transferred to another plastic box containing 2 X sse, 1 % SDS and washed at room temperature by gentle rocking for 15 minutes. The buffer was then changed and the washing continued at 60 in a shaking water bath for 30 minutes. Depending on the homology between the probe and the immobil ised DNA, the washing conditions were varied. The stringency ranged from 1 X sse, 1 % SDS, at 65°e to 0.2 X sse, 1 % SDS, at 65°e. After the washing, the filters were immediately sealed into plastic bags and put for autoradiography. Special care was taken to not to allow the filters to dry during any stage which might otherwise cause permanent binding of the probe to the filter preventing the reprobing of the same filter with a different probe at a later time. For autoradiography, the plastic bag containing the washed filter was fixed on a 3 MM Whatman sheet and placed securely ins ide a X ray cassette with one or two intensifying screens, and a X -ray film was placed over the filter in a dark room. The cassette was kept at -7o0e for the desired length of exposure. The film was taken out in the dark room, developed for approximately 3 minutes, washed in water for one minute to wash off all the developer adhering to the film, and fixed for 5 minutes. Finally, the film was washed in cold water for 10 minutes and air dried

The prehybridisation and hybridisation of the Southern filters was carried out as described by Maniatis et al., ( 1982 ), with some modifications. In all stages, the SDS concentration was maintained at 1 % to minimise the background likely to occur on the nylon membrane. Prehybridisation was done at 68°C, for 4 - 6 hours, with 0.1 ml of prehybridisation buffer for each square centimeter of the membrane. The probe was denatured by immersing the eppendorf tube in a boiling water bath for 10 minutes and added directly to the bag containing prehybridisation mix. Hybridisation was done in aqueous system, at 68°e, without the use of formam ide, for 18 - 2 4 hours, in a plastic bag kept submerged in a water bath, without any shaking. At the end of hybridisation, the filter was taken out of the bag and quickly immersed in a plastic box containing 5 X sse, 1 % SDS at room temperature. After 15 minutes, the filter was

Hybridisation of southern filters.

eppendorf tube was put at the bottom of the column to collect the eluate. The column was respun as before and the purified probe collected in the eppendorf tube, the unincorporated nucleotides remaining within the column. One ul aliquot from the purified probe was diluted 100 fold, mixed well and 1 ul aliquots were put in triplicate into 3 ml scintillation fluid containing vials which were counted in a Beckman Liquid Scintillation Counter. The total radioactivity of the probe was calculated by multiplying the mean radioactivity of the three diluted samples with a factor of 104 ( dilution factor, 102, total reaction volume, 102 ). The specific activity of the probes ranged from 1 X 107 to 5 x·1o7 cpm 1 ug DNA. The probe purified by the above method did not require any further purification.

The nick translated probe was purified by a spun column procedure to remove the unincorporated nucleotides. A sterile 1 ml syringe was plugged at the lower end with siliconised glass wool. The syringe was then filled with Bio-gel P-4 Bio Rad Laboratories, USA ) equilibrated in advance with TE. For doing this, 30 grammes of Bio-gel P-4 was slowly added into 250 ml of TE ensuring a good dispersion of the powder. This was then autoclaved at 15 psi for 20 minutes. After cooling, the supernate was decanted and replaced with an equal volume of sterile TE. The slurry was stored at 4°C. The slurry was poured upto the 1 ml mark in the syringe. The syringe was placed into a centrifuge tube and spun at 2000 · rpm for 3 minutes. The column was packed by repeating this process till the packed column volume reached 1 ml mark. Next, 50 ul of 2 mg 1 ml denatured salmon sperm DNA was loaded as carrier and the column spun as before. 100 ul of TE was then added to the column and it was respun as before. Finally, the nick translation reaction was diluted to 100 ul with TE and loaded on to the column. A sterile 1.5 ml

Purification of the probe.

Following electrophoretic resolution of total RNA, the gels were blotted on to GeneScreen membrane as described by Maniatis et al., 1982 ) .. The RNA gel to be used for blotting was not stained with ethidium bromide. The blotting was performed in 20 X sse or 20 X SSPE, OIN.

Northern blot.

bands seen in the DNA size marker, were marked with a ball -point pen at the places where small holes had been pierced in the gel earlier ( see above ). Thus it was easy to monitor the size of the fragments showing hybridisation to the probe. The gel was then peeled off and the membrane w~shed in 6 X sse with gentle rocking for 10 minutes to wash away any residual agarose sticking to the membrane. After air drying at room temperature, the membrane was baked at so0e for two hours. The baked filter was stored at room temperature in a dessicator, if not used immediately. The dehydrated gel was restained in water containing 0.5 ug I ml ethidium bromide for 30 minutes and examined on a short wave UV transilluminator to check for the presence of any DNA fragments that escaped blotting. The absence of any residual bands indicated that the transfer was complete.

Restriction fragments of DNA resolved on agarose gel were transferred to nylon membrane ( GeneScreen or GeneScreen Plus by the capillary blotting procedure of Southern ( 1975 ) as described by Maniatis et al., ( 1982 ) . After the completion of electrophoresis, the gel was stained and photographed as described earlier. Position of the various bands obtained in the DNA size marker lane were marked by piercing small holes at the two ends of each band in the gel with a yellow tip. The gel was then denatured, neutralised and blotted essentially as described by Maniatis et al., ( 1982 ) . Locally available coarse absorbent paper was used to make the paper towels of the appropriate size. In case of genomic DNA from mammalian cells, the agarose gel was first treated with 0.25 M HCl for 10 minutes, followed by the rest of the procedure as mentioned above. The transfer buffer was 20 X SSPE in all cases. To prevent the absorption of fluid from the 3 MM paper under the gel directly to the blotting paper atop the nylon membrane, the gel was surrounded with polythene sheets to minimise the direct contact between the blotting paper and the 3 MM paper placed under the gel. The blotting was performed for 18 -24 hours. After the transfer was over, the paper towels and the 3 MM papers on top of the nylon filter were peeled off. The gel along with the attached membrane, was turned over and kept on a clean sheet of 3 MM paper with the gel side up. The position of the gel slots was marked with a ball -point pen. Also, the positions of the

southern blot.

Transformation was performed in chilled 1.5 ml eppendorf tubes, using 200 ul of competent cells and about 50 ng of ligated plasmid DNA. Frozen competent cells were thawed in ice and the DNA was added immediately after thawing. The DNA volume was always kept under 30 ul. The DNA was mixed well with the cells by gentle tapping, and the tube incubated in ice for 3 0 minutes with occasional gentle shaking. The tube was then immersed in a 42°C water bath for 2 minutes, to give a heat shock to the cells. The cells were then incubated in ice for 10 minutes. Next, 1 ml LB was to the cells, and the cells incubated in a 37°C water bath without shaking, for one hour. 50 ul aliquots were plated in triplicate from the transformed cell mixture on suitable antibiotic containing agar plates and incubated 0/N at 37°C to select the transformants. In case of JM105 cells, the transformed cells were plated on antibiotic containing agar plates on which 50 ul of 2 % X-gal ( made in dimethyl formamide ) , and 10 ul of 100 mM IPTG had been spread in advance, to select for the lac-phenotype. The lac-colonies appeared colourless while the lac+ colonies were blue. For each batch of transformations, a negative control was included in which no DNA was added to the cells while keeping the rest of the procedure the same as for the test transformations.

Transformation procedure.

containing 2. 2 M formaldehyde and 50 % V /V formamide. The samples were chilled on ice for 5 mins. and loading buffer added. A Taq I digest of phi X 174 DNA, filled-in wi~h Klenow polymerase using 32P-dCTP, was used as size marker for electrophoresis. The gels were run at <5 Vjcm.

Total RNA was resolved in formaldehyde -agarose gels as described by Maniatis et al., ( 1982 ) • In general, the electrophoresis was performed using 1.2 ~ 0 agarose gels containing 2.2 M formaldehyde and 1 X running buffer 0.04 M rnorpholinopropanesulfonic acid -MOPS, pH 7.0; 0.01 M sodium acetate; 0.001 M EDTA ). RNA samples upto 20 ug in 5 ul ) were incubated at 55°c for 15 minutes in 5 X gel buffer

Electrophoresis of RNA.

lectrophoresed on 0.7 % -1.2 % agarose gels in TAE or TBE buffer. Choice of the percentage of agarose and the electrophoresis buffer system was made following the guidelines of Maniatis et al., ( 1982 ). In general, upto 1 kb fragments were resolved on 1.2 % agarose gels using TBE buffer. For most other purposes, TAE buffer was used. Agarose gel electrophoresis was carried out as described by Maniatis et al., ( 1982 ) . The run was stopped when the bromophenol blue dye migrated to within 1 em -1.5 em from the edge of ' the gel, except when the sample had fragments smaller than 500 bp, in which case the elctrophoresis was terminated at an earlier stage. The gel was immersed in water containing 0.5 ug I ml ethidium bromide, for 30 minutes, to stain the DNA. When detecting very low amounts of DNA, the staining was done for 60 minutes followed by destaining in 1 mM Mgso4 for one hour at room temperature. The DNA bands were visualised on a short wavelength UV transilluminator ( Fotodyne, Inc., USA and photographed with a Polaroid MP-4 camera using Polaroid type 667 film.

DNA digested with restriction enzymes was

Digestions involving more than one restriction endonuclease were carried out with 2 - 4 ug DNA in a final reaction volume of up to 50 or 100 ul. In these cases, if the two enzymes had radically different optimal assay conditions, the DNA was digested first with the enzyme requiring a lower salt concentration. After incubating for one hour, a 5 ul aliquot from the digestion reaction was electrophoresed on a mini gel to monitor the extent of digestion. Once the digestion was complete, appropriate amount of salt and the

second enzyme were added and the incubation continued in an increased final reaction volume, to offset any increase in the glycerol concentration in the new reaction. Alternatively, the DNA was extracted once with phenol/chloroform, once with chloroform, and then precipitated with one half volume of 7.5 M ammonium acetate and two volumes of ethanol. The precipitation was done for 30 minutes at room temperature, and the DNA spun down for 30 minutes at room temperature. The supernate was discarded, pellet washed with 70% ethanol, recentrifuged, dried briefly under vacuum and finally resuspended in 18 ul distilled water. The DNA purified in this manner could then be used for setting up digestion with a second enzyme or for setting up a ligation. For those double digestions where one of the enzymes was known to be active over a broad range of ionic strength conditions, including those required for the optimal activity of the second enzyme, both the enzymes were added simultaneously in the digestion reaction, which was carried out using the optimal conditions of the second enzyme having more stringent assay requirements.

was added followed by gentle shaking for 90 minutes at room temperature. This DNA was stored at 4°C. The DNA prepared by this method was of sufficient purity for restriction endonuclease cleavage and Southern blotting, but because of RNA contamination, this DNA could not be used for accurate absorbance measurements. However, typically a 30 ul aliquot was expected to contain approximately 10 ug DNA.

al., ( 1986). Briefly, about 108 cells were pelleted and the pellet washed twice with 10 mM phosphate buffered saline, pH 7. 4. The pellet was resuspended in 2 ml of a sol uti on containing 0.1 M NaCl, 0.2 M sucrose, 0.01 M EDTA, and 0.3 M Tris, pH 8.0. To this, 125 ul of 10 % SDS was added, mixed by vortexing and the sample incubated at 65°c for at least 30 minutes. Next, 350 ul of 8 M potassium acetate was added, the contents vortexed to mix and incubated on ice for 60 minutes. The lysate was centrifuged at 5000g for 10 minutes at 4 °c. The supernate was transferred to a new tube and extracted with 2 ml of phenol ( saturated previously with TE) and 2 ml of chloroform I isoamyl alcohol ( 24:1 ). The extraction was done by gentle rocking or by inverting the tube. The tube was spun at 1500g for 5 minutes to separate the two phases, and the upper aqueous phase was collected. This was re -extracted with 2 ml of chloroform I isoamyl alcohol as described above and the aqueous phase collected. Then 5 ml of ethanol was added to the aqueous phase to precipitate the DNA. The two layers were mixed slowly to prevent shearing of DNA. The DNA was pelleted by centrifugation at 1500g for 10 minutes at 4°C. The supernate was discarded very carefully, to minimise the loss of the loose DNA pellet. The DNA pellet was washed gently with 5 ml of 80 % ethanol. Again, the tube was centrifuged at 1500g to pellet the DNA and the supernate was discarded. The final DNA pellet was dried partially by letting the tube stand at room temperature for 30 minutes. To resuspend the DNA, 300 ul TE

Genomic DNA from cultured mammalian cells was isolated by a rapid procedure, essentially as described by Davis et

Isolation of genomic DNA from mammalian cells.

ecanted and the pellet dried briefly under vacuum. The final DNA pellet was resuspended in 500 ul of TE. A 1:50 dilution of the sample was used to measure the absorbance at 260 nm and at 280 nm. The A260 and A280 values were used to estimate the concentration and purity of the sample as described by Maniatis et al., ( 1982).

further purified by centrifugation to equilibrium in a 30 ml cesium chloride -ethidium bromide density gradient, as described by Maniatis et al., ( 1982 ) . The band corresponding to closed circular plasmid DNA was collected and further purified by a second centrifugation to equilibrium in a 6. 5 ml cesium chloride -ethidium bromide density gradient. The final DNA band collected from the gradient was extracted with an equal volume of isopropanol which had been previously saturated with TE and cesium chloride. This extraction was repeated twice to completely remove the ethidium bromide from the DNA sample. The DNA was then dialysed against one liter of TE for at least 8 hours, at 4 °c, with several changes of TE. To the dialysed sample, one tenth volume of 3 M sodium acetate, pH 5.2, was added and the DNA precipitated with two volumes of chilled ethanol. The precipitation was carried out 0/N at 0 -20 c. The precipitated centrifugation at 10, 000 rpm, DNA was collected by for 10 minutes. The supernate was carefully d

resuspended in 20 ml of Tris -Glucose solution ( 25 mM Tris. HCl, pH 8. 0; 50 mM Glucose ) . The cells were vortexed followed by repeated pipetting to obtain a uniform cell suspension. To this, 6.0 ml of a freshly prepared lysozyme solution ( 10 mg 1 ml, prepared freshly in sterile distilled water ) was added. The cell suspension was swirled to mix thoroughly and incubated for 5 minutes at room temperature. Next, 0.5 M EDTA was added to a final concentration of 10 mM, the contents swirled to mix and incubated in ice for 20 minutes. Next, 40 ml of a lytic mix containing 0. 1 % SDS and 0. 2 N NaOH was added. This was prepared freshly by mixing 4 ml of 10 % SDS solution into 36 ml of 0.22 N NaOH solution. The solution was mixed by vigorous but brief shaking till the cell lysate became clear, followed by incubation on ice for 5 minutes. Finally, 20 ml of 5 M potassium acetate solution, pH 4.8 was added. Again the contents were swirled to mix, followed by incubation in ice for at least 1 - 2 hours. The lysate was centrifuged at 10,000 rpm for 30 minutes at 4°c. The supernate was filtered through sterilised glass wool kept in a funnel, and collected in a graduated cylinder. The measured volume of the cell lysate was transferred into another centrifuge bottle and two volumes of 95 % ethanol added to precipitate the DNA, at 0 -20 c, 0/N. The DNA was pelleted by centrifugation at 10,000 rpm at 4 °c for 30 minutes. The supernate was carefully poured off and the pellet res~spended in 25 ml of TE ( 10 mM Tris.HCl, pH 8.0; 1 mM EDTA ). The plasmid DNA was

Plasmid DNA was isolated using the alkaline lysis method of Birnboim ( 1979 ) with slight modifications. One liter of TB supplemented with ampicillin @ 50 ug 1 ml was inoculated with 10 ml of a freshly grown primary culture and the culture incubated 0/N at 37°c, in an incubator -shaker. The cells were pelleted by centrifugation at 4000g for 10 minutes at 4 °c. The supernate was discarded and the pellet

Isolation of plasmid DNA.

Large scale isolation of DNA.

Bacto -tryptone, Bacto -agar and Bacto -yeast extract were from Difco Laboratories, Detroit, USA. Fetal calf I serum, Ham s F-12 medium ( DMEM ) , I Iscove s Laboratories, USA.

Media.

The membranes were suspended (1.4 x 108 cell equivalent) in 250 III of incorporation buffer (50 mM HEPES, pH = 7.4, 25 mM KCI, 5 mM MgCb, 5 mM MnCI2, 0.1 mM TlCK, 1 Ilg/ml leupeptin, 1 mM ATP, 0.5 mM dithiothreitol and 0.4 Ilg/ml tunicamycin). Each assay tube was prepared by adding 12.5 III of 1 % Chaps, 2.8 III of 200 IlM GOP-Man, 10 III of GOP-[3H]-Man (1IlCi) and 25 nmol of synthetic substrate (49). The contents were lyophilized and 250 III of membrane suspension (1 .4 x 108 cell equivalent in incorporation buffer) were added to each tube. The tubes were incubated at 28°C for 20 minutes, cooled to 0 °C and the membranes were pelleted at 4 °C for 10 minutes in a microcentrifuge. The eH] mannosylated products, that were recovered in the supernatant, were mixed with 0.5 ml 100 mM ammonium acetate and applied to a C18 Sep-pak cartridge that had been washed with 5 ml 80% propan-1-01 and 5 ml 100 mM ammonium acetate. The cartridge was washed with 1.5 ml of 100 mM ammonium acetate and then the eluate was reapplied to the same cartridge. The cartridge was subsequently washed with 5 ml of 100 mM ammonium acetate, after which the bound material was eluted with 5 ml of 60% propan-1-01. The final eluate was concentrated and redissolved in 100 III of 60% propan-1-01. One tenth of this volume was taken for scintillation counting. The above assay was then carried out with a range of concentrations of OMJ to assess it's effect on the activity of eMPT enzyme parse.

eMPT inhibition assay

Membrane protein suspension (5.2 x 109 cells) was centrifuged (3000 g, 4°C, 10 min). The debris was discarded and the supernatant was subjected to ultracentrifugation (100000 g, 4°C, 1 h). The pellet thus obtained was dissolved in 400 ~L of loading buffer (50 mM HEPES-NaOH, pH = 7.4, 0.25 M sucrose, 1 mM ATP,1 mM EOTA, 2 mM OTT, 2 mM leupeptin, 0.2 mM TLCK, 0.1 mM PMSF), and loaded onto a linear sucrose gradient. The gradient was prepared by layering eight 200. ~L fractions (0.25-2 M sucrose in 25 mM HEPES-NaOH, pH = 7.4) over a sucrose cushion (2.5 M) in Ultraclear centrifuge tube (Beckman) followed by centrifugation at 218000 g for 1 h. Organelles in the buffer were fractionated by centrifugation at 218000 g for 4 h at 4 °c in a Beckman L-80 Ultracentrifuge using a SW41 rotor. Each layer was carefully separated out and diluted with 500 ~L of 50 mM HEPES-NaOH buffer. Protein was estimated for each fraction separately using standard BCA assay. ~-1 ,4 Galactosyl transferase was used as a positive marker for golgi and vesicle integrity was determined by measuring the latency of galactosyltransferase catalyzed transfer of [3H] galactose from UOP-[3H]-Gal to GlcNAc

Organelle separation of L.donovani 93

mixture was concentrated and the residue was repeatedly lyophilized to yield 7S; ESMS (mlz): 263.1 (M-Hr. Guanosine 5'-diphospho-4,S-di-deoxy-4,S-difluoro-a-D-talose mono triethyl amine salt) 77. A mixture of 4-morpholine-N,N'-dicyclohexylcarboxaminidium guanosine 5'-monophosphomorpholidate (27 mg, 34.4 Ilmol) and 7S (10 mg, 21.5 Ilmol) was coevaporated with anhydrous pyridine (3 x 500 Ill). 1 H-tetrazole (5 mg, 68.7 Ilmol) and anhydrous pyridine (1 ml) were added and the mixture was stirred under argon atmosphere for 2 days. Water was added and the mixture was concentrated under reduced pressure to afford 77; ESMS (mlz): 608.3 (M-Hr.

