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  1. Last 7 days
    1. Riding over the hills, and eating their fill,7 lying a little too long; these things are, perhaps, enough to explain what happened. How­ever, that may be: they woke suddenly from a sleep they had never meant to take. The standing stone was cold, and it cast a long pale shadow. The sun was gleaming through the mist; north, south, and east, the fog was thick, cold and white. The air was silent, heavy and chill.The hobbits8 sprang to their feet in alarm, and ran to the western rim. They found that they were upon an island in the fog. Even as they looked out in dismay towards the setting sun, it sank before their eyes into a white sea, and a cold grey shadow sprang up in the East behind. The fog rolled up to the walls and rose above them, and as it mounted it bent over their heads until it became a roof. They felt as if a trap was closing about them. They packed up as quickly as their chilled fingers would work.Soon they were leading their ponies in single file9 over the rim and down the long northward slope of the hill, down into a foggy sea. As they went down the mist became colder and damper, and their hair hung lank and dripping on their foreheads. When they reached the bottom it was so cold that they halted and got out cloaks and hoods, which soon became bedewed with grey drops. Then, mounting their ponies, they went slowly on again. To prevent their getting separated and wandering in different directions they went in file, with Frodo leading. Suddenly Frodo saw a hopeful sign. On either side ahead a darkness began to loom through the mist; and he guessed that they were at last approaching the gap in the hills. 'Come on! Follow me!' he called back over his shoulder, and he hurried forward. His pony reared, and he fell off. When he looked back he found that he was alone: the others had not fol­lowed him.

      main body

    2. Riding over the hills, and eating their fill,7 lying a little too long; these things are, perhaps, enough to explain what happened. How­ever, that may be: they woke suddenly from a sleep they had never meant to take. The standing stone was cold, and it cast a long pale shadow. The sun was gleaming through the mist; north, south, and east, the fog was thick, cold and white. The air was silent, heavy and chill.The hobbits8 sprang to their feet in alarm, and ran to the western rim. They found that they were upon an island in the fog. Even as they looked out in dismay towards the setting sun, it sank before their eyes into a white sea, and a cold grey shadow sprang up in the East behind. The fog rolled up to the walls and rose above them, and as it mounted it bent over their heads until it became a roof. They felt as if a trap was closing about them. They packed up as quickly as their chilled fingers would work.Soon they were leading their ponies in single file9 over the rim and down the long northward slope of the hill, down into a foggy sea. As they went down the mist became colder and damper, and their hair hung lank and dripping on their foreheads. When they reached the bottom it was so cold that they halted and got out cloaks and hoods, which soon became bedewed with grey drops. Then, mounting their ponies, they went slowly on again. To prevent their getting separated and wandering in different directions they went in file, with Frodo leading. Suddenly Frodo saw a hopeful sign. On either side ahead a darkness began to loom through the mist; and he guessed that they were at last approaching the gap in the hills. 'Come on! Follow me!' he called back over his shoulder, and he hurried forward. His pony reared, and he fell off. When he looked back he found that he was alone: the others had not fol­lowed him.
  2. May 2019
    1. The clear cell-free supernatants were used as the source of crude recombinant xylanase.
    2. Quantitative screening for determination of xylanase in shake flask
    3. 2 mL of an overnight culture of E. coli cells was inoculated into 100 mL LB medium and incubated with vigorous shaking at 30 °C until A600 of 0.8 was reached. •Cells were collected in 50 mL plastic (Falcon) tubes, cooled for 15 min on ice and centrifuged in a pre-cooled centrifuge (4,000 rpm for 10 min at 4 °C). •The pellet was suspended in 20 mL of ice-cold 50 mM CaCl2-15% glycerol solution, maintained on ice for 15 min and centrifuged again at 4,000 rpm for 10 min at 4 °C. •Pellet was resuspended in 2 mL of ice-cold 50 mM CaCl2-15 % glycerol solution, kept on ice for 30 min and aliquoted in 400 μL in microcentrifuge tubes. These were stored at -80 °C until required.
    4. Preparation of calcium-competent cells
    5. Two hundred μL of alkaline-SDS solution was added to the above suspension, mixed by inverting the tubes up and down 3 times and incubated for 5 min at room temperature. ƒTo the above mixture, 250 μL of 3 M Na-acetate (pH 4.8) was added, mixed by inverting the tubes up and down 3 times, and centrifuged at 12,000 x g for 10 min. ƒThe supernatant was collected in another micro centrifuge tube (MCT), 200 μL of phenol:chloroform solution was added, inverted two times and centrifuged at 12, 000 x g for 8 min at room temperature. ƒThe aqueous phase was transferred to new tubes and 500 μL of chilled (-20 °C) ethanol (96 %) was added. ƒThe tubes were centrifuged at 13,000 x g for 25 min at 4 °C, supernatant discarded and pellet dried for 15 min at room temperature. ƒThe pellet was washed with 500 μL of chilled 70 % (v/v) ethanol and centrifuged at 13, 000 rpm for 4 min at 4 °C. ƒThe pellet was dried at room temperature and dissolved in 50 μL of 1X TE buffer (pH 8.0) containing RNase and stored at -20 °C till further use.
