Transfection.

with glutamine. Rat-2 and FWIL cells were cultured in IMDM supplemented with glutamine. All media were supplemented with 10 % fetal calf serum~ The cells were maintained in a 5 % co2 atmosphere and were split after 72 hours in culture, at a ratio of 1 : 15 approximately. For splitting the adherent cells, the cells were washed once with HBSS and 0.1 % trypsin in PBS added to the cells. The flask was shaken briefly to ensure a uniform distribution of trypsin over the cells. The cells were incubated in trypsin for 1 - 2 minutes after which 2 ml of FCS was added to the cells to inactivate the trypsin. The trypsin was carefully aspirated from the flask and fresh culture medium was added into the flask. The cells were dislodged from the bottom of the flask by gently tapping the flask against the working bench. Alternately, the cells were resuspended by vigorous pipetting up and down of the medium. The cells were centrifuged at 1500g for 5 minutes at room temperature and the supernate was discarded aseptically. The cells were resuspended in a known volume of fresh culture medium, an aliquot counted on a haemocytometer, and then accordingly seeded at the desired density in a fresh flask. For long term storage, the cells were frozen in a mixture of 95 ~ 0 culture medium and 5 ~ 0 DMSO in liquid nitrogen.

eHO-Kl cells were cultured in Ham s F-12 med1a. NIH3T3, mouse LMtk-and HeLa cells were cultured in DMEM supplemented

Growth and maintenance of cell lines.

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

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

Hybridisation of southern filters.

hours. The NC filters having bound DNA liberated from bacterial colonies, were set up for hybridisation with radioactive probes as described by Maniatis et al., ( 1982 ). The filters were washed thoroughly with a solution containing 50 mM Tris.Cl, pH 8.0, 1 M NaCl, 1 mM EDTA, 1 % SDS, at 42°C, for 1 hour, to wash off any residual bacterial debris and agar etc. Prehybridisation and hybridisation was performed in aqueous solution without formamide in 5 X SSPE. The filters were washed up to a stringency of 0.2 X sse at 65°e.

Colonies bound to nitrocellulose filter ( NC ) were lysed to liberate the DNA which was hybridised as described by Maniatis et al., 1982 ) . To obtain sharper autoradiography signals, the nitrocellulose filter bearing colonies was first overlaid on a 3 MM Whatman paper impregnated with 10 % SDS till the NC wetted evenly. The NC was peeled off and overlaid on another 3 MM paper impregnated with the denaturing solution. In this manner, the NC was successively treated with denaturing and neutralising solutions. Finally, the NC filter was air dried, sandwiched between two sheets of 3 MM paper and baked at 80°C for two

Colony hybridisation.

Hybridisa.tion of DNA L RNA bound to nylon membranes.

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

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

Purification of the probe.

400 ci 1 mmole to 3000 ci 1 mmole. The nick translation reaction was set up as recommended by the manufacturer of the kit, using about 0.5 ug DNA. The reaction was incubated at 12 -14 °C for 90 minutes, except in the case of small fragments ( 500 bp ) when the reaction was incubated for 45 minutes only. The reaction was terminated by the addition of stop buffer supplied with the kit.

DNA was labelled using the nick translation kits supplied by BRL or NEN, USA, or Amersham, UK. The 32P-deTP was from either NEN or Amersham, UK, at a concentration of 10 mei I ml. The specific activity of the label ranged from

Nick translation.

32~ -labelling of DNA.

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

Northern blot.

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

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

southern blot.

Colony lifts were performed essentially as described by Maniatis et al. , 1982 ) . Recombinant colonies were grown 0/N at 37°C to have well separated colonies. The colonies were overlaid with 80 mm diameter nitrocellulose filter circles BA 85, S & S and after the filter became wet throughout, it was peeled off in a single, smooth motion, avoiding the smearing of the bacterial colonies. The plate was reincubated at 37°C for a few hours to regenerate the colonies. The colonies transferred to the filter were lysed to bind the liberated DNA to the nitrocellulose.

Colony lifts.

Transfer of DNA.

Imrnobilisation of DNA L RNA on~ solid support.

lysed directly in 1. 5 ml of solution D ( 4 M guanidium thiocyanate, 25 rnM sodium citrate, pH 7.0, 0.5 % sarcosyl and 0.1 M 2-mercaptoethanol ) . For every 2 ml of the lysate, 0.2 ml of chloroform was added, followed by vigorous mixing for 15 seconds, and incubation on ice for 15 minutes. The lysate was spun at 12, OOOg, at 4 °c for 15 mins. , and the aqueous phase transferred to another tube. RNA was precipitated with an equal volume of isopropanol and incubation at -2o0c for 45 mins. The samples were then spun at 12,000g for 15 mins. at 4°c, and the supernate discarded. The RNA pellet was washed twice with 75 % ethanol. Finally, the pellet was dried briefly under vacuum for 10 -15 mins. and dissolved in 0.5 % SDS. All chemicals and glassware used for handling RNA were treated with diethylpyrocarbonate ( DEPC ) .

Total RNA was isolated from cultured mammalian cells by the method of Chornczynski and Sacchi ( 1987 ), with slight modifications. Briefly, cells from a 3.5 ern petri-dish were

Isolation of RNA

From an 0/N grown culture, 1 ml cells were pelleted in a 1.5 rnl eppendorf tube. The cells wer~ washed once with 100 ul of solution I ( 50 rnM glucose in 25 rnM Tris. HCl ,· pH 8. 0 ) . The cells were pelleted again and resuspended in 70 ul of solution I. To this, 20 ul of a freshly prepared solution of lysozyme 10 rng 1 ml in distilled water was added. The tube was vortexed to mix the contents and incubated in ice for 5 minutes. Next, 10 ul of 0.1 M EDTA, pH 8.0, was added, vortexed and the tube incubated in ice for 5 minutes. Next, 200 ul of solution IV ( 0.2 N NaOH + 1 % SDS was added, the contents vortexed quickly but briefly to mix and incubated in ice for 5 minutes. Finally, 150 ul of 5 M potassium acetate, pH 4. 8 was added and the tube incubated in ice. After 60 minutes, the tube was centrifuged for 10 minutes at 10,000 rpm, at 4°C. 450 ul of the supernate was removed to another tube and DNA precipitated with two volumes of ethanol at -7 0°C for 15 minutes. The DNA pellet was collected by centrifugation and after draining off the supernate, the pellet was washed with 80 % ethanol. The pellet was dried briefly under vacuum and finally resuspended in 150 ul TE. From this, a 10 ul aliquot was used for checking on gel or for•setting up digestions with restriction endon~cleases.

Plasmid DNA minipreps.

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

Transformation procedure.

were stored at -70°C for at least six months without any significant loss in the competence.

A single ~.coli colony taken from an agar plate was used to inoculate 10 ml of LB and incubated 0/N at 37°C in an incubator-shaker. Next day, 0. 5 ml of this freshly grown culture was used to inoculate 100 ml of LB in a 500 ml flask. The culture was incubated at 37°C in an incubator -shaker and absorbance of the growing culture was monitored at 620 nm. When the A620 reached 0. 4 -0. 5 ( in about 120 -150 minutes), the flask was rapidly chilled by shaking in ice. The cells were harvested in sterile, chilled centrifuge bottles at 4, ooog for 10 minutes at 4 °c. The pellet was gently resuspended in 50 ml sterile, ice cold 100 mM cacl2 and the cells incubated in ice for 30 minutes. The cells were again centrifuged as above and the pellet resuspended in 6.5 ml of sterile, chilled, 100 mM cac12 containing 15 % glycerol. The cells were resuspended very gently, and a 200 ul aliquot was transformed with a standard plasmid DNA to check the competence of the cells. Meanwhile, the rest of the competent cells were incubated in ice for 16 -18 hours, to increase the competence of the cells a further few fold. After ascertaining high transformation efficiency of the competent cells, the cells were dispensed as 200 ul aliquots into prechilled, sterile 1.5 ml eppendorf tubes. These cells

Preparation of competent E.coli cells.