6 Hz), 4.85 (1H, s); 13C NMR 853.28,65.12 (15 Hz, C3), 67.3 (24 Hz, C5), 69.72 (C2), 81.1 (JCF = 168 Hz, C4), 89.9 (JCF = 171 Hz, C4), 101.47 (C1). 1 ,2,3-Tri-O-acetyl-4,6-di-deoxy-4,6-difluoro-a-D-talopyranoside (73). To compound 72 (100 mg, 0.543 mmol) was added 2% sulfuric acid solution in acetic anhydride (1.2 ml). The mixture was stirred at rt for 90 minutes. The contents were diluted with saturated sodium bicarbonate solution. The mixture was extracted with ethyl acetate. The organic phase was thoroughly washed with water, dried over sodium sulfate and concentrated to afford 73. 2,3-Di-O-acetyl-4,6-di-deoxY-4,6-difluoro-a-D-talo-di-O-benzyl phosphate (75) : Compound 73 ( 70 mg, 0.225 mmol) was dissolved in anhydrous CH3CN saturated with dimethylamine (5 ml ) at -20°C and stirred for 3h after which TlC confirmed the disappearance of starting material. Excess of dimethylamine was removed under reduced pressure at 30°C and the reaction mixture was concentrated to afford 2,3, di-O-acetyl-4,6-di-deoxy-4,6-difloro-a-D-talopyranoside (74). To a stirred solution of compound 74 and 1 H-tetrazole (21 mg, 0.3 mmol) in anhydrous CH2CI2 (400 Ill) was added dibenzyl-N,N-diisopropylphosphoramidite (99.4 Ill, 104.3 mg, 0.3 mmol) and the mixture was stirred under argon atmosphere for 2 h at rt. Subsequently, the reaction mixture was cooled to -40°C and m-CPBA (87 mg, 0.504 mmol) was added and stirring was continued for another 30 minutes at rt. The reaction was quenched by the addition of a solution of saturated sodium bicarbonate. The mixture was extracted with CH2CI2. The organic phase was thoroughly washed with water, dried over Na2S04 and concentrated to afford 75, which was purified by running a silica coated preparative TlC plate; Rf = 0.24 (50% ethyl acetate in hexane); 1H NMR characterstic ¢ 5.67 (1 H, dd, J = 6.3 Hz and 1.8 Hz, H-1); 13C NMR: ~ 20.5-20.6 (OAc), 64.77, 64.99, 66.28, 66.43, 69.9 (24 Hz, C5), 79.96 (JCF = 169 Hz, JCH = 7.1 Hz, C6), 84.08 (JCF= 180, JCH = 5.4 Hz, C4), 95.68,126.85-128.7,169.50,169.77; 31p NMR 8 -3.03; ESMS (mlz): 551.2 (M+Nat. 4,6-Di-deoxy-4,6-difluoro-a-D-talosyl phosphate (76). To a solution of 75 (30 mg, 0.056 mmol) in CH30H (1 ml) was added palladium on charcoal (10%, 280 mg) and formic acid (100 Ill). The mixture was stirred at 50°C for 3h. The catalyst was filtered off and the solvent was evaporated. The residue was taken in a mixture of CH30H:water:triethylamine (5:3:2, 1.6 ml) and stirred for 2 days at rt. The reaction

Methyl-4,6-di-deoxy-4,6-difluoro-a-D-talopyranoside (72). DAST (750 j.!L, 5.6 mmol) was added with stirring at -40 °c, to a suspension of methyl-a-D-mannopyranoside 62 (200 mg, 1 mmol) in anhyd CH2CI2 (4 mL). The mixture was stirred at -40 °c for another 30 minutes and then at rt for 3 h. After cooling to -200C, the excess of reagent was destroyed by addition of CH30H (600 j.!L) and sodium bicarbonate (200 mg). The cooling bath was removed, and the mixture was filtered once effervescence ceased. The filtrate was concentrated, loaded onto a silica column and eluted out with CH2CI2 to yield 72; Rf= 0.7 in 12.5% CH30H in CH2CI2; 1H NMR (CDCI3) 83.40 (3H, s, OCH3), 4.19 (1 H, m), 4.52 (1 H, d, 6 Hz), 4.68 (1 H, d,

Synthesis of [4,6-Dideoxy-4,6-difluoro]-GDP Talose (Scheme 16 of Results and Discussion)

Cultured promastigotes were harvested by centrifugation of suspension culture (500 ml) in falcon tubes at 3000 g for 10 min at 20°C in a cooling centrifuge (Rota 4R; Plastocraft). The clear spun media was carefully decanted and the pellet was resuspended in ice-cold phosphate buffered saline (PBS, 20mM, pH = 7.2). Centrifugation was done again as earlier and washings were collected in a separate falcon. The washing step with PBS was repeated twice. The promastigotes in PBS were then counted using a Neubauer chamber. For this an aliquot was taken and diluted with PBS (normally 10 J..ll original suspension was mixed with 60 J..ll PBS) and then formaldehyde was added to this (30 J..ll to give a final dilution of 1:10). After 10 minutes of fixing in formaldehyde, 10 J..ll of this diluted suspension was put under the coverslip on Neubauer chamber and counted. Total cell count was determined using the standard formula. For breaking cells to get membrane preparation,93 the cell pellet (6.5 x 109 cells) was suspended in 5 ml of hypotonic buffer (0.1 mM TlCK and 1 J..lg/ml leupeptin) and sonicated in ice (6 x 10 s pulses with 3 s intervals). Breaking of cells were assessed by a light microscope. The membrane protein was further processed as per the requirement of the experiment.

Preparation of Cell-free system of L.donovani

N-Butyl-4-~-galactopyranosyl-a-D-glucopyranosyl ~-amino lactam (61). To a solution of 5 (12 mg) in CH30H (1 ml) was added palladium on carbon (10%, 35 mg) and formic acid (100 Ill). The mixture was stirred at 50°C overnight. The catalyst was filtered off and solvent was evaporated to afford 61; 1H NMR: 80.72-0.77 (t, 3H, CH2-CH3), 1.14-1.22 (m, 4H, CHz-CHz-CH3),1.40-1.45 (t, 2H, N-CH2), 4.31 (d, J = 7.8 Hz, 1H, H-1'), 5.38 (d, J = 4.2 Hz, 1H, H-1); 13C NMR: 8 12.72,19.69,28.77,40.65, 52.84, 61.09, 67.48, 68.50, 70.88, 72.56, 75.17, 77.66, 79.12, 103.19, 169.83; ESMS (mlz): 430.37 (M+Nat.

3,6··Di-O-benzyl-4-(2,3,4,6-tetra-O-benzyl-~-galactopyranosyl)-a-D-glucopyrano syl ~ amino lactam (58). To a solution of hexa-O-benzyl lactal (32, 300 mg, 0.36 mrnol) in CHCI3 (0.36 ml) was added trichloroacetyl isocyanate (90 Ill, 0.74 mmol). The mixture was stirred at rt for 18 h to afford the intermediate 57. This intermediate was characterized by 1 H NMR: 06.04 (1 H, d, J = 5.4 Hz, H-1, gluco isomer), 5.96 (1 H, d, J = 3.3 Hz, man no isomer). The reaction mixture was then cooled to -20°C and treated with benzylamine (0.13 ml, 1.17 mmol) and the flask was gradually brought to rt. The organic phase was thoroughly washed with water, dried over Na2S04 and concentrated. The residue was purified by silica column chromatography 1,30% ethyl acetate in hexane) to afford 58 (275 mg, 87%); Rt = 0.33 in 50% ethyl acetate in hexane; 1H NMR: & 3.37-3.46 (m, 5H, H-2,6,6'), 3.58-3.7 (m, 3H, H-3,4,5), 3.77-3.89 (m, 3H, H-2',3',5'), 4.34 (d, J = 4.2 Hz, 1 H, H-1 '),4.44 (d, 1 H, H-4'), 5.4 (d, J = 4.5 Hz, H-1), 6.24 (s, 1 H, NH), 7.22-7.36 (m, 30H, Ph); 13C NMR: & 54.27,68.43, 69.39, 71.48, 72.65, 73.06, 73.12, 73.37, 74.56, 75.05, 75.35, 75.95, 79.47, 82.31, 102.98,127.42-128.33,138.07-138.83,166.90; ESMS (mlz): 914.5 (M+Nat. 4-~-Galactopyranosyl-a-D-glucopyranosyl ~ amino lactam (59). To a solution of 58 (30 mg, 0.035 mmol) in CH30H (3 ml) was added palladium on carbon (10%, 170 mg) and formic acid (

Synthesis of anomeric ~-Iactam analogues of eMPT substrate91•92 (Scheme 14 of Results and Discussion)

mg, 0.03 mmol) in 95% aqueous pyridine (1 ml) was added. After 30 min CH2Cb was added and the solution was washed successively with cold 1 M Na2S203 (2 x 5 ml) and cold 1 M TEA hydrogen carbonate (2 x 5 ml), dried over Na2S04 and concentrated. The residue was purified by silica column chromatography (1.5% CH30H in CH2Cb with 0.1 % Et3N); Rf = 0.54 in 20% CH30H in CH2CI2; 1 H NMR: 8 -0.01 (s, 6H, Me~iCMe3), 0.84 (s, 9H, Me2SiCMe3), 1.95-2.11 (m, 18H, OAc), 3.62 (m), 3.88 (m), 4.2 (m), 4.5 (m), 4.9 (m, 2H, H-2', 3'), 5.28 (m, 3H, H-1, 2, 3), 5.44 (m, 1 H, CH=CH2); 31 P NMR .8-2.68; ESMS (mlz) : 925.3 (M-Et3N-H)". Dec-9-enyl-6-dihydroxyl-4-~-D-galactopyranosyl-a-D-mannopyranosyl phospha te triethylammonium salt (55). A solution of aqueous HF (48%) in CH3CN (5:95, 400 Ill) was added to compound 54 (10 mg, 0.009 mmol) at 0 aC. The solution was stirred at 0 aC for 2 h. The reaction was quenched by the addition of the aqueous NaHC03 solution until effervescence ceased and diluted with CH2CI2. The organic layer was extracted with water and TEAS solution thoroughly, dried over Na2S04 and concentrated to give dec-9-enyl-2,3,4-tri-O-acetyl-4-~-D-galactopyranosyl-a-D­mannopyranosyl phosphate triethylammonium salt; ESMS (m/z): 811.4 (M-EtsN-H)". A solution of oxalyl chloride (0.38 mg, 1.5 Ill, 0.003 mmol) in anhydrous CH2CI2 (50 Ill) was cooled to -78 aC and DMSO (0.47 mg, 1.7 Ill, 0.006 mmol) was added, followed by the addition of a solution of dec-9-enyl-2,3,4-tri-O-acetyl-4-~-D­galactopyranosyl-a-D-mannopyranosyl phosphate (7 mg, 0.007 mmol) in CH2CI2 (100 Ill). The mixture was stirred for another 30 minutes and then triethylamine (10 Ill) was added. The solution was brought to rt, water was added and the mixture was extracted with CH2Cb. The organic layer was dried over Na2S04 to give the aldehyde 55. Dec-9-enyl-6-dihydroxyl-4-~-D-galactopyranosyl-a-D-mannopyranosyl phosphate triethylammonium salt (56). The residue was taken in a mixture of CH30H:water:triethylamine (5:3:2, 1.6 ml) and stirred for 2 days at rt. The reaction mixture was concentrated and the residue was repeatedly lyophilized to yield 56.

Dec-9-enyl-2,3,4-tri-O-acetYI-[6-0-(t-butYldimethYlsilyl)-4-~-D-galactopyranosyl] -a-D-mannopyranosyl phosphate tri ethylammonium salt (54). A mixture of H-phosphonate 6 (from scheme 1, 50 mg, 0.057 mmol) and dec-9-en-1-01 (30 Ill, 0.172 mmol) was dried by evaporation of pyridine (2 x 0.5 ml). The residue was dissolved in anhydrous pyridine (1 ml), pivaloyl chloride (22 Ill, 0.172 mmol) was added, and the mixture was stirred at rt for 1 h whereafter a freshly prepared solution of iodine (6

(Scheme 13 of Results and Discussion)

Synthesis of S'-hemiacetal analogue90 of Gal 1,4~-Man-a­phosphate acceptor

Design and Synthesis of mechanism based inhibitors of elongating MPT enzyme of LPG biosynthesis

was diluted with water and the aqueous layer was thoroughly extracted with ethyl acetate (15 ml x 2). The organic layer was dried over Na2S04, concentrated and dried to yield C4C] labelled stearyl alcohol 51. [14C]-Stearyl-2,3,6-tetra-O-acetyl-4-0-(2,3,4 ,6-tretra-O-acetyl-~-D-gal actopyrano syl)-a-D-mannopyranosyl phosphate triethylammonium salt (52). A mixture of H-phosphonate 47 (296 mg, 0.37 mmol) and [14C] stearyl alcohol (51,100 mg, 0.37 mmol) was dried by evaporation of pyridine (2 x 3 ml). The residue was dissolved in anhydrous pyridine (5 ml), adamantane carbonyl chloride (160 mg, 0.8 mmol) was added, and the mixture was stirred at rt for 1 h whereafter a freshly prepared solution of iodine (160 mg, 0.63 mmol) in 95% aqueous pyridine (5 ml) was added. After 30 min CH2Cb was added and the solution was washed successively with cold 1 M Na2S203 (2 x 10 ml) and cold 1 M TEA hydrogen carbonate (2 x 10 ml), dried over Na2S04 and concentrated. The residue was purified by silica column chromatography (2.5% CH30H in CH2CI2 with 1 % Et3N) to afford 52. [14C]-Stearyl-4-~-D-galactopyranosyl-a-D-mannopyranosyI phosphate triethyl ammonium salt (53). To a solution of compound 4 (75 mg, 0.07 mmol) in anhydrous CH30H (12.5 ml) was added anhydrous sodium carbonate (80 mg, 0.75 mmol). The mixture was stirred at rt for 2 h, whereafter sodium carbonate was removed by filtration. The solvent was evaporated and residue concentrated to yield 53; R,= 0.55 in 10: 1 0:3 CH30H:CH2CI2:O.25% KC!.

[14C]-Stearyl alcohol (51). Stearic acid (50,100 mg) in anhydrous THF (1 mL) was diluted with C4C] stearic acid (1.2 mL, 120 !lCi). To this was added THF-borane complex (4 mL). The mixture was refluxed at 90°C for 36 h. The contents were then poured onto CH3COOH:H20 (8 mL, 1:1), taken in a separating funnel. The mixture

Synthesis of [14C] labeled Stearyl linked Gal 1,4 f3 Man phosphate (Scheme 12 of Results and Discussion)

3.37 (t, J = 2 Hz, 1 H), 3.34 (s, 3H, OMe); 13C NMR (020, 75 MHz) 8 103.01, 102.17, 100.70,99.71,78.64,77.46,77.21,77.05,75.50,75.31, 75.17, 73.79, 72.92, 72.60, 72.48, 72.24, 70.82, 70.23, 69.94, 68.40, 68.08, 66.62, 60.99, 60.79, 60.46, 56.83; HRMS(FAB): Calculated for [ M+ Nat C25H44021Na 703.227279, found 703.226277.