    6. The cells of E. coli DH10B having p18GFP vector were cultivated for overnight at 37 °C in LB medium containing ampicillin (100 μg mL-1). ƒThe E. coli culture having p18 GFP vector (~1.5 mL) was taken in Eppendorf tubes and centrifuged at 10, 000 x g for 5 min. ƒThe pellet was homogenized by vortex mixing in 100 μL of homogenizing solution
    7. Plasmid isolation from miniprep method
    8. An attempt was made to study the effect of storage of DNA extracts on DNA yield and purity. The DNA extracts were centrifuged and the supernatants were dispensed into 2.0 mL Eppendorf tubes and stored at -20 oC for a month. DNA precipitation and its quantification were carried out at a week intervals.
    9. Effect of storage on soil/sediment DNA extracts
    10. Attempts have been made to amplify the signature sequences of bacterial, archaeal and fungal specific regions by using respective sets of primers shown in Table2.2. The reactions were carried out in 50 μL reaction mixtures in a Thermal Cycler (Bio-Rad, USA) using respective primers (Table 2.2). The PCR conditions were optimized as follows: for Bacterial 16S rDNA, initial denaturation of 3 min at 94 oC followed by 30 cycles of 30 sec at 93 oC, 60 sec at 55 oC and 90 sec at 72 oC; Archaeal 16S rDNA, 5 min at 95 oC, 35 cycles of 50 sec at 94 oC, 60 sec at 62 oC and 60 sec at 72 oC; fungal specific ITS regions, 3 min at 95 °C, 30 cycles of 60 sec at 94 °C, 56 °C at 45 sec and 50 sec at 72 °C. Final extension time was 7 min at 72 °C in all PCR runs. Amplifications were visualized on 1.2 % w/v agarose gels
    11. PCR amplification of microbial population
    12. Purity of the DNA extracted from various environmental samples was confirmed by subjecting the extracted DNA to restriction digestion. DNA was digested with Sau3AI (New England Biolabs). One μg of metagenomic DNA in 20 μL reaction mixture was treated with 0.5 U of Sau3AI and incubated at 37 °Cfor 10 min. The reaction was terminated at 80 °C for 20 min and the digested DNA was fractionated on 1.2 % (w/v) agarose gel.
    13. Restriction digestion
    14. VALIDATION OF METAGENOME OBTAINED BY THE PROTOCOL DEVELOPED IN THIS INVESTIGATION
    15. as well as commercial methods (MN kit, Germany; Mo-Bio kit, CA, USA; Zymo soil DNA kit, CA, USA) according to the manufacturer’s protocols and compared in terms of DNA yield and purity.
    16. The soil DNA from Pantnagar and Lonar soil samples were also extracted by various manual (Desai and Madamwar, 2007; Agarwal et al., 2001; Yamamoto et al., 1998
    17. Comparison of yield and purity of crude DNA
    18. Various strains of Escherchia coli (DH5α, XL1Blue, DH10B) were used as hosts for the propagation of recombinant vectors. In addition, Bacillus subtilis was used as a host for the expression of xylanase gene from the recombinant vector pWHMxyl. Different vectors used in this investigation are listed in
    19. BACTERIAL STRAINS
    1. Parasites from synchronized cultures were harvested at different time points of growth to obtain ring, trophozoite and schizont stage parasites. RNA was isolated from these stages by using RNAeasy kit (Qiagen) following manufacturer's protocol. The concentration of total RNA was determined by measuring the absorbance at 260 nm. Purity of nucleic acid preparations were determined by calculating OD26onm / OD28onm ratio, a value of near ~ 1.6-1.8 was taken as a standard of purity. To get stage specific cDNA from RNA, reverse transcription was performed using RT-PCR kit (Invitrogen) that contained random hexamers. Subsequently, the gene of interest was amplified using gene specific primers
    2. Isolation of the parasite RNA
    3. Human 0+ or AB+ RBC was obtained from a donor and mixed with heparin (50 units/ml of blood) and centrifuged at 500 g for 10 min with minimu1p. de-acceleration. The supernatant was removed carefully and the pelleted RBCS were washed 3 times with RPMI 1640 to remove serum and buffy coat. Equal amount of RPMI 1640 media was added to packed RBC volume to achieve 50% hem~tocrit and stored at 4°C till: further use
    4. Preparation of RBCsfor culture
    1. proteins of interest were pooled and 1 mM TCEP was added. The protein of interest was collected and stored at -80°C for further use after adding 1 mM TCEP.