All glassware I plasticware used for transformation procedure was sterile and prechilled.

Transformation of E.coli.

minigel alongwith unligated vector to test the ligation. The ligated DNA was used to transform competent ~.coli cells.

Wherever possible, the cloning of DNA fragments was achieved by ligation of compatible sticky ends generated on the vector as well as the insert by digestion with the same enzyme. Self ligation of the linearised vector with compatible sticky ends was minimised by dephosphorylation of the vector DNA using bacterial alkaline phosphatase. The ligation conditions for each batch of T4 DNA ligase were standardised using Hind III generated fragments of lambda DNA as a test sample for sticky end ligation. Routinely, 200 ng of vector DNA was mixed with 2 - 5 fold molar excess of the insert fragment DNA, 2 ul each of the 10 X ligase buffer 500 mM Tris. HCl, pH 7. 5, 100 mM Mgcl2 ) , 10 mM ATP, and 200 mM DTT. The final reaction volume was adjusted to 15 - 2 0 ul with sterile double distilled water, and 0.5 - 1 ul of T4 DNA ligase ( 103 units I ml ) was added. The contents were mixed well and incubated at 13°C for 12 -16 hours. An aliquot of 2 ul was electrophoresed on a

Ligation of DNA fragments.

a 1.5 ml eppendorf tube and the gel slice put into the paper cone. The tube was centrifuged for 10 minutes at room temperature, to elute the DNA into the filtrate. The filtrate was extracted with one volume of phenol I chloroform ( 1:1 vlv ) , and the DNA precipitated from the aqueous phase by the addition of 5 M NaCl to a final concentration of 1 M, and 2 - 3 volumes of ethanol at -20°C, for a few hours. The centrifuged DNA pellet was dissolved in an appropriate volume of TE.

After digestion of the plasmid DNA with appropriate restriction enzymes, the DNA fragments were resolved by electrophoresis on preparative agarose gels of a suitable percentage, and stained with ethidium bromide as described above. Depending upon the amount of DNA to be resolved on the gel, the size of the sample well varied from 1.5 em - 5 em x 0.3 em, such that the desired fragment could be cut out with a minimum of agarose accompanying it. The DNA bands were visualised under long wave UV ( 366 nm ), using a hand held monitor model UVGL-58 Mineralight Lamp, UVP, Inc., California, USA), and the desired fragment cut out as a thin agarose slice keeping the size of the slice as small as possible ) . DNA was eluted from the agarose slice by the method of Zhu et al., ( 1985). Briefly, a GeneScreen ( NEN ) or Durapore ( Millipore, GVWP 04 700 membrane was wetted with 200 ul of elution buffer ( 0.1 % SDS +50 mM Tris. HCl, pH 7.5 ), and folded over to form a cone. Meanwhile, the conical lower half of an eppendorf tube was cut off and a hole pierced in the bottom with a hot wire or needle. The membrane was placed into this cone, pushing it as far as possible. This assembly was then transferred to

Isolation of restriction fragments of DNA

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

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

Electrophoresis of RNA.

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

DNA digested with restriction enzymes was

For rapid electrophoretic analysis of plasmid DNA prepared by miniprep protocol, or to monitor the progress of digestion during various cloning procedures, the DNA was resolved on short agarose gels, taking less than one hour for the run. The electrophoresis was carried out in TAE buffer using 8 em long gels with a comb of teeth size 0.4 x 0.2 em. The width of the gel was variable, depending on the number of samples to be analysed. Gels were run at 50 100 volts, till the bromophenol blue dye migrated to within 0.5 em of the edge of the gel.

Mini gel electrophoresis

Electrophoresis of DNA

Agarose ~ electrophoresis

mixed with the sample by vortexing, and the DNA loaded on a preparative agarose gel. When digesting vector DNA in preparation for a ligation, the DNA was first purified from the digestion reaction as described in 3.2.4.8. This DNA was then treated with bacterial alkaline phophatase as described by Maniatis et al., 1982 ) • The dephosphorylated DNA was run on a preparative agarose gel to purify the linearised, dephosphorylated vector DNA. The efficiency of dephosphorylation was monitored by self ligation, followed by transformation of competent E.coli cells. Only after achieving efficient dephosphorylation of the vector DNA, was it used for ligation with the insert DNA.

For digestions aimed at purification of restriction fragments, 10 -20 ug of DNA was digested in a reaction mixture of about 100 -200 ul volume. Aliquots from the digestion reaction were checked on a minigel after one hour to monitor the extent of digestion. After the digestion was complete, one tenth volume of the 10 X tracking dye was

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

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

Routinely, with sterile double 0.2 - 1 ug DNA was made up to 18 ul distilled water in an autoclaved eppendorf tube. 2 ul of 10 X buffer and 2 - 5 unitp of restriction endonuclease were added. The reaction components were mixed well and incubated in a 37°C water bath for 1 - 2 hours. The digestion reaction was terminated by the addition of 2 ul of 10 X tracking dye ( 0.25 % xylene cyanol, 0.25 % bromophenol blue, 0.1 M EDTA, pH 8.0, and 50 % glycerol followed by brief vortexing to mix, after which the sample was loaded on to the gel.

All fine chemicals and the thermal cycler used for PCR, were kindly provided by Cetus Corporation, California, USA. 3.2.4. Digestion of DNA with restriction enzymes. DNA samples were digested with restriction endonucleases in the appropriate digestion buffers as recommended by BRL. The digestion buffers were in most cases, supplied by BRL. Composition of the 1 X buffers is given in Table 4.

Figure !• Thermal cycle profile of a typical polymerase chain reaction ( PCR ). A typical PCR consists of repititive cycles of multiple temporal segments ( designated here as A - G ) with distinct target temperatures. After making the desired cocktail of template DNA, primers, Taq polymerase and the enzyme buffer, the reaction tube is incubated in a programmable thermal cycler, to incubate the reaction contents at pre-set temperatures for designated periods of time. Segments A -B, template denaturation; c -D, primer annealing; E -F, strand synthesis; G, ramp to the completion of the first cycle prior to the start of the next cycle ( dotted line ) . The duration and target temperature for each segment in an amplification cycle can be varied to suit the desired objectives.

plasmid DNA in a 100 ul mixture having 10 ul of 10 X PCR buffer, 10 ul of 10 mM dNTPs, 3 ul of each primer to give a final primer concentration of 1 uM, and 2.5 units of the Thermus aquaticus thermostable DNA polymerase. PCR reaction buffer ( 10 X ) contains 500 mM KCl, 100 mM Tris. Cl, pH 8 . 3 ' 15 mM MgC12 and 0.1 9.,-0 gelatin. The mixture was subjected to PCR amplification in a programmed thermal cycler block set for 3 0 cycles. The procedure is diagramatically outlined in Fig. 1 and involved four steps : a) The reaction was heated to 95°C for 30 seconds to separate the two strands of the target DNA; b) the reaction was then cooled to 37°C for one minute to allow annealing of the two primers to the template DNA to occur ; c) next, the temperature was raised to 72°C and the reaction maintained at this temperature for 10 minutes, for primer extension to occur; d) at the end of the cycle, the temperature was again raised to 95°C as in step (a) to start a new cycle. In between the steps (a) to (d), one minute ramp times were used to allow the efficient realisation of the set temperature. In the last cycle, the duration of step (c) was extended to ensure the conversion of all single strands into double stranded DNA. At the end of 30 cycles of PCR, a 10 ul aliquot from the PCR reaction was electrophoresed on a 1 % SeaKem I 3 % NuSieve agarose gel in TBE buffer, to resolve the PCR products. The amplified DNA was purified by electrophoresing the entire PCR mixture on a 1 % preparative agarose gel in TAE buffer.