MR ( 020, 300 MHz) 8 5.23.(q, J = 1.46 Hz, 1 H, H-1"'), 5.20 (d, J = 1.22 Hz, 1 H, H-1"), 4.86 (bs, 1H, H-1), 4.26 (d, J = 8.51 Hz, 1H, H-1'), 3.95 (d, J = 1.83 Hz, 1 H, H-2"), 3.93 (m, 1 H), 3.9 (d, J= 2.53 Hz, 1 H, H-2), 3.76 (bs, 1 H), 3.75 (bs, 1 H, H-2"), 3.54 (d, J = 1.8 Hz, 3H), 3.40 (d, J = 7.91 Hz, 1 H, H-2'),

at -30°C when TlC showed complete disappearance of the reactants. The mixture was quenched with pyridine (2 ml), filtered through celite pad and the filtrate was co-concentrated with toluene. The residue was purified by silica column using ethyl ace'late-hexane (32:68) to provide fully protected tetrasaccharide cap (43, 0.019 g, 63~~) domain of LPG; R, = 0.236 in 50% ethyl acetate-hexane; [a]D +12.06 (c 0.058, CHCI3); 1H NMR (COCI3, 300 MHz) 8 7.29-7.14.(m, 30H, ArH), 5.33 (d, J = 1.8 Hz, 11-l), 5.38-5.32 (m, 3H), 5.28-5.23 (d, J = 9.9 Hz, 2H), 5.18 (dd, J = 1.8, 1.5 Hz, 1 H), 4.93-4.89 (d, J = 11 Hz, 2H), 4.75-4.74 (d, J = 2.1 Hz, 1 H, H-1'or H-1"'), 4.66-4.60 (rn, 2H), 4.62-4.61 (d, J = 1.64 Hz, 1H, H-1"' or H-1"), 4.54-4.50 (d, J = 12 Hz, 2H), 4.44-4.42 (d, J = 6.9 Hz, 1H, H-1'), 4.40-4.38 (d, J = 6.1 Hz, 1H), 4.35-4.31 (d, J = 9.9 Hz, 2H), 4.25 (m, 1 H), 4.29-4.17 (m, 4H), 4.14-4.07 (m, 4H), 4.04 (m, 2H), 4.00 (m, :2H), 3.89 (d, J = 2.7 Hz, 1 H), 3.83-3.80 (m, 1 H), 3.74-3.68 (m, 1 H), 3.57-3.52 (m, 1 H), 3.46 (s, 3H, OMe), 3.44-3.32 (m, 4H), 2.09, 2.03, 2.01, 2.00, 1.99, 1.97, 1.96 (7 x s, 21 H, 7 x OCOCH3); 13C NMR (COCI3, 75 MHz) 8 174.9, 173.5, 171.2, 170.3, 169.6, 169.5, 169.3, 146.6, 138.9, 138.6, 132.6, 130.9, 129.8, 128.4, 128.2, 128.1, 128.07,128.0,127.76,127.72,127.65,127.54,127.4, 127.34, 127.3, 126.9, 109.15, 103, 100.6, 98.56, 74.7, 74.5, 73.2, 72.9, 72.5, 69.6, 68.1, 66.1, 62.3, 62.1, 61.9, 56.8,20.76,20.58; HRMS(FAS): Calcd. for [M+Nar C81H94028Na 1537.58290, found 1537.58270. Methyl 0-( a-D-man nopyranosyl )-( 1--72)-O-a-D-mannopyranosyl-(1--72)-0-[J3-D-galactopyranosyl-(1--74)]-a-D-mannopyranoside (44). Solution of the fully protected tetrasaccharide cap 43 (2 mg, 0.0014 mmol) in absolute EtOH (3 ml) and palladium hydroxide (5 mg, 20 wt %) was stirred under slight pressure of hydrogen for 4 h. The reaction mixture was filtered through celite and the filtrate concentrated under reduced pressure to obtain debenzylated product. This was dissolved in anhydrous CH30H (1.5 ml), catalytic amount of sodium methoxide (0.8 mg) was added and the solution was stirred for 2 h at rt. The reaction mixture was quenched with 3 drops of 0.5% HCI solution and excess of CH30H was removed under reduced pressure and the residue was lyophilized three times with the addition of water (500 Jll) to remove traces of HC!. This provided pure methyl glycoside of the tetrasaccharide cap 44 in quantitative yield; R, = 0.276 in nPrOH:acetone:H20 (first run 9:6:5, second run 5:4:1); 1H N

eves (4 A, 150 mg) under nitrogen for 30 min. The mixture was then cooled to -30°C and trimethylsilyltriflate solution (TMSOTf, 3.66 III dissolved in 1 ml CH2CI2) was added dropwise keeping the reaction temperature at -30°C. The reaction mixture was stirred for another 15 min

0.09 in 50% ethylacetate-hexane; [aJo +19.54 (c 0.22, CHCI3); 1H NMR (CDCI3, 300 MHz) .85.40-5.42 (dd, J = 2.4,3.3 Hz, 1H, H-3), 5.38-5.37 (d, J = 3.3 Hz, 1H, H-1), 5.36-5.33 (m, 2H, H-4', H-4), 5.30-5.23 (m, 2H, H-2', H-3), 4.92 (d, J = 1.8 Hz, 1 H, H-1'),4.23-4.19 (m, 2H, H-6a', H-6b'), 4.17-4.14 (dd, J = 3.7,5.1 Hz, 1H, H-2), 4.13-4.11 (m, 2H, H-6a,6b), 4.08-4.05 (ddd, J = 2.7,2.4 Hz, 1 H, H-5), 3.65-3.59 (m, 1 H, H-5'), 2.13-1.99 (7 x s, 21 H, COMe); 13C NMR (CDCI3, 75 MHz) 8 171.0, 170.6, 170.3, 169.7, 169.6, 169.4, 169.37, 169.3, 98.6, 92.5, 77.2, 70.4, 69.97, 69.6, 69.5, 68.97, 68.3, 66.2, 66.0, 62.27, 62.1,20.77-20.54; HRMS(FAB): Calcd for [M+Hr C26H37018 637.197990, found 637.200305. 3,4,6-Tri-O-acetyl-2-0-(2,3,4,6-tetra-O-acetyl-(a-D-mannopyranosyl)-(3-D-manno pyranosyl trichloroacetimidate (42). To a solution of heptaacetate 41 (254 mg, 0.4 mmol) in anhydrous CH2CI2 (3 ml) at O°C was added successively, trichloroacetonitrile (10.0 equiv, 400 Ill) and DBU (0.0325 equiv, 20 Ill). After stirring for 1 h at 0 °C TlC showed completion of the reaction. Solvent was evaporated under reduced pressure and the residue was flash chromatographed (30:70, ethyl acetate-hexane) to give the disaccharide donor 42 (O.185g, 60%); [aJo +31.21, (c 1.36, CHCI3); 1H NMR (CDCb, 300 MHz) 8 8.71 (1 H, s, NH), 6.41 (d, J = 1.86 Hz, 1H, H-1), 5.49-5.46 (bd, J = 9.9 Hz, 1H, H-4), 5.43-5.38 (dd, J = 3.45,10.2 Hz, 1H, H-3'), 5.35-5.31 (dd, J = 3.15, 10.2 Hz, 1 H, H-3), 5.28-5.23 (m, 1 H, H-4'), 5.28-5.26 (dd, J = 1.8, 3.3 Hz, 1 H, H-2'), 4.98 (d, J = 1.5 Hz, 1 H, H-1 '), 4.29-4.27 (1 H, dd, J = 2.55,5.1, H-2), 4.24-4.14 (5H, m, H-6'a, 6'b, 5', 6a, 6b), 4.12-4.10 (ddd, J = 3,3.6,3.6 Hz, 1 H, H-5), 2.14-2.00 (7 x s, 21 H, COMe); 13C NMR (CDCb, 75 MHz) 8 170.6, 170.5, 170.2, 169.78, 169.59, 169.37, 169.10,99.1,95.4,76.4,75.4,74.8,71.1, 69.7, 69.5, 69.4, 68.2, 66.0, 65.2, 62.1, 61.5, 20.74-20.53; HRMS(ESMS): Calcd for [M+Nar C28H36018NCI3Na 802.0896, found 802.0801. Methyl 0-(2,3,4,6-tetra-O-acetyl-a-D-mannopyranosYI)-(1-72)-0-(3,4,6-tri-0-acetyl-a-D-mannopyranosYI)-(1-72)-0-[(2,3,4,6-tetra-O-benzyl-(3-D-galactopyra nosyl)-(1-74)]-3,6-di-O-benzyl-a-D-mannopyranoside (43). A solution of the protected Gal-Man acceptor 35 (0.018 g, 0.020 mmol) and mannobiose trichloroacetamidate donor 42 (0.031 g, 0.04 mmol) in anhydrous CH2CI2 (2 ml) was stirred with freshly activated molecular si

NMR (CDCI3, 300 MHz) /55.78 (d, J = 0.9 Hz, 1 H, H-1), 5.46-5.50 (dd, J = 9.9 Hz, 1H, H-4', H-2'), 5.29-5.31 (dd, J = 3.6,1.95 Hz, 1H, H-4), 5.09-5.13 (dd, J = 9.6, 3 Hz, 1 H, H-3), 5.00 (d, J = 1.8 Hz, 1 H, H-1'), 4.40 (m, 1 H, H-5'), 4.14-4.16 (dd, J = 3,1.2 Hz, 1H, H-2), 4.01-4.34 (m, 4H, H-6', H-6), 3.76-3.80 (m, 1H, H-5), 2.01-2.15 (8 x s, 24H, COMe); 13C NMR (CDCI3, 75 MHz) 820.3-20.77,60.2, 61.6, 62.1, 65.6, 66.0, 68.2, 68.7, 69.8, 72.0, 73.1, 74.5, 75.1, 77.1, 90.8, 98.2, 168.2, 169.1, 169.3, 169.5, 169.6, 170.4, 170.7, 171. HRMS(FAB): Calcd. for [M+Nar C2sH3S019Na 701.190499, found 701.187839. 3,4,6-Tri-O-acetyl-2-0-(2,3,4,6-tetra-O-acetyl-a-D-mannopyranosyl)-j3-D-manno pyranose (41). The mannobiose octaacetate 40 (300 mg, 0.47 mmol) was dissolved in anhydrous acetonitrile saturated with dimethylamine (39 mL) at -20°C and stirred for 5 h after which TLC confirmed disappearance of the starting material. Excess of dimethylamine was removed under reduced pressure at 30°C and the reaction mixture was concentrated. Flash column chromatography of the crude product with 40% ethyl acetate-hexane resulted in pure heptaacetate 41 (0.225 g, 91 %); R, =

mixture was left at rt for 90 min. after which a solution of sodium acetate (42.5 g) in water (53 mL) at 5 °C was slowly added, keeping the internal temperature of the mixture around 35°C. The resultant solution was then poured onto ice, and the mixture was extracted with CH2CI2 (60 mL x 3). The organic layer was thoroughly washed with cold water and saturated aqueous NaHC03 solution, dried over Na2S04 and concentrated. The residue was crystallized from dry ether to afford pure 39 (3.5 g), and was characterized by comparison with data of commercially available material from Sigma. 1 ,3,4,6-Tetra-O-acetyl-2-0-(2,3,4,6-tetra-O-acetyl-a-D-mannopyranosyl)-j3-D-mannopyranose (40). A solution of man nose trichloroacetimidate donor 38 (985 mg, 2 mmol) and freshly prepared 39 (348 mg, 1 mmol) in anhydrous CH2CI2 (20 mL) was stirred with activated molecular sieves (10 g, 4 A) under nitrogen for 30 min. The reaction mixture was cooled to -30°C and a solution of trimethylsilyltriflate (TMSOTf, 220 ilL, 1.2 mmol) in anhydrous CH2CI2 (10 mL) was added dropwise. The temperature was maintained below -30°C for 15 min when TLC indicated completion of the reaction. The mixture was quenched with pyridine (5 mL), filtered through celite, and the filtrate was co-concentrated with toluene. Flash chromatography of the crude product with 50 % ethyl acetate-hexane afforded pure mannobiose octaacetate 40 as amorphous solid (0.406 g, 60%); R, = 0.208 in 50% ethylacetate-hexane; [a]D +9 (c 0.25, CHCI3); 1H

2,3,4,6-Tetra-O-acetyl-a-D-mannopyranosyl-trichloroacetimidate (38) 1,2,3,4,6-penta-O-acetyl-a-D-mannopyranose (36, 500 mg, 128 mmol) was dissolved in dry CH3CN saturated with dimethylamine (35 mL) and stirred at -20°C for 1 h after which TLC confirmed complete disappearance of the starting material. Extra dimethylamine was removed under reduced pressure at room temperature and the reaction mixture was concentrated. Flash column chromatography (25:75 ethyl acetate-hexane) provided 2,3,4,6-tetra-O-acetyl-a-D-mannopyranose (37, 445 mg) in quantitative yield. To a solution of compound 37 (0.335 g, 0.962 mmol) in anhydrous CH2CI2 (3 mL) was added trichloroacetonitrile (CI3CCN, 10.0 equiv, 1 mL) and 1,8-diaza bicyclo[5.4.0]undec-7-ene (DBU, 74.7 uL, 0.05 equiv) at O°C. After stirring for 75 min, the solvent was evaporated under reduced pressure and the residue purified by flash chromatography with 20 % ethyl acetate-hexane to give pure 38 (331 mg,70%); 1H NMR (CDCI3, 300 MHz) 8 8.77 (s, 1 H, NH), 6.26 (d, J = 1.8 Hz, 1 H, H-1), 5.45 (dd, J = 2.3 Hz, 1 H, H-2), 5.39-5.37 (dd, J = 2,5 Hz, 1 H, H-3), 5.42-5.33 (m, 1 H, H-4 ), 4.18 (m, 1 H, H-5), 4.12-4.29 (m, 2H, H-6); 13C NMR (CDCI3, 75 MHz) 8 170.45, 169.68, 169.60, 169.50, 159.62, 94.39, 71.08, 68.67, 68.13, 67.73, 65.26, 61.91, 20.54; HRMS(ESMS): Calcd. for [M+Hr C1sH2101ONCI3 491.0153, found 491.0187. 1,3,4,6-Tetra-O-acetyl-f3-D-mannopyranose (39). Few crystals of D-mannose were added to acetic anhydride (53 mL), followed by the addition of 6-7 drops of 60% perchloric acid. This solution was maintained at 45°C and to this was added 0-mannose (14 g) portionwise with constant stirring for 20 min. the mixture was then left at rt for 1 h and subsequently cooled to 15°C. Phosphorus tribromide (13.4 mL) was added dropwise to this mixture, followed by the addition of water (4.8 mL). The

using 20% ethyl acetate in hexane to yield methyl 0-(2,3,4,6-tetra-O-benzyl-a-D-galactopyranosyl)-(1-74)-3,6-di-O-benzyl-a-D-mannopyranoside 35 (149 mg, 64.5%); R, = 0.27 in 50% ethyl acetate-hexane; [a]o +3.891, (c 0.257, CHCI3 ); 1H NMR (CDCI3, 300 MHz) 87.7-6.89 (m, 30H, Ph), 4.96-4.31 (m, 12H, PhCH2), 4.47-4.45 (d, J = 5 Hz, 1H, H-1'), 4.35 (d, J = 2.1 Hz, 1H, H-1), 4.08 ("t", J = 8.7 Hz, 1H, H-4), 4.03 (bs, 1 H, H-2), 3.9-3.89 (d, J = 2.4 Hz, 1 H, H-4'), 3.84-3.80 (dd, J = 2.5, 8.1 Hz, 1 H, H-3), 3.78-3.73 (dd, J = 5.4, 10.4 Hz, 1 H, H-2'), 3.55-3.47 (m, 7H), 3.52 (s, 3H, OMe); 13C NMR (CDCI3, 75 MHz) .8138.85, 138.63, 138.39, 138.33, 137.88, 135.52 (6 ipso C), 128.30-127.29 (ArC's), 103.07, 100.65,82.48,79.75,76.91,75.27,75.06,74.49, 73.39, 73.07, 72.54, 72.33, 68.52, 68.29, 56.87; HRMS(FAB): calcd for [M+Nar CSSHSOOllNa 919.4033333, found 919.400521.

(232 mg, 91 %); Rt= 0.16 in 50% ethyl acetate-hexane; [a]o +21.84 (c 0.238, CHCI3); 1H NMR (COCI3, 300 MHz) 8 5.0 (d, J = 2.1 Hz, 1H, H-1), 4.55 (d, J = 12.9 Hz, 1H, H-3), 4.55 (m, 1 H, H-4), 4.40-4.22 (m, 3H, H-1', 2', 4'), 3.85 (m, 2H, H-5, 5'), 3.50 (m, 2H, H-6), 3.40 (m, 2H, H-6'), 3.11 (d, J = 2.1 Hz, 1 H, H-2); 13C NMR (COCI3, 75 MHz) 8 138.6-138.2 (6 ipso C), 128.3-127.4 (ArC's), 102.51,82,4,79.7,76.6,75.2, 74.6, 73.5, 73.47, 73.41, 73.0, 72.69, 72.61, 69.17, 68.36; HRMS(ESMS): calcd for [M+Nat CS4Hs601O Na 887.3771 found 887.3761. Methyl 0-(2,3,4,6-tetra-O-benzyl-a-D-galactopyranosYI)-(174)-3, 6 -di-O-benzyl-a-D-glucopyranoside (34). The a-epoxide (33, 232 mg, 0.268 mmol) was dissolved in anhydrous CH30H (150 mL) and allowed to stir at rt for 4 h. The solvent was evaporated and the residue dried under vacuum to yield B-methyl lactoside 34 (231 mg, 96.08%); Rt= 0.37 in 50% ethyl acetate in hexane; [a]o +12 (c 0.200, CHCI3); 1H NMR (COCh, 300 MHz) 8 7.32-7.20 (m, 30H, Ph), 5.11-4.34 (m, 12H, PhCH2), 4.29 (s,1H, H-1'), 4.25 (s, 1H, H-2'), 4.22-4.19 (dd, J = 3.6,7.5 Hz, 1H, H-1), 3.95 (m,1H, H-3), 3.90 (d, J = 2.7 Hz, 1 H, H-4'), 3.8-3.78 (dd, J = 4.35, 11 Hz, 1 H, H-3'), 3.76-3.69 (m, 2H, H-2, H-4), 3.54 (s, 3H, OMe), 3.57-3.33 (m, 6H); 13C NMR (COCb, 300 MHz) 8 138.92, 138.82, 138.68, 138.39, 138.22, 137.92 (6 ipso C), 128.29-127.35 (ArC's), 103.40, 102.69, 82.65, 74.62, 74.54, 73.36, 73.32, 73.06, 72.94, 72.52, 68.09, 56.87; HRMS(FAB): calcd for [M+Nat CSSH60011Na 919.4033, found 919.4023. Methyl 0-(2,3,4,6-tetra-O-benzYI-a-D-galactopyranosyl)-(174)-3,6 -di-O-benzyl-a-D-mannopyranoside (35). A solution of oxalyl chloride (95.8 JlL, 0.177 mmol) in anhyd CH2CI2 (7 mL) was cooled to -78°C, and anhydrous OMSO (154.3 JlL, 0.349 mmol) was added dropwise. The mixture was stirred at -78°C for 10 min and solution of methyl glycoside 34 (231 mg, 0.258 mmol) in CH2CI2 (11.5 mL) was added over 10 min. The cloudy solution was stirred for 40 min followed by addition of triethylamine (5 mL) to give a clear solution. The mixture was brought to rt, diluted with cold water (30 mL) and extracted with CH2CI2• The organic layer was dried over Na2S04 and concentrated under reduced pressure to yield the oxidised 2-ulose intermediate (Rt = 0.53 in 50% ethyl acetate in hexane). This product was dissolved in 50% CH2CI2 in CH30H (4 mL) and NaBH4 (150 mg, 3.96 mmol) was added at 0 °C. The reaction mixture was brought to rt and after 4 h it was diluted with CH2CI2 and washed with cold water. The organic layer was collected, dried over Na2S04 and concentrated to give a crude product which was purified by flash chromatography

