(3R,4S,5R,6R)-6-(hydroxymethyl)oxane-2,3,4,5-tetrol

Conditions: temp:0.00; pressure:0.00; stir:; See reaction.notes.procedure_details.

Procedure: To a 250 ml round bottom flash, charged with 10.0 g (0.041 mole) of N ε-(tert-butoxy carbonyl) L-lysine 27, commercially available from BACHEM Bioscience Inc. (subsidiary of BACHEM Switzerland, 3700 Horizon Drive, Renaissance at Gulph Mills, King of Prussia, Pa., 19406, USA), and 100 ml of methanol is added 10.0 g (0.07 mole) of ethyl trifluoroacetate followed by 20.0 ml (0.144 mole) of triethylamine. The mixture is stirred at 20-300° C. for 20 hours and then concentrated via rotary evaporation. The residue is diluted with 50 ml of diethyl ether and then filtered. The filtrate is then washed with 3×30 ml aliquots of 1N aqueous HCL, 20 ml of deionized water, and finally with 20 ml of deionized water. The organic phase is dried over anhydrous magnesium sulfate, filtered and concentrated. The residue is distilled under vacuum to yield product 28 in good yield. N ε-(tert-butoxy carbonyl) N1-α-trifluoroacetyl L-lysine is reacted with tri-o-acetyl tet-Gal-NAC 1-a-S-Pentylamine in the presence of a BOP reagent. The activated carboxylic acid reacts with the clustered galactosamine in DBIF and Triethylamine to afford galactose cluster lyEsine adduct 29 in good yield. The N a-trifluoroacetyl protection of the lysine is removed to afford Primary α-amino lysine-galactose cluster 29a in moderate yield.

Conditions: temp:0.00; pressure:0.00; stir:; See reaction.notes.procedure_details.

Procedure: To a 250 ml round bottom flash, charged with 10.0 g (0.041 mole) of N ε-(tert-butoxy carbonyl) L-lysine 27, commercially available from BACHEM Bioscience Inc. (subsidiary of BACHEM Switzerland, 3700 Horizon Drive, Renaissance at Gulph Mills, King of Prussia, Pa., 19406, USA), and 100 ml of methanol is added 10.0 g (0.07 mole) of ethyl trifluoroacetate followed by 20.0 ml (0.144 mole) of triethylamine. The mixture is stirred at 20-30° C. for 20 hours and then concentrated via rotary evaporation. The residue is diluted with 50 ml of diethyl ether and then filtered. The filtrate is then washed with 3×30 ml aliquots of 1N aqueous HCL, 20ml of deionized water, and finally with 20 ml of deionized water. The organic phase is dried over anhydrous magnesium sulfate, filtered and concentrated. The residue is distilled under vacuum to yield product 28 in good yield. N ε-(tert-butoxy carbonyl) N1α-trifluoroacetyl L-lysine is reacted with tri-o-acetyl tet-Gal-NAC 1-a-S-Pentylamine in the presence of a BOP reagent. The activated carboxylic acid reacts with the clustered galactosamine in DMF and Triethylamine to afford galactose cluster lysine adduct 29 in good yield. The N α-trifluoroacetyl protection of the lysine is removed to afford Primary α-amino lysine-galactose cluster 29a in moderate yield.

Conditions: temp:0.00; pressure:0.00; stir:; See reaction.notes.procedure_details.