    2. The mutant proteins were expressed and purified analogous to wild type RaPt protein. Mutant clones pAC36, pAC50 and pAC38 were transformed in BL21 strain of E. coli. Analogous to the wild type RGPL protein the cells harbouring the mutant expression plasmids were cultured at 37°C to an O.D6oonm of 0.6 and uninduced at 30°C for 6-8 hrs. After harvesting, the cells were resuspended in lysis buffer (1 00 mM phosphate pH: 7 .0, I 0% glycerol) and disrupted using french press at 1100 psi pressure. Cell debris was removed by centrifugation at 50,000 g for 40 min at 4°C. 0. 75 ml L.1 of Ni2+ -NT A slurry was added to the supernatant and incubated at 4°C for 1 hr. This suspension was loaded onto a column working under gravity flow. The resin was washed with wash buffer (100 mM phosphate pH: 7.0, 10% glycerol and 5 mM imidazole) till all unbound proteins were removed. The protein was eluted using elution buffers containing increasing concentration of imidazole. Fractions containing the
    3. Expression and purification of RcPL mutant proteins
    1. Hair from the skin overlying the left and right dorsal flanks were removed using electrically operated razor. The skin overlying the abdomen was sterilized by wiping with 70% ethanol. Ketamine (1 00 mg/kg) and xylocaine (2%) (20 mg/kg) were mixed and administered intraperitoneally. The mice were returned to the cage and the onset of anesthetic effect was monitored. The mice were considered to be in surgical anesthesia when there was loss of palpebral reflex, righting reflex, and toe pinch reflex. Respiratory rate and heart rate were monitored continuously.
    2. General anesthesia:
    3. treatment were harvested by centrifugation at 250 x g for 5 min following which they were resuspended in 1x PBS (pH 7.5). PI was added at a final concentration of 1 J.tg/mL and incubated for 5 minutes following which the cells were pelleted by centrifugation and washed once with PBS. These cells were analyzed for uptake of PI by either flow cytometry in FL2 channel (570 nm) or by fluorescence microscopy using a G2A filter block.
    4. Propidium iodide (PI) is a DNA intercalating fluorescent dye which is excluded by viable cells with intact membranes, however, dead and dying cells with damaged membranes take up the dye. To assess viability, cells after appropriate
    5. population was determined by analyzing cells immunostained with an antibody against CD14 conjugated to FITC and the purity obtained was approximately 85% monocytes, the remaining being lymphocytes. The monocytes were further cultured in the presence of human AB serum for 7 days to allow differentiation to macrophages. At the end of 7 days post-isolation, greater than 95% of cells in culture are monocytes, with the majority of lymphocytes undergoing neglect induced death.
    6. Peripheral blood (30 mL) was collected by venipuncture from healthy male volunteers after obtaining an informed consent and in accordance to the regulations of the Institutional Human Ethics Committee (National Institute of Immunology, New Delhi, India). The peripheral blood mononuclear cell (PBMC) population was isolated by density gradient centrifugation using Histopaque 1077, where, human whole blood was layered on Histopaque 1077 and centrifuged at 400 x g for 35 min at 25°C. The mononuclear cell population was isolated from the plasma-histopaque interface, and the monocytes were further purified by washing off the non-adherent cells after incubating the total PBMC for 1 h at 3 7°C. The homogeneity
    7. Peripheral blood monocy.te isolation and macrophage differentiation
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    1. include SORTING, that sorts, packs and assesses the quality of the experimentally measured diffraction data, and is run in the first step. The program TABLING calculates the continuous Fourier coefficients from the model placed in the artificial cell. The cross-rotation function is carried out by the program ROTING, which uses Crowther's algorithm (Crowther, 1972). TRAING is used to calculate the translation function. Finally FITING is used to refine the orientational and positional parameters of the molecule corresponding to the potential solutions, as a rigid body.
    2. To carry out MR, the AMoRe package can be used. AMoRe constitutes a suite of programs written by Jorge Navaza (Navaza, 1993; Navaza, 1994). These
    3. Automated molecular replacement package (AMoRe)
    4. mounted on goniometer heads, which were in turn fixed on the oscillator dial of the image plate. However since our crystals suffered significant radiation damage at room temperature we decided to attempt cryo-crystallography and collected data at low temperature. Radiation damage to protein crystals is greatly reduced at lower than room temperatures (D. J. Haas, 1970; Low et al., 1966). Primary radiation damage is largely caused by interactions between the molecules in the crystal and the beam. This energy is dissipated in at least two ways; it produces thermal vibrations (heat) and it provides the necessary energy to break bonds between atoms in the molecules. Secondary damage to the crystals is caused by the diffusion of reactive radicals produced due to damage to the protein. This diffusion is aided by the presence of thermal energy. At cryo-temperature of around 1 OOK, thermal damage is limited and also the reactive products are immobilized and do not cause extensive secondary damage in areas of the crystal which are not exposed to the beam (Garman, 1999). For low temperature data collection, the crystals were initially soaked in a cryo-protectant, which was basically the mixture of the mother liquor and antifreeze. We added 30% glycerol to our mother liquor, in which the crystals were soaked from between 1 to 5 minutes to achieve cryo-protection. The crystals were then picked up using a 20Jl nylon loop, which was immediately flash frozen in a stream of nitrogen at 120k at a flow rate of 6 liters/min (Oxford cryo-systems). The crystals were centered in the beam using the two arcs and translations on the goniometer head and by viewing the crystal on the monitor of the attached CCD camera. The collimation, crystal to detector distance, oscillation angle and the exposure time per frame were optimized after a few trial frames in each case.