am of nitrogen gas, and further dried under vacuum to provide a semisolid hexa-O-benzyl lactal 1,2a-epoxide 33

dissolved in water (100 mL) and extracted with CH2Cb (3 x 60 mL). All the organic extracts were combined, dried over Na2S04, and concentrated to provide hexa-O-acetyllactal (30, 7.2 g, 87.8%) as amorphous solid [a]o -18 (c 1.0, CHCI3)84. Hexa-O-benzyl lactal (32). A solution of hexa-O-acetyl lactal (30, 7.26 g, 0.013 mmol), dry sodium carbonate (9 g, 0.085 mol) in anhyd CH30H (150 mL) was stirred at rt for 90 min. The suspension was filtered to remove extra Na2C03 and the filtrate was concentrated under reduced pressure to give deacetylated lactal 31 (same as described in Scheme-1) as an amorphous solid (3.87 g, 97.7%); R, = 0.2 in 7:3 CHCkCH30H; [a]o +27 (c 1.6, H20)84. Compound 31 (500 mg, 1.62 mmol) dissolved in anhydrous DMF (5 mL) was added dropwise at 0 °C to a suspension of NaH (1.3 g, 60% dispersion in paraffin) in DMF (5 mL), followed by addition of benzyl bromide (2 mL, 16.8 mmol) and few crystals of tetrabutyl ammonium iodide. The reaction mixture was brought to rt and stirred for 3 h. After completion of the reaction, the mixture was cooled to 0 °C and quenched with CH30H (5 mL), diluted with cold water (50 mL) and extracted with diethyl ether (3 x 30 mL). The ethereal layer was dried over Na2S04 and concentrated to give a crude product which was flash chromatographed using 5% ethyl acetate in hexane to provide compound 32 (792 mg, 60.2%); R, = 0.6 in 50% ethyl acetate-hexane; [a]o -2.1 (c 0.726, CHCI3); 1H NMR (CDCI3, 300 MHz) () 6.43 (dd, J = 6.2,1.1 Hz, 1H, H-1), 4.92 (brd, J = 10.8 Hz, 1 H, H-3'), 4.86 (m, 1 H, H-2), 4.53 (dd, J = 10.5, 1.2 Hz, 1 H, H-4'), 4.35 ( d, J = 4.2 Hz, 2H, H-1'), 4.29 (brs, 1 H, H-4), 4.26 (m, 1 H, H-3), 3.86-3.74 (m, 2H, H-5, 5'), 3.65 (m, 2H, H-6), 3.45 (d, J = 4.2 Hz, 2H, H-1'); 13C NMR (CDCI3, 75 MHz) () 138.6-138.2 (6 ipso C), 128.3-127.4 (ArC's), 102.51, 82.4,79.7, 76.6, 75.2, 74.6, 73.5, 73.47, 73.41,73.0,72.69,72.61,69.17,68.36; HRMS (FAB): calcd for [M+Nat CS4Hs60sNa 871.382204, found 871.386586. Hexa-O-benzyl-lactal-1,2a-epoxide (33). A solution of 3,3-dimethyl dioxirane (DMD) in acetone was freshly prepareds3.s4 by adding potassium monoperoxy sulphate (Oxone, DuPont, 25 g, 0.041 mol) into a mixture of water (20 mL), acetone (13 mL, 0.177 mol), sodium bicarbonate (12 g) in a two neck flask with vigorous stirring at rt. The DMD solution was received through a water condenser (5°C) by application of slight vacuum into a flask cooled to -50°C. DMD was added dropwise to a solution of compound 32 (250 mg, 0.3 mmol) in anhydrous CH2CI2 (2 mL) at 0 °C. After 2 h the reaction mixture was concentrated with a strea

Hexa-O-acetyl lactal (30). A solution of Vitamin B12 (310 mg, 0.22B mmol) in anhydrous CH30H (BO ml) was thoroughly purged with nitrogen for 30 min and zinc powder (17.5 g, 267.6 mmol) and ammonium chloride (14.2 g, 266.25 mmol) were added to the solution. The reaction mixture was stirred for another 45 min and heptaacetyl lactosyl bromide (29), freshly prepared from lactose [peracetylation using acetic anhydride and sodium acetate, followed by anomeric bromination (4B% hydrobromic acid in acetic acid)], was dissolved in CH30H (30 ml) and added. Immediately after addition of the bromide, the dark red solution changed to reddish yellow and then back to dark red in 5 min. The solution was filtered through celite to remove zinc, and the celite pad was washed with CH30H and the filtrate was concentrated under reduced pressure to give a white and red solid product. This was

Synthesis of Tetrasaccharide Cap Domain of LPG

Polycondensation. Compound 26 (25 mg, 0.033 mmol) was dried by evaporation of pyridine (500 III x 3) therefrom. The residue was dissolved in 10:1 pyridine:triethylamine (40 Ill), and pivaloyl chloride (9 Ill, 0.073 mmol) was added. Another lot of pivaloyl chloride (6 Ill, 0.04B mmol) was added in 45 min. After 3 h, the mixture became viscous, and a freshly prepared solution of iodine (220 Ill, 35 mg, 0.137 mmol in pyridine-water, 95:5) was added. After 2 h, CHCI3 was added and the organic layer was successively washed with cold 1 M aqueous Na2S203 solution and 1 Mice-cold TEAB buffer, dried over Na2S04 and concentrated to dryness to afford 27. For final deprotection, above residue was dissolved in 0.1 M NaOMe solution in CH30H (440 Ill), 1,4-dioxane (BOO Ill), and CHCI3 (BOO Ill). The mixture was stirred at rt for 7 h and left at 4 °C for 16 h, then diluted with CH30H, deionized with Dowex 50W-X4 (H+) resin, filtered and immediately neutralized with drops of triethylamine. The mixture was concentrated to dryness to afford fully deprotected phosphoglycans (28). 31 P (D~O): 8 -1.73, O.BB. Preliminary CD analysis of Phosphoglycans. The above polycondensation product (28) was lyophilized repeatedly and then redissolved in H20 (400 Jll). This solution was taken in a glass cuvette (300 Ill, 1 mm pathlength). It's CD spectra was recorded on a spectropolarimeter (JASCO, J-710) between 175-250 nm at 25°C. For reference, the CD spectra of agarose (15% W/V)87 was also recorded under the same conditions as mentioned above.

Triethylammonium 2,3,6-tri-o.acetyl-4-o.(2,3,4-tri-o.acetyl-~-D-galactopyrana syl)-a-D-manno pyranosyl hydrogen phosphonate (26). Compound 6 (30 mg, 0.034 mmol) was dissolved in a mixture of acetic acid-water-THF (3:1:1,2.5 ml). The mixture was stirred at 40°C for 9 h, after which the solvent was evaporated off under vacuo at rt. To remove excess of acid, water (1 ml) was added and evaporated off twice to afford 26 in quantitative yield; 1H NMR (CDCI3, 300 MHz) 0 1.95-2.09 (m, 21 H), 3.49-3.68 (m, 4H), 3.88 (m, 1 H), 4.14 (m, 1 H), 4.36 (d, J = 4.5 Hz, 1 H), 4.47 (d, J = 7.8 Hz, 1 H), 4.95 (dd, J = 3_3 and 7_8 Hz, 1 H), 5.05 (dd, J = 2_1 and 7.8 Hz, 1 H), 5.21 (dd, J = 2.1 and 3.6 Hz, 1 H), 5.41 (d, J = 3.3 Hz, 1 H), 5.48 (dd, J = 2.1 and 7.8 Hz, 1 H), 7.99 ( d, JH,p = 637_0 Hz, 1 H); 13C NMR (CDCI3, 75 MHz) 0 20.48-20.76, 60.10, 62.42, 66.57, 69.36, 69.53, 69.69, 71.20, 73.30, 73.86, 91.59, 92.54, 101_09, 169.13-170.49; 31p (CDCI3): 00.22; ESMS mlz657.3 (M-EhN-Hr.

Synthesis of phosphoglycans by polycondensation

Selective cleavage of phosphoglycans from the resin. This was accomplished by taking the PG loaded resin (3 mg) and Wilkinson's catalyst (1 mg) in argon-purged solvent mixture (300 Ill, toluene-PrOH-H20, 2:1 :0.08 containing 0.01 N HCI) and shaking it for 7 h at rt. The cleavage after first cycle of coupling provided 2,3,6-tri-0-acetyl-4-0-[2,3,4-tri-O-acetyl-6-0-(t-butyldimethylsilyl)-~-D-galacto pyranosyl]-a-D-mannopyranosyl-phosphate. This intermediate was subjected to full deprotection to provide ~-D-galactopyranosyl-a-D-mannopyranosyl phosphate (25) and compared with authentic sample earlier reported86 by our laboratory; [a]D = +10° (c 0.1, H20); lH NMR (D20, assignments by 2D COSY and TOCSY experiments) 0 3.45 (dd, J = 6.67 and 1.5 Hz, 1 H, H-2'), 3.46 (m, 1 H, H-5), 3.60 (m, 1 H, H-5'), 3.53-3.56 (m, 2H, H-2,3'), 3.68 (m, 2H, H-6), 3.76 (t, J = 7.11 and 2.64 Hz, 1 H, H-3), 3.83 (m, 2H, H-6'), 3.83 (m, 1 H, H-4'), 3.94 (m, 1 H, H-2), 4.38 (d, J = 9.65 Hz, 1 H, H-4), 4.38 (d, J = 7.6 Hz, 1 H, H-1'), 5.27 (dd, J1H-P = 6.8 Hz and J1•2 = 1.9 Hz, 1 H, H-1); 31p NMR 0 -2.07; ESMS, 421.2 [M-1 Ht; HRMS (ESMS): calcd for [M-Hr C12H22014P 421.2720 found 421.2718. Similar procedure was used to cleave phosphotetrasaccharide 22 from resin followed by complete deprotection, which provided compound 23 that was characterized by its comparison with standard prepared by solution method.

opyranosyl phosphate] triethylammonium salt (22). The butenediol-linker functionalized Merrifield resin (19, 50 mg, 0.43 mmol/g, 0.021 mmol) was swollen in anhydrous pyridine (100 Ill) for 15 min, followed by addition of phosphoglycan H-phosphonate donor 6 (26 mg, 0.03 mmol) dissolved in anhydrous pyridine (500 Ill). Now pivaloyl chloride (20 Ill) was added and the resin mixture was shaken for 2 h. Thereafter a 200 III solution of iodine (4 mg) in 95% aqueous pyridine was added and stirring continued for another hour. The resin was then thoroughly washed with CH30H (700 III x 3) and dried over P20S overnight to afford acceptor-functionalized resin (20, 50 mg). ~ The coupled intermediate was characterized by positive ion ESMS after cleaving it off from the resin (2 mg) by treatment with 0.1 N HCI (100 Ill) at 100°C for 1 min. The product that got cleaved under this condition was characterized as 2,3,6-tri-0-acetyl-4-0-[2,3,4-tri-O-acetyl-6-0-(t-butyldimethylsilyl)-~-D-galactopyranosyl-a-D­mannopyranose which was identical to compound 5, already synthesized by solution method described earlier; ESMS m/z 731.3 (M+Nat. This compound on full deprotection with 48% aqueous HF-CH3CN (5:95) and CH30H-H20-EhN (5:2:1) provided disaccharide Gal1 ,4~Man (24); lH NMR 8 5.12 (d, J = 1.67 Hz, 1 H, H-1 a), 4.85 (d, 1 H, J = 0.98 Hz, 1 H, H-1 ~), 4.40-4.36 (m, 2H, H-1' and H-4), 3.75 (dd, 1 H, H-2'),3.94-3.92 (m, 2H, H-4' and H-2), 3.89-3.83 (m, 2H, H-6'), 3.81-3.79 (dd, 1H, J= 6 and 2 Hz, 1 H, H-3), 3.75-3.71 (m, 2H, H-6), 3.63-3.59 (dd, 1 H, H-3'), 3.51-3.46 (m, 2H, H-5, H-5'); ESMS: m/z 341.0 [M-Hr. To a part of the PG loaded resin 20 (15 mg), 48% aqueous HF-CH3CN (5:95,500 Ill) was added at 0 °C and the mixture was stirred on a orbital shaker for 3 h. The resin was then washed with CH30H (500 III x 2) and dried under vacuum to afford acceptor bound resin (21) with free 6' hydroxyl groups. This intermediate was again characterized by ESMS after cleaving it off from a small part of the resin (2 mg) by treatment with 0.1 N HCI (100 Ill) at 100°C for 1 min. The product that got cleaved under this condition was characterized as 2,3,6-tri-O-acetyl-4-0-(2,3,4-tri-O-acetyl-~­D-galactopyranosyl)-a-D-mannopyranose. Authenticity of this compound was confirmed by its comparison (TlC, NMR, ESMS) with standard separately prepared via solution synthesis by deprotection (HF-CH3CN) of TBDMS group from compound 5 (Scheme-1). A second cycle of PG coupling was carried out with identical procedure given above to afford phosphotetrasaccharide (22).

and water (150 mL). The organic layer was dried (Na2S04) and concentrated. The crude product was purified by silica column chromatography (20% ethyl acetate in hexane with 1% EhN) to afford 17 (4.2 g, 80%); Rf = 0.3 in 50% ethyl acetate in hexane; 1H NMR (CDCI3, 300 MHz): <52.03 (s, 1 H), 3.68 (d, J = 4.8 Hz, 2H), 3.78 (s, 6H), 4.03 (d, J = 5.4 Hz, 2H), 5.73-5.75 (m, 2H), 6.82 (tt, J = 1.2 and 9.0 Hz, 4H), 7.25-7.44 (m, 9H); 13C NMR (CDCb, 75 MHz): 55.12, 55.13, 58.75, 59.93, 113.05, 126.68,127.76,127.99,128.95,129.87,130.92, 136.07,144.79,158.37; ESMS m/z 413.39 (M+Nat Preparation of functionalized resin by coupling of linker (19). 4-(4,4'-Dimethoxytrityl)-2-cis-butenol (17, 1 g, 2.56 mmol) was dissolved in anhydrous DMF (8 mL). Upon cooling to 0 °C, sodium hydride (60% dispersion in mineral oil, 150 mg, 3.75 mmol) was added and the solution was stirred for 1 h. Merrifield's resin (18, 650 mg, chloromethylated polystyrene cross-linked with 1 % divinylbenzene, Fluka-63865) was added along with tetra-butylammonium iodide (95 mg, 0.256 mmol) and shaking was continued for an additional hour at 0 °C after which the reaction mixture was brought to rt and shaken for another 12 h. The capping of unreacted sites on resin was accomplished by addition of CH30H (100 ilL) and sodium hydride (100 mg) and shaking the contents for another 4 h, after which more CH30H (5 mL) was added and the resin was washed sequentially with 1:1 CH30H: DMF (10 mL), THF (10 mL x 3) and CH2CI2 (10 mL x 3). The resin was dried over P20s under vacuum to afford 836 mg of the linker-attached resin (19). To quantify loading8S of linker onto the solid support, a stock solution of 3% TFA in CH2CI2 (10 ml) was prepared which contained effectively 0.167 mg of the protected resin. The resulting orange colour liberated by the release of dimethoxytrityl (DMTr) cation was measured by UV at 503 nm, and the loading of the linker onto the resin was calculated to be 0.43 mmol/g of resin. The deprotection of the entire DMTr-linker functionalized resin was then carried out by treating the resin with 1 % TFA in CH2CI2 (10 mL). Further washing with CH2CI2 (20 mL x 3), 1% EhN in CH2CI2 (10 mL) and CH2CI2 (10 mL) and drying under vacuum afforded 640 mg of deprotected resin ready for coupling with phosphoglycan donors. Solid Phase Synthesis of 2,3,4-Tri-O-acetyl-~-D-galactopyranosyl-(1 ~4)-2,3,6-tri-O-acetyl-a.-D-mannopyranosyl phosphate 6-[2,3,4-tri-O-acetyl-6-0-(t-butyldi methylsi lyl)-~-D-galactopyranosyl-(1 ~4 )-1 ,2,3,6-tetra-O-acetyl-a.-D-mann

Synthesis of SOlid-phase linker, 4-(4,4'-Dimethoxytrityl)-cis-2-butenol (17). To a solution of cis-butene-1,4-diol (16, 4.7 mL, 5 g, 56.7 mmol) in anhydrous pyridine (100 mL) at 0 °C was added 4,4'-dimethoxytrityl chloride (6.4 g, 18.9 mmol). The reaction mixture was gradually brought to rt over 3 h and stirred for additional 12 h. Ethyl acetate (200 mL) was added and the organic phase was washed with water (150 mL), saturated aqueous NaHC03 (200 mL), saturated aqueous NaCI (200 mL)

Solid phase phosphoglycan synthesis

(250 ~L) was added dropwise. The mixture was stirred at 0 °C for 2 h and quenched with 1 M TEAS solution (pH=7, 1 mL). The clear solution was stirred for 15 min. after which CH2CI2 was added and the organic layer was washed with ice cold water (1 mL x 2), cold 1 M TEAS buffer (1 mL x 2), dried over Na2S04, and concentrated to yield compound 13 (5.1 mg, 86%); ESMS m/z 1'427.9 (M-Et3N-H): 2,3,4-Tri-O-acetyl-~-D-galactopyranosyl-(1 ~4 )-1 ,2,3,6-tetra-O-acetyl-a-D-manno pyranoside 6-{2,3,4-tri-O-acetyl-6-0-(t-butyldimethylsilyl)-~-D-galactopyranosyl -(1~4)-2,3,6-tri-O-acetyl-a-D-mannopyranosyl phosphate 6-[2,3,4-tri-O-acetyl-~­D-galactopyranosyl-(1~4)-2,3,6-tri-O-acetyl-a-D-mannopyranosyl phosphate] } bistriethylammonium salt (14). Mixture of compounds 13 (5.1 mg, 0.003 mmol) and 6 (5 mg, 0.007 mmol) was dried by evaporation of pyridine (500 ~L x 2). The residue was dissolved in anhydrous pyridine (200 ~L) and pivaloyl chloride (2.4 ~L, 0.02 mmol) was added. The mixture was stirred at rt for 1 h and a freshly prepared iodine solution (200 ~L, 4 mg, 0.015 mmol in pyridine-water, 95:5) was added. After 30 min CH2CI2 was added and the solution was washed successively with cold 1 M aqueous Na2S203 solution (2 mL x 2), ice-cold 1 M TEAS buffer (2 mL x 2), dried over Na2S04 and concentrated to afford 14 (4.5 mg, 61%); Rf = 0.11 in 10% CH30H in CH2CI2; ESMS m/z2061.44 (M-2EhN-H), 2062.35 (M-2EhN). ~-D-Galactopyranosyl-(1~4)-a-D-mannopyranoside {6-~-D-galactopyranosyl­(1~4)-a-D-mannopyranosyl phosphate 6-[ ~-D-galactopyranosyl-(1~4)-a-D­mannopyranosyl phosphate]} bis-triethylammonium salt (15). The global deprotection of fully protected phosphohexasaccharide 14 was carried out by same method as given for preparation of compound 9, and this compound was identical to PG oligomer 12 prepared by upstream extension described earlier.

