cyclohexane-1,2,3,4,5,6-hexol

Conditions: temp:0.00; pressure:0.00; stir:

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

Procedure: Angyal, S. J. et al. Carbohydr. Res. 76:121 (1979); Posternak, T., Biochem. Prep. 2:57 (1952). A solution containing sorbitol (1.0 g) and yeast extract (0.05 g) in 10 mL distilled, deionized water was autoclaved for 25 min and cooled to room temperature. After inoculation with Gluconobacter oxydans ATCC 621 the culture was incubated in an orbital shaker at 200 rpm for 24, h at 30° C. This G. oxydans culture was subsequently added to a second sterile solution containing myo-inositol (12.0 g, 66.7 mmol), D-sorbitol (0.4 g), and yeast extract (2.0 g) in 400 mL distilled, deionized water. After incubation in an orbital shaker at 200 rpm for 48 h at 30° C., cells were removed by centrifugation. The resulting culture supernatant was concentrated to 75 mL, MeOH (400 mL) added, and the solution maintained at −20° C. for 12 h. Precipitate which formed was filtered, washed with MeOH, and dried to afford myo-2-inosose as a white powder (8.17 g, 69%). A second crop of myo-2-inosose (3.09 g, 26%) was obtained after maintaining the filtrate at −20° C. for an additional 12 h. mp 188-192° C. 1H NMR (D2O): δ4.25 (d,J=10 Hz, 2 H), 3.66 (dd, J=9, 9 Hz), 3.26 (m, 2 H). 13C NMR (D2O): δ206.0, 94.3, 76.2, 74.5, 74.1, 74.0, 73.3, 73.2. extract (0.05 g) in 10 mL distilled was incubated in an orbital shaker at 200 rpm for 24, h at 30° C oxydans culture was subsequently added to a second sterile solution extract (2.0 g) in 400 mL distilled After incubation in an orbital shaker at 200 rpm for 48 h at 30° C. cells were removed by centrifugation The resulting culture supernatant was concentrated to 75 mL, MeOH (400 mL) added the solution maintained at −20° C. for 12 h Precipitate which formed was filtered washed with MeOH dried

Conditions: temp:97.51; pressure:0.00; stir:; See reaction.notes.procedure_details.

Procedure: The first step for the preparation of scyllitol diborate involves a sponge nickel mediated isomerization of myo-inositol in water near reflux to yield a mixture of inositols consisting of roughly 22% scyllitol (as judged by HPLC). After separating the crude reaction mixture from the nickel, scyllitol is selectively converted to scyllitol diborate by treatment with sodium tetraborate at 80-90° C. and pH 9-10. Scyllitol diborate is then crystallized by cooling and is isolated in 17-20% yield and >99% HPLC purity (AUC). All six batches of diborate proceeded as expected (Table 1). The isomerization reactions required 2-3 hours to heat to 95-100° C. and each were sampled for analysis after 1, 2, and 3 hours. All five reactions resulted in myo/scyllo ratios of 2-3:1 (typical) by 1H NMR and all but one batch resulted in >20% AUC (HPLC Analysis) scyllo-inositol (Table 2). The diborate formations resulted in myo/scyllo ratios of >60:1 (typical) by 1H NMR (Table 2) within one hour and the subsequent crystallizations were allowed to proceed over 14-16 hours (overnight). The resulting diborate slurries filtered very fast and were isolated as large white crystals. The diborate filter cakes were found to have consistent 1H NMR spectra, HPLC purities of 98.7%-99.2% AUC and moisture levels of 46.8%-56.4%. Nickel and aluminum analysis revealed levels ranging from <1 ppm to 17 ppm for nickel and <1 ppm to 28 ppm for aluminum. The yields corrected for moisture content ranged from 17.1% to 20.3%. near reflux to yield After separating the crude reaction mixture from the nickel, scyllitol Scyllitol diborate is then crystallized by cooling is isolated in 17-20% yield and >99% HPLC purity (AUC) each were sampled for analysis after 1, 2, and 3 hours All five reactions resulted in myo/scyllo ratios of 2-3:1 (typical) by 1H NMR