    5. Data collection for macromolecular crystallography involves exposure of the crystal to X-rays and recording the intensities of the resultant diffraction patterns. Rapid advances in this field have made available sophisticated electronic detectors like the Image plate detector, high power X-ray generators and synchrotrons. Successful data set collection is followed by data processing to extract the hkl indices with corresponding intensities, along with an estimate of the errors involved. At the core of the Image Plate detector is an amorphous thin film made of Barium, Europium and Bromium. This material that is coated on to a motorized plate absorbs X-rays to form F-centers. These F-centers are the regions that store photon energy as excited electrons. After the exposure is complete the plate is read by a He-Ne (2eV) red laser. Absorption of photons induces excited electrons to return to ground state with the emission of blue light (4eV) which is quantitatively read by a photomultiplier. Exposing it to intense white radiation erases the plate. While the basic technology behind the image plates remains the same, improvements in electronics and computers has led to greater automation and faster data collection cycles. The X-ray intensity data for various Fab-peptide complexes of 36-65 were collected on the Mar345dtb, installed on a rotating anode X-ray source (RIGAKU, Japan) operating at 50kV and 1 OOmA (CuKa. radiation) with Osmic mirrors (RIGAKU, Japan). While the Mar225 image plate installed at BM14 (ESRF, Grenoble, France) was used to record three Fab-peptide complexes of BBE6.12H3. Data for antigen free BBE6.12 H3 Fab and its complex with Ppy peptide was recorded on Mar345dtb image plate (Mar research, Germany), installed on the home source. For data collection at room temperature, the crystals were mounted in 0.5 mm quartz capillary tube along with some mother liquor. The capillaries were then
    6. X-ray intensity data collection
    7. solution was injected into the HPLC. A salt gradient of 0 to 0.2 M NaCl over a period of 120 minutes was run and fractions for each peak, as detected by measurement of UV absorbance at 220nm, were collected. An aliquot of each fraction was subjected to acetone precipitation and the obtained precipitate was analyzed on SDS-PAGE to ascertain which fraction corresponds to IgG. The IgG fractions from different runs were pooled and concentrated to -1 mg/ml which was then dialyzed against the digestion buffer (0.15 M NaCl, O.lM Tris-Cl, pH 7.1).
    8. The collected ascitic fluid was centrifuged to remove cell debris and fat. Mouse monoclonal ascites, was filtered through glass wool to remove lipid like material left over after centrifugation. The supernatant was then subjected to (Nr4)zS04 fractionation. Saturated (Nlit)zS04 solution (SAS) at pH 7.0 was gradually added to the ascites in an ice bath with continuous stirring till a concentration of 40% (v/v) was achieved. The mixture thus, obtained was centrifuged to get the protein pellet and the pellet was re-suspended in buffer (0.01 M Tris-Cl, pH 8.5). The crude antibody solution obtained from ammonium sulfate fractionation was dialyzed against the wash buffer (0.0 1 \1 Tris-Cl, pH 8.5) and then subjected to ion-exchange chromatography using 5PW-DEAE (60x150 mm) column on a Waters3000 preparative HPLC (Waters, L:SA), to purify IgG. All solutions used during chromatography were filtered (0.451-lm) and then degassed. Following equilibration of the column with wash buffer, a 2 ml aliquot of the crude antibody
    9. Antibody purification
    10. The peptides were purified using reverse-phase HPLC. Binding occurs through hydrophobic interactions between peptide and the column support. Decreasing the ionic nature or increasing the hydrophobicity of eluant such that it competes with peptide for hydrophobic groups on the column accomplishes elution. Crude peptides were purified on a Waters Xbridge™ BEH 130 reverse-phase C 18 column ( 19x250mm, 1 O~m, spherical) on a semi-preparative HPLC system (Waters, USA) using a linear gradient of 0.1% trifluoroacetic acid (Sigma) and acetonitrile (Merck). The absorption was monitored at 214nm. After purification, the peptides were lyophilized. The purity of the peptide was checked by determination of molecular mass using single quadruple mass analyzer (Fisons Instruments, UK). Circular dichroism studies were performed on the peptides to determine secondary structural state, if any, in solution. 50 )lM peptide concentrations in water were used and data was accumulated for 1 0 scans at a temperature of 10° C using a JASCO 710 spectropolarimeter. 1.0 nm bandwidth and O.lnm resolutions were used, with the sample being placed in a 2mm path length cuvette.
    11. Peptide purification
    12. All the peptides used in this study were synthesized by solid phase method on an automated peptide synthesizer (Applied Biosystems, Model 431A), using F-moc (9-fluorenylmethyloxycarbonyl) chemistry on a p-hydroxymethyl phenoxymethyl polystyrene resin (Nova Biochem). For the peptide synthesis, 0.1 mmol of the resin was used and deprotected using 20% piperidine in N-methyl-pyrrolidone (NMP). Subsequently 0.5nmol of the first amino acid was added and coupling was performed usmg DCC-HoBt (dicyclohexylcarbodiimide-hydroxybezotriazole) ester formation method. All other amino acids were coupled by DCC ester coupling. Amino acids and solutions required for peptide synthesis were procured from Nova Biochem and Applied Biosystems, respectively. After completion of synthesis, deprotection was carried out in 20% piperidine/DMF. Finally, the resin was shrunk using ether and dried under vacuum for a minimum of four hours. The cleavage was performed in dark using 94% TF A, 5% anisole, EDT and water accompanied by continuous stirring for two hours. The resin was then filtered and washed with DCM and the solution was evaporated on a rotary evaporator (Buchi, Switzerland) till only a small quantity of DCM/cleavage mixture is left. Cold anhydrous diethyl ether was added to the filtrate to aid in the separation of scavengers from the mixture. The peptides were then extracted with water using a separating funnel. Extraction was followed by evaporation of residual diethyl ether on the rotary evaporator. Total aqueous layer was then frozen as a thin film and lyophilized.