Procedure: Eight co-integrants each from the crosses summarized in Table 2 were streaked for single colonies (from cultures cryo-preserved in 10% glycerol; described in Section 6.4)) on Luria broth plates with ampicillin (100 μg/ml), spectinomycin (50 μg/ml) and gentamycin (10 μg/ml). Single colonies were inoculated into Luria broth liquid (without drug) and incubated overnight at 37° C. with gyratory shaking. Ten microliters (each) from these cultures were spread on a MacConkey Agar (base; Becton Dickson, Difco Division, Franklin Lakes, N.J.) plated with 2% galactose and 100 μg/ml ampicillin. Following overnight growth three types of galactose-resistant colonies appeared on the agar surface: red colonies (with various morphologies), white opaque colonies and white translucent colonies, in numbers varying from a few dozen to several hundred. Resolved co-integrants were among the white translucent colonies, and a single white colony was picked from each spot, and re-streaked for purification on the same MacConkey agar. These purified white colonies were tested for spectinomycin resistance (50 μg/ml) and gentamycin resistance (10 μg/ml). The resultant colony types are summarized below in Table 4 (Gent=gentamycin, Spec=spectinomycin, R=resistance, and S=sensitivity) were inoculated into Luria broth liquid (without drug) and incubated overnight at 37° C. with gyratory shaking re-streaked for purification on the same MacConkey agar

Conditions: temp:0.00; pressure:0.00; stir:; See reaction.notes.procedure_details.

Procedure: To a 250 ml round bottom flash, charged with 10.0 g (0.041 mole) of N ε-(tert-butoxy carbonyl) L-lysine 27, commercially available from BACHEM Bioscience Inc. (subsidiary of BACHEM Switzerland, 3700 Horizon Drive, Renaissance at Gulph Mills, King of Prussia, Pa., 19406, USA), and 100 ml of methanol is added 10.0 g (0.07 mole) of ethyl trifluoroacetate followed by 20.0 ml (0.144 mole) of triethylamine. The mixture is stirred at 20-30° C. for 20 hours and then concentrated via rotary evaporation. The residue is diluted with 50 ml of diethyl ether and then filtered. The filtrate is then washed with 3×30 ml aliquots of 1N aqueous HCL, 20ml of deionized water, and finally with 20 ml of deionized water. The organic phase is dried over anhydrous magnesium sulfate, filtered and concentrated. The residue is distilled under vacuum to yield product 28 in good yield. N ε-(tert-butoxy carbonyl) N1α-trifluoroacetyl L-lysine is reacted with tri-o-acetyl tet-Gal-NAC 1-a-S-Pentylamine in the presence of a BOP reagent. The activated carboxylic acid reacts with the clustered galactosamine in DMF and Triethylamine to afford galactose cluster lysine adduct 29 in good yield. The N α-trifluoroacetyl protection of the lysine is removed to afford Primary α-amino lysine-galactose cluster 29a in moderate yield.

Conditions: temp:0.00; pressure:0.00; stir:; See reaction.notes.procedure_details.

Procedure: Eight co-integrants each from the crosses summarized in Table 2 were streaked for single colonies (from cultures cryo-preserved in 10% glycerol; described in Section 6.4)) on Luria broth plates with ampicillin (100 μg/ml), spectinomycin (50 μg/ml) and gentamycin (10 μg/ml). Single colonies were inoculated into Luria broth liquid (without drug) and incubated overnight at 37° C. with gyratory shaking. Ten microliters (each) from these cultures were spread on a MacConkey Agar (base; Becton Dickson, Difco Division, Franklin Lakes, N.J.) plated with 2% galactose and 100 μg/ml ampicillin. Following overnight growth three types of galactose-resistant colonies appeared on the agar surface: red colonies (with various morphologies), white opaque colonies and white translucent colonies, in numbers varying from a few dozen to several hundred. Resolved co-integrants were among the white translucent colonies, and a single white colony was picked from each spot, and re-streaked for purification on the same MacConkey agar. These purified white colonies were tested for spectinomycin resistance (50 μg/ml) and gentamycin resistance (10 μg/ml). The resultant colony types are summarized below in Table 4 (Gent=gentamycin, Spec=spectinomycin, R=resistance, and S=sensitivity) were inoculated into Luria broth liquid (without drug) and incubated overnight at 37° C. with gyratory shaking re-streaked for purification on the same MacConkey agar

Conditions: temp:0.00; pressure:0.00; stir:; See reaction.notes.procedure_details.