Polymerase chain reaction was carried out as described by Scharf et al., ( 1986 ) , witD. some modifications. 23 mer oligonucleotide pri~ers synthesised by the solid phase triester method were designed to flank the target DNA desired to be amplified. A primer was designed to be complementary to , the 5 end of the phCG eDNA (+) strand and I was termed 5 primer ( also see Fig. 20 ) . Another primer was designed to be complementary to the sequence flanking the translation termination codon ( TAA ) of HBsAg (-) strand and this was I 1 I , termed 3 pr1mer. The 5 pr1mer also included the recognition sequence for restriction endonuclease Sal I as an overhang, while the I 3 primer included the recognition sequence for Hind III as an overhang. In addition, two extra bases ( G or C ) flanking the restriction site were included in the sequence of the primers, to improve the enzyme digestion. Thus, the nucleotide sequence of I the 5 primer read as : I I I 5 -GGCGTCGACATGGAGATGTTCCA-3 , while that for the 3 primer read I I as : 5 -CCAAGCTTTTAAATGTATACCCA-3 . A standard PeR· reaction contained 500 ng to 1 ug

Polymerase chain reaction ( PCR 1·

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

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

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

Isolation of genomic DNA from mammalian cells.

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

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

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

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

Isolation of plasmid DNA.

Large scale isolation of DNA.

yeast extract, and 10 g NaCl in distilled water, pH adjusted to 7.5 with NaOH and final volume made up to one liter (Maniatis et al., 1982). Cultures of ~.coli cells transformed with plasmid DNA were grown in media supplemented with 50 ug/ml of ampicillin. For large scale plasmid DNA isolation, ~.coli cells were grown in an enriched medium, Terrific Broth ( TB ) . One liter of TB was prepared by adding 100 ml of a sterile solution of 0.17 M KH2Po4 and 0.72 M K2HPo4 to a sterile solution containing 12 g Bacto -tryptone, 24 g Bacto -yeast extract, 4.0 ml glycerol and water to a final volume of 900 ml ( Tartof and Hobbs, 1987 ) . The media were sterilised by autoclaving at 15 psi for 20 minutes. Heat labile compounds and antibiotics were sterilised by filtration through a 0.45 u nitrocellulose membrane and added to autoclaved media after cooling the same to 55°C. Solid media was prepared by adding 1. 5 % bacto -agar prior to autoclaving. Storage of ~.coli was carried out essentially as described by Maniatis et al., ( 1982).

Composition of growth media used for culturing ~. coli is given in Table 3. For routine propagation, ~.coli cells were grown in Luria Bertani medium LB ) . LB was prepared by dissolving 10 g Bacto -tryptone, 5 g Bacto -

Growth and storage of bacteria.

The inhibition potency of our synthetic substrate, lactal (31), was determined by measuring the hydrolytic activity of ~-D-galactosidase on o-nitrophenyl ~-D­galactopyranoside.97 Each assay tube contained 2 ml of o-nitrophenyl ~-D galactopyranoside solution (500 IlM), 0.25 units of ~-D-galactosidase (source: Aspergillus oryzae; purchased from Sigma, cat. no. G-5160) and a certain concentration of lacta!. The tube was incubated at 30°C for 13 min, after which 200 III of the mixture was added to a tube containing 100 III of acetate buffer (25 mM, pH = 4.5) and 200 III of sodium carbonate solution (200 mM). The colour liberated was read spectrophotometrically at 400 nm. The control tube contained everything except the substrate, lacta!.

~-D-Galactosidase inhibition by Lactal

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

eMPT inhibition assay

mixture). These samples were lyophilized and 125 III of the reaction mixture was added to each tube. The tubes were then incubated at 25°C for 1 h and the biosynthetic LPG was extracted as described above. 10 III of the solvent E extract was taken for scintillation counting.

1. Mild acid hydrolysis: 0.6 ml of the pooled solvent E soluble fractions was dried with a stream of nitrogen and then suspended in 0.02 N HGI (200 Ill). The mixture was then placed in a 100 °G water bath for 5 minutes. After hydrolysis, the sample was again dried under nitrogen and codried thrice with toluene (0.5 ml). The residue was suspended in 0.6 ml of 0.1 M NaGI in 0.1 M glacial acetic acid, loaded onto phenyl sepharose column and elution done in the same manner as described before. Fractions of 0.6 ml each were collected and assayed for radioactivity. 2. Nitrous acid deamination: 0.6 ml of the pooled solvent E soluble fractions was dried with a stream of nitrogen and then suspended in 0.2 ml of 0.125 M sodium acetate (pH = 4.0) and 0.25 M sodium nitrite. The mixture was incubated at 25 °G for 40 h. The sample was dried under nitrogen, suspended in 0.6 ml of 0.1 M NaGI in 0.1 M glacial acetic acid, loaded onto phenyl sepharose column and elution done in the same manner as described before. Fractions of 0.6 ml each were collected and assayed for radioactivity. 3. PI-PLC treatment: 0.6 ml of the pooled solvent E soluble fractions was dried with a stream of nitrogen and suspended in 0.4 ml of PI-PlG buffer (0.1 M Tris chloride, pH = 7.4 with 0.1 % deoxycholate) and 0.2 ml of PI-PlG concentrate (B.subtifis culture supernatant) was added. The mixture was then incubated at 37 °G for 16 h. The sample was dried under nitrogen, suspended in 0.6 ml of 0.1 M NaGI in 0.1 M glacial acetic acid, loaded onto phenyl sepharose column and elution done in the same manner as described before. Fractions of 0.6 ml each were collected and assayed for radioactivity. The effect of deoxymannojirimycin (Sigma, Gat. no. 0-9160) on the cell free biosynthesis was carried out. OMJ (5 mg) was dissolved in 1 ml of MQ water and 2.5, 5, 25 and 50 III were transferred to eppendorf tubes separately (which corresponded to 0.5, 1, 5 and 10 IlM concentrations of OMJ in 125 III ofthe reaction

Characterization of biosynthetic LPG

NaGI in 0.1 M glacial acetic acid, 1.2 ml of 0.1 M glacial acetic acid, 0.6 ml of water and 3.6 ml of solvent E. Fractions of 0.6 ml each were collected and assayed for radioactivity.