    13. Procedure for peptide synthesis
    14. the peptide and the resin is cleaved using trifluoroacetic acid (TF A) to release the polypeptide.
    15. Solid phase peptide synthesis was introduced by Merrifield in 1963, and includes successive assembly of amino acid residues to build the peptide chain on an insoluble polymeric support. The C-terminal residue, with protected a-amino and side chain functional groups, is chemically attached to the insoluble resin via a flexible linker. Subsequently, in the coupling step, the a-amino group is deprotected and the next protected amino acid is reacted with the resin-bound first amino acid. This cycle of deprotection and coupling is repeated till the complete peptide chain is synthesized. After the synthesis of the desired peptide, the anchoring bond between
    16. Peptide synthesis
    1. Competent cells of the different strains of E. coli were prepared as described by Cohen et al. ( 1972). An LB-agar plate was streaked with the desired strain, and a single colony was inoculated into 5 ml of LB medium. The culture was grown at 37 °C with continuous shaking at 200 rpm for 6 hours. A small inoculum from this culture was used to start a I 00 ml culture in the same medium. At an OD600 of 0.3-0.4, when the culture reached early Jog phase, it was chilled on ice for 30 min .. and centrifuged at 2000 g for 15 min. at 4 °C. The pellet was gently resuspended in 50 ml of chilled 50 mM calcium chloride and incubated on ice for 60 min. The cell suspension was centrifuged at 2000 g for 15 min. at 4 °C, and the pellet was gently resuspended in 5 ml of chilled 50 mM calcium chloride containing 20% glycerol. The competent cell suspension was immediately aliquoted in prechilled vials and stored at -70 °C.
    2. Preparation of Competent Bacterial Cells
    3. Cancer cell lines of human origin, HUT102, T-cell leukemia; K562, erythroleukemia; COL0205; colon adenocarcinoma; MCF7, breast adenocarcinoma; A431, epidermoid carcinoma; A549, lung carcinoma and HeLa, cervical carcinoma and J774A.I, mouse monocyte-macrophage; and L929, mouse fibroblast were obtained from ATCC. All the cell Jines were maintained in RPMI 1640 supplemented with antibiotic antimycotic solution, 2 mM glutamine and I 0% heat inactivated foetal calf serum (Life Technologies, Maryland, USA). E. coli strain DH5a was used for DNA manipulation, cloning and mutagenesis. Strains CJ236 and DH5aF' were used. for oligonucleotide mediated site directed mutagenesis. BL21 (A.DE3) strain containing T7 RNA polymerase gene under the control of lac promoter, was used for expression of the recombinant proteins.
    4. Cell Lines and Bacterial Strains
    1. administered at two sites. In addition, the primary dose also contained 500 J..Lg of SPLPS as an additional adjuvant. This was followed by 2 booster at 4 weekly intervals with an equal amount of r-bZP3-DT conjugate.
    2. Female New Zealand White rabbit (Small Animal Facility, National Institute of Immunology, New Delhi, India), 6 months of age was immunized intramuscularly with r-bZP3-DT conjugate equivalent to 125 Jlg of r-bZP3 (expressed in SG13009[pREP4] cells) in 0.9% saline emulsified with Squalene and Arlacel "A" in a ratio of 4: I and
    3. Immunization of Rabbit
    4. 650C in 0.2X SSC, 0.1 % SDS for I 0 min. The membrane was wrapped in Saran wrap and exposed to an X-ray film. The colonies that were positive by colony hybridization were inoculated in a 3 ml culture and used for preparing DNA for analysis by restriction digestion and Southern blotting. The digested DNA was resolved on a 0.8% agarose gel as described above. The gel was soaked in 4 volumes of denaturing solution (1.5 M NaCI and 0.5 M NaOH) for 1 h at RT with shaking followed by neutralization (1 M Tris HCI, pH 8 and 1.5 M NaCI) for 1 hat RT. The DNA was transferred to a Nylon membrane, UV crosslinked and hybridized with the full length 32p labeled· bZP3 probe as described above.
