing the presence
of a de novo inositol biosynthesis in the parasite. This result
was corroborated by a separate biosynthetic experiment using
-[6-13C]glucose as precursor. This led to the identification of
MIP synthase activity in the parasite which could be exploited
for drug design. We have also demonstrated the potential of the
combined application of stable isotope labelling and ESMS for
the elucidation of biosynthetic pathways in parasites where this
approach has not been exploited.
Fig. 2 ESMS (negative-ion) data from experiment on biosynthetic
incorporation of -[6-13C]glucose in Leishmania donovani: (D1) ESMS
of glycero-PI purified from control experiment; (D2) glycero-PI purified
from labelling experiment; (E1) ESMS of inositol obtained by
hydrolysis of glycero-PI from control experiment; (E2) inositol
obtained by hydrolysis of glycero-PI from labelling experiment.
and -[2-3H]mannose (15 Ci mmolϪ1) and myo-[3H]inositol (83
Ci mmolϪ1) from Amersham. The enzymes phospholipase A2
(bee venom), PI-specific phospholipase C (Bacillus cereus) and
other biochemicals were from Sigma. The negative-ion ESMS
data was obtained on a VG Platform-II quadrupole mass
spectrometer equipped with a MassLynxTM data system and
pneumatic-nebuliser-assisted electrospray LC/MS interface.
Acetonitrile–water (1:1 mixture) was used as carrier solvent at
a flow rate of 10 µl minϪ1 and the analyte was infused through a
rheodyne injector into the ESMS probe. NMR spectra were
recorded on a Bruker DRX spectrometer operating at 300 MHz
1
for H, 75 MHz for 13C and 125 MHz for 31P nuclei. Unless
stated otherwise, the spectra were recorded in CDCl3 and
chemical shifts are in ppm relative to residual solvent reson-
ance. J-Values are given in Hz. The NMR spectral assignments
were made using COSY, HETCOR and HMQC experiments.
Experimental
General methods
The chemicals and reagents used for synthesis of labelled
materials were from Aldrich and Fluka, -[6-13C]glucose (99
atom-% enriched) was from Aldrich. The precoated TLC and
HPTLC plates were from Merck. Culture media (Medium-199,
DMEM) and other additives/vitamins/co-factors used in
Leishmania parasite culture were from Gibco-BRL. Radio-
labelled metabolic precursors, [U-14C]glycerol (147.8 mCi
mmolϪ1), -[1-14C]glucosamine (52.6 mCi mmolϪ1), N-acetyl-
-[1-3H]glucosamine (3.0 Ci mmolϪ1), [1-14C]palmitic acid (54
mCi mmolϪ1), and En3Hance spray were from DuPont-NEN,
Enzymic synthesis of myo-inositol
Glucose 6-phosphate 2. -Glucose 1 (77.4 mg, 0.43 mmol)
was dissolved in deoxygenated water (2 ml) and MgCl2ؒ6H2O
(5.1 mg, 0.25 mmol), KCl (3.73 mg, 0.05 mmol), dithiothreitol
(DTT) (2.3 mg, 0.015 mmol) and sodium azide (0.65 mg, 0.01
mmol) were added. The mixture was stirred for 10 min at room
temperature and then ATP (272.3 mg, 0.45 mmol; USB) was
added, the pH was adjusted to 7.6 with 1 M KOH and the
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solution was purged with nitrogen for 5 min. Now, hexokinase
(Type VI from baker’s yeast, 40 U; Sigma) solution in water
(200 µl) was added and the mixture was incubated at 37 ЊC. The
progress of the phosphorylation was checked by TLC in 9.5
mM tetrabutylammonium hydroxide in 80% aq. acetonitrile
and spots were visualised by ammonium molybdate–cerium()
sulfate reagent. The reaction mixture turned turbid at the end
of the reaction, and the mixture was brought to pH 9.0 with
1 M KOH, and barium acetate (200 mg in 600 µl of water)
was added with stirring. The precipitate was removed by centri-
fugation (3500 rpm for 5 min at 20 ЊC) and the supernatant was
mixed with cation-exchange resin (100 mg, Dowex 50 W, HCR
W2 activated with 1 M HCl, and washed with water), and the
resultant clear solution was decanted and its pH adjusted to
9.0. MeOH was then added dropwise to the brink of precipit-
ation. This solution was applied to an anion-exchange resin
column (Dowex 1X8, 25–50 mesh; 1 × 10 cm bed, washed with
water, 50% aq. methanol, 65 mM NH4HCO3 and again with
water). Elution was performed with water (50 ml), 50% aq.