Parasites (6 X 109) were harvested, pelleted at 3000 g for 10 min, washed with PBS, repelleted and suspended in 10 mL of HEPES buffer (100 mM HEPES-NaOH, pH = 7.4, 50 mM KCI, 10 mM MnCI2, 10 mM MgCI2, 0.1 mM TLCK, 1 Jlg/mL leupeptin) containing 10% glycerol. The cells were disrupted in a Parr nitrogen cavitation bomb (1500 psi, 25 min, 4°C, 3 cycles). The debris was removed by centrifugation at 3000 g for 5 min and the supernatant was centrifuged at 100,000 g for 1 h at 4°C. The resulting membrane pellet was resuspended in 10 mL of HEPES buffer without glycerol and centrifuged again at 100,000 g for 1 h at 4°C. The membranes were finally suspended in 1 mL (13 mg/mL) of HEPES buffer without glycerol. The incubation mixture per reaction contained membrane protein (2 mg) in 125 JlL of 50 mM HEPES-NaOH buffer, pH = 7.2 containing supplements (25 mM KCI, 5 mM MgCI2, 5 mM MnCI2, 0.1 mM TLCK, 1 JlglmL leupeptin, 0.8 mM ATP, 0.4 mM On) with 2 JlM UOP-[3H]-galactose (2 JlCi) and 10 JlM GOP-mannose. The mixture was incubated at 25°C for 1 h, terminated by the addition of CHCI~CH30H (3:2) to give a final ratio of CHCI~CH30H/H20 (3:2:1) and sonicated. The layers were then allowed to separate out after which the lower layer was removed with the aid of a micropipette. The tube containing the upper and intermediate layer was centrifuged (10,000 rpm, 4°C, 5 minutes). The supernatant was discarded and the resultant pellet (membranes) was suspended in 1 mL of CHCI~CH30H/H20 (1:1 :0.3). The solution was again centrifuged (10,000 rpm, 4°C, 5 minutes) and the pellet was extracted with 1 mL of solvent E (H20/ethanol/diethylether/pyridine/NH40H 15:15:5:1 :0.017) thrice. The solvent E extracts were pooled and dried under a stream of nitrogen, suspended in 0.6 mL of 0.1 M NaCI in 0.1 M glacial acetic acid and chromatographed over a 1 mL column of phenyl sepharose. Phenyl Sepharose Column of Biosynthetic LPG. The solvent E extract suspended in 0.6 mL of 0.1 M NaCI in 0.1 M glacial acetic acid was applied to a column (0.5 x 2 cm) of phenyl sepharose (Pharmacia BioteCh), preequilibrated with 0.1 M NaCI in 0.1 M glacial acetic acid. The column was then washed sequentially with 3 mL of 0.1 M

Cell-free Biosynthesis42 of LPG using Leishmania membranes

Inhibition of LPG biosynthesis: Identification of deoxymanno-jirimycin (DMJ) as an inhibitor that targets elongating MPT enzyme

Transport assays were done by mixing 100 III of vesicle suspension (200 Ilg of protein) with 100 III of reaction buffer (10 mM Tris-HCI, pH=7.4, 10 mM MnCI2, 4mM MgCI2, 0.1 mM TlCK, 1 Ilg/ml leupeptin, 1 Ilg/ml pepstatin A, 1 mM dithiothreitol, 0.5 mM phenylmethylsulfonyl fluoride, 0.5 mM 2,3-dimercaptopropanol and 16 11M (0.16 IlCi) of GDP-[3H]-Man) . After incubation at 28°C for 6 min, the samples were placed on ice, diluted with 1.5 ml of washing buffer (10 mM Tris-HCI, pH=7.4, 0.25 M sucrose), and applied to a filtration apparatus containing HA filters. The filters were washed with 20 ml of washing buffer, and the radioactivity bound onto the filters was measured by scintillation counting. The amount of GDP-[3H]-Man that was non specifically bound to the outside of the vesicles was determined by measuring the radioactivity associated with the vesicles at 0 time of incubation of vesicles with solute.

GOP Mannose transporter assay 49

ingredients were present except N-acetylglucosamine. This value represented the galactose released. The difference in the counts between the tube in which acceptor was present and control represented transferase activity.

A mixture of sodium cacodylate (20 ~L, 0.2 M, pH = 6.5 adjusted with Hel) , MnCI2 (3 ~L, 1 M), mercaptoethanol (3 ~L, 1 M) and Triton X-100 (5 ~L, 10% w/v) was added to a solution containing N-acetylglucosamine (3 ~L, 1 M) and protein (1 00 ~g). The reaction was started with the addition of UOP-galactose (15 ~L, 10 mM with 1 ~Ci of eH] UOP-galactose). The mixture was incubated at 37°C for 60 minutes after which the reaction was stopped by the addition of EOTA (17 ~L, 0.3 M, pH = 7.4 adjusted with NaOH) and placing the tube on ice. The mixture was then passed through a column of Oowex 2X8 (200-400 mesh in cr form) already washed thoroughly with water. The unreacted UOP-galactose remained bound to the column while galactose which had been transferred to N-acetylglucosamine to form lactosamine, as well as free galactose, was eluted out with 1.5 mL of distilled water. One tenth of volume was taken for scintillation counting. For each assay, a control tube was run in which all

1,4 ~ Galactosyl transferase assay 96

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

Organelle separation of L.donovani 93

Water was added and the mixture was concentrated under reduced pressure to afford 89; ESMS (mlz): 604.1 (M-Hr.

ixture was stirred under argon atmosphere for 2 days.

were diluted with ice cold water. The mixture was extracted with CH2CI2. The organic layer was thoroughly washed with water, dried over Na2S04 and concentrated to yield 84. 2,3,4,6-Tetra-O-acetyl-a-L-manno-di-O-benzyl phosphate (86). Compound 84 (50 mg, 0.128 mmol) was dissolved in anhydrous CH3CN saturated with dimethylamine (5 ml ) at -20 DC and stirred for 3h after which TlC confirmed the disappearance of starting material. Excess of dimethylamine was removed under reduced pressure at 30 DC and the reaction mixture was concentrated to afford 2,3,4,6-tetra-O-acetyl-a-l-mannose (85). To a stirred solution of compound 85 and 1 H-tetrazole (9.5 mg, 0.138 mmol) in anhydrous CH2CI2 (400 Ill) was added dibenzyl-N,N'-diisopropyl phosphoramidite (56.5 Ill, 59.4 mg, 0.172 mmol) and the mixture was stirred under argon atmosphere for 2 h at rt. Subsequently, the reaction mixture was cooled to-40 DC and m-CPBA (40 mg, 0.23 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 bicarbonate. The mixture was extracted with CH2CI2. The organic phase was thoroughly washed with water, dried over Na2S04 and concentrated to afford 86, which was purified by running a silica coated preparative TlC plate; Rf = 0.16 in 50% ethyl acetate in hexane; 1H NMR: 8 3.9-4.22 (m, 4H), 5.02-5.06 (m, 4H), 5.21-5.28 (m, 2H), 5.59 (1 H, dd, JHP = 6.3 Hz, JHH = 1.8 Hz, H-1); 13C NMR: 8 20.49, 20.60, 61.68,65.19,68.14,68.68,69.75,69.92,70.31, 95.09, 127.89-128.72, 169.43; 31p NMR 8 -3.2; ESMS (mlz): 631.2 (M+Nat. a-L-mannosyl phosphate (88). To a solution of 86 (25 mg, 0.04 mmol) in CH30H (1 ml) was added palladium on charcoal (10%, 200 mg) and formic acid (100 Ill). The mixture was stirred at 50 DC for 3 h to afford compound 87. The catalyst was filtered off and the solvent was evaporated. The residue was taken in a mixture of CH30H:H20: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 88; ESMS (mlz): 259.19 (M-H)". Guanosine 5'-diphospho-a-L-mannose ( mono triethylamine salt) 89. A mixture of 4-morpholine-N,N'-dicyclohexylcarboxaminidium guanosine 5'-monophospho morpholidate (56 mg, 0.071 mmol) and 88 (16 mg, 0.034 mmol) was coevaporated with dry pyridine (3x500 Ill). 1 H-tetrazole (10 mg, 0.137 mmol) and dry pyridine (1.2 ml) were added and the m