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

Procedure: Meanwhile, enzyme reaction product was measured as follows: 10 mg of scyllo-inosose, 40 mg of NADPH, and 10 U of the enzyme were allowed to react in 1.0 ml of 100 mM Tris buffer (pH 8.0) at 36° C. for 4 hours, and a heat treatment was performed at 80° C. for 10 min, followed by cooling. Then, 100 μl of a strong base cation exchange resin, 100 μl of a strong acid anion exchange resin, and 10 mg of activated carbon were added, and the mixture was stirred and centrifuged. Then, the supernatant was diluted 2-fold, and measurement was performed by HPLC (Shodex Asahipak NH2P-50 4E Φ 4.6×250 mm: Shodex) using an RI detector under conditions of a column temperature of 40° C. and a mobile phase flow rate of 1.5 ml (80% acetonitrile). As a result, the Atu4375 gene product and the Atu3234 gene product of Agrobacterium tumefaciens C58, the BG14057 gene product of Bacillus subtilis 168, the Xcc3438 gene product of Xanthomonas campestris pv. campestris, and the Atu4375 gene product, and the Atu3234 gene product of AB10121 strain were found to have a high enzyme activity, and 100% of the product was scyllo-inositol obtained by the reduction, while myo-inositol that is an isomer thereof was not detected. As a result, recombinant enzymes derived from the above-mentioned genes were found to have a high scyllo-inositol dehydrogenase activity, and the gene products were scyllo-inositol dehydrogenase. Meanwhile, in the products obtained by the reduction reaction of scyllo-inosose, only scyllo-inositol was detected, and the enzymes were found to stereospecifically reduce scyllo-inosose into scyllo-inositol. by cooling Then, the supernatant was diluted 2-fold under conditions of a column temperature of 40° C.

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

Procedure: Meanwhile, the enzyme reaction product was measured as follows: 10 mg of scyllo-inosose, 40 mg of NADPH, and 10 U of the enzyme were allowed to react in 1.0 ml of 100 mM Tris buffer (pH 8.0) at 36° C. for 4 hours, and a heat treatment was performed at 80° C. for 10 min, followed by cooling. Then, 100 μl of a strong base cation exchange resin, 100 μl of a strong acid anion exchange resin, and 10 mg of activated carbon were added, and the mixture was stirred and centrifuged. Then, the supernatant was diluted 2-fold, and measurement was performed by HPLC (Shodex Asahipak NH2P-50 4E Φ 4.6×250 mm: Shodex) using an RI detector under the condition of a column temperature of 40° C. and a mobile phase flow rate of 1.5 ml (80% acetonitrile). As a result, the ydgJ gene product was found to have a high scyllo-inositol dehydrogenase activity, and 100% of the product was scyllo-inositol obtained by reduction, while myo-inositol that is an isomer thereof was not detected. As a result, the solution of a recombinant enzyme derived from the ydgJ gene was found to have a high scyllo-inositol dehydrogenase activity, and the gene product was scyllo-inositol dehydrogenase. Meanwhile, in the product obtained by the reduction reaction of scyllo-inosose, only scyllo-inositol was detected, and the enzyme was found to stereospecifically reduce scyllo-inosose into scyllo-inositol. Meanwhile, the sequence of the ydgJ gene is shown in SEQ ID NO: 1, and the amino acid sequence corresponding thereto is shown in SEQ ID NO: 2. by cooling Then, the supernatant was diluted 2-fold under the condition of a column temperature of 40° C.