    5. The ligation mixture was used for transformation of DH5a cells as described earlier. Transformed bacterial colonies growing on LB Amp plates were screened by colony hybridization. Briefly, colonies were grown for 6-8 h on a Nylon membrane placed on a LB Amp plate. The colonies were lysed by placing the membrane on a Whatman® 3MM paper soaked in I 0% SDS for 3 min, followed by treatment with denaturing solution (0.5 N NaOH, I.5 M Nael) for 5 min and neutralization solution (0.5 M Tris Hel pH 8, 1.5 . M Nael) for 5 min in the same manner. The membrane was dried, UV cross linked (Ultraviolet crosslinker, Amersham) and processed for prehybridization and hybridization. Stocks of 20X sse (174 giL NaCI, 88.2 giL sodium citrate, pH 7.0) and 50X Denhardt's (I% ficoll, I% PVP, I% BSA) were prepared. The membrane was prehybridized for 4-6 h in the prehybridization solution (5X SSe, 5X Denhardt's, 0.5% SDS, I 0 J..Lg/ml sheared and denatured salmon sperm DNA). The bZP3 DNA was labelled using the Multiprime DNA labeling system using 50 ng of purified bZP3 DNA. For hybridization with the probe, I o6 cpm/ml of the denatured 32p labeled bZP3 probe was added to the prehybridization solution and incubation was further carried out for I4-I6 h. For removing the non specifically bound probe, the membrane was washed successively at RT in 2X sse for 10 min, at 55°e in 0.2X sse, 0.1% SDS for 10 min and finally at
    6. Screening of the Recombinant Transfer Vector
    7. Peptide antisera were generated in the laboratory against peptides PI, 23-45 aa residues with an extra lysine at the N-terminus (KQPFWLLQGGASRAETSVQPVL VE), P2, 300-322 aa residues (CSFSKSSNSWFPVEGPADICQCC) and P3, 324-347 aa residues (KGDCGTPSHSRRQPHVVSQWSRSA) corresponding to bZP3 precursor protein in rabbits and were used to determine their reactivity with the r-bZP3 protein expressed in E. coli in an enzyme linked immunosorbent assay (ELISA). Microtitration plates were coated with 200 ng of r-bZP3 or I J.tg/well of the peptide. HRPO conjugated goat anti-rabbit Ig at I :5000 dilution was used as revealing Ab.
    8. Reactivity with Anti-peptide Sera
    9. E. coli strains deficient in specific proteases were used to study their influence on the expression of r-bZP3. BL21 (DE3) and BL21 (pLysS) deficient in ompT and ion proteases and DF5 carrying a targeted mutation of the ptr gene, were transformed with the pQE-bZP3 plasmid. Colonies obtained were grown 0/N and subcultured next morning and grown till A6oo=0.7. Cultures were then induced with 0.5 mM IPTG for 3 h. Harvested cells were checked by SDS-P AGE and immunoblotting.
    10. Expression of r-bZP3 in Different E. coli Strains
    11. Conditions for expression of r-bZP3 in SG 13009[pREP4] cells transformed with the pQE-bZP3 plasmid were standardized. Cells were grown till A6oo=0.7 and induced with different concentrations of IPTG (0.5, 1, 2, or 4 mM) for a constant time period (3h) or induced with a 0.5 mM IPTG for different time periods (0, 1, 2, 3 or 5 h). Cells were harvested and analyzed by SDS-PAGE and immunoblot as described above.
    12. Standardization of Expression Conditions
    13. A I 00 ml culture was grown and induced according to the procedure mentioned above. The culture was divided into 2 aliquots and cells were pelleted down. For cytosolic localization, one pellet was resuspended in 5 ml of sonication buffer (50 mM Na-phosphate, pH 7.8, 300 mM NaCI). The sample was frozen and then thawed in ice-water and cells lysed by brief sonication. The sample was centrifuged at I 0,000 g for 20 min. The soup and the pellet represent the soluble and insoluble components of the cell pellet. In order to check for periplasmic localization, the 2nd aliquot of cells was resuspended in I 0 ml of hypertonic solution (30 mM Tris, pH 8, 20% sucrose, 1 mM EDT A) and incubated at RT for 10 min with shaking. Cells were centrifuged at 8,000 g for 10 min. The pellet was subjected to osmotic shock in 5 mM MgS04. Cells were stirred for 10 min in an ice water bath, centrifuged at 8000 g at 4°C for I 0 min. The soup collected represented the periplasmic fraction. The fractions were analyzed by 0.1% SDS-1 0% PAGE and Western blotting as described above.
    14. Intracellular Localization
    15. The cell pellet obtained from l ml culture was solubilized by boiling for 5 min in 100 J..Ll of 2X sample buffer (0.0625 M Tris, pH 6.8, 2% SDS, 10% glycerol, 5% BME and 0.001% bromophenol blue) and the proteins were resolved on a 0.1% SDS-10% PAGE (Laemmeli, 1970). The gel was stained with Coomassie brilliant blue for staining total cellular proteins. For immunoblotting, the proteins were electrophoretically transferred to 0.45 J..Lm nitrocellulose membrane overnight at a constant voltage of 15 V in Tris glycine buffer with 20% methanol (Towbin et al., 1979). Nonspecific sites on the membrane were blocked by incubation with 5% BSA in 50 mM phosphate buffered saline (PBS), pH 7 .4, for 1 h followed by 3 washes (15 min each) with PBS containing 0.1% Tween-20 (PBST). For detection of bZP3, a murine monoclonal antibody (MAb), MA-451, generated against the pZP3P and recognizing a cross reactive epitope (166-171 aa residues) within the bonnet sequence was used (Afzalpurkar and Gupta, 1997). The membrane was incubated for 1 h with a 1 :5 dilution of MA-451 culture supernatant, followed by 3 washes in PBST. Horseradish-peroxidase (HRPO) conjugated goat anti-mouse immunoglobulin (lg) was used to reveal bound Ab. Colour was developed with 0.6% (w/v) 4-chloronaphthol in 50 mM PBS, pH 7.4, containing 25% methanol and 0.06% H202. The reaction was stopped by washing the membrane with PBS.