methanol (50 ml), 65 mM NH4HCO3 in 50% aq. methanol and
finally with 200 mM aq. NH4HCO3. This last fraction was
lyophilised to constant weight to give -glucose 6-phosphate as
its diammonium salt (103 mg, 81%) as a white powder. This
compound was analyzed by TLC [9.5 mM tetrabutylam-
monium hydroxide in 80% aq. acetonitrile; spots visualised by
ammonium molybdate–cerium() sulfate reagent, Rf 0.33];
1H NMR (D2O) δ 5.07 (1H, d, J 3.6, H-1α), 4.49 (1H, d, J 8.0,
H-1β), 4.08–3.92 (2H, m, H2-6), 3.59–3.33 (3H, m); 31P NMR
δ 4.20, assignments confirmed by COSY; ESMSϪ m/z 259.2
[M Ϫ H]Ϫ.
microscope for the extent of disruption. The cell debris was
removed from the lysate by centrifugation at 13 000 g (Kontron)
for 20 min and the clear supernatant was taken. The nucleic
acids were precipitated by slow addition of 2.22 ml of 25%
(w/v) streptomycin sulfate solution to this supernatant at 0 ЊC
and storage for 30 min. The precipitate was removed by centri-
fugation at 27 000 g for 20 min. To the clear supernatant (30 ml)
was added powdered ammonium sulfate (6.87 g) slowly with
stirring at 0 ЊC to a final concentration of 229 g lϪ1 (40% satur-
ation). After being stirred for another 30 min the solution was
left at 4 ЊC for 2 h and precipitated material was removed by
centrifugation (27 000 g; 20 min). Salt saturation was increased
to 75% (483 g lϪ1) by adding ammonium sulfate (6.68 g), and
after stirring for another 30 min the solution was left at 4 ЊC for
2 h and the precipitated material was obtained by centri-
fugation (27 000 g, 20 min). This final pellet (600 mg) was
resuspended in 15 ml of buffer A and dialysed against buffer A
(1 l × 4) at 4 ЊC using 12 000 MW dialysis membrane
(Sigma). Final dialysed material was checked for enzyme activ-
ity, protein concentration (BCA method) and SDS-PAGE and
stored at Ϫ20 ЊC.
myo-Inositol 1-phosphate 3. -Glucose 6-phosphate di-
ammonium salt 2 (90 mg, 0.306 mmol) was dissolved in 10 ml
of buffer B (50 mM TrisؒHCl, pH 7.4, containing 5% ethanol,
0.02% sodium azide, 14.4 mM NH4Cl, 22 mM NaF and 10 mM
DTT). Nicotinamide adenine dinucleotide (β-NADϩ, 114.7 mg,
0.173 mmol; Sigma) and partially purified MIP synthase (2.5 ml
solution) was added and the mixture was incubated at 37 ЊC
for 24 h. The progress of the reaction was monitored by TLC
(9.5 mM tetrabutylammonium hydroxide in 80% aq. aceto-
nitrile). At the end of the reaction, the mixture was heated at
95 ЊC for 3 min and the denatured precipitated protein was
removed by centrifugation for 5 min at 5000 g. The supernatant
was applied to an anion-exchange resin column [AG1-X8; Bio-
Rad, bed 1 × 15 cm, resin washed with water and equilibrated
with buffer C (100 mM ammonium formate containing 20 mM
sodium tetraborate, pH 8.5)]. Elution was performed with a
linear gradient (100 ml) of buffer C to buffer D (625 mM
ammonium formate containing 20 mM sodium tetraborate, pH
8.5). Fractions (5 ml) were collected, and checked by TLC, and
the fractions containing inositol 1-phosphate were pooled.
From this desired fraction, cations were removed by treatment
with a cation-exchange resin (Dowex HCR W2; Sigma, bed
1 × 10 cm, resin washed with 2 M HCl and water). Elution was
performed with water and a single fraction was obtained from
effluent pH 1.0 to 6.0. This fraction was lyophilised and the
residue was repeatedly dissolved in methanol and lyophilised to
constant weight. The resulting product 3 (17.2 mg, 21.7%)
showed 1H NMR (D2O) δ 4.23 (1H, t, J 2.7, H-2), 3.90 (1H, td,
J 9.7 and 2.7, H-1), 3.74 (1H, t, J 9.5, H-6), 3.64 (1H, t, J 9.8,
H-4), 3.58 (1H, dd, J 9.8 and 2.7, H-3), 3.34 (1H, t, J 7, H-5)
and assignments were confirmed by a COSY experiment;
31P NMR δ 2.73; ESMSϪ m/z 259.2 [M Ϫ H]Ϫ.