Penta-O-acetyl-a-L-Mannose (84): To a solution of l-mannose (30 mg, 0.16 mmol) in pyridine (300 Ill) was added acetic anhydride (500 Ill) at 0 °C. The flask was left at 4 °C for 12 h. The mixture was then stirred at rt for 1 h, following which the contents

Synthesis of L-Mannose analogue of GOP Mannose (Scheme 18 of Results and Discussion)

(1 ml) was added palladium on charcoal (10%, 176 mg) and formic acid (100 Ill). The mixture was stirred at 50°C for 3h after which 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 mixture was concentrated and the residue was repeatedly lyophilized to yield 82; ESMS (mlz): 387.34 (M-H)'. Guanosine 5'-diphospho-6-deoxy-6-fluoro-a-D-mannose (mono-triethylamine salt) 83. Mixture of 4-morpholine-N,N'-dicyclohexylcarboxaminidium guanosine 5'-monophosphomorpholidate (43 mg, 0.054 mmol ) and 82 (16 mg, 0.034 mmol) was coevaporated with dry pyridine (3 x 500 Ill). 1 H-tetrazole (8 mg, 0.108 mmol ) and dry 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 yield 83; ESMS (mlz): 606.11 (M-Hr.

solution of compound 80 and 1 H-tetrazole (7 mg, 0.102 mmol) in anhydrous CH2CI2 was added dibenzyl-N,N'-diisopropylphosphoramidite (42 Ill, 43.8 mg, 0.127 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 (30 mg, 0.17 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 81, which was purified by running a silica coated preparative TlC plate; R, = 0.12 (twice run in 30% ethyl acetate in hexane); 1H NMR: characterstic () 5.6 (1 H, dd, JHP = 6.3 Hz and JHH = 1.8 Hz); 13C NMR: () 20.50, 20.53, 20.60, 64.75, 68.11, 68.58, 69.86, 70.67, 70.93, 81.87, 95.01, 128-128.72, 169.38, 169.50, 169.67; 31 P NMR () -3.11; ESMS (m/z): 591.34 (M+Nat. 6-Deoxy-6-fluoro-a-D-mannosyl phosphate (82). To a solution of 81 (20 mg, 0.035 mmol) in CH30H

Methyl-S-deoxy-S-difluoro-a-D-mannopyranoside (78). DAST (134 Ill, 1 mmol) was added with stirring at -40 °c, to a suspension of methyl-a-D-mannopyranoside S2 (200 mg, 1 mmol) in anhydrous CH2Cb (4 ml). The mixture was stirred at -40 °c for another 30 minutes and then at rt for 3h. After cooling to -20°C, the excess of reagent was destroyed by addition of CH30H (600 Ill) and sodium bicarbonate (200 mg). The cooling bath was removed, and the mixture was filtered once effervescence ceased. The filtrate was concentrated and purified by silica column chromatography (3% CH30H in CH2CI2) to yield 78; Rf = 0.21 in 12.5% CH30H in CH2CI2• 1 ,2,3,4-Tetra-O-acetyl-S-deoxy-S-fluoro-a-D-mannopyranoside (79). To compound 78 (100 mg, 0.51 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 Na2S04and concentrated to afford 79; Rf = 0.35 in 50% ethyl acetate in hexane. 2,3,4-Tri-O-acetyl-S-deoxY-S-fluoro-a-D-manno-di-O-benzyl phosphate (81). Compound 79 ( 30 mg, 0.085 mmol) was dissolved in anhydrous acetonitrile saturated with dimethylamine (5 ml ) at -20°C and stirred for 3 h 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 2,3,4-tri-O-acetyl-6-deoxY-6-floro-a-D-mannopyranoside (80). To a stirred

Synthesis of [6-Deoxy-6-fluoro]-GDP Mannose95 (Scheme 17 of Results and Discussion)

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

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

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

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

(34 mg, 0.198 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 bicarbonate. The mixture was extracted with CH2CI2. The organic phase was thoroughly washed with water, dried over Na2S04 and concentrated to afford 69, which was purified by running a silica coated preparative TLC plate; Rf = 0.23 in 50% ethyl acetate in hexane; 1H NMR characterstic.8 5.72 (1 H, dd, JHP = 6.9 Hz, JHH = 1.8 Hz, H-1), 5.83 (1 H, t, JHF = 53.4 Hz, H-6); 31p NMR 8 -2.81; ESMS (mlz): 753.36 (M+Nat. 6-Deoxy-6,6-difluoro-a-D-mannosyl phosphate (70). To a solution of 69 (25 mg, 0.034 mmol) in CH30H (1 mL) was added palladium on charcoal (10%, 170 mg) and formic acid (100 j.!L). The mixture was stirred at 50°C overnight. The catalyst was removed by passing the mixture through a pad of celite. A few drops of triethylamine were added and the solution was stirred for 15 minutes. The solvent was evaporated and the product was repeatedly lyophilized to afford 70; ESMS (mlz): 279.22 (M-H)". Guanosine 5'-diphospho-6-deoxy-6,6-difluoro-a-D-mannose (mono-triethyl amine salt) 71. A mixture of 4-morpholine-N,N'-dicyclohexylcarboxaminidium guanosine 5'-monophosphomorpholidate (29 mg, 0.037 mmol) and 70 (11 mg, 0.023 mmol) was coevaporated with dry pyridine (3 x 200 j.!L). 1 H-tetrazole (5.5 mg, 0.074 mmol) and anhydrous pyridine (900 j.!L) 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 71; ESMS (mlz): 624.15 (M-H)"

Methyl-6-deoxy-6,6-difluoro-2,3,4-tri-O-benzyl-a-D-mannopyranoside (66). A solution of oxalyl chloride (54.62 mg, 37.6 Ill, 0.43 mmol) in anhydrous CH2CI2 (15 ml) was cooled to -78°C and DMSO (67.2 mg, 62 Ill, 0.86 mmol) was added dropwise, followed by the addition of a solution of 65 (500 mg, 1.07 mmol) in CH2CI2 (5 ml) over a period of 5 minutes. The mixture was stirred for another 30 minutes and then triethylamine (1.2 ml) was added. The solution was brought to room temperature, water was added and the mixture was extracted with CH2CI2. The organic layer was dried over Na2S04 to give the intermediate aldehyde. A solution of DAST (112.8 mg, 92.5 Ill, 0.7 mmol) in anhydrous CH2CI2 (1.5 ml) was cooled to -78°C. To this was added a solution of the aldehyde (325 mg, 0.7mmol) in anhydrous CH2CI2 (1.5 ml) dropwise. The mixture was stirred at rt for 90 minutes. After cooling to -20°C, excess of reagent was destroyed by the addition of CH30H and sodium bicarbonate. The mixture was filtered once effervescence ceased. The filtrate was concentrated and the residue was purified by silica column chromatography (5% ethyl acetate in hexane) to afford 66; Rt = 0.34 in 25% ethyl acetate in hexane; 1H NMR characterstic 8 5.97 (1 H, t, JHF = 52.6 Hz, H-6); 19F NMR &-132.65 (dd, J = 57 and 10.9 Hz), -132.90 (d

d, J = 57 and 16.4 Hz); ESMS (mlz): 507.2 (M+Nat. Acetyl-2,3,4-tri-O-benzyl-6-deoxY-6,6-difluoro-a-D-mannopyranoside (67). To compound 66 (70 mg, 0.144 mmol) was added 1 % sulfuric acid solution in acetic anhydride (1 ml). The mixture was stirred at rt for 1 h. 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 Na2S04 and concentrated to afford 67 which was purified by silica column chromatography (5% ethyl acetate in hexane); Rt = 0.34 (30% ethyl acetate in hexane). 2,3,4-Tri-O-benzyl-6-deoxy-6,6-difluoro-a-D-manno-di-O-benzyI phosphate (69). Compound 67 (50 mg, 0.105 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 2,3,4-tri-O-benzyl-6,6-difluoro-a-D-mannopyranoside (68). To a stirred solution of compound 68 (46 mg, 0.097 mmol) and 1 H-tetrazole (8.5 mg, 0.118 mmol) in anhydrous CH2CI2 (400 Ill) was added dibenzyl-N,N-diisopropylphosphoramidite (39 Ill, 40.9 mg, 0.118 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