Procedure: Solutions of Glu (40 mM-4.0 μM), Fru (0.4 mM), Gal (0.4 mM) and Man (0.4 mM) in deionized water were prepared. An aliquot (2.0 mL) of the solution of TPEDB (0.1 mM) in the carbonate buffer containing 4 vol % DMSO was added into a 10 mL volumetric flask, followed by the addition of 1.0 mL of a solution of Fru (0.4 mM) and 1.0 mL of a solution of Glu (40 mM). After shaking for a few seconds, a solution of TPEDB (50 μM) with 10 mM of Glu and 0.1 mM of Fru in the carbonate buffer containing 2% DMSO was obtained. The solution was allowed to stand for 30 min before its FL measurement. The solution of TPEDB and Glu containing another saccharide (Gal or Man) was prepared by similar procedures. was added into a 10 mL volumetric flask was obtained

Conditions: temp:0.00; pressure:0.00; stir:; See reaction.notes.procedure_details.

Procedure: To a 250 ml round bottom flash, charged with 10.0 g (0.041 mole) of N ε-(tert-butoxy carbonyl) L-lysine 27, commercially available from BACHEM Bioscience Inc. (subsidiary of BACHEM Switzerland, 3700 Horizon Drive, Renaissance at Gulph Mills, King of Prussia, Pa., 19406, U.S.A.), and 100 ml of methanol is added 10.0 g (0.07 mole) of ethyl trifluoroacetate followed by 20.0 ml (0.144 mole) of triethylamine. The mixture is stirred at 20-30°0 C. for 20 hours and then concentrated via rotary evaporation. The residue is diluted with 50 ml of diethyl ether and then filtered. The filtrate is then washed with 3×30 ml aliquots of 1N aqueous HCL, 20 ml of deionized water, and finally with 20 ml of deionized water. The organic phase is dried over anhydrous magnesium sulfate, filtered and concentrated. The residue is distilled under vacuum to yield product 28 in good yield. N ε-(tert-butoxy carbonyl) N1 -α-trifluoroacetyl L-lysine is reacted with tri-o-acetyl tet-Gal-NAC 1-a-S-Pentylamine in the presence of a BOP reagent. The activated carboxylic acid reacts with the clustered galactosamine in DMF and Triethylamine to afford galactose cluster lysine adduct 29 in good yield. The N α-trifluoroacetyl protection of the lysine is removed to afford Primary α-amino lysine-galactose cluster 29a in moderate yield.

Conditions: temp:0.00; pressure:0.00; stir:; See reaction.notes.procedure_details.

Procedure: Lactose was contacted with UDP-N-acetylglucosamine and a β-galactoside β1-3 N-acetylglucosaminyl transferase purified from pig serum in a 0.1M HEPS buffered aqueous solution at 37° C. The product tetrasaccharide was then contacted with UDP--Gal and a β-N-acetylglucosaminoside β1-4 Galactosyltransferase isolated from bovine milk, in a 0.1M HEPS buffered aqueous solution at 37° C. The product tetrasaccharide was then contacted with CMP--NAN and a β-galactoside α2-6 sialyltransferase isolated from rat liver in a 0.1M HEPES buffered aqueous solution at 37° C. The title pentasaccharide was isolated by conventional methods. a β-galactoside β1-3 N-acetylglucosaminyl transferase purified from pig serum in a 0.1M HEPS at 37° C

Conditions: temp:0.00; pressure:0.00; stir:; See reaction.notes.procedure_details.

Procedure: Man 32.8; Glc 188.3

Conditions: temp:0.00; pressure:0.00; stir:; See reaction.notes.procedure_details.