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

Procedure: Meanwhile, the enzyme reaction product was measured as follows: 10 mg of scyllo-inosose, 40 mg of NADPH, and 10 U of the enzyme were allowed to react in 1.0 ml of 100 mM Tris buffer (pH 8.0) at 36° C. for 4 hours, and a heat treatment was performed at 80° C. for 10 min, followed by cooling. Then, 100 μl of a strong base cation exchange resin, 100 μl of a strong acid anion exchange resin, and 10 mg of activated carbon were added, and the mixture was stirred and centrifuged. Then, the supernatant was diluted 2-fold, and measurement was performed by HPLC (Shodex Asahipak NH2P-50 4E Φ 4.6×250 mm: Shodex) using an RI detector under the condition of a column temperature of 40° C. and a mobile phase flow rate of 1.5 ml (80% acetonitrile). As a result, the ydgJ gene product was found to have a high scyllo-inositol dehydrogenase activity, and 100% of the product was scyllo-inositol obtained by reduction, while myo-inositol that is an isomer thereof was not detected. As a result, the solution of a recombinant enzyme derived from the ydgJ gene was found to have a high scyllo-inositol dehydrogenase activity, and the gene product was scyllo-inositol dehydrogenase. Meanwhile, in the product obtained by the reduction reaction of scyllo-inosose, only scyllo-inositol was detected, and the enzyme was found to stereospecifically reduce scyllo-inosose into scyllo-inositol. Meanwhile, the sequence of the ydgJ gene is shown in SEQ ID NO: 1, and the amino acid sequence corresponding thereto is shown in SEQ ID NO: 2. by cooling Then, the supernatant was diluted 2-fold under the condition of a column temperature of 40° C.

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

Procedure: In a preferred embodiment of the process the roots are cut into 4-7 mm small explants; are sterilized by treatment with 1% cetabelon (v/v) for a period of about 5-15 minutes, then with 70% (V/v) ethanol for 30 sec., followed by treatment with 0.1% HgCl2 (w/v) for about 1-2 min. The basal medium in step (a) is modified Murashige and Skoog's (1962) medium supplemented with 200 mg/l myoinositol, 10 mg/l each of thiamine hydrochloride and pyridoxine hydrochloride and 5 mg/l of nicotinic acid (Medium I), and medium II used in step (b) is obtained by addition of 2,4-dichlorophenoxyacetic acid (0.1-2.0 mg/l) and Kn (0.1-0.5 mg/l), that are preferably added to the basal medium for optimal callus growth.

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

Procedure: Angyal, S. J. et al. Carbohydr. Res. 76:121 (1979); Posternak, T., Biochem. Prep. 2:57 (1952). A solution containing sorbitol (1.0 g) and yeast extract (0.05 g) in 10 mL distilled, deionized water was autoclaved for 25 min and cooled to room temperature. After inoculation with Gluconobacter oxydans ATCC 621 the culture was incubated in an orbital shaker at 200 rpm for 24, h at 30° C. This G. oxydans culture was subsequently added to a second sterile solution containing myo-inositol (12.0 g, 66.7 mmol), D-sorbitol (0.4 g), and yeast extract (2.0 g) in 400 mL distilled, deionized water. After incubation in an orbital shaker at 200 rpm for 48 h at 30° C., cells were removed by centrifugation. The resulting culture supernatant was concentrated to 75 mL, MeOH (400 mL) added, and the solution maintained at −20° C. for 12 h. Precipitate which formed was filtered, washed with MeOH, and dried to afford myo-2-inosose as a white powder (8.17 g, 69%). A second crop of myo-2-inosose (3.09 g, 26%) was obtained after maintaining the filtrate at −20° C. for an additional 12 h. mp 188-192° C. 1H NMR (D2O): δ4.25 (d,J=10 Hz, 2 H), 3.66 (dd, J=9, 9 Hz), 3.26 (m, 2 H). 13C NMR (D2O): δ206.0, 94.3, 76.2, 74.5, 74.1, 74.0, 73.3, 73.2. extract (0.05 g) in 10 mL distilled was incubated in an orbital shaker at 200 rpm for 24, h at 30° C oxydans culture was subsequently added to a second sterile solution extract (2.0 g) in 400 mL distilled After incubation in an orbital shaker at 200 rpm for 48 h at 30° C. cells were removed by centrifugation The resulting culture supernatant was concentrated to 75 mL, MeOH (400 mL) added the solution maintained at −20° C. for 12 h Precipitate which formed was filtered washed with MeOH dried

Conditions: temp:97.51; pressure:0.00; stir:; See reaction.notes.procedure_details.