Methyl-6-0-(triphenylmethyl)-a-D-rnannopyranoside (63). Methyl-a-D-manno pyranoside (62, 5g, 25.7 mmol) was dissolved in DMF (17 mL). Trityl chloride (7.9 g, 28.3 mmol), DMAP (515 mg, 2.06 mmol) and triethylamine (3.9 mL, 28.3 mmol) were added, and the mixture was stirred at room temperature overnight. The reaction mixture was concentrated and the residue was purified by silica column chromatography (5% CH30H in CH2CI2) to give 63 (8 g, 71.4%); R, = 0.14 in 5% CH30H in CH2CI2; ESMS (mlz): 459 (M+Nat. Methyl 2,3,4-tri-O-benzyl-6-0-(triphenylmethyl)-a-D-mannopyranoside (64). Compound 63 (5.8 g, 13.3 mmol) was dissolved in DMF (80 mL), followed by addition of sodium hydride (60% dispersion, 2.12 g, 53.2 mmol) and benzyl bromide (6.3 mL, 53.2 mmol) dropwise at 0 °C. The reaction mixture was stirred overnight at rt and the excess of sodium hydride was destroyed by addition of CH30H and water. The mixture was extracted with CH2CI2. The organic phase was washed thoroughly with saturated NaHC03 solution and water, dried over Na2S04 and concentrated to give 64; R,= 0.45 in 20% ethyl acetate in hexane; 1H NMR: 83.25 (dd, 1 H, H-2), 3.39 (s, 3H), 3.7-4.0 (m, 5H), 4.29-4.82 (7H, m, 3 x PhCH2 and H-1), 6.9-7.54 (m, 30H, Ph). Methyl 2,3,4-tri-O-benzyl-a-D-mannopyranoside (65). To a solution of compound 64 (1 g, 1.415 mmol) in CH2CI2 : CH30H (1 :2, 9 mL) was added p-toluene sulfonic acid (14 mg) and the mixture was stirred at rt for 2 h. Excess of acid was neutralized by the addition of triethylamine. The mixture was concentrated and purified by silica column chromatography (40% ethyl acetate in hexane) to yield 65 (4.5 g, 72.5%); R, = 0.13 in 30% ethyl acetate in hexane; 1H NMR: 8 3.29 (s, 3H, OMe), 3.61-3.96 (m, 5H), 3.93 (dd, J = 9 and 7.5 Hz, 1 H, H-3), 4.69 (d, J = 3 Hz, H-1), 4.63-4.95 (m, 6H, 3 x Ph CH2) , 7.25-7.34 (m, 15H, Ph); 13C NMR: 8 54.68, 62.34, 71.95, 72.89, 74.67, 74.79,75.10,80.13,99.27,127.50-128.30; ESMS (mlz): 487.3 (M+Nat.

Synthesis of [6-0eoxy-6,6-difluoro]-GOP Mannose94 (Scheme 15 of Results and Discussion)

Synthesis of GOP Mannose analogues

Design and Synthesis of GOP Man analogues and Evaluation of Golgi-specific GOP-Man transporter activity of L.donovan

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 [3H] 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 membranes were suspended (1.4 x 108 cell equivalent) in 250 J..ll of incorporation buffer (50 mM HEPES, pH = 7.4, 25 mM KCI, 5 mM MgCb, 5 mM MnCI2, 0.1 mM TlCK, 1 J..lg/ml leupeptin, 1 mM ATP, 0.5 mM dithiothreitol and 0.4 J..lg/ml tunicamycin). Each assay tube was prepared by adding 12.5 J..ll of 1% Chaps, 28 J..ll of 200 J..lM GDP-Man, 10 J..ll of GDP-[3H]Man (1 J..lCi) and 25 nmol of synthetic

Elongating mannosyl phosphate transferase (eMPT) assay45

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

Preparation of Cell-free system of L.donovani

incubated at 23°C in a cooling incubator (CI-12S; Remi). Fresh passaging was done weekly in a similar fashion. After about 15 passages, a fresh cryostock from liquid nitrogen was expanded and passaging done as mentioned before. Random samples from culture flasks free from any visible microbial contamination and full of all healthy, motile parasites under microscopic examinations formed the basis of selection of the culture suitable for further use. After culturing, used flasks, pipettes, glassware etc were decontaminated by immersing them in 5% formaldehyde solution and then discarded. All other routine standard cell culture practices were observed.

For both routine as well as bulk culture of L.donovani 008 strain promastigotes, medium dMEM was used. This media was prepared by dissolving one sachet of powdered media dMEM (GIBCO BRl) in 800 ml of distilled water. To this was added 25 mM HEPES and other supplements (0.05 mM adenosine, 0.05 mM xanthine, 1 mg biotin, 0.04% tween 80, 5 mg hemin, 0.5% triethanolamine, 0.3% bovine serum albumin, 50 mg gentamycin sulfate). pH of the media was adjusted to 7.2 , volume made upto one litre and the media was sterilized using bell filter (0.22 Il, Sterivex GV; Millipore). The media was used within two months of preparation. To this media, as per requirement of routine culture, heat inactivated fetal bovine serum (HI-FBS) was added @ 10%. In the present study Leishmania donovani, 008 strain, promastigotes were used throughout obtained from Prof. K.P.Chang, Chicago Medical Centre, USA. These were initially isolated from patients native to central Bihar. Upon arrival these promastigotes were expanded in medium 199 and cell bank was raised where -107 viable parasites were taken in 1 ml of complete medium 199 containing 10% glycerol. These were stored in liquid nitrogen. The revival capacity of these frozen cells was checked after one week storage by snap thawing the contents of one vial at 37°C, inoculating 50 ml of dMEM media with the entire contents and incubation at 23°C for one week. A luxuriant growth with healthy viable parasites was observed under the microscope. Routinely, L.donovani promastigotes were cultured in T-125 culture flasks having 50 ml of dMEM media each supplemented with 10% FBS. Media was inoculated with 100 III of a previous culture containing _106 promastigotes. These flasks were

Maintenance and revival of L.donovani culture

Preparation of Microsomal Membranes of L.donovani parasite

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

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

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

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

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

(Scheme 13 of Results and Discussion)