Culture of mutant (opi 1ꢀ) strain of yeast, and partial purifi-
cation of MIP synthase. The mutant yeast strain (Saccharo-
myces cerevisiae) was obtained from American Type Culture
Collection (ATCC) maintained on agar slants. This is an over-
producer of inositol with the genotype MATa, opi1 (also
termed opi 1δ). The prepared agar slants were streaked with the
yeast and maintained at 4 ЊC. For YEPD medium preparation,
yeast extract (1 g), peptone (2 g), and agar (1.3 g) were dissolved
in 90 ml of water, and the solution was boiled for 5 min, allowed
to cool to about 70 ЊC, and glucose (2 g in 10 ml of water) was
added. The medium was dispensed at 6 ml tubeϪ1 in autoclaved
tubes which were laid in a slanting position and allowed to
solidify at ambient temperature. For bulk culture of the yeast,
an inositol-deficient medium containing yeast extract–peptone–
dextrose (YEPD medium) was prepared, because in inositol-
rich medium the turnover of MIP synthase was low. For this
reason, yeast extract (1.2 g, 0.1%) and peptone (24 g, 2%) were
dissolved in water (1100 ml; pH 6.0) in a conical flask fitted with
an air-bubbling assembly. The assembly and medium was auto-
claved at 121 ЊC at 15 psi for 15 min and the sterilised solution
was cooled to ≈75 ЊC and glucose solution (24 g in 100 ml of
filter-sterilised water) was added. The medium was inoculated
with mutant yeast and the culture was incubated at 30 ЊC for 48
h with bubbling of air. After 48 h of incubation, the cells were
harvested (centrifugation at 4 ЊC and 4000 rpm for 30 min) and
washed twice with 100 ml of buffer A [TrisؒHCl (20 mM,
pH 7.2; Sigma) containing ammonium chloride (20 mM),
2-mercaptoethanol (10 mM) and phenylmethanelsulfonyl
fluoride (PMSF, 0.5 mM; Sigma)], each time pelleting the cells
at 6000 rpm for 10 min at 4 ЊC. The final washed pellet was
resuspended in 40 ml of buffer A. An aliquot of this suspension
was diluted 20-fold and used for microscopic examination for
intactness of cells and any possible presence of microbial con-
taminants. The suspension was subjected to disruption by a
French Press (SLM-AMINCO Spectronic Instruments). The
disruption chamber was pre-cooled to below 0 ЊC into which
the suspension (20 ml) was added, a pressure of 40 000 psi was
applied, and the cycle was repeated thrice with each 20 ml part.
The final disrupted material was pooled and checked under a
Dephosphorylation of myo-inositol 1-phosphate. Compound
3 (17.2 mg, 66 mmol) was dissolved in buffer (3 ml; 0.1 M
TrisؒHCl, pH 8.0) and alkaline phosphatase (4 U; 8.4 ml, stock
82 U 170 mlϪ1, Type III from E. coli; Sigma) was added. The
reaction mixture was incubated at 25 ЊC for 16 h after which
the entire reaction mixture was loaded onto a silica column
(1 × 25 cm, packed in 20% methanol in chloroform). Elution
was as follows: fractions 1–4 (7.5 ml each) with 20% meth-
anol in chloroform, fractions 5–12 (7.5 ml each) with 30%
methanol in chloroform, fractions 13–19 (7.5 ml each) with
50% methanol in chloroform, fractions 20–21 (10 ml each) with
methanol, and fraction 22 (10 ml) with water. The fractions
were analyzed by TLC (9.5 mM tetrabutylammonium hydrox-
ide in 80% aq. acetonitrile; spots visualised by potassium per-
manganate reagent, Rf 0.15). Only water fractions contained
J. Chem. Soc., Perkin Trans. 1, 2000, 1283–1290
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myo-inositol 4, which was lyophilised (6.7 mg, pure, 63.6%); 1H
NMR (D2O) δ 3.96 (1H, t, J 2.7, H-2), 3.53 (2H, t, J 9.8, H-4,
-6), 3.44 (2H, m, H-1, -3), 3.18 (1H, t, J 9.2, H-5), with assign-
ments confirmed by a COSY experiment; ESMSϪ m/z 179
[M Ϫ H]Ϫ.
(peaks clustered together), 123.02, 99.70, 81.20, 79.02, 77.71,
75.58, 74.40, 73.62, 73.39, 73.16, 72.51, 56.14, 20.49; ESMSϩ
m/z 527 [M ϩ Na]ϩ.
1-O-Acetyl-3,4,5-tri-O-benzyl-6-deoxy-6-oxo-myo-inositol 9.