(19 x OCOCH3), 3.50 (m, 6H, H2-6 Gal/Gal'/Gal"), 3.87-3.94 (m, 3H, H-5, Gal/Gal'/Gal"), 4.14-4.07 (m, 3H, 5-H, Man/Man'/Man"), 4.30-4.35 (m, 3H, 4-H, Man/Man'/Man"), 4.39 (m, 6H, H2-6, Man/Man'/Man"), 4.48 (m, 2H, 3-H, Man'/Man"), 4.52 (m, 1 H, 3-H, Man), 4.94 (d, J = 7.7 Hz, 3H, H-1, Gal/Gal'/Gal"), 5.28 (m, 6H, 2-H Man, H-4 Gal/Gal'/Gal", H-3 Gal'/Gal"), 5.29 (m, 1 H, H-3, Gal), 5.43 (m, 2H, H-2 Gal'/Gal"), 5.45 (dd, JHH = 1.9 and JHP = 7.0 Hz, 2H, H-1, Man'/Man"), 5.46 (m, 3H, H-2, Gal/Gal'/Gal"), 6.01 (d, J = 1.9 Hz, 1 H, 1-H, Man); 31p_NMR: 8 -1.94; ESMS m/z2061.44 (M-2Et3N-H), 2062.35 (M-2Et3N). ~-D-Galactopyranosyl-(1 ~4)-a-D-mannopyranoside {S-~-D-galactopyranosyl­(1~4)-a-D-ma nnopyranosyl phosphate S-[ ~-D-galactopyranosyl-(1~4)-a-D­mannopyranosyl phosphate]) bis-triethylammonium salt (12). The global deprotection of fully protected phosphohexasaccharide 11 was carried out by same method as given for preparation of compound 9 earlier; 1 H-NMR (020), due to Oligomeric nature of the molecule (three identical PG repeats), all NMR peaks could not be assigned,: 3.45 (m, 3H, H-2, Gal/Gal'/Gal"), 3.46 (m, 2H, H-5, Man'/Man"), 3.55 (m, 1 H, H-5, Man), 3.56-3.53 (m, 3H, H-3, Gal/Gal'/Gal"), 3.60 (m, 3H, H-5, Gal/Gal'/Gal"), 3.68 (m, 6H, H2-6, Man/Man'/Man"), 3.76 (m, 3H, H-3, Man/Man'/Man"), 3.80 (m, 6H, H2-6, Gal/Gal'/Gal"), 3.83 (m, 3H, H-4, GaVGal'/Gal"), 3.85 (m, 1 H, H-2, Man), 3.94 (m, 2H, H-2, Man'/Man"), 4.32 (m, 1 H, H-4, Man), 4.37 (d, J= 7.6 Hz, 2H, H-1, Gal'/Gal"), 4.35 (d, J= 7.6, 1H, H-1, Gal), 5.09 (d, J= 1.8, 1 H, H-1, Man), 5.36 (dd, JHH = 1.9 and JHP = 6.8 Hz, 2H, H-1, Man'/Man"); 31p_NMR: -1.29; ESMS: m/z 574.12 ([M-2Et3N-2Hf 2,3,4-Tri-O-acetyl-~-D-galactopyranosyl-(1 ~4 )-1 ,2,3,S-tetra-O-acetyl-a-D-manno pyranoside S-[2,3,4-tri-O-acetyl-S-0-(t-butyldimethylsilyl)-~-D-galactopyrano syl-(1 ~4 )-2,3,S-tri-O-acetyl-a-D-mannopyranosyl-H-phosphonate] triethylamm onium salt (13). Compound 8 (5 mg, 0.003 mmol) was dissolved in saturated solution of Me2NH in anhydrous CH3CN (2 mL) at -20°C and the solution was stirred for 3 h during which TLC confirmed disappearance of the starting material. Excess of Me2NH was removed und

= 1.9 and JHP = 6.8 Hz, 2H, H-1, Man'/Man"); 31p_NMR: -1.29; ESMS: m/z 574.12 ([M-2Et3N-2Hf 2,3,4-Tri-O-acetyl-~-D-galactopyranosyl-(1 ~4 )-1 ,2,3,S-tetra-O-acetyl-a-D-manno pyranoside S-[2,3,4-tri-O-acetyl-S-0-(t-butyldimethylsilyl)-~-D-galactopyrano syl-(1 ~4 )-2,3,S-tri-O-acetyl-a-D-mannopyranosyl-H-phosphonate] triethylamm onium salt (13). Compound 8 (5 mg, 0.003 mmol) was dissolved in saturated solution of Me2NH in anhydrous CH3CN (2 mL) at -20°C and the solution was stirred for 3 h during which TLC confirmed disappearance of the starting material. Excess of Me2NH was removed under reduced pressure below 30°C and the reaction mixture was concentrated to give the anomeric deprotected product in quantitative yield. To a stirred solution of imidazole (6 mg, 0.87 mmol) in anhydrous CH3CN (250 J!L) at 0 °C was added PCI3 (10 J!L, 0.112 mmol) and EhN (30 J!L, 0.215 mmol). The mixture was stirred for 20 min, after which a solution of the above compound in anhydrous CH3CN

Man), 61.37 (C-6, Man'), 62.30 (C-6, Gal'), 65.53 (C-6, d, Jcp = 5.5 Hz, Gal), 69.28 (C-4, Gal), 69.83 (C-4, Gal' and C-3, Man'), 70.84 (C-3, Man and C-2, Man), 71.08 (C-2, d, Jcp = 7.4 Hz, Man'), 72.13 (C-2, Gal' and C-2, Gal), 72.34 (C-5, Man), 73.69 (C-3, Gal', C-3, Gal and C-5, Man'), 74.89 (C-5, d, JcP = 7.5 Hz, Gal), 76.52 (C-5, Gal'), 77.05 (C-4, Man'), 78.14 (C-4, Man), 97.03 (C-1, d, Jcp = 5.5 Hz, Man'), 100.76 (C-1, Man), 104.20 (C-1, Gal'), 104.42 (C-1, Gal); 31p-NMR: -1.29; ESMS m/z 745.38 (M-Et3N-H)"; HRMS (ESMS): calcd for (M-Et3N-H)" C24H42024P 745.1804, found 745.1830. 2,3,4-Tri-O-acetyl-(3-D-galactopyranosyl-(1 ~4)-1 ,2,3,6-tetra-O-acetyl-a-D-mann opyranoside 6-(2,3,4-tri-O-acetyl-(3-D-galactopyranosyl-(1~4)-2,3,6-tri-O-acetyl­a-D-mannopyranosylphosphate ) triethylammonium salt (10). A solution of 48% aqueous HF in CH3CN (5:95, 5 ml) was added to compound 8 (20 mg, 0.015 mmol) at 0 DC and stirred at 0 DC for 2 h. The reaction was quenched by the addition of the aqueous NaHC03 solution until effervescence ceased and diluted with CH2CI2 (5 ml). The organic layer was washed with water, dried over Na2S04 and concentrated to give compound 10 (15.6 mg, 85%); ESMS m/z 1290.4 (M-EhN-H)" 2,3,4-Tri-O-acetyl-(3-D-galactopyranosyl-(1 ~4 )-1 ,2,3,6-tetra-O-acetyl-a-D-manno pyranoside 6-{2,3,4-tri-O-acetyl-6-0-(t-butyldimethylsilyl)-(3-D-galactopyrano syl-(1~4)-2,3,6-tri-O-acetyl-a-D-mannopyranosyl phosphate 6-[2,3,4-tri-O-acetyl -(3-D-galactopyranosyl-(1 ~4)-2,3,6-tri-O-acetyl-a-D-mannopyranosyl phosphate ]) bis-triethylammonium salt (11). Mixture of phosphotetrasaccharide acceptor 10 (15.6 mg, 0.015 mmol) and H-phosphonate donor 6 (20.8 mg, 0.024 mmol) was dried by evaporation of pyridine (500 III x 3). The residue was dissolved in anhydrous pyridine (500 Ill), and pivaloyl chloride (10 Ill, 0.083 mmol) was added. The mixture was stirred for 1 h at rt after which a freshly prepared solution of iodine (500 Ill, 16 mg, 0.06 mmol in pyridine-water, 95:5) was added. After 30 min, CH2CI2 was added and the solution was washed successively with cold 1 M aq Na2S203 solution (5 ml x 2) and ice-cold 1 M TEAS buffer (5 ml x 2), dried over Na2S04 and concentrated. The silica column purification using 5% CH30H in CH2CI2 with 1 % EhN afforded compound 11 (16 mg, 63%); R, = 0.11 in 10% CH30H in CH2Cb; lH-NMR (CDCI3); assignments by 1 H_l H COSY and HMQC experiments. Due to repeating nature (three repeats of phosphoglycan) of the molecule, all NMR peaks could not be assigned:1H NMR 0 0.01 (s, 6H, OSiM~CMe3), 0.84 (s, 9H, OSiMe2CMe3), 2.15-1.96

2.15 (13 x OCOCH3), 3.50 (m, 4H, H2-6 Gal and Gal'), 3.87 (m, 1 H, H-5, Gal'), 3.94 (m, 1H, H-5, Gal), 4.07-4.10 (m, 1H, H-5, Man'), 4.07-4.14 (m, 1H, H-5, Man), 4.35 (m, 1 H, H-4, Man'), 4.39 (m, 4H, 4-H, H2-6, Man and H2-6, Man'), 4.40 (m, 1 H, H-4, Man), 4.48 (m, 1 H, H-3, Man'), 4.52 (m, 1 H, H-3, Man), 4.94 (d, J = 7.7 Hz, 2H, H-1 ,Gal and H-1, Gal'), 5.28 (m, 4H, H-2 Man, H-4 Gal, H-3 Gal' and H-4 Gal'), 5.29 (m, 1 H, H-3, Gal), 5.43 (m, 1 H, H-2 Gal'), 5.45 (dd, JHH= 1.9 and JHP = 7.0 Hz, 1 H, H-1, Man'), 5.46 (m, 1 H, H-2, Gal), 6.01 (d, J = 2.7 Hz, 1 H, H-1, Man); 13C NMR: 0 -5.75, 17.95 and 25.57 (for TBOMS group), 20.48-20.79 (CH~02 x 13), 60.06 (C-6, Gal'), 60.42 (d, Jcp = 8 Hz, C-6, Gal), 62.22 (C-6, Man), 62.63 (C-6, Man'), 66.55 (d, C-2, Man'), 67.46 (d, C-5, Gal), 68.27 (C-4, Gal), 68.64 (C-4, Gal'), 69.37 (C-3, Man'), 69.66 (C-5, Man), 69.84 (C-3, Man), 70.14 (C-5, Man'), 70.75 (C-2, Gal'), 70.88 (C-2, Gal), 71.20 (C-2, Man), 73.31 (C-3, Gal'), 73.76 (C-3, Gal), 74.24 (C-4, Man'), 77.15 (C-4, Man), 78.95 (C-5, Gal'), 90.41 (d, C-1, Man'), 91.69 (C-1, Gal), 101.08 (C-1, Man), 101.29 (C-1, Gal'), 168-171 (CH3CO x 13); 31p_NMR: 0 -2.90 (dt, JPH 7.5 and 10); ESMS m/z 1405.2 (M-EhN-Hf; HRMS (ESMS): calcd for (M-Et3N-Hf C56H82037PSi 1405.4042, found 1405.4105. J3-D-Galactopyranosyl-(1 ~4)-a-D-mannopyranoside 6-[J3-D-galactopyranosyl-(1~)-a-D-mannopyranosyl phosphate] triethylammonium salt (9). A solution of 48% aqueous HF in CH3CN (5:95, 1.5 ml) was added to compound 8 (15 mg, 0.01 mmol) at 0 °C. The solution was stirred at 0 °C for 2 h. The reaction was quenched by the addition of aqueous NaHC03 solution until effervescence ceased, and diluted with CH2CI2 (5 ml). The organic layer was washed with water, dried over Na2S04 and concentrated. The residue was dissolved in anhydrous CH30H (500 Ill) and NaOMe (15 mg) was added, the solution was stirred overnight at rt, deionized with AG-X8 resin (H+), filtered and immediately neutralized with Et3N. After concentration, water (500 III x 3) was evaporated off from the residue to afford tetrasaccharide phosphodiester 9 (7.9 mg, 94%); [a]o = 34° (c 0.15, H20); lH-NMR (020), lH_1H_ COSY assignments: 3.45 (m, 2H, H-2, GaVGal'), 3.46 (m, 1 H, H-5, Man'), 3.55 (m, 1 H, H-5, Man), 3.56-3.53 (m, 2H, H-3, Gal/Gal'), 3.60 (m, 2H, H-5, Gal/Gal'), 3.68 (m, 4H, H2-6, Man/Man'), 3.76 (m, 2H, H-3, Man/Man'), 3.80 (m, 4H, H2-6, Gal/Gal'), 3.83 (m, 2H, H-4, GaVGal'), 3.85 (m, 1 H, H-2, Man), 3.94 (m, 1 H, H-2, Man'), 4.32 (m, 1 H, H-4, Man), 4.37 (d, J = 7.6 Hz, 1 H, H-1, Gal'), 4.35 (d, J = 7.6 Hz, 1 H, H-1, Gal), 5.09 (d, J = 1.8 Hz, 1 H, H-1, Man), 5.36 (dd, JHH = 1.9 Hz and JHP = 6.8 Hz, 1 H, H-1, Man'); 13C-NMR, assignment made by 20 lH_13C HETCOR experiment, 61.37 (C-6,

66.57 (C-4'), 69.36 (C-3), 69.53 (C-5), 69.69 (C-2'), 71.20 (C-2). 73.30 (C-3'), 73.86 (C-5'), 91.59 (C-4), 92.54 (C-1), 101.09 (C-1'), 169.13-170.49 (COMe); 31p NMR: 8= 0.13; ESMS m/z 771.26 (M-Et3N-Hr; HRMS (ESMS): calcd for (M-EbN-Hr C30H48019PSi 771.2297, found 771.2276. 1 ,2,3,6-Tetra-O-acetyl-4-0-(2,3,4-tri-O-acetyl-j3-D-galactopyranosyl)-a-D-manno pyranose (7). A solution of 48% aqueous HF in CH3CN (5:95, 8 ml) was added to compound 4 (100 mg, 0.132 mmol) at 0 °C and the solution was stirred for 2 h. The reaction was quenched with aqueous NaHC03 solution until effervescence ceased, and diluted with CH2CI2. The organic layer was washed thoroughly with water, dried over Na2S04 and concentrated to give 7 (72 mg, 85.7%); Rt = 0.3 in 70% ethyl acetate in hexane; [a]o = +4.6° (c 0.3, CHCI3); 1H NMR (CDCI3, 300 MHz) 81.97-2.16 (m, 21 H, 7 x OAc), 3.67-3.74 (m, 3H, H-5',6), 4.08-4.14 (m, 3H, H-5,6'), 4.58 (d, J = 7.8 Hz, 1H, H-1'), 5.16 (dd, J = 2.1 and 7.8 Hz, 1H, H-2'), 5.23 (dd, J = 2.1 and 3.6 Hz, 1 H, H-2), 5.32 (d, J = 3.3 Hz, 1 H, H-4), 5.41 (dd, J = 3.6 and 4.5 Hz, 1 H, H-3), 6.01 (d, J = 2.1 Hz, 1 H, H-1); 13C NMR (CDCI3, 75 MHz) 8 20.42-20.77 (7 x COMe), 60.74 (C-6'), 62.25 (C-6), 67.56 (C-4'), 68.31 (C-3), 69.35 (C-5), 69.43 (C-2'), 70.77 (C-2), 70.83 (C-3'), 73.98 (C-5'), 74.32 (C-4), 90.45 (C-1), 101.30 (C-1'), 168.32-170.80 (7 x COMe),; ESMS m/z659.28 (M+Nar; HRMS (ESMS): calcd for (M+NH4r C26H40N018 654.2245, found 654.2272. 2,3,4-Tri-O-acetyl-j3-D-galactopyranosyl-(1-?4)-1 ,2,3,6-tetra-O-acetyl-a-D-manno pyranoside 6-[2,3,4-tri-O-acetyl-6-0-(t-butyldimethylsilyl)-j3-D-galactopyranosyl -(1 ~4)-1 ,2,3,6-tetra-O-acetyl-a-D-mannopyranosyl phosphate] triethyl ammonium salt (8). Mixture of H-phosphonate donor 6 (32 mg, 0.036 mmol) and acceptor 7 (23 mg, 0.036 mmol) was dried by evaporation of pyridine (500 III x 3). The residue was dissolved in anhydrous pyridine (600 Ill) and pivaloyl chloride (15 Ill, 0.123 mmol) was added. The reaction mixture was stirred for 1 h at rt and a freshly prepared iodine solution (600 Ill, 18 mg, 0.078 mmol in pyridine-water, 95:5) was added. After 30 min. CH2CI2 (10 ml) was added and the solution was washed successively with cold 1 M aqueous solution of Na2S203 (5 ml x 2) and ice-cold 1 M TEAS buffer (5 ml x 2), dried over Na2S04 and concentrated. Column chromatography on silica gel (3% CH30H in CH2CI2 with 1 % EbN) afforded product 8 (40 mg, 73.8%); Rt= 0.21 in 10% CH30H in CH2CI2; [a]o = -6.1° (c 0.18, CHCI3); 1H_ NMR (CDCI3, 300 MHz); assignments confirmed by 1H_1H COSY and HMQC experiments: 1 H NMR 8 0.01 (5, 6H, OSiM9:2CMe3), 0.84 (s, 9H, OSiMe2CMe3). 1.96-