Procedure: To a 250 ml round bottom flash, charged with 10.0 g (0.041 mole) of N ε-(tert-butoxy carbonyl) L-lysine 27, commercially available from BACHEM Bioscience Inc. (subsidiary of BACHEM Switzerland, 3700 Horizon Drive, Renaissance at Gulph Mills, King of Prussia, Pa., 19406, USA), and 100 ml of methanol is added 10.0 g (0.07 mole) of ethyl trifluoroacetate followed by 20.0 ml (0.144 mole) of triethylamine. The mixture is stirred at 20-300° C. for 20 hours and then concentrated via rotary evaporation. The residue is diluted with 50 ml of diethyl ether and then filtered. The filtrate is then washed with 3×30 ml aliquots of 1N aqueous HCL, 20 ml of deionized water, and finally with 20 ml of deionized water. The organic phase is dried over anhydrous magnesium sulfate, filtered and concentrated. The residue is distilled under vacuum to yield product 28 in good yield. N ε-(tert-butoxy carbonyl) N1-α-trifluoroacetyl L-lysine is reacted with tri-o-acetyl tet-Gal-NAC 1-a-S-Pentylamine in the presence of a BOP reagent. The activated carboxylic acid reacts with the clustered galactosamine in DBIF and Triethylamine to afford galactose cluster lyEsine adduct 29 in good yield. The N a-trifluoroacetyl protection of the lysine is removed to afford Primary α-amino lysine-galactose cluster 29a in moderate yield.

Conditions: temp:0.00; pressure:0.00; stir:; See reaction.notes.procedure_details.

Procedure: To a 250 ml round bottom flash, charged with 10.0 g (0.041 mole) of N ε-(tert-butoxy carbonyl) L-lysine 27, commercially available from BACHEM Bioscience Inc. (subsidiary of BACHEM Switzerland, 3700 Horizon Drive, Renaissance at Gulph Mills, King of Prussia, Pa., 19406, USA), and 100 ml of methanol is added 10.0 g (0.07 mole) of ethyl trifluoroacetate followed by 20.0 ml (0.144 mole) of triethylamine. The mixture is stirred at 20-30° C. for 20 hours and then concentrated via rotary evaporation. The residue is diluted with 50 ml of diethyl ether and then filtered. The filtrate is then washed with 3×30 ml aliquots of 1N aqueous HCL, 20ml of deionized water, and finally with 20 ml of deionized water. The organic phase is dried over anhydrous magnesium sulfate, filtered and concentrated. The residue is distilled under vacuum to yield product 28 in good yield. N ε-(tert-butoxy carbonyl) N1α-trifluoroacetyl L-lysine is reacted with tri-o-acetyl tet-Gal-NAC 1-a-S-Pentylamine in the presence of a BOP reagent. The activated carboxylic acid reacts with the clustered galactosamine in DMF and Triethylamine to afford galactose cluster lysine adduct 29 in good yield. The N α-trifluoroacetyl protection of the lysine is removed to afford Primary α-amino lysine-galactose cluster 29a in moderate yield.

Conditions: temp:0.00; pressure:0.00; stir:; See reaction.notes.procedure_details.

Procedure: Eight co-integrants each from the crosses summarized in Table 2 were streaked for single colonies (from cultures cryo-preserved in 10% glycerol; described in Section 6.4)) on Luria broth plates with ampicillin (100 μg/ml), spectinomycin (50 μg/ml) and gentamycin (10 μg/ml). Single colonies were inoculated into Luria broth liquid (without drug) and incubated overnight at 37° C. with gyratory shaking. Ten microliters (each) from these cultures were spread on a MacConkey Agar (base; Becton Dickson, Difco Division, Franklin Lakes, N.J.) plated with 2% galactose and 100 μg/ml ampicillin. Following overnight growth three types of galactose-resistant colonies appeared on the agar surface: red colonies (with various morphologies), white opaque colonies and white translucent colonies, in numbers varying from a few dozen to several hundred. Resolved co-integrants were among the white translucent colonies, and a single white colony was picked from each spot, and re-streaked for purification on the same MacConkey agar. These purified white colonies were tested for spectinomycin resistance (50 μg/ml) and gentamycin resistance (10 μg/ml). The resultant colony types are summarized below in Table 4 (Gent=gentamycin, Spec=spectinomycin, R=resistance, and S=sensitivity) were inoculated into Luria broth liquid (without drug) and incubated overnight at 37° C. with gyratory shaking re-streaked for purification on the same MacConkey agar