Enol acetate 8 (50.4 mg, 0.1 mmol) was dissolved in acetone (2
ml)–water (800 µl), and mercury() acetate (310 mg, 0.99 mmol)
was added. The solution was stirred for 45 min and then satur-
ated aq. NaCl (1.2 ml) was added, and the mixture was stirred
for 24 h at room temp. The solvents were evaporated off and the
residue was extracted with ethyl acetate. The organic layer was
dried, concentrated and purified by silica column chromato-
graphy to provide desired 914 (21 mg, 42%); 1H NMR δ 7.4–7.2
(15H, m, Ph), 5.17 (1H, br s, H-1), 4.95–4.7 (5H, m), 4.52 (1H,
d, J 11), 4.34 (1H, t, J 2.6), 4.17–4.05 (2H, m), 3.88–3.84 (1H,
dd, J 6 and 2.4), 2.24 (3H, s); 13C NMR δ 197.90, 169.85,
128.53–127.67 (peaks clustered together), 83.44, 81.72,
78.87, 76.05, 74.94, 73.48, 73.21, 69.21, 20.43; ESMSϩ m/z 513
[M ϩ Na]ϩ.
Chemical synthesis of D-myo-inositol
Methyl
2,3,4-tri-O-benzyl-6-O-(triphenylmethyl)-D-gluco-
pyranoside 5. A solution of -glucose 1 (200 mg. 1.11 mmol) in
anhydrous MeOH (5 ml) was mixed with cation-exchange resin
(Dowex 50W; 200 mg, resin pre-washed with water, 2 M HCl,
water and MeOH) and the mixture was refluxed overnight. It
was cooled to room temperature and the resin was removed by
filtration, and the filtrate was concentrated to give viscous, solid
methyl α--glucopyranoside (208 mg, 96%). Methyl glucoside
(194 mg, 1 mmol) was dissolved in DMF (0.6 ml), trityl chloride
(307 mg, 1.1 mmol), DMAP (20 mg, 0.08 mmol) and triethyl-
amine (153 µl, 1.1 mmol) were added, and the mixture was
stirred overnight at room temp. After completion of the
reaction the mixture was concentrated, and purified by silica
column chromatography to give methyl 6-O-(triphenylmethyl)-
-glucopyranoside (270 mg, 62%). This intermediate (218 mg,
0.5 mmol) and NaH (144 mg, 3.6 mmol) were dissolved in
DMF (1.5 ml), and benzyl bromide (522 mg, 363 µl, 3.05 mmol)
was added dropwise at 0 ЊC. The reaction mixture was stirred
overnight at room temp. and excess of NaH was destroyed by
addition of MeOH and water, and the mixture extracted with
CHCl3. The organic phase was washed with water and dried
(Na2SO4), concentrated, and purified by silica column chrom-
atography to give 517 (251 mg, 71%); 1H NMR δ 7.55–6.8 (30H,
m, Ph), 5.1–4.2 (7H, m, 3 × PhCH2 and H-1), 4.15–3.35 (5H,
m), 3.40 (3H, s), 3.20 (1H, dd, J 10 and 4, H-2); ESMSϩ m/z 729
[M ϩ Na]ϩ.
1-O-Acetyl-3,4,5-tri-O-benzyl-myo-inositol 10. To a solution
of 1-O-acetyl-3,4,5-tri-O-benzyl-6-deoxy-6-oxo-myo-inositol 9
(19.6 mg, 0.04 mmol) in anhydrous acetonitrile (1.2 ml)
was added sodium triacetoxyborohydride (86.4 mg, 0.4 mmol,
freshly prepared) and 200 µl of glacial acetic acid. The mixture
was stirred at room temp. for 45 min and then 0.5 M sodium
hydrogen sulfate (720 µl) was added dropwise to destroy excess
of hydride reagent. The mixture was extracted with ethyl acet-
ate and the organic phase was washed successively with 0.5 M
sodium hydrogen sulfate and saturated aq. disodium hydrogen
phosphate, dried over sodium sulfate, and concentrated to give
1
1014 (13.5 mg, 68.6%); H NMR δ 7.4–7.2 (15H, m, Ph), 5.18
(1H, dd, J1,2 3, J1,6 9, H-1), 4.90–4.50 (6H, m, 3 × PhCH2),
4.38 (1H, t, J 3, H-2), 4.15 (1H, t, J6,5 10, J6,1 9, H-6), 3.97
(1H, t, J4,5 9, J4,3 10, H-4), 3.59 (1H, dd, J3,4 10, J3,2 3, H-3),
4.26 (1H, dd, J5,4 9, J5,6 10, H-5), 2.24 (3H, s, COMe); 13C
NMR δ 170.82, 128.50–127.59 (peaks clustered together),
82.90, 80.83, 80.05, 75.72, 75.55, 73.10, 72.72, 70.32, 67.67,
22.62; chemical shifts and stereochemical assignments were
confirmed by COSY and NOESY experiments; ESMSϩ m/z
515 [M ϩ Na]ϩ.