    16. SDS-PAGE and Immunoblot
    17. The pQE-bZP3 plasmid was transformed in Ml5[pREP4] and SG13009[pREP4] bacterial strains provided with the kit. The transformed colonies were analyzed for expression. A single transformed colony was inoculated and grown overnight at 37oc in 1 ml of LB containing 100 J..Lg/ml of ampicillin and 25 J..Lg/ml of kanamycin. Cells were subcultured 1:10, next morning and grown until cell density reached an A600 of approximately 0.6-0.7. The cells were further grown in the presence of isopropyl P-D thiogalactopyranoside (IPTG) to induce expression of the fusion protein under the T -5 promoter. The cells were collected by centrifugation at 13,000 g for 60 sec and the resulting pellet was stored at -700C until used.
    18. Expression in MJS[pREP4] and SG13009[pREP4] E. coli Strains
    19. vector, under the phage T7 promoter, in BL21 (DE3) cells, and under the T5 phage promoter, in the pQE30 vector for expression in SG13009[pREP4] and M15[pREP4] cell strains. For cloning in pRSET B, the full length bZP3 initially subcloned in the pBacPAK8 vector at the Kpn I and Sac I sites was released after digestion with Kpn I and EcoR I and cloned in a similarly restricted pRSETB vector inframe with an N-terminal His6 tag. For cloning in the pQE30 vector, the pBacPAK8 carrying the full length bZP3 was initially digested with Not I, filled in with Klenow and then digested with Kpn I. The purified bZP3 fragment was then cloned in the vector digested with Kpn I and Sma I in frame with an N-terminal His6 tag. Though transformants positive for the bZP3 insert in the right reading frame were recovered, no expression could be detected by SDS-PAGE or immunoblots in either case. An alternate strategy was then devised in which an internal fragment of the gene, excluding the signal sequence and the transmembrane-like domain, following the putative furin cleavage site, was amplified by PCR using the forward primer 5'-CGGGATCCCAACCCTTCTGGCTCTTG-3' incorporating a BamH I site and the reverse primer 5'-CCGAGCTCAGAAGCAGACCTGGACCA-3' incorporating a Sac I site. The PCR was done in a 50 J!l volume using 50 pM of each primer and Vent polymerase for extension. The pBluescript-bZP3 (1 0 ng) having a full length bZP3 insert was used as the template and was initially denatured at 95°C for 10 min. Amplification was carried out for 35 cycles of denaturation at 95°C for 2 min, primer annealing at 600C for 2 min and extension at 72°C for 3 min followed by a final extension at 72oc .for 15 min. The amplified bZP3 fragment was digested with BamH I and Sac I and cloned in frame downstream of a His6 tag under the T5 promoter-lac operator control in the pQE30 vector. The authenticity of the construct was confirmed by N-terminal sequencing using an upstream sequencing primer GGCGT ATCACGAGGCCCTTTCG.
    20. Our initial attempts to express the full length gene in E. coli as a His6 fusion protein failed. Attempts were initially made to express the His6-bZP3 protein in the pRSET B
    21. PCR Amplification and Cloning in pQE30 Vector
    22. CLONING AND EXPRESSION IN E. coli
    23. ligation reactions were carried out usmg conditions and buffers specified by the manufacturer.
    24. The PCR amplified eDNA fragment corresponding to bZP3 was resolved on a 0.8% agarose gel run using IX TAE buffer (0.04 M Tris-acetate, O.OOI M EDTA) and purified using the Geneclean® II kit. The PCR amplified bZP3 was digested with Kpn I and Sac I and ligated into the pBluescriptll SK(+) vector at the same sites. The digestion and
    25. Agarose Gel Electrophoresis, Digestion and Ligation
    1. The RIA for alpha hCG was similar to that used to estimate ~hCG. A monoclonal antibody specific to alpha hCG ( Gupta et al. , 1985 was used for the assay. The standard used was total hCG.
    2. RIA for alpha hCG.
    3. PBS and then replenished with the complete medium. Two days following transfection, the cells were subcultured into the appropriate selective medium for selection of stable clones as described below.
    4. Calcium phosphate mediated stable transfections were performed by the method of Graham and Van der Eb ( 1973 with modifications as described by Gorman ( 1986 ). For each plasmid, two petri dishes each containing 0. 5 x 106 CHO-K1 cells were used, with 10 ug of cesium purified DNA for each transfection. A mock transfection which did not contain any DNA, was performed simultaneously as negative control. Precipitation of the DNA was done with great care to ensure the obtention of a fine, translucent precipitate rather than a dense and opaque precipitate. The calcium phosphate I DNA precipitate was added in 4 ml medium to the cells and the cells incubated for 3 hours at 37°C. At this stage, the cells were examined under the microscope and a fine precipitate appeared as small grains all over the cells. The cells were washed once with serum free medium and a glycerol shock given for 3 minutes at 37°C. The cells were washed twice again with
    5. Using calcium phosphate.
    6. Stable transfection was performed into CHO-K1 cells by the following procedures
    7. stable transfection.