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

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

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

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

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

5.2 (m, 3H, H-1, 4, 3), 5.28 (dd, J = 2.1 and 3.6 Hz, 1 H, H-2), 7.95 (d, JHP=637 Hz, 1 H); 31 P NMR f> 0.129; ESMS (mlz) 699.27 (M-Et3N-H)" Stearyl-2,3,6-tetra-O-acetyl-4-0-(2,3,4,6-tetra-O-acetyl-~-D-galactopyranosyl)-a­D-mannopyranosyl phosphate triethylammonium salt (48). A mixture of H-phosphonate 47 (25 mg, 0.031 mmol) and stearyl alcohol (11 mg, 0.04 mmol) was dried by evaporation of pyridine (2 x 0.5 mL). The residue was dissolved in anhydrous pyridine (1 mL), adamantane carbonyl chloride (16 mg, 0.08 mmol) was added, and the mixture was stirred at rt for 1 h whereafter a freshly prepared solution of iodine (16 mg, 0.063 mmol) in 95% aqueous pyridine (3 mL) was added. After 30 min CH2CI2 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 (2.5% CH30H in CH2CI2 with 1 % Et3N) to afford 48; Rt = 0.46 in 20% CH30H in CH2CI2; 1H NMR: 8 0.84 (t, 3H, CH3), 1.23-1.45 (lipid protons), 1.85-2.12 (m, 21 H, OAc), 3.84-4.16 (m), 4.51 (d, J = 7.8 Hz, 1H, H-1'), 4.85-5.01 (m, 2H, H-2', 3'), 5.25 (m, 3H, H-1, 4, 3), 5.52 (dd, J = 2.1 and 3.6 Hz, 1 H, H-2), 5.69 (dd, 1 H, JHP = 6.8 and J1,2 =1.9 Hz, H-1); 13C NMR: 8 13.99, 20.48-20.77, 22.56, 27.8-29.59, 31.80, 36.44, 38.78, 52.82, 60.69, 68.99, 69.48, 70.23, 70.91, 76.52, 93.26, 100.93, 168.99-170.42; 31p NMR: 8 -2.90; ESMS (mlz): 967 (M-Et3N-H)' Stearyl-4-~-D-galactopyranosyl-a-D-mannopyranosylphosphate triethylammo nium salt (49). To a solution of compound 48 (15 mg, 0.014 mmol) in anhydrous CH30H (2.5 mL) was added anhydrous sodium carbonate (16 mg, 0.15 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 49 in quantitative yield; Rt= 0.55 in 10:10:3 CH30H:CH2CI2:O.25% KCI; 31p NMR.8 -1.72; ESMS (mlz): 673 (M-Et3N-H)'

1 ,2,3,6-Tetra-O-acetyl-4-0-(2,3,4,6-tetra-O-acetyl-~-D-galactopyranosyl)-a.-D­mannopyranose (46). Acetic anhydride (4 ml) was added dropwise to a stirring solution of Gal (1-4)~ Man (45, 700 mg, 2.04 mmol) in anhydrous pyridine (6 ml) at 0 °C. The reaction mixture was gradually brought to room temperature and stirred for 16 h. After completion of the reaction, the mixture was poured over ice and the product crystallized out to afford 46 in quantitative yield. Triethylammonium 2,3,6-tri-O-acetyl-4-0-[2,3,4,6-tetra-O-acetyl-~-D-galacto pyranosyl]-a.-D-mannopyranosyl hydrogen phosphonate (47). Compound 46 (600 mg, 0.89 mmol) was dissolved in anhydrous CH3CN saturated with dimethylamine (40 mL) at -20°C and stirred for 3 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 to provide 2,3,6-tri-O-acetyl-4-0-(2,3,4,6-tetra-O-acetyl-~-D-galactopyranosyl)-a.-D-mannopyra nose. To a stirred solution of imidazole (1 g, 14.68 mmol) in anhydrous CH3CN (20 mL) at 0 °C was added phosphorus trichloride (0.8 ml, 9.14 mmol) and triethylamine (2.4 mL, 0.86 mmol). The mixture was stirred for 20 min, after which a solution of the above anomeric deprotected compound (500 mg, 0.786 mmol) in anhydrous CH3CN (20 mL) was added dropwise. The mixture was stirred at 0 °C for 2 h and quenched with 1 M triethylammonium (TEA) hydrogen carbonate solution (pH=7.2, 10 mL). The clear solution was stirred for 15 min. CH2CI2 was added and the organic layer was washed with ice cold water (2 x 10 ml) and cold 1 M TEA hydrogen carbonate solution (2 x 10 ml), dried over Na2S04 and concentrated to yield 47 (500 mg, 86.2%); Rt = 0.35 in 20% CH30H in CH2CI2; 1H NMR: 8 1.9-2.08 (m, 21 H, 7 x OAc), 3.84-4.13 (m, 6H, H-5, 5', 6, 6'), 4.35 (d, J = 4.5 Hz, 1 H, H-4), 4.47 (d, J = 7.8 Hz, 1 H, H-1 '), 4.9 (dd, J =3.3 and 7.8 Hz, 1 H, H-3'), 5.05 (dd, J = 2.1 and 7.8 Hz, 1 H, H-2'),

Synthesis of Stearyl linked Gal 1,4 ~ Man phosphate (synthetic substrate for elongating-MPT activity)

Synthesis of Radiolabeled Exogenous Precursor of Phosphoglycan Biosynthesis

Design, Synthesis and Evaluation of Inhibitors of Phosphoglycan biosynthesis

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

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

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

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

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

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

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

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

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

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

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

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

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

Synthesis of Tetrasaccharide Cap Domain of LPG

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

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

Synthesis of phosphoglycans by polycondensation

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

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

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

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

Solid phase phosphoglycan synthesis

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

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

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

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

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

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

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

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

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

Solution Phase Synthesis of Phosphoglycans

Synthesis of Phosphoglycan Repeats of Lipophosphoglycan

The NMR spectra of the compounds were obtained on a 300 MHz (for 1H) NMR spectrometer (Avance-DRX 300; Bruker), equipped with a quadrinuclear probe (QNP) and an inverse gradient probe, using XWIN NMR software. Both these probes were 5 mm probes. Deutrated solvents (CDCI3, CD30D, 020 etc) were used for dissolving samples and locking the instrument. Tetramethylsilane (TMS, SiMe4) was used as the internal reference for 1H NMR and 80% ortha-phosphoric acid as external reference for 31 P-NMR. The chemical shifts have been expressed in terms of parts per million (ppm, 6) relative to TMS and coupling constants (J-values) have been expressed in Hertz (Hz). Molecular masses of the compounds were determined by mass spectrometry. Depending on the nature of the compound, electrospray-ionization (ES-MS) was obtained in negative or positive ion mode on a quadrupole mass spectrometer (VG Platform II; VG BioTech, Fisons Instruments, Altrincham, UK) using MassLynx software. High resolution mass spectra were obtained from IICT, Hyderabad and University of Kansas mass spectrometry facility. Optical rotations were obtained using a Perkin-Elmer 241 Spectropolarimeter. Measurements were made at 25°C using sodium D-line. The [alo values have been expressed in the units of 10.1 deg cm2 gm-1. The entire radioactivity operation was carried out in a fume-hood devoted to radiochemical work. Disposable items were discarded at a defined and instructed place. All other necessary precautions for handling radioactivity were taken. Liquid scintillation counting of samples was done on a scintillation counter using preset program for 1H ~ emitter. A suitable aliquot in triplicate was mixed with 5 mL scintillation fluid (Cocktail W-10 g PPO, 0.25 g POPPO and 100 g naphthalene per litre of 1,4-dioxan; SRL) in scintillation vials. For solvent blank, vial containing 5 mL scintillation cocktail was used.