Methyl 2,3,4-tri-O-benzyl-D-glucopyranoside 6. To a solution
of compound 5 (212 mg, 0.3 mmol) in dichloromethane–
methanol (1:2; 3 ml) was added p-TsOH (3 mg) and the mixture
was stirred overnight. Usual work-up and purification on a sil-
ica column gave a colourless, syrupy material 614 (128 mg, 92%);
1H NMR δ 7.5–7.1 (15H, m, Ph), 5.1–4.5 (6H, m, 3 × PhCH2),
4.57 (1H, d, J 2, H-1), 4.02 (1H, dd, J 9 and 7.5, H-3), 3.84–
3.37 (5H, m), 3.37 (3H, s, OMe); 13C NMR δ 183.03, 128.38–
127.52 (7 peaks clustered together), 98.09, 81.85, 79.91, 77.34,
75.64, 74.92, 73.32, 70.58, 61.78, 55.09; ESMSϩ m/z 487
[M ϩ Na]ϩ.
1D-1-O-Acetyl-myo-inositol
11.
1-O-Acetyl-3,4,5-tri-O-
benzyl-myo-inositol 10 (12.3 mg, 0.025 mmol) and 10% Pd/C
(10 mg) as a mixture in ethanol (1.5 ml) was hydrogenated (10
atm) in a Parr cavitation apparatus for 24 h. After completion
of the reaction, the catalyst was filtered off through a Celite pad
and washed with ethanol–water (2:1) and the pH of the filtrate
was brought to 8 by NH4OH. The solution was concentrated to
afford 1-O-acetyl-myo-inositol 11 (5.1 mg, 92%); 1H NMR
(D2O) δ 4.65 (1H, br s, H-1), 4.03 (1H, t, J 2.7, H-2), 3.9–3.2
(4H, m, H-3, -4, -5 and -6), 2.03 (3H, s); ESMSϪ m/z 221.3
[M Ϫ H]Ϫ.
Methyl
2,3,4-tri-O-benzyl-6-O-acetyl-D-xylo-pent-5-eno-
pyranoside 8. A solution of oxalyl dichloride (10.16 mg, 7 µl,
0.08 mmol) in anhydrous CH2Cl2 (2.78 ml) was cooled to
Ϫ78 ЊC and DMSO (12.5 mg, 11.4 µl, 0.16 mmol) was added
dropwise, followed by addition of a solution of 6 (93 mg, 0.2
mmol) in CH2Cl2 (926 µl) over a period of 5 min. The mixture
was stirred for 30 min and then triethylamine (222 µl) was
added. The solution was brought to room temp., water (2 ml)
was added, and the mixture was extracted with CH2Cl2. The
organic layer was dried over sodium sulfate to give intermediate
aldehyde 7 which was used directly and dissolved in dry
acetonitrile (2.5 ml) and anhydrous K2CO3 (171 mg, 1.24 mmol)
was added. After stirring of the mixture for 10 min at room
temp., Ac2O (111 µl, 1.17 mmol) was added and the reaction
mixture was heated overnight under nitrogen at 80 ЊC. After
completion, water (2.5 ml) was added and the mixture was
extracted with diethyl ether. The organic layer was concentrated
and subjected to silica column chromatography and elute
with 20–30% ethyl acetate–hexane to provide the desired
product methyl 2,3,4-tri-O-benzyl-6-O-acetyl--xylo-pent-5-
D-myo-Inositol 4. 1-O-Acetyl-myo-inositol (5 mg, 22.5 µmol)
was dissolved in dry methanol (2 ml) and sodium methoxide
(2.4 mg, 45 µmol) was added. The mixture was heated at
70 ЊC for 3 h after which the excess of NaOMe was destroyed
by addition of water (200 µl). The contents were evaporated
to dryness and then reconstituted in water (1 ml), cation-
exchange resin (Amberlite IR-120, pre-treated with 2 M HCl,
and water) was added, and the mixture was vortexed. The resin
was removed by filtration and the filtrate was lyophilised to
yield white, powdery -myo-inositol (3.8 mg, 94%), Rf 0.15 in
9.5 mM tetrabutylammonium hydroxide in 80% aq. aceto-
nitrile; spot visualised by ammonium molybdate–cerium()
1
sulfate; H NMR (D2O) δ 3.96 (1H, t, J 2.7, H-2), 3.53 (2H,
1
enopyranoside14 8 (54 mg, 53%); H NMR δ 7.4–7.2 (15H, m,
t, J 9.2 and 9.8, H-4, -6), 3.44 (2H, dd, J 9.8 and 2.7, H-1, -3),
3.18 (1H, dd, J 9.2 and 9.8, H-5); 13C NMR δ 74.14 (C-5), 72.19
(C-4, -6), 71.97 (C-2), 70.92 (C-1, -3); assignments were con-
Ph), 4.9–4.6 (8H, m), 4.0–3.9 (2H, m), 3.6–3.5 (1H, m), 3.47
(3H, s), 2.15 (3H, s); 13C NMR δ 167.21, 134.92, 128.46–127.56
1288
J. Chem. Soc., Perkin Trans. 1, 2000, 1283–1290

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firmed by COSY and HMQC experiments; ESMSϪ m/z 179
[M Ϫ H]Ϫ.