    8. 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
    9. 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
    10. Hybridisation of southern filters.
    11. 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.
    12. 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
    13. Purification of the probe.
    14. 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.
    15. Northern blot.
    16. 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.
    17. 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
    18. southern blot.
    19. 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.
    20. Transformation procedure.
    21. 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.
    22. 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
    23. Electrophoresis of RNA.
    24. 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.
    25. DNA digested with restriction enzymes was
    26. 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
    27. 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.
    28. 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.
    29. 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
    30. Genomic DNA from cultured mammalian cells was isolated by a rapid procedure, essentially as described by Davis et
    31. Isolation of genomic DNA from mammalian cells.
    32. 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).
    33. 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
    34. 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
    35. 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
    36. Isolation of plasmid DNA.
    37. Large scale isolation of DNA.
    38. 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.
    39. Media.
    1. 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.
    2. eMPT inhibition assay
    3. 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
    4. Organelle separation of L.donovani 93
    5. 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. 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
    7. 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,
    8. Synthesis of [4,6-Dideoxy-4,6-difluoro]-GDP Talose (Scheme 16 of Results and Discussion)
    9. 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.
    10. Preparation of Cell-free system of L.donovani
    11. 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.
    12. 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 (
    13. Synthesis of anomeric ~-Iactam analogues of eMPT substrate91•92 (Scheme 14 of Results and Discussion)
    14. 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.
    15. 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
    16. (Scheme 13 of Results and Discussion)
    17. Synthesis of S'-hemiacetal analogue90 of Gal 1,4~-Man-a­phosphate acceptor
    18. Design and Synthesis of mechanism based inhibitors of elongating MPT enzyme of LPG biosynthesis
    19. 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!.
    20. [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
    21. Synthesis of [14C] labeled Stearyl linked Gal 1,4 f3 Man phosphate (Scheme 12 of Results and Discussion)
    22. 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.
    23. 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'),
    24. 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
    25. 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
    26. 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
    27. 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, =
    28. 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
    29. 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
    30. 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.
    31. (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
    32. am of nitrogen gas, and further dried under vacuum to provide a semisolid hexa-O-benzyl lactal 1,2a-epoxide 33
    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
    34. 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
    35. Synthesis of Tetrasaccharide Cap Domain of LPG
    36. 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.
    37. 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.
    38. Synthesis of phosphoglycans by polycondensation
    39. 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.
    40. 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).
    41. 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
    42. 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)
    43. Solid phase phosphoglycan synthesis
    44. (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.
    45. (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
    46. = 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
    47. 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
    48. 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,
    49. 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-
    50. 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),
    51. 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
    52. 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
    53. Solution Phase Synthesis of Phosphoglycans
    54. Synthesis of Phosphoglycan Repeats of Lipophosphoglycan
    1. 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.
    2. Antibody isotyping
    3. 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.
    4. 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.
    5. 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
    6. Plasmid DNA adsorbed onto gold microcarriers
    7. 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.
    8. Plasmid DNA administered by electroporation
    9. 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.
    10. Plasmid DNA administered in saline
    11. 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:
    12. 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.
    13. Purification in refolded form
    14. 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
    15. 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
    16. Detection of the expressed recombinant protein following i11 vitro transfection of mammalian cells with the plasmid DNA.
    17. 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.
    18. 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'-
    19. PCR amplification of dZP3 eDNA
    1. Thespleenswereremovedandhomogenisedindividuallythroughanylonnet(80mesh).Forisolationoflymphocytes,thetissuehomogenatewaslayeredonatwo-stepPercolldensitygradientandcentrifugedfor30minat400xg.Thelymphocyteswerecollectedatthe1.040-1.080g/cm3interface,washedtwice(400xg,10min)withrHbss,andresuspendedin2mlrRPMl.Cellswerecountedbytrypanblueexclusioninahaemocytometer(viability>95%)andthenumberoflymphocytesfrombloodandspleenwereadjustedto2x106/ml
    2. Spleen
    3. ultrapureHNO3andtissuesamplesweredissolvedin70%HNO3;microwavedfor5minat90W,180W,270Wand360W,untiltotaldigestionhadoccurredandthendilutedwithMilli-Qgradewater(Millipore,Acton,Massachusetts,U.S.A)
    4. Totalsodium,potassiumandcalciumconcentrationsweredeterminedwithatomicabsorptionspectrophotometry.Tothispurpose,plasmasamplesweredilutedwith1%
    5. Ionconcentrations
    6. Aftereffluentexposure,thetissuesweredissectedout,weighedandhomogenizedin0.25Msucrose.Thehomogenateswerecentrifugedat10,000rev/minatatemperaturebelow8°C.AcetylcholinesteraseactivityofthesampleswasdeterminedatpH7.0usingafinalhomogenateconcentrationof25mgmf1at10°CwithmMacetylcholineiodideassubstrate and0.001or0.002NsodiumhydroxideastitrantfollowingHestron’smethodasgivenbyMetcalf(1951).ProteindeterminationsforalltheChEanalyseswereconductedonaliquotsofthehomogenatesusingamodificationoftheLowryetal.(1951)method.AchEactivityisexpressedinpmolesofacetylcholinechloridehydrolysedmgtissue'1hr'1