2,3,6-Tri-O-acetyl-4-0-[2,3,4-tri-O-acetyl-6-0-(t-butyldimethylsilyl)-(3-D-galactop yranosyl]-a-D-mannopyranose (5). Compound 4 (100 mg, 0.132 mmol) was dissolved in saturated Me2NH solution in anhydrous CH3CN (20 ml) at -20°C and stirred for 3 h after which TlC confirmed disappearance of the starting material. Excess of Me2NH was removed under reduced pressure below 30°C and the reaction mixture was concentrated to give the desired anomeric deprotected compound 5 in quantitative yield; R, = 0.25 in 70% ethyl acetate in hexane; [a]D = +3.75° (c 0.16, CHCI3); 1H NMR (CDCI3, 300 MHz) 80.01 (s, 6H, M~SiCMe3), 0.84 (s, 9H, Me2SiCMSJ), 1.95-2.19 (m, 18H, 6 x OAc), 3.56-3.66 (m, 4H, H-6,6'), 3.91 (m, 1H, H-5), 4.12-4.16 (m, 2H, H-5', OH), 4.40 (d, J= 4.5 Hz, 1H, H-4), 4.40 (d, J= 7.8 Hz, 1 H, H-1'), 4.99 (dd, J = 3.3 and 7.8 Hz, H-3'), 5.09 (dd, J = 2.1 and 7.8 Hz, 1 H, H-2'), 5.17 (dd, J = 2.1 and 3.6 Hz, 1 H, H-2), 5.23 (dd, J = 3.6 and 4.5 Hz, 1 H, H-3), 5.43 (m, 2H, H-4',1); 13C NMR (CDCI3, 75 MHz) 8 -5.77 (M~SiCMe3), 17.98 , (Me2SiCMe3)" 20.40-21.38 (OAc), 25.58 (Me2SiCMe3), 60.06 (C-6'), 62.62 (C-6), 66.56 (C-4'), 68.78 (C-3), 69.30 (C-5), 69.51 (C-2'), 70.06 (C-2), 71.21 (C-3'), 73.37 (C-5'), 74.15 (C-4), 91.82 (C-1), 101.04 (C-1'), 169.10-170.52 (COMe); ESMS m/z 731.3 (M+Nat. Triethylammonium 2,3,6-tri-O-acetyl-4-0-[2,3,4-tri-O-acetyl-6-0-(t-butyldimethyl silyl)-(3-D-galactopyranosyl]-a-D-mannopyranosyl hydrogen phosphonate (6). To a stirred solution of imidazole (224 mg, 3.28 mmol) in anhydrous CH3CN (5 ml) at o °C was added PCI3 (160 Ill, 1.8 mmol) and EhN (480 Ill, 3.44 mmol). The mixture was stirred for 20 min, after which a solution of compound 5 dissolved in anhydrous CH3CN (5 ml) was added dropwise. The mixture was stirred at 0 °C for 3 hand quenched with 1 M triethylammonium bicarbonate (TEAS) buffer (pH 7, 2 ml). The clear solution was stirred for 15 min, diluted with CH2CI2 (20 ml), and the organic layer was washed with ice cold water (10 ml x 2) and cold 1 M TEAS solution (10 ml x 2) successively, dried over Na2S04 and concentrated to yield phosphoglycan donor 6 (100 mg, 86%); R, = 0.45 in 20% CH30H in CH2CI2; [a]D = -4.5° (c 0.27, CHCb); 1H NMR (CDCI3, 300 MHz) 8 0.01 (s, 6H, M~SiCMe3), 0.82 (s, 9H, M~SiCMSJ), 1.95-2.09 (m, 18H, 6 x OAc), 3.49-3.68 (m, 4H, H-6,6'), 3.88 (m, 1 H, H-5), 4.14 (m, 1 H, H-5'),4.36 (d, J = 4.5 Hz, 1 H, H-4), 4.47 (d, J = 7.8 Hz, 1 H, H-1'), 4.95 (dd, J = 3.3 and 7.8 Hz, 1H, H-3'), 5.05 (dd, J = 2.1 and 7.8 Hz, 1H, H-2'), 5.21 (dd, J = 2.1 and 3.6 Hz, 1 H, H-2), 5.41 (d, J = 3.3 Hz, 1 H, H-4'), 5.48 (dd, J = 1.8 and 8 Hz, 1 H, H-1), 6.92 (d, JH,p= 637.0 Hz, 1H, H-1); 13C NMR (CDCI3, 75 MHz) 8 -5.80, (M~SiCMe3), 17.98 (Me2SiCMe3), 20.48-20.76 (OAc), 25.57 (Me2SiCMSJ), 60.10 (C-6'), 62.42 (C-6),

chromatography (8% CH30H in CH2CI2) to provide compound 2 (10.8 g, 79.5%); Rf = 0.47 in 15% CH30H in CH2CI2; [a]o = +3.45° (c 0.29, CH30H); 1H NMR (020, 300 MHz) 00.01 (s, 6H, M~SiCMe3), 0.82 (s, 9H, Me2SiCM~), 3.48 (m, 1 H, H-2'), 3.58 (m, 1 H, H-3'), 3.65 (m, 1 H, H-5), 3.76 (m, 4H, H-6,6'), 3.82 (d, J = 3.1 Hz, 1 H, H-4'), 3.92 (m, 1 H, H-5'), 4.38 (m, 1 H, H-3), 4.31 (d, J = 5.7 Hz, 1 H, H-4), 4.46 (d, J = 7.8 Hz, 1 H, H-1'), 4.76 (dd, J = 3.6 and 6.3 Hz, 1 H, H-2), 6.37 (dd, J = 1.1 and 6.2 Hz, 1 H, H-1); 13C NMR (020, 75 MHz) 0 -4.84 (M~SiCMe3), 25.23 (Me2SiCM~), 59.57 (C-6'), 60.89 (C-6), 67.14 (C-4'), 68.45 (C-3), 70.87 (C-5), 72.52 (C-2'), 75.23 (C-2), 76.68 (C-3'), 77.43 (C-5'), 101.73 (C-4), 102.87 (C-1'), 143.88 (C-1); ESMS m/z 445.10 (M+Naf; HRMS (FAB): calcd for (M+Lif C18H3409SiLi 429.2132, found 429.2126. 1,2,3,6-Tetra-O-acetyl-4-0-[2,3,4-tri-O-acetyl-6-0-( t-butyldimethylsilyl)-~-D-gala ctopyranosyl]-a-D-mannopyranose (4). A solution of 2 (5 g, 11.8 mmol) in water (50 mL) was stirred, to which was added a solution of m-CPBA (6.5 g, 36 mmol) in diethyl ether (50 mL) dropwise at -10 °C. The reaction mixture was brought to 0 °C and stirred for 4 h, and aqueous layer was extracted thoroughly with ether, Iyoph iii zed to afford 4-0-[6-0-( t-butyldi methylsilyl)-f3-0-galactopyranosyl]-a-0-mannopyranose (3) . This was dissolved in anhydrous pyridine (25 mL) and acetic anhydride (25 mL) was added dropwise at 0 °C. The mixture was gradually brought to rt and stirred for 16 h, and after completion of the reaction it was quenched with ice and diluted with CH2CI2. The organic layer was washed with water, dried (Na2S04) and concentrated to give a syrup which was purified by silica column (20% ethyl acetate in hexane) to provide compound 4 as white amorphous solid (7.5 g, 84%); [a]o = +6.72° (c 0.55, CHCI3); Rf = 0.69 in 70% ethyl acetate in hexane; 1H NMR (COCI3, 300 MHz) 0 0.01 (s, 6H, M~SiCMe3)' 0.84 (s, 9H, Me2SiCMe3), 1.95-2.14 (m, 21 H, 7 x OAc), 3.56-3.64 (m, 4H, H-6,6'), 4.17-5.04 (m, 2H, H-5,5'), 4.53 (d, J = 7.8 Hz, 1H, H-1'), 5.01 (dd, J = 3.3 and 7.8 Hz, 2H, H-4), 5.12 (dd, J = 2.1 and 7.8 Hz, 1H, H-2'), 5.21 (dd, J = 2.1 and 3.6 Hz, 1H, H-2), 5.34 (dd, J = 3.6 and 4.5 Hz, 1H, H-3), 5.41 (d, J = 3.3 Hz, 1H, H-4'), 6.01 (d, J = 2.1 Hz, 1H, H-1); 13C NMR (COCI3, 75 MHz) 8 -5.85 (M~SiCMe3), 17.94 (Me2SiCMe3), 20.40-20.86 (OAc), 25.54 (Me2SiCMe3), 60.01 (C-6'), 62.14 (C-6), 66.45 (C-4'), 68.18 (C-3), 69.25 (C-5), 69.39 (C-2'), 70.58 (C-2), 70.79 (C-3'), 73.38 (C-5'), 73.62 (C-4), 90.25 (C-1), 101.14 (C-1'), 168.08-170.23 (7 x CO); ESMS m/z 773.24 (M+Naf; HRMS (ESMS): calcd for (M+NH4f C32Hs4 N018 Si 768.3110, found 768.3139

Lactal (1). A solution of cyanocobalamin83 (Vitamin B12, 1.5 g, 1.14 mmol) in anhydrous CH30H (400 mL) was thoroughly purged with nitrogen gas for 30 min and zinc powder (87.5 g, 1.338 mol) and ammonium chloride (71 g, 1.33 mol) were added to the solution. The reaction was stirred for another 45 min and hepta-O-acetyl lactosyl bromide (47 g, 67.5 mmol), freshly prepared from lactose [peracetylation using acetic anhydride and sodium acetate, followed by anomeric bromination (48% hydrobromic acid in acetic acid)], was dissolved in CH30H (150 mL) and added. Immediately after addition of the bromide, the dark red solution changed to reddish-yellow and then back to dark red in 5 min. The solution was filtered through celite to remove zinc, the celite pad was washed with CH30H and the filtrate was concentrated to give a white and red solid. This mixture was dissolved in water (500 mL) and extracted with CH2CI2 (300 mL x 3). Organic extracts were combined, dried over Na2S04, and concentrated to provide hexa-O-acetyl lactal (36 g, 87%) as an amorphous solid, mp 113° (lit84 mp 114°); [a)D = -18° (c 1.0, CHCI3) (Iit84, -18°, c 1.0, CHCI3). In the next step of complete deacylation, hexa-O-acetyl lactal (36 g, 64.5 mmol) and freshly dried Na2C03 (45 g, 425 mmol) were suspended in anhydrous CH30H (750 mL) and stirred for 90 min at rt. The suspension was filtered to remove excess of Na2C03 and the filtrate was concentrated under reduced pressure to give deprotected lactal (1) as an amorphous solid (19.4 g, 98%); R,= 0.2 in 30% CH30H in CH2CI2; mp 191-193°; [a]D = +27° (c 1.6, H20) (lit84, +27°, c 1.6, H20). 6'-0-(f-butyldimethylsilyl)-lactal (2). A solution of lactal (1, 10 g, 32.4 mmol) and BU2SnO (8 g, 32.5 mmol) in anhydrous CH30H (1000 mL) was heated to reflux for 4 h followed by removal of solvent which provided a yellow powder. The dibutyltin complex was dissolved in anhydrous THF (1000 mL) and TBDMSCI (4.9 g, 32.3 mmol) was added, and the solution was stirred for 48 h at rt. After the completion of reaction, the solvent was evaporated to give a residue which was purified by silica

Solution Phase Synthesis of Phosphoglycans

Synthesis of Phosphoglycan Repeats of Lipophosphoglycan

The antibody isotypes in the immune sera were determined, by indirect ELISA, using mouse MAb isotyping reagents (Sigma). The microtitration plates coated with r-dZP3 (400 ng/well) and blocked with 1% BSA, were incubated with doubling dilution of pooled serum samples of a group of immunized animals. All the incubations were carried out at 37°Cand were followed by three washings with PBST. The incubation was followed by addition of goat anti-mouse isotype specific antibodies at 1:1000 dilution. The binding was revealed by rabbit anti-goat lgG-HRPO conjugate (Pierce) at an optimized dilution of I: 10,000 and processed for enzymatic activity estimation as described earlier.

Antibody isotyping

d) Particle delivery using the Helios gene gun A day prior to immunization, hair were removed from the abdominal region of mice using a commercial depilatory agent (Anne French cream). Two cartridges/mouse ( ~ 2 Jlg DNA) were shot under pressurized helium gas ( 400 psi) intradermally at the shaven area of the abdomen of mice using the Helios gene gun. Two boosters comprising of two cartridges each were given on days 21 and 35. On day 45, mice in each group received i.m. injection of E. coli expressed recombinant protein (20 Jlglmouse in saline). Mice were bled retro-orbitally on days 0, 45 and 52 for analysis of antibody response.

tubing, which was cut into 0.5 inch pieces (cartridges). These cartridges were used to deliver DNA into epidermis of male/female mice. a) Preparation of DNA-gold microcarrier suspension Twenty five mg of gold microcarriers were weighed in a 1.5 ml eppendorf tube to which 100 J..Ll of 0.05 M spermidine was added and vortexed for 10 sec. To the above mixture 100 J..Ll of DNA (0.5 mg/ml) was added and vortexed for another 10 sec. While vortexing, 100 J..Ll of 1 M CaCh was added dropwise to the mixture and left at RT for 10 min to allow precipitation of DNA onto gold microcarriers. The DNA-gold pellet was collected by centrifuging at 12,000 X g for 1 min at RT. The pellet was washed thrice with 100% ethanol (freshly opened bottle), resuspended in 3 ml of 0.1mg/ml polyvinylpyrollidone (PVP) in ethanol and stored at -20°C till further use. b) Loading the DNA/microcarrier suspension into gold-coat tubing using the tubing prep station A 25 inch length of tubing was cut and fixed on tubing prep station, air dried by passing nitrogen gas through it for 15 min. The DNA/microcarrier suspension was vortexed and injected into the tubing using a 5 ml syringe and the microcarriers allowed to settle in the tubing for 3 min. Ethanol from the tubing was removed by slowly sucking into the syringe. The tubing was rotated, while passing the nitrogen gas, using the tubing prep station, for 20-30 sec to allow the microcarriers to evenly coat the inside of the tubing. c) Preparation of cartridges using the tubing cutter The tubing was cut into 0.5 inch long pieces (cartridges) by using the tubing cutter and cartridges stored at 4°C in vials containing desiccant pellets till further use.

Suspension of DNA adsorbed onto gold microcarriers at 0.5 Microcarrier Loading Quantity (MLQ; 50 J.lg DNA/25 mg gold microcarriers) was prepared and coated inside Tefzel

Plasmid DNA adsorbed onto gold microcarriers

A day prior to immunization, hair were removed from both the hind limbs of the mice using a commercial depilatory agent (Anne French cream, Geoffrey Manners & Co. Ltd, Mumbai, India). Mice were immunized in a similar way as in the saline group but in addition, ten very short electric pulses were given at the site of injection immediately after DNA administration using a gas igniter (Upadhyay, 2001). Voltage delivered in each trigger was 18kV for 10-7s.

Plasmid DNA administered by electroporation

Inbred male BALB/c.T mice (6-8 week, Small Experimental Animal Facility, National Institute of Immunology, New Delhi, India) were immunized intramuscularly (i.m.) with 100 J.lg of respective plasmid DNA or VR1020 vector in 100 J.ll saline (0.9% NaCl) in the anterior tibialis muscle in the hind limbs (each receiving 50 J.ll). Two booster injections of 100 J.lg DNA in saline were given on day 21 and 35. On day 45, mice in each group received i.m. injection of E. coli expressed recombinant protein (20 J.lg/mouse in saline). Mice were anesthetized and bled retro-orbitally on days 0, 45 and 52 for analysis of respective antibody responses.

Plasmid DNA administered in saline

IV. IN-VIVO IMMUNIZATION STUDIES These experiments were carried out with the approval of Institutional Animal Ethics Committee. Three different modes of administration were used:

a) Purification oLin elusion bodies For the purification of inclusion bodies, the bacterial cell pellet from 1 liter culture was resuspended in 10 ml of Tris-HCl buffer (50 mM; pH 8.5) containing 5 mM EDTA and sonicated using Branson sonifier-450 for 8 cycles of 90 sec each (30 watt output; Branson Ultrasonic Corp., Danbury, CT, USA) on ice. The inclusion bodies were collected by centrifugation of the sonicate at 8000 X g for 30 min at 4°C. The pellet was washed twice with 15 ml of 50 mM Tris-HCl buffer with 5 mM EDTA containing 2% sodium deoxycholate in order to remove loosely bound E. coli proteins from the inclusion bodies. Subsequently, the inclusion body pellet was washed with 50 mM Tris-HCI buffer (pH 8.5), followed by a washing with the double distilled water. All the buffers used for the purification contained 20 mM of phenylmethyl sulphonyl fluoride (PMSF). b) Solubilization and renaturation The purified inclusion bodies were solubilized in 100 mM Tris-HCl (pH 12.0) containing 2M urea at RT for 30 min, and centrifuged at 8000 X g for 30 min at 4°C. The pH ofthe supernatant was brought down immediately to 8.5 with 1 N HCl and then extensively dialyzed against renaturation buffer (50 mM Tris-HCl buffer; pH 8.5, 1 mM EDT A, 0.1 mM reduced glutathione, 0.01 mM oxidized glutathione and 10% sucrose). The protein was finally dialyzed against 20 mM Tris-HCl, pH 8.5 and its concentration estimated using BCA.