(1:2, v/v). The butanol phase was removed and the lower aque-
ous phase was re-extracted with water-saturated butan-1-ol.
The pooled butanol phase containing PI/GPIs was concen-
trated and analysed by HPTLC using a CHCl3–MeOH–13 M
NH3–1 M NH4OAc–H2O (180:140:9:9:23) solvent system.
The presence of PI and GPI bands was confirmed by exposing
the plates to Bial’s reagent (0.9% FeCl3 and 0.55% orcinol in
ethanol–H2SO4) for the presence of sugars, DPH reagent
(0.03% 1,6-diphenylhexa-2,3,5-triene in CHCl3) for lipids, and
Molybdenum Blue spray reagent (zinzade reagent) for phospho-
lipids. The identities of the isolated parasitic PI and GPI
molecules were confirmed by comparison with authentic
samples. To locate positions of radio-labelled glycolipids, the
HPTLC plate was sprayed with En3Hance (DuPont NEN) and
then used for fluorography on Kodak film (exposed at Ϫ70 ЊC).
Isolated compounds were subjected to PI-specific phospho-
lipase C (PI-PLC, Bacillus cereus), phospholipase A2 enzyme
treatment, nitrous acid deamination, negative-ion ESMS, and
NMR for confirmation of the structure.
Synthesis of 13C-labelled myo-inositol 4
myo-[1-13C]Inositol was synthesised from -[6-13C]glucose as
starting material (200 mg, 99 atom-%, Aldrich) which was
taken through all the steps described above for the unlabelled
enzymic as well as chemical syntheses. The progress of reac-
tions was monitored by comparing TLC results with the
corresponding unlabelled compounds. From the chemical route
22.7 mg of labelled inositol could be obtained from 200 mg
of -[6-13C]glucose. The final labelled myo-[1-13C]inositol was
1
3
characterised by H NMR (D2O) δ 4.0 (1H, m, H-2, JH-H 2.7,
2JC-H 5.2 due to coupling of H-2 with 13C-enriched C-1), 3.50
(2H, m, H-4 and -6, J 9.8, 9.2 and 2JC-H 5.2 due to coupling of
H-6 with enriched 13C-1), 3.40 (2H, m, H-1, -3, JC-H 141.5 Hz
due to direct coupling of enriched 13C-1 and H-1), 3.10 (1H, t,
J 9.2, H-5); 13C NMR (proton-decoupled) δ 71.0 (13C-enriched
C-1); 13C NMR (proton-coupled) δ 71.0 (d, J 141.5 due to direct
13C-1/H-1 coupling; each peak of this doublet was further split
2
Preparation of glycero-PI by mild alkali hydrolysis. Parasitic
PI fraction (50 µl) was dried with a stream of nitrogen gas and
4 M NH4OH (100 µl) was added. The mixture was incubat-
ed at 37 ЊC for 16 h. At the end of reaction butan-1-ol (100 µl)
was added, the contents were thoroughly mixed, and the phases
were separated by centrifugation (10 000 rpm for 5 min at
20 ЊC). The aqueous layer was re-extracted with water-
saturated butan-1-ol (100 µl). The final aqueous phase was
freeze-dried to obtain glycero-PI which was used for ESMS
(continuum data, m/z 200–500).
Similarly, mild alkaline hydrolysis of PI (Sigma) (PC, egg
yolk), lecithin (soybean, USB) and a 1:1 mixture of pure PI
and PC was carried out with 4 M NH4OH, and at the end of the
reaction CHCl3 (100 µl) was added to each tube and the con-
tents mixed thoroughly. The organic and aqueous phases were
separated by centrifugation, concentrated, and used for ESMS.
by 5.2 Hz due to JC-H coupling of 13C-1 with H-2 and -6); the
1H and 13C assignments were confirmed by COSY, HETCOR
and HMQC experiments; ESMSϪ m/z 180 [M Ϫ H]Ϫ.