Procedure: The first step for the preparation of scyllitol diborate involves a sponge nickel mediated isomerization of myo-inositol in water near reflux to yield a mixture of inositols consisting of roughly 22% scyllitol (as judged by HPLC). After separating the crude reaction mixture from the nickel, scyllitol is selectively converted to scyllitol diborate by treatment with sodium tetraborate at 80-90° C. and pH 9-10. Scyllitol diborate is then crystallized by cooling and is isolated in 17-20% yield and >99% HPLC purity (AUC). All six batches of diborate proceeded as expected (Table 1). The isomerization reactions required 2-3 hours to heat to 95-100° C. and each were sampled for analysis after 1, 2, and 3 hours. All five reactions resulted in myo/scyllo ratios of 2-3:1 (typical) by 1H NMR and all but one batch resulted in >20% AUC (HPLC Analysis) scyllo-inositol (Table 2). The diborate formations resulted in myo/scyllo ratios of >60:1 (typical) by 1H NMR (Table 2) within one hour and the subsequent crystallizations were allowed to proceed over 14-16 hours (overnight). The resulting diborate slurries filtered very fast and were isolated as large white crystals. The diborate filter cakes were found to have consistent 1H NMR spectra, HPLC purities of 98.7%-99.2% AUC and moisture levels of 46.8%-56.4%. Nickel and aluminum analysis revealed levels ranging from <1 ppm to 17 ppm for nickel and <1 ppm to 28 ppm for aluminum. The yields corrected for moisture content ranged from 17.1% to 20.3%. near reflux to yield After separating the crude reaction mixture from the nickel, scyllitol Scyllitol diborate is then crystallized by cooling is isolated in 17-20% yield and >99% HPLC purity (AUC) each were sampled for analysis after 1, 2, and 3 hours All five reactions resulted in myo/scyllo ratios of 2-3:1 (typical) by 1H NMR

Conditions: temp:97.51; pressure:0.00; stir:; See reaction.notes.procedure_details.

Procedure: The first step for the preparation of scyllitol diborate involves a sponge nickel mediated isomerization of myo-inositol in water near reflux to yield a mixture of inositols consisting of roughly 22% scyllitol (as judged by HPLC). After separating the crude reaction mixture from the nickel, scyllitol is selectively converted to scyllitol diborate by treatment with sodium tetraborate at 80-90° C. and pH 9-10. Scyllitol diborate is then crystallized by cooling and is isolated in 17-20% yield and >99% HPLC purity (AUC). All six batches of diborate proceeded as expected (Table 1). The isomerization reactions required 2-3 hours to heat to 95-100° C. and each were sampled for analysis after 1, 2, and 3 hours. All five reactions resulted in myo/scyllo ratios of 2-3:1 (typical) by 1H NMR and all but one batch resulted in >20% AUC (HPLC Analysis) scyllo-inositol (Table 2). The diborate formations resulted in myo/scyllo ratios of >60:1 (typical) by 1H NMR (Table 2) within one hour and the subsequent crystallizations were allowed to proceed over 14-16 hours (overnight). The resulting diborate slurries filtered very fast and were isolated as large white crystals. The diborate filter cakes were found to have consistent 1H NMR spectra, HPLC purities of 98.7%-99.2% AUC and moisture levels of 46.8%-56.4%. Nickel and aluminum analysis revealed levels ranging from <1 ppm to 17 ppm for nickel and <1 ppm to 28 ppm for aluminum. The yields corrected for moisture content ranged from 17.1% to 20.3%. near reflux to yield After separating the crude reaction mixture from the nickel, scyllitol Scyllitol diborate is then crystallized by cooling is isolated in 17-20% yield and >99% HPLC purity (AUC) each were sampled for analysis after 1, 2, and 3 hours All five reactions resulted in myo/scyllo ratios of 2-3:1 (typical) by 1H NMR