Purification in refolded form

albumin (BSA) in PBS for 2 hat 4°C. For detection of r-bmZPI, a murine monoclonal antibody (MAb), MA-813, generated against E. coli expressed r-bmZP1 (Govind et al., 2000), was used as the primary antibody. The cells were incubated with 1 :500 dilution of MA-813 ascites fluid for 2 hat 4°C. Cells were washed 5 times with PBS and incubated for 1 h with a 1:800 dilution of goat anti-mouse Ig-fluorescein isothiocyanate (FITC) conjugate (Sigma) at 4°C. After washing with PBS, coverslips with the cells were mounted in glycerol : PBS (9 : 1 ), and examined under an Optiphot fluorescent microscope (Nikon, Chiyoda-Ku, Tokyo, Japan). For detecting r-dZP3, MAb, MA-451 (1 :500 dilution of ascites fluid), generated against porcine ZP3f3 (a homologue of dZP3) and immunlogically cross-reactive with dZP3 (Santhanam et al., 1998) was used. For detecting r-rG, rabbit polyclonal antibodies (1:1000 dilution) against E. coli expressed r-rG, was used as primary antibody. The polyclonal antibody was provided by Dr. Sangeeta Choudhury, Project Associate, Gamete Antigen Laboratory, National Institute of Immunology, New Delhi. Goat anti-mouse immunoglobulins-FITC conjugate (1 :800) and goat anti-rabbit immunoglobulins-FITC conjugate (1 :2000; Pierce) were used for detecting anti-dZP3 and anti-rG antibodies respectively

Initial standardization of transfection conditions was done using VRbmZPl plasmid DNA and COS-I mammalian cell line. In brief, cells were cultured in T-25 tissue culture flasks in Dulbecco's Modified Eagle's Medium (DMEM) supplemented with 10% fetal calf serum (FCS) at 37°C with 5% C02. For subculturing, cells were trypsinized (0.5% trypsin + 0.2% EDTA in DMEM without FCS), centrifuged at 250 X g for 10 min, resuspended in DMEM supplemented with 10% FCS and aliquoted into T-25 flasks. For transfection, cells were seeded on coverslips in a 24-well tissue culture plate at a density of 5x 104 cells/well, a day prior to transfection. To standardize in vitro transfection conditions for optimum expression of bmZP1, varying amount of plasmid DNA was mixed with lipofectamine in DMEM devoid ofFCS (final reaction volume 200 f.!l) and incubated at RT for 45 min. The cells on the coverslips were washed twice with plain DMEM devoid of FCS. DNA-Iipofectamine complex was added dropwise to the cells and the plate incubated for 8 h at 3 7°C in humidified atmosphere of 5% C02• Subsequently, 1 ml of DMEM containing 10% FCS was added per well and cells allowed to grow for 48 h. After incubation, cells were processed for visualization of r-bmZPl by indirect immunofluorescence assay. Cells were washed twice with phosphate buffer saline (PBS; 50 mM Phosphate and 150 mM NaCI, pH 7.4), fixed in chilled methanol (-20°C) for 3 min and blocked with 3% bovine serum

Detection of the expressed recombinant protein following i11 vitro transfection of mammalian cells with the plasmid DNA.

GAAGATCTCAGACCATCTGGCCAACT-3' as the forward pnmer, and 5'-GAAGATCTT-TAAGTGTGGGAAACAGACTT-3' as the reverse primer as described for bmZPl except that primer annealing was performed at 53°C for 1 min.

The dog ZP3 ( dZP3) eDNA, excluding the SS and the TD, was cloned in prokaryotic expression v~ctor, pQE30 (QIAGEN) as described previously (Santhanam et al., 1998). To clone dZP3 eDNA in mammalian expression vector, VR1020, the pQE30-dZP3 clone was used as a template to PCR amplify dZP3 eDNA (79-1056 nt; 978 bp) using 5'-

PCR amplification of dZP3 eDNA

Thespleenswereremovedandhomogenisedindividuallythroughanylonnet(80mesh).Forisolationoflymphocytes,thetissuehomogenatewaslayeredonatwo-stepPercolldensitygradientandcentrifugedfor30minat400xg.Thelymphocyteswerecollectedatthe1.040-1.080g/cm3interface,washedtwice(400xg,10min)withrHbss,andresuspendedin2mlrRPMl.Cellswerecountedbytrypanblueexclusioninahaemocytometer(viability>95%)andthenumberoflymphocytesfrombloodandspleenwereadjustedto2x106/ml

Spleen

ultrapureHNO3andtissuesamplesweredissolvedin70%HNO3;microwavedfor5minat90W,180W,270Wand360W,untiltotaldigestionhadoccurredandthendilutedwithMilli-Qgradewater(Millipore,Acton,Massachusetts,U.S.A)

Totalsodium,potassiumandcalciumconcentrationsweredeterminedwithatomicabsorptionspectrophotometry.Tothispurpose,plasmasamplesweredilutedwith1%

Ionconcentrations

Aftereffluentexposure,thetissuesweredissectedout,weighedandhomogenizedin0.25Msucrose.Thehomogenateswerecentrifugedat10,000rev/minatatemperaturebelow8°C.AcetylcholinesteraseactivityofthesampleswasdeterminedatpH7.0usingafinalhomogenateconcentrationof25mgmf1at10°CwithmMacetylcholineiodideassubstrate and0.001or0.002NsodiumhydroxideastitrantfollowingHestron’smethodasgivenbyMetcalf(1951).ProteindeterminationsforalltheChEanalyseswereconductedonaliquotsofthehomogenatesusingamodificationoftheLowryetal.(1951)method.AchEactivityisexpressedinpmolesofacetylcholinechloridehydrolysedmgtissue'1hr'1

Acetylcholinesterase(AchE

Salineextracts(0.89%)oftissueswerepreparedinTeflonglasshomogenizer.ThecarbohydratecontentoftheextractwasdoneaccordingtothemethodofShibkoetal.(1967)

Carbohydrates

HaemoglobinwasdeterminedbySahlimethod.HaemoglobinisconvertedtoacidhaematinbytheactionofHC1.Theacidhaematinsolutionisfurtherdilutedwiththeaciduntilitscolormatchesexactlythatofthepermanentstandardofthecomparatorblock.TheHbconcentrationisreaddirectlyfromthecalibrationcurve.

Haemoglobin(Hb)determination

Theexperimentalfishwasacclimatedtoglassrespirometersforabout24hrandtheywerenotgivenanyfoodduringthisperiod.TheeffluentexposedfishesalongwiththecontrolsweresubjectedtoO2consumptionseparately.Theexperimentswereperformedinaninsulatedroombetween8to10AMwithlightson.TherateoftotalO2uptakethroughgillsfromflowingwaters(DO=7.2mg021'1)wasmeasuredinfishesofdifferent body weights.Forthis,acylindricalglassrespirometerof2litrecapacitywasused.Thefishwasintroducedintherespirometerwhichwasconnectedtoalargeconstantlevelwatertanktomaintaintheflowofwaterunderconstanthydrostaticpressure.Thewaterenteredtherespirometeratonesideanditsflowperminutewasmeasuredasitlefttheotherside.Theflowwasadjustedaccordingtothesizeofthefish.Thefishwasacclimatizedtotherespirometeratleast12hrbeforereadingswere taken.ConcentrationofdissolvedoxygeninthesampleswasmeasuredbyWinkler’svolumetricmethod(Welch,1948).ThedifferenceinO?levelsbetweentheambientwaterandthatsuppliedtotherespirometeraswellaswiththerateofwaterflowandtheweightofthefishwasusedtocalculatetherateof O2uptakeintermsoftime(ml02hr'1)withthehelpoftheequation:V02=Vw(Ci02CE02)Where,VO2=02uptake(ml02hr'1)Vw=water(mlm'1)andCi02-CE02respectivelythe02concentrationofinletandoutletwaters.Arespirometercontainingnoanimalsservedasacontrolforadjustingcalculationsfor02uptakeinthewater.Uponremoval,fisheswereblottedwithpapertoweling, andweighed

Aquaticrespiration

Theywereheld(28°±0.5°C;naturalphotoperiod)in240litretapwatertanks(1x1x0.3m)andacclimatedtothelaboratoryconditionsforaweekbeforeexperimentation.Agentlecontinuouswaterflowwasmaintainedthroughtheaquariaforconstantwaterrenewal.

Holdingtanks

Percentage lethality was calculated as:100×((number of non-CyO/ number CyO)×100)

Flies were maintained at 18°C or 25°C as appropriate. Through out this thesis, flies defined as wild-type were yellow white of the genotype: y67c23w118. BEAF32 null lines BEAF32AB-KO/CyOGFP, kindly provided by Craig Hart, University of Illinois (Roy et al., 2007a). Homozygous BEAF32AB-KOlines were obtained by selection against the CyOGFPmarker at the 3rdinstar larvae stage, using a Leica M165 FC with a GFP filter. Lethality of the BEAF32AB-KOallele was assessed against the dppHr27hypersensitive allele (genotype: dppHr27,cn1,bw1/CyO P{dpp-P23}). For this embryos were collected from the following crosses as set up by Catherine Sutcliffe:BEAF32AB-KO/+ ×dppHr27,cn1,bw1/CyO P{dpp-P23}and+/+ ×dppHr27,cn1,bw1/CyO P{dpp-P23}

Fly Stocks and Crosses

eppendorf tube had DNA of interest. The purified DNA fragments were checked on an agarose gel, with an appropriate marker, before setting up the ligation reaction

The plasmid DNAs for cloning, were digested with the respective enzymes, checked on an appropriate percentage of agarose gel along with 100 bp ladder or .A Hind ill marker (Promega, USA) and the required fragments were eluted from the gel using the Qiagel Gel Extraction kit (Qiagen, U.K.). According to the manufacturer's directions, the area of the gel containing the DNA fragment was excised using a clean and sharp blade, minimizing the amount of surrounding agarose excised with the fragment. The gel slice was weighed and placed in a microfuge tube. Three volumes of Gel Solubilization Buffer (QG) was added for every one volume of gel. The gel piece was then vortexed and incubated at 500C for 10 min. The contents were mixed in between, by inverting the tube few times, to ensure gel dissolution. It was then centrifuged at 13,000 rpm for 1min. The flow through in the discard column was carefully removed. Then 500 p.l of buffer PB was added to remove the traces of gel and the tube was centrifuged at 13,000 rpm for 1 min. The flow through in the discard column was removed and 750 p.l of Wash Buffer (PE) (containing ethanol) was added and the tube was centrifuged at 13,000 rpm for 1 min. The flow through was discarded and another spin at maximum for 2 min was given to remove the traces of wash buffer. The column was then put on a fresh tube and finally, to elute the DNA, 40 p.l of Tris-EDTA buffer (TE) or RNase-DNase-free water was added and then centrifuged at 13,000 rpm for 1 min. The flow through in the

Gel elution of DNA fragments:

was used to amplify the oligonucleotides (Promega Biotech, Madison, USA). pGEMT-Easy and p-TARGET cloning vectors were also obtained from Promega. In vitro transcription was carried out using Riboprobe transcription system (Promega Biotech, Madison, U.S.A.). BCA protein assay kit was obtained from Pierce Biotechnology (Rockford, IL, U.S.A.). Reverse transcription was carried out using lmProm-TI™ Reverse Transcriptase kit from Promega. Luciferase activity in the cell extracts was measured using Luciferase assay System (Promega Biotech., U.S.A.).

Qiaprep spin mini kit and Qiagen plasmid midi kit (West Sussex, U.K.) were used for isolation of DNA. Isolation of DNA fragments from gel was carried out using QiaGel extraction kits or PCR products were purified using nucleotide removal kit from Qiagen (West Sussex, U.K.). PCR core system I

Kits

1X PBS diluted in distilled water 1X fixative solution diluted in distilled water 2.4.12.3 Staining Solution25 μl Solution A 25 μl Solution B 25 μl Solution C 125 μl 20 mg/ml X-gal in DMF

Working Solutions:

Procedure:

β- galactosidase assay was performed in a 96 well format. Briefly, 4000-5000 cells were plated in 96 well tissue culture coated plate. Cells were transfected with reporter plasmid after 18 -24 hrs and after 48 hrs the cells were washed once with D-PBS. 50μl of lysis buffer was added to the well and cells were lysed by freezing plate at -70°C and thawing at 37°C. Cells were pipette up and down and then the plate was centrifuged at 9000 X g for 5 minutes. The supernatant from each plate was transferred to clean eppendorf tube. Immediately prior to assay the ONPG cocktail was prepared as below: 47 μl 0.1 M sodium phosphate (pH 7.5)22 μl 4 mg/ml ONPG1 μl 100X Mg solution30μl of each well extract was added to microtitre well plate and70μl of ONPG cocktail was added to each well. The plate was kept on ice throughout the procedure. After addition of ONPG cocktail the plate was transferred to 37°C and the development of colour was monitored every 10 minutes for development of color. After development of yellow colour, the reaction was stopped by addition of 150μl of 1M sodium carbonate to each well

ethanol has dried. The pellet was resuspended in 20 μl of milliQ water and 20 μg/ml RNase added. The tube was incubated at 50°C for 45 min. the tube was vortexed for few seconds. Quality of the plasmid DNA was then accessed by running 1% agarose gel.

Overnight Grown culture was pelleted by centrifugation at 10,000g at 4°C for 3 min and the supernatant was discarded. Pellet was resuspended in 250 μl of ice-cold alkaline lysis solution 1. 300 μl of alkaline solution 2 was then added and the tube was inverted gently 3-4 times and incubated at room temperature for 5 min. 350 μl of ice cold solution 3 was added and mixed by inverting the tube rapidly for 3 or 5 times. Suspension was incubated on ice for 10 min. Bacterial lysate was spun at 10,000g for 12 min at 4°C. Supernatant was transferred to a fresh tube. 0.4 volume of phenol: chloroform was added to the supernatant and the contents mixed. It was then spun at 10,000g at 4°C for 12 min. Aqueous phase was taken out in a fresh tube and 0.6 volume of isopropanol was added, mixed properly and incubated at room temperature for half an hour followed by spinning at 10,000g at RT for 20 min. Supernatant was discarded. Pellet was washed with 70% ethanol. The tube was stored at room temperature until the

Preparation of Plasmid DNA by alkaline lysis

250 mM KCl 55 mM MnCl2.4H20 50 x TAE (1 litre): 242 g of tris base 57.1 ml of glacial acetic acid 100 ml of 0.5 M EDTA Alkaline Lysis Solution 1: 50mM tris-HCl (pH 8.0) 10.0 mM EDTA 50 mM glucose Alkaline Lysis Solution 2: 0.2M NaOH 1% SDS Alkaline Lysis Solution 3: 3.0M Potassium acetate

2.7 mM KCl 10 mM Na2HPO42 mM KH2PO4Tris Buffer Saline (TBS) (10X): 12.1gm Trizma Base 40.0gm NaCl Adjust PH to 7.6; make up the volume to 1 lit with milli Q water. HEPES Buffer Saline: 20 mM HEPES (pH 7.5) 150 mM NaCl Blocking Buffer: 5% fat free milk or 2% BSA in PBST or TBST. Stripping Buffer: 100 mM β-mercaptoethanol 2% (w/v) SDS 62.5 mM Tris-HCl (pH6.7) Luria Broth: 10g tryptone 10g NaCl 5g yeast extract, make up the volume to 1 lit with water. TB buffer for preparation of competent cells: 10 mM PIPES (free acid) 15 mM CaCl2.2H20

2.5 ml of 1.5 M Tris-Cl (pH 8.8) 4.0 ml of 30% acrylamide; bisacrylamide (29:1) mix 50.0 μl of 20% SDS 3.35 ml of milli-Q water 100 μl of 10% APS 10.0 μl of TEMED. 2X SDS loading Buffer: 130 mM Tris-Cl (pH 8.0) 20% (v/v) glycerol 4.6% (w/v) SDS 0.02% bromophenol blue 2% DTT SDS PAGE Running Buffer: 25mM Tris base, 0.2M glycine 1% SDS Western Blot: 1 x Blotting Buffer (2Litres): 25mM tris base, 0.2M glycine 20% methanol Phosphate Buffer saline (PBS): 137 mM NaCl

Whole cell lysis buffer: 20mM Tris (PH 7.5) 150mM NaCl 1mM EDTA 1mM EGTA 1 % triton X 100 2.5mM sodium pyrophosphate 1mM β-glycerophosphate 1mM Na3VO41μg/ml aprotinin, 1μg/ml leupeptin and 1μ.ml pepstatin SDS-PAGE: Stacking Gel Mix (4ml, 5%): 380μl of 1M Tris-Cl (pH 6.8) 500μl of 30% acrylamide ; bisacrylamide (29:1) Mix 15 μl of 20% SDS 2.1 ml of milli-Q water 30 μl of 10% APS 5 μl of TEMED. 12% Resolving Gel Mix (10ml):

Buffers and Media

Monoclonal antibody against KRAS were purchased from Merck Research Laboratories, phospho p44/42 (ERK1/2)and total p44/42 (ERK1/2)antibodies were purchased from Cell Signaling Technologies. Anti tubulin antibody was obtained Sgima-Aldrich Chemicals. HRP conjugated anti-mouse and anti-rabbit secondary antibodies were purchased from Bangalore Genei Pvt. Ltd.