Microbiological methods
Parasite culture. Leishmania donovani promastigotes (DD8
strain, obtained from Professor K.-P. Chang of Chicago Med-
ical School) were grown at 23 ЊC in Medium-199 supplemented
with 10% HI-FBS (heat-inactivated foetal bovine serum),
Hepes (25 mM) and gentamycin sulfate (50 mg lϪ1) to a density
of 4 × 107 cells mlϪ1. The biosynthetic viability of the cell culture
was evaluated by metabolic labelling of L. donovani promasti-
gotes with 2.5 µCi of -[1-14C]glucosamine, 25 µCi of N-acetyl-
-[1-3H]glucosamine, 2.5 µCi of [1-14C]palmitic acid, 21.2 µCi
of -[2-3H]mannose and 15 µCi of myo-[3H]inositol. In the case
of labelling with mannose, glucosamine and N-acetylglucos-
amine, the precursors were added to three tubes separately, each
containing 109 promastigotes (no starvation) in 2 ml of DMEM.
The modified medium (dDME) supplemented with 0.3%
bovine serum albumin (fraction V), adenosine (0.05 mM),
xanthine (0.05 mM), biotin (10 mg lϪ1), Tween 80 (40 mg lϪ1),
haemin (5 mg lϪ1), and triethanolamine (0.5 ml lϪ1) was
prepared according to the method of Orlandi and Turco.20 In
the case of inositol labelling, promastigotes were starved in
dDME (without inositol and with fat-free BSA; prepared from
inositol-free DMEM) for 48 h at 23 ЊC and then to this washed,
resuspended cell pellet (4.75 × 1010 promastigotes in 5 ml of
dDME) was added myo-[3H]inositol. Similarly, for labelling
with palmitic acid, cells were starved in dDME (with fat-free
BSA; prepared from pyruvate-free DMEM) for 48 h at 23 ЊC
and then to the washed, resuspended cell pellet (109 promastig-
otes in 5 ml of the same dDME) was added [1-14C]palmitic acid.
Suitable controls were used in each of the above metabolic
labelling experiments. Incubations were carried out overnight at
23 ЊC and cell viability was checked before and after incubation.
For biosynthetic incorporation, exponentially growing para-
site cells from Medium-199 were harvested by centrifuging at
4000 rpm for 8 min at 23 ЊC. The cells were starved in 20 ml of
inositol-free dDME with fat-free BSA at 23 ЊC for 24 h after
which the culture was harvested. The cell suspensions were
checked for parasite viability before and after incubation. After
completion of incubation, the parasites were collected and
PI/GPIs isolated. The cell pellet was washed three times with
phosphate-buffered saline (PBS) (50 mM, pH 7.2). The washed
cell pellet was extracted twice with 10 volumes of chloroform–
methanol–water (1:2:0.8, v/v) and the insoluble material was
removed by centrifugation (10 000 g; 15 min). The combined
supernatants were dried under a stream of nitrogen and the
extracted lipids were partitioned between water and butan-1-ol
Hydrolysis of glycero-PI to inositol. The glycero-PI from the 4
M NH4OH digest of the parasite PI was lyophilised in a glass
tube, 10% NH4OH (100 µl) was added, and the tube was sealed
under nitrogen before being heated at 150 ЊC for 18 h; after
cooling, the tube was broken and the contents were mixed with
CHCl3 (100 µl). The aqueous layer was freeze-dried and further
purified for isolation of labelled inositol by PLC (9.5 mM
tetrabutylammonium hydroxide in 80% acetonitrile–water; Rf
inositol 0.15, and glucose 0.34).
Biosynthetic incorporation of 1D-myo-[1-13C]inositol in
L. donovani
Exponentially growing L. donovani promastigotes were har-
vested from Medium-199, washed thrice, and resuspended in
inositol-free Medium-199 with fat-free BSA. The promastigotes
were starved for 24 h at 23 ЊC after which the culture was div-
ided into two equal parts and each was harvested separately to
give 2 × 109 cells partϪ1. To the first part was added 5 ml of
normal medium with fat-free BSA (control experiment) and to
the second part were added 5 ml of myo-[1-13C]inositol-rich
medium with fat-free BSA and 2 mg of labelled inositol (50
atom-% 13C) (labelling experiment). The cells were incubated at
23 ЊC for 24 h, after which harvesting, initial processing, and
extraction of PI were carried out. The PI fraction in each case
was divided into two equal parts, one of which was treated with
4 M NH4OH at room temp., and the other part was treated with
10% NH4OH at 150 ЊC in a sealed tube. After both treatments
the aqueous phases were separated, washed with CHCl3–
MeOH, and purified for glycero-PI and inositol, respectively.