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

Procedure: Meanwhile, enzyme reaction product was measured as follows: 10 mg of scyllo-inosose, 40 mg of NADPH, and IOU of the enzyme were allowed to react in 1.0 ml of 100 mM Tris buffer (pH 8.0) at 36° C. for 4 hours, and a heat treatment was performed at 80° C. for 10 min, followed by cooling. Then, 100 μl of a strong base cation exchange resin, 100 μl of a strong acid anion exchange resin, and 10 mg of activated carbon were added, and the mixture was stirred and centrifuged. Then, the supernatant was diluted 2-fold, and measurement was performed by HPLC (Shodex Asahipak NH2P-50 4E Φ4.6×250 mm: Shodex) using an RI detector under conditions of a column temperature of 40° C. and a mobile phase flow rate of 1.5 ml (80% acetonitrile). As a result, the Atu4375 gene product and the Atu3234 gene product of Agrobacterium tumefacience C58, the BG14057 gene product of Bacillus subtilis 168, the Xcc3438 gene product of Xanthomonas campestris pv. campestris, and the Atu4375 gene product, and the Atu3234 gene product of AB10121 strain were found to have a high enzyme activity, and 100% of the product was scyllo-inositol obtained by the reduction, while myo-inositol that is an isomer thereof was not detected. As a result, recombinant enzymes derived from the above-mentioned genes were found to have a high scyllo-inositol dehydrogenase activity, and the gene products were scyllo-inositol dehydrogenase. Meanwhile, in the products obtained by the reduction reaction of scyllo-inosose, only scyllo-inositol was detected, and the enzymes were found to stereospecifically reduce scyllo-inosose into scyllo-inositol. by cooling Then, the supernatant was diluted 2-fold under conditions of a column temperature of 40° C.

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

Procedure: Meanwhile, the enzyme reaction product was measured as follows: 10 mg of scyllo-inosose, 40 mg of NADPH, and 10 U of the enzyme were allowed to react in 1.0 ml of 100 mM Tris buffer (pH 8.0) at 36° C. for 4 hours, and a heat treatment was performed at 80° C. for 10 min, followed by cooling. Then, 100 μl of a strong base cation exchange resin, 100 μl of a strong acid anion exchange resin, and 10 mg of activated carbon were added, and the mixture was stirred and centrifuged. Then, the supernatant was diluted 2-fold, and measurement was performed by HPLC (Shodex Asahipak NH2P-50 4E Φ4.6×250 mm: Shodex) using an RI detector under the condition of a column temperature of 40° C. and a mobile phase flow rate of 1.5 ml (80% acetonitrile). As a result, the ydgJ gene product was found to have a high scyllo-inositol dehydrogenase activity, and 100% of the product was scyllo-inositol obtained by reduction, while myo-inositol that is an isomer thereof was not detected. As a result, the solution of a recombinant enzyme derived from the ydgJ gene was found to have a high scyllo-inositol dehydrogenase activity, and the gene product was scyllo-inositol dehydrogenase. Meanwhile, in the product obtained by the reduction reaction of scyllo-inosose, only scyllo-inositol was detected, and the enzyme was found to stereospecifically reduce scyllo-inosose into scyllo-inositol. Meanwhile, the sequence of the ydgJ gene is shown in SEQ ID NO: 1, and the amino acid sequence corresponding thereto is shown in SEQ ID NO: 2. by cooling Then, the supernatant was diluted 2-fold under the condition of a column temperature of 40° C.