Antibodies

1μl of the cell lysate was mixed with 200 μl of 5X Bradford reagent and 800 μl of water. O.D was measured at 595 nm. Standard curve of BSA was plotted using various dilutions of BSA protein by Bradford method. Protein estimation of the cell lysate samples was performed using the standard curve equation y=0.0695x + 0.0329 μg/μl

Protein estimation by Bradford method

Overnight cell culture raised in LB medium was subcultured 1:100 in LB with 20 mM MgCl2. When the A600 reached 0.4-0.6, the culture was centrifuged at 2800g for 5 min at 4 ̊C. To the cell pellet 0.4 volumes of ice-cold TBF-I buffer was added and incubated on ice for 15 min. The cell suspension was centrifuged at 2800g for 5 min at 4 ̊C and the cells recovered were dissolved in 0.04 volume of ice-cold TBF-II buffer and kept on ice for 45 min. 100 μl aliquots of these competent cells were used for transformation using the normal transformation protocol

TBF method for preparation of high competency cells

Quiagen/HiPura following the manufacturer's protocols

The rapid alkaline lysis method of plasmid isolation, as described by Sambrook and Russel (2001), was followed with minor modifications. Bacterial pellet from 3 ml of stationary-phase culture was resuspended in 200 μl of ice-cold solution I (50 mM glucose, 25 mM Tris-Cl pH 8.0, 10 mM EDTA pH 8.0 containing 1 mg/ml lysozyme) by vortexing. After 5 min incubation at room temperature, 400 μl of freshly prepared solution II (0.2 N NaOH, 1% SDS) was added and the contents were mixed, by gently inverting the tube several times. This was followed by the addition of 300 μl of ice-cold solution III (5 M potassium acetate, pH 4.8) and gentle mixing. The tube was incubated on ice for 5 min and centrifuged at 20,0000g for 15 min at 4°C. The clear supernatant was removed into a fresh tube and, if required, was extracted with an equal volume of phenol:chloroform mixture. The supernatant was precipitated with either two volumes of cold 95% ethanol or 0.6 volumes of isopropanol at room temperature for 30 min. The nucleic acids were pelleted by centrifugation, washed with 70% ethanol, vacuum dried, and dissolved in appropriate volume of TE buffer. If required, the sample was treated for 30 min with DNase free RNase at a final concentration of 20 μg/ml. The plasmid DNA was checked on a 0.8% agarose gel and stored at −20°. The plasmid DNA thus isolated was suitable for procedures such as restriction digestion, ligation, and preparation of radiolabeled probes. Plasmid isolation was also done with any of the commercially available kits from

Isolation of plasmid DNA

dependent transcription termination within the untranslated region of trpE. Anthranilate is a precursor of tryptophan, which is the product of trpE-encoded anthranilate synthase. Therefore, in trpE(fs) strains, growth on minimal glucose plates supplemented with anthranilate (100 μg/ml) reflects transcriptional polarity relief at the trp locus, and the same was scored after streaking the relevant strains on such medium

The trpE9777 is a frameshift (fs) mutationconfers Trp auxotrophy and also polarity on the downstream trpDCBA genes in the operon due to premature Rho-

trpE(fs) assay

This test was therefore used for two purposes: (i) to distinguish relA+ from relA− strains, and (ii) as a qualitative measure of transcriptional polarity relief at the ilv locus. Growth in the presence of amino acids Serine, Methionine, and Glycine (SMG) was scored on glucose-minimal A plates supplemented with each of the amino acids at 100 μg/ml and compared with the growth on non-supplemented glucose-minimal A plates to score for SMG phenotype

The E. coli relA mutants exhibit SMG-sensitive (SMGS) phenotype i.e. growth-inhibition in the presence of Serine, Methionine and Glycine at 1 mM concentration each (Uzan and Danchin, 1978) and is proposed to be a consequence of transcriptional polarity exerted by a frameshift mutation in the ilvG gene on the expression of downstream genes of the ilvGMEDA operon (Lopes et al., 1989). It was observed in another study that the rho and nusG mutants that are defective for transcription termination conferred SMG-resistant (SMGR) phenotype in a relA1 strain (Harinarayanan and Gowrishankar, 2003)

SMG resistance

Plasmids constructed in this study

The bacteriophage P1kc was from our laboratory collection and is referred to as P1 throughout this thesis. Phage λcI857 was also from our laboratory collection. Other bacteriophages that were used in this study included the following: (i) λNK1098 carries a Tn10 transposon with a tertracycline (Tet) ressistance marker. (ii) λNK1324 carries a mini-Tn10 transposon Tn10dCm with a chloramphenicol (Cm)-resistance marker, Cmr. The lambda phage vectors above (Kleckner et al., 1991) were used to make random transposon insertions in the chromosome either for the purpose of insertional mutagenesis or for tagging antibiotic resistance markers to point mutations

Bacteriophages

Western blotting by adding 2X sample buffer as described in 3.2.B.19 and 3.2.B.20 respectively

Triton X-114 has a property of a low cloud point (23°C). At temperatures above the cloud point, detergent solutions separate into aqueous and detergent-enriched phases thus separating membrane proteins (detergent phase) from cytosolic ones (aqueous phase). Promastigotes in the logarithmic phase of growth were harvested and dead cells pelleted at 129 x g. 109 live promastigotes were resuspended into 10mL of 0.5% vI v Triton X-114 containing protease inhibitor cocktail. The cells were homogenized using a 30-40 strokes of dounce homogenizer. The homogenate was incubated on ice for 90 min with intermittent stirring. Following this insoluble material was pelleted by centrifugation at 3700 x g for 35 min at 4°C. The supernatant was incubated at 37°C for 2 hr., till layers separated well. The top aqueous layer was separated from the bottom detergent layer. Samples of both these were prepared for SD

Triton X-114 extraction of membrane proteins

First strand synthesis of mRNA into e-DNA was performed using First strand e-DNA synthesis kit from Invitrogen (Carlsbad, CA) following manufacturer's protocol. Briefly, 4 !lg of total RNA was denatured at 65°C for 5 min in the presence of Oligo dT12-18 and dNTPs and then cooled on ice for 1 min. DTT, MgCb and RNaseOUT in lOX reverse transcriptase buffer added to the above mixture and incubated at 42°C for 2 min. 1!!L/reaction of the Superscript Reverse Transcriptase enzyme was added to the denatured RNA and incubated at 42°C for 50 min. The enzyme was denatured by heating at 70°C for 15 min. The reaction was completed with degradation of the complementary RNA strand by incubating with RNase H for 20 min at 37°C. The DNA preparation was stored at -20°C.

First strand synthesis by reverse transcription

Murine macrophage cell line J774A.1 (ATCC no. TIB-67) was maintained in phenol red free DMEM supplemented with 10% heat inactivated (45 min at 65°C) foetal bovine serum at 37°C in 5% C02 and 95% air. The cultures were sub-cultured every three days or at the attainment of 80 % confluency.

In vitro J774A.l murine macrophage cultur

and a colourless upper aqueous phase. The upper aqueous phase in which RNA existsexclusively, was transferred to a fresh microfuge tube and RNA was precipitated byadding 0.5 ml of isopropyl alcohol for each ml of Trizol used. Samples were incubatedat 15 to 30ºC for 10-min and centrifuged at 12000 rpm for 10-min at 4ºC. RNA formeda gel like precipitate at the bottom of the tube. Supernatant was removed and RNA waswashed with 75% ethanol (by adding 1 ml of ethanol per ml of Trizolemployed). RNAcould be stored after this step in –20 or –70ºC for more than a year. RNA pellet was airdried for 15-to 30-min following which it was dissolved in nuclease free water. Theconcentrations and purity of RNA samples were determined spectroscopically as wellas by visual inspection on formaldehyde-agarose gel in MOPS buffer (Goodet al., 1996). Before loading onto the gel, RNA was mixed with loading buffer and heated at90ºC for 3-min

For isolation of RNA, cells were grown in minimal A medium supplemented with 0.2%glucose upto A600of 0.6. Cells were harvested by centrifugation and total RNA wasisolated by using Trizol (Invitrogen) according to manufacturer’s instructions. 1 ml ofTrizol was used to lyse cells equivalent of approximately 4 ml of overnight culture.Homogeneous lysis was achieved by gentle pipetting repeatedly. The homogenized samples were incubated at room temperature for 5-min to permit complete dissociationof nucleoprotein particles. Following homogenization, 0.2 ml of chloroform for each 1ml Trizol reagent was added and vigorously shaken with hand for 15-sec and incubatedfurther for 3-min at RT. It was then centrifuged at 12000 rpm for 10-min at 4ºC, whichseparates out the homogenate into lower phenol chloroform phase (red), an interphase

Isolation of total cellular RNA

Protein concentrations were estimated by the method of Bradford (1976). The A595wasmeasured after complexation with Bradford reagent. Bovine serum albumin was usedas standard against whichthe unknown protein concentrations were estimated

Protein estimation

Typically 200-300 ng of DNA was used in each ligation reaction. The ratio of vector toinsert was maintained between 1:3 to 1:5 for cohesive end ligation and 1:1 for blunt endligation. The reaction was generally performed in 10 μl volume containing ligationbuffer (provided by the manufacturer) and 0.05 Weiss unit of T4-DNA ligase, at 16ºCfor 14-to 16-hrs. On using the rapid ligation kitfrom Fermentas, incubation was at 22ºC for 1-2 hrs

Ligation of DNA

PCR products were purified using the PCR Purification Kit (Qiagen) as per the manufacturer's instructions

DNA fragments to be used for specific purposes like ligation or radioactive labeling were eluted from the agarose gel after electrophoresis. The gel piece containing thedesired band was sliced out from the gel and the DNA was purified using commerciallyavailable purification kits for this purpose. The efficiency of elution was determined bychecking a small aliquot of DNA sample on the gel

Purification of PCR products

Purification of DNA by gel elution

Around 0.5 to 1 μg of DNA was regularly used for each restriction digestion. 2to 5units of restriction enzyme were used in the total reaction volume of 20 μl containing 2μl of the corresponding buffer supplied at 10 X concentration by the manufacturer. Thereaction was incubated for 2 hrs at the temperature recommended by the manufacturer.The DNA fragments were visualised by ethidium bromide staining after electrophoresison a 0.8 to 1% agarose gels. Commercially available DNA size markers were run alongwith the digestion samples to compare with and to estimate the sizes of the restrictionfragments

Restriction enzyme digestion and analysis

TheDNA samples were mixed with appropriate volumes of 6 X loading dye (0.25%bromophenol blue and 0.25% xylene cyanol and 30% glycerol in water) and subjectedto electrophoresis through 0.8 to 1 % agarose gel in TAE buffer. The gel was stained in1 μg/ml ethidium bromide solution for 15-min at room temperature and visualised byfluorescence under UV-light in a UV-transilluminator

Agarose gel electrophoresis

werethen recovered by centrifugation at 12,000 rpm for 30-min. The pellet was washed oncewith 70% ethanol, air-dried and re-suspended in 100 μl of TE-buffer. It was treatedwith RNase at a concentration of 20 μg/ml by incubating at 37ºC for 1-hr. It was furtherextracted with an equal volume of phenol:chloroform mixture followed bychloroform:isoamyl alcohol (24:1) mixture. After centrifugation, the clear supernatantwas used for recovering the nucleic acids. The nucleic acids were precipitated with 200μl of alcohol in presence of 0.3 M sodium acetate (Sambrook and Russell, 2001). In casewhere high purity plasmid preparations are required (DNA sequencing) the plasmidisolation was carried out with the commercially available kits following themanufacturer’s instruction. Plasmids were observed on 1% agarose gel

1.5 ml of stationary phase culture wascentrifuged and cell pellet was re-suspended in 567 μl of TE buffer. To this 30 μl of10% SDS, and 3 μl of proteinase K (20 mg/ml) were added in that order and the cellsuspension was mixed and incubated at 37ºC for 1-hr. When the suspension was clear, 100 μl of 5 M NaCl was added and thoroughly mixed followed by the addition of 80 μlCTAB/NaCl (10% cetyl trimethyl ammonium bromide in 7 M NaCl). The suspensionwas incubated at 65ºC for 10-min, brought to room temperature and extracted with anequal volume (780 μl) of chloroform isoamyl alcohol (24:1), and aqueous phasetransferred to fresh tube. The aqueous phase was further extracted successively, firstwith phenol:chloroform:isoamyl alcohol (25:24:1) and then with chloroform isoamylalcohol (24:1). DNA was precipitated fromthe clear supernatant by the addition of 0.6volumes of iso-propanol. The chromosomal DNA was either spooled out or pelleted atthis stage and washed with 70% ethanol air dried and dissolved in 100 μl of TE-buffer

Isolation of chromosomal DNA

1.5 ml of cells from an overnight culture waspelleted by centrifuging in cold (4ºC) for10-min at 6000 rpm. The cells were re-suspended in 200 μl solution I (50 mM glucose; 25 mM Tris-Cl, pH-8; 10 mM EDTA, pH-8) with vortexing. 400 μl of freshly preparedsolution II (0.2% NaOH, 1% SDS) was added and mixed by gently inverting the tubes.Subsequently, 300 μl of solution III (prepared by mixing 60 ml of 5 M CH3COOK,11.5 ml glacial acetic acid, 28 ml water) was added and the tubes were invertedrepeatedly and gently for homogeneous mixing followed by incubation for 5-min onice. After centrifuging at 12,000 rpm for 15-min, supernatant wasdecanted into a freshtube, an equal volume of iso-propanol was added, the precipitated nucleic acids

Isolation of plasmid DNA

Recombinant DNA techniques

The colonies to be tested were streaked on the surface of minimal A-glucose plates containing either 0.4-0.7 M NaCl with 1 mM glycine betaine, and incubated at 37oC. NaCl-tolerant strains grew toform single colonies in 36-60 hrs whereas NaCl-sensitive ones did not. As controls, MC4100 (WT) and other previously identified NaCl sensitive mutants were streakedfor comparison

NaCl-sensitivity testing

Competent cells for high efficiency transformations were prepared by a method ofInoue et al. (1990) with few modifications. An overnight culture of the strain (routinelyDH5α) was sub-cultured into fresh sterile LB-brothin 1:100 dilutions and grown at 18ºC to an A600of 0.55. The cells were harvested by centrifugation at 2500 rpm for 10-min at 4ºC. This was re-suspended in 0.4 volumes of INOUE buffer and incubated inice for 10 min. The cells were recovered by centrifugation at 2500 rpm at 4ºC for 10-min and finally re-suspended in 0.01 volume of the same buffer. Sterile DMSO wasadded to a final concentration of 7%. After incubating for 10-min in ice, the cells werealiquoted in 100 μl volumes, snap frozen in liquid nitrogen and stored at –70ºC

Preparation of high efficiency competent cells

the infection mixture was centrifuged, washed in 5 ml of citratebuffer and plated without phenotypic expression

To 2 ml of fresh overnight culture of recipient strain, 108pfu equivalent of phage lysatewas added and incubated at 37ºC without shaking for 15-min to facilitate phageadsorption. The un-adsorbed phage particles were removed by centrifugation at 4000rpm for 5-min and pellet of bacterial cells was re-suspended in 5 ml of LB-brothcontaining 20 mM sodium citrate to prevent further phage adsorption. This wasincubated for 30-min at 37ºC without shaking to allow the phenotypic expression of theantibiotic resistance gene. The mixture was then centrifuged, and the pellet was resuspendedin 0.3 ml citrate buffer. 100 μl aliquots were plated on appropriate antibioticcontaining plates supplemented with 2.5 mM sodium citrate. A control tube withoutaddition of P1 lysate was also processed in the same way. In the case of selection ofnutritional requirement,

Phage P1 transduction

bindingsite lie upstream of the MCS to ensure the high level expression of any genecloned in MCS. A stretch of hexa-histidine (His6)-encoding codons followed by stopcodon is incorporated downstream of MCS to give a C-terminally His6-taggedrecombinant protein (EMD Biosciences).6. pBAD18:It is an expression vector with a pMB9derived origin of replication and allows for tightly regulated expression of genes cloned under the PBADpromoter of the araBADoperon (Guzman et al.,1995). The vector also carries thearaCgene, encoding the positive and negative regulator of this promoter.7. pCP20: pSC101-based Ts replicon, chloramphenicol resistant, ampicillin resistant, for in vivoexpression of Flp recombinase (Datsenko and Wanner, 2000)Plasmid DNA preparations were routinely made from recAstrainDH5αandwerestored in 10 mM Tris-Cl (pH-8.0) with 1 mM EDTA at –20ºC. The plasmid constructsused in this study are given in Table 2.2.Table 2.2Plasmid constructs

The plasmid vectors used in this study were as follows:1.pCU22: It is a derivative of pUC19 used to prepare supercoiled DNA for in vitrotranscription where two strong phage fdtranscription terminators flank MCS. This ensures that the transcripts originated from vector based promoters will not interferewith the transcription from the cloned promoter and that the transcript originated fromthe cloned promoter will be terminated after the MCS (Ueguchi and Mizuno,1993).2.pMU575: It is an IncW-based, single-copy, trimethoprim resistance bearingpromoter probe vector. It carries its MCS upstream of a promoterless galK’-lacZfusion. This fusion has the first 58 codons of galKfused to the 8th codon oflacZ, andthe resultant hybrid polypeptide possesses functional β-Galactosidase activity(afterassembly as a tetramer). Translation of the hybrid gene is controlled by the ribosomebinding site of galK. There are stop codons in all the three reading frames between MCS and initiation codon of galKso that there is no interference caused bytranslational read-through from inserts cloned into MCS region. A strong pheRterminator located upstream of the MCS prevents read through from any vector-basedpromoter into the lacZgene (Andrews et al.,1991).3. pTrc99A:It is an expression vector with ColE1 origin of replication and ampicillin resistance marker. It provides IPTG dependent induction of the cloned gene (Amann et al., 1988)4. pCL1920: It is a pSC101-based, low-copy-number vector with spectinomycin and streptomycin resistance marker carrying the MCS in lacZαregion and henceprovides the advantage of screening the insertions using α-complementation (Lernerand Inouye,1990).5. pET21b: It is a ColE1-based, high-copy-number expression vector bearing ampicillinresistance marker. A strong T7 RNAP-recognised promoter and an efficient ribosome

Plasmids

For TEM, C. glabrata cells were digested with zymolyase 20T for 3 h at 30◦C, centrifuged at 1,000 g and washed with YPD medium. Cell fixation was performed as described for SEM and dehydrated samples were embedded in araldite 6005 resin. After complete polymerization at 80 ̊C for 72 h, ultra-thin (50-70 nm) sections were preparedwith a glass knife on Leica Ultra cut (UCT-GA-D/E-1/00)microtomeand mounted on copper grids. Aqueous uranyl acetate-stained and Reynolds lead citrate-counterstained samples were viewed under Hitachi H-7500 transmission electron microscope

Transmission electron microscopy

Cell wall β-glucan measurement was carried out as describedpreviously with some modifications(Kapteynet.al.,2001). Briefly, cell wall fractions were washed multiple times with 1 N NaCl. Washed cell walls were boiled twice in 50 mM Tris-HCl(pH 7.8) containing 2% SDS, 100 mM Na-EDTA and 40 mM β-mercaptoethanol for 5 min to remove non-covalently linked proteins and other contaminants. SDS-treated cell wall fraction was collected and rinsed thrice with water. For β-glucan isolation, cell wallswere extracted three times, each for 1 h, in 0.5 ml 3% NaOH at 75 ̊C and centrifuged at 1,200 g.All 3% NaOH supernatant fractions were saved for isolation of mannan as described below. 3% NaOH-extractable cell wall pelletwasneutralized twice in 100 mM Tris-HCl (pH 7.5) and once in 10 mM Tris-HCl (pH 7.5) and digested with 5 mg/ml zymolyase-20T in 10 mM Tris-HCl (pH 7.5) for 14-16 h at 37 ̊C. This treatment liberates approximately 90-95% glucose into the supernatant. Total glucan content in the cell wall was measured by estimating glucose from both the solubilised supernatant and zymolyase-20T insoluble pellet fractions with phenol-sulphuric acid carbohydrate estimation method using purified glucose as the standard