At zero time of incubation, a 50 µl aliquot of the culture was
removed and processed as described above for the isolation of
free inositol, which was further subjected to PLC (9.5 mM
J. Chem. Soc., Perkin Trans. 1, 2000, 1283–1290
1289

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2 (a) T. B. McNeely and S. J. Turco, J. Immunol., 1990, 144, 2745;
tetrabutylammonium hydroxide in H2O–CH3CN 20:80). This
was required to separate inositol (Rf 0.15) from glucose (Rf
0.34) present in the parasite culture. The inositol band was
scraped from the plates and eluted in 20% methanol in water by
sonication. The silica particles were removed by centrifugation
at 13 000 rpm for 20 min and the supernatant was used for
ESMS.
(b) A. Descoteaux, G. Matlashewski and S. J. Turco, J. Immunol.,
1992, 149, 3008; (c) J. R. Giorgione, S. J. Turco and R. M. Epand,
Proc. Natl. Acad. Sci., USA, 1996, 93, 11634; (d) A. Descoteaux,
S. J. Turco, D. L. Sachs and G. Matlashewski, J. Immunol., 1991,
146, 2747; (e) M. Desjardins and A. Descoteaux, J. Exp. Med., 1997,
185, 2061.
3 (a) M. J. Berridge and R. F. Irvine, Nature, 1989, 341, 197; (b) M. J.
Berridge, Nature, 1993, 361, 315; (c) N. Divecha and R. F. Irvine,
Cell, 1995, 80, 269.
Biosynthetic incorporation of D-[113C]glucose in L. donovani
4 (a) Inositol Phosphates and Derivatives. Synthesis, Biochemistry
and Therapeutic Potential, ACS Symposium Series 463, American
Chemical Society, Washington, DC, 1991; (b) B. V. L. Potter and
D. Lampe, Angew. Chem., Int. Ed. Engl., 1995, 34, 1933.
5 (a) T. F. Donahue and S. A. Henry, J. Biol. Chem., 1981, 256, 7077;
(b) M. D. Johnson and S. A. Henry, J. Biol. Chem., 1989, 264, 1274;
(c) A. L. Majumdar, M. D. Johnson and S. A. Henry, Biochim.
Biophys. Acta, 1997, 1348, 245.
Exponentially growing L. donovani promastigotes were har-
vested from Medium-199 and washed thrice with glucose-free
DMEM. The washed pellet was resuspended in 50 ml of
glucose-free dDME. The cells were starved for 24 h at 23 ЊC
after which the suspension was divided into two equal parts and
each part was harvested separately to give 6 × 108 cells partϪ1
.
6 J. P. Hirsch and S. A. Henry, Mol. Cell. Biol., 1986, 6, 3320.
7 (a) D. E. Kiely and W. R. Sherman, J. Am. Chem. Soc., 1975, 97,
6810; (b) T. Maeda and F. Eisenberg Jr., J. Biol. Chem., 1980, 255,
8458; (c) F. A. Loewus and M. W. Loewus, Annu. Rev. Plant
Physiol., 1983, 34, 137.
8 (a) M. E. Migaud and J. W. Frost, J. Am. Chem. Soc., 1995, 117,
5154; (b) M. E. Migaud and J. W. Frost, J. Am. Chem. Soc., 1996,
118, 495.
To each part was added 5 ml of glucose-free dDME. Further,
the first part (control ) was fortified with 22.5 mg of -glucose
while the other part (labelling) was fortified with 22.5 mg of
-[6-13C]glucose (99 atom-%). These cell suspensions were
incubated for 24 h at 23 ЊC, and labelled glycero-PI and inositol
were isolated as described above and analysed by ESMS.
9 W. R. Sherman, in Inositol Lipids in Cell Signalling, ed. R. H.
Michell, A. H. Drummond and C. P. Downes, Academic Press,
New York, 1989, p. 39.
10 P. Sahai and R. A. Vishwakarma, J. Chem. Soc., Perkin Trans. 1.,
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Acknowledgements
We thank Professor K.-P. Chang (Chicago) for providing
L. donovani strains, Professor Sam Turco (Kentucky) and
Dr Malcolm McConville (Melbourne) for authentic samples of
parasitic GPI molecules, and Dr Sandip Basu for helpful dis-
cussions and encouragement. The Department of Science and
Technology (Government of India) provided funding support
for the work (Grant No. SP/SO/D-31/95) and the Department
of Biotechnology provided core grant and infrastructure
support to our Institute.
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