All the reagents used in chemical syntheses and biosynthetic experiments were of the highest purity grade available. Glass-backed and aluminium TLC plates (Kieselgel 60 F254) were procured from Merck. Silica coated preparative glass-backed TLC plates were purchased from AnalTech. AG1-X8 and Dowex 2X8 anion exchange resins were obtained from Bio-Rad. All dry solvents were prepared in the laboratory using standard procedures for drying. Milli Q UF (Reverse Osmosed, ion exchanged and Ultra-filtered; Millipore Corporation, USA) grade water was used. For refluxing an oil-bath (high boiling silicone oil) was used and the temperature was controlled by a variostate and sensor. Stirring of the contents of reaction flasks, oil bath etc., were done using appropriate sized magnetic bars and Magnetic stirrer (Remi). For filtration of materials, Whatman #1 paper and Celite (Fluka) was used. For removing solvent from compounds, a flash evaporator (Rotavapour R-114, Buchi) was used which was connected to a water-chiller circulator and at times with a high vacuum pump to remove high boiling solvents. Monitoring the progress of reactions, analysis of column fractions and identification of reaction intermediates were done by thin layer chromatography on glass backed precoated TLC plates. The developed plates were air-dried and subjected to the following detection system: 1. Iodine vapors: Sublime iodine crystals were mixed with silica gel in an air-tight chamber. When the chamber was full of iodine vapors, the plates were exposed to this when yellowish brown spots were visible against white background. 2. Ultraviolet light: U.V. absorbing compounds were visualized by a hand-held UV lamp (Spectroline Model ENF-260C/F) employing both long and short wavelength UV. 3. Ammonium molybdate-ceric sulfate reagent: This reagent was prepared by dissolving ammonium molybdate (2.5 g) and ceric sulfate (1 g) in water (90 mL) and conc. Sulfuric acid (10 mL). The developed plates were immersed in this reagent and heated with a heat-gun (HEJET Model, Aldrich). Blue spots were observed when the compounds reacted with this reagent.

General procedures

RFFIT is used for detennination of rabies virus neutralizing antibody (RVNA) titers. It is an in vitro cell culture based technique in which foci of virus-infected cells are observed by fluorescent antibody staining. In brief, mouse neuroblastoma (MNA) cells were cultured in T-25 tissue culture flasks in DMEM supplemented with 10% FCS at 35°C in humidified atmosphere of0.5% C02. For subculturing, cells were trypsinized (0.5% trypsin + 0.2% EDT A in DMEM without FCS), centrifuged at 150 X g for 10 min, resuspended in DMEM supplemented with 10% FCS and aliquoted into T -25 flasks. Sera from immunized mice were heat inactivated at 56°C for 30 min and the RVNA titers were determined by RFFIT as described previously (Smith . et al., 1996). Briefly, 100 111 of various dilutions of the reference (Standard Rabies Immune Globulin, Biological Research and Reviews, FDA, Maryland, US) and the test sera were mixed with 100 111 Challenge Virus Strain-11 of rabies virus (containing 50 FFD50) in 8-well tissue culture chamber slides and incubated at 35°C in presence of 0.5% C02 for 90 min. After the incubation period, 0.2 ml of MNA cells ( 1 x 1 05) were added to each well and the slides incubated for 40 h following which these were fixed in chilled acetone and stained with FITC conjugated anti-rabies MAb (Centocor Inc, USA) for 45 min. The slides were washed three times with PBS, mounted in glycerol : PBS (9 : 1 ), and examined under fluorescence microscope (Optiphot, Nikon, Japan). Data was expressed as neutralizing antibody titer that is the reciprocal of the serum dilution resulting in a 50% reduction in the number of the virus infected cells in the presence of the test serum.

RAPID FLUORESCENT FOCUS INHIBITION TEST (RFFIT)

Turku, Finland). Data were expressed as mean counts per minute (cpm) ± SE of triplicate cultures.

Single cell suspensions of splenocytes in RPMI-1640 medium were prepared from plasmid DNA immunized mice, on day 45, by mechanical disruption of the spleen. Red blood cells were lysed by exposing the cell pellet to 1 OX concentration of 50 mM PBS and immediately bringing the concentration to IX PBS by addition of water. Cells were diluted to a final concentration of 3 x 106 cells/ml in RPMI-1640 medium supplemented with l 0% FCS. A 100 J.ll aliquot of splenocytes was added to each well of a 96-well microtitration plate containing serial dilutions of refolded recombinant proteins (r-bmZP1, r-dZP3 orr-rG), diluted in the same medium, as a source of antigen. All assays were carried out in . triplicates. Three days after the addition of the cells, culture were pulsed with 1 J.lCi/well of eH] thymidine (NEN, Life Science Products, Boston, MA) for 16 h. Cells were lysed and harvested onto glass fibre filaments for liquid scintillation counting (Betaplate; Wallac,

T CELL PROLIFERATION ASSAY

weight) as a general anesthetic and ovaries were snap frozen in liquid nitrogen. Ovarian sections of 5 J.lm thickness were cut in a cryostat at -20°C and fixed in chilled methanol for 15 min at RT. Sections passing through follicles were selected, washed with 50 mM PBS and blocked with 3% normal goat serum (NGS) in PBS (v/v) at RT for 1 h. Sections were then washed two times with PBS and incubated with 1: 1 0 dilution of immune serum samples. Ovarian sections incubated with 1:10 dilution of mouse preimmune or immune sera from mice immunized with VR1020 vector served as negative controls. After incubation, the sections were washed three times with PBS and incubated with 1:800 dilution of goat anti-mouse IgG conjugated to FITC (Sigma) for 1 hat RT. The slides were washed three times with PBS, mounted in glycerol : PBS (9 : 1 ), and examined under fluorescence microscope (Optiphot, Nikon, Japan).

Ability of mouse polyclonal antibodies, generated subsequent to immunization with VRbmZP1 and VRdZP3 plasmid DNA, to recognize native ZP was evaluated by indirect immunofluorescence assay. A normal cycling female bonnet monkey and a female dog were ovariectomized after administration of ketamine hydrochloride (5 mg/kg body

REACTIVITY WITH NATIVE ZP OF THE IMMUNE SERUM SAMPLES OBTAINED FROM MICE IMMUNIZED WITH VRbmZPl AND VRdZP3 PLASMID DNA

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

Antibody isotyping

492 run with 620 nm as the reference filter. The antibody response generated was represented as the geometric mean of the absorbance of individual mice sera in a group of immunized animals. b1 addition, the antibody titer against r-dZP3 was also determined by ELISA. The assay was carried out as described above except that 100 ~l of doubling dilutions of the serum samples (dilutions made in PBST supplemented with 0.1% BSA) were added per well in duplicate. For each serum sample tested, a reciprocal of dilution giving an absorbance of 1.0 was calculated by regression analysis and represented as antibody units (AU).

Microtitration plates were coated with optimized concentration of r-bmZP1 (250 ng/well), r-dZP3 (400 ng/well) or r-rG (500 ng/well) in 50 mM PBS, pH 7.4, at 37°C for 1 h and then at 4°C, 0/N. The plates were washed once with PBS and incubated with 1% BSA, (200 Jll/ well) in PBS for 2 h at 37°C for blocking the non-specific sites. All subsequent incubations were carried out for 1 h at 37°C and each incubation was followed by three washings with PBS containing 0.05% Tween-20 (PBST). Post-blocking, the plates were incubated with 1 :50 dilution of either the preimmune or the immune serum samples obtained from mice immunized with the respective plasmid DNA. Antibodies bound tor-bmZP 1, r-dZP3 and r-rG were revealed with 1:2000 dilution of goat anti-mouse IgG (whole molecule) HRPO (Dako). Estimation of the enzymatic activity was carried out with 0.05% OPD in 50 mM citrate phosphate buffer, pH 5.0, containing 0.06% H202 as the substrate. The reaction was stopped with 50 Jll of 5 N H2S04 and the absorbance read at

Enzyme Linked Immunosorbant Assay (ELISA)

CHARACTERIZATION OF THE ANTIBODIES GENERATED IN RESPONSE TO PLASMID DNA IMMUNIZATION

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

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

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

Plasmid DNA adsorbed onto gold microcarriers

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

Plasmid DNA administered by electroporation

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

Plasmid DNA administered in saline

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

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