1-(9-FLUORENYLMETHYLOXYCARBONYL-AMINO)-3,6-DIOXA-8-OCTANEAMINE HYDROCHLORIDE

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

Procedure: 1-(9-fluorenylmethyloxycarbonyl-amino)-3,6-dioxa-8-octanamine hydrochloride (Fmoc-EDOBEA, Iris Biotech GmbH) was added to an anhydrous DMSO solution (5 mg/mL) of HA-TBA synthesized using HA-Na (99 kDa) as a starting material in Example 1-2 in an amount of 0.1 molar equivalents relative to the HA unit. Next, DMT-MM was added in an amount of 0.2 molar equivalents relative to the HA unit, and stirring was performed at room temperature for 6 hours. Then, L-asparagine methyl ester hydrochloride and DMT-MM were added in this order in amounts of 5 and 3-6 molar equivalents, respectively, relative to the HA unit, and stirring was performed at room temperature overnight. The reaction solution was dialyzed for purification against DMSO. Piperidine (Wako Pure Chemical Industries, Ltd.) was added to the resulting dialyzate to g a concentration of 20%, and stirring was performed for 2 hours to remove the Fmoc group. Thereafter, the mixture was dialyzed for purification against DMSO, an aqueous solution of 0.3 M NaCl, and distilled water in this order. 5N NaOH solution was added to the resulting dialyzate to adjust the pH to at least 12.5, and stirring was performed for an hour to perform ester hydrolysis and carboxy deprotection. Thereafter, the mixture was neutralized with 5N HCl solution and further dialyzed for purification against distilled water and ultrapure water in this order, and then the dialyzate was freeze-dried to give HA-Asn/EDOBEA. synthesized respectively, relative to the HA unit, and stirring was performed at room temperature overnight The reaction solution was dialyzed for purification against DMSO Piperidine (Wako Pure Chemical Industries, Ltd.) was added to the resulting dialyzate stirring was performed for 2 hours to remove the Fmoc group Thereafter, the mixture was dialyzed for purification against DMSO an aqueous solution of 0.3 M NaCl, and distilled water in this order 5N NaOH solution was added to the resulting dialyzate stirring was performed for an hour ester hydrolysis and carboxy deprotection further dialyzed for purification against distilled water and ultrapure water in this order the dialyzate was freeze-dried

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

Procedure: 1-(9-fluorenylmethyloxycarbonyl-amino)-3,6-dioxa-8-octanamine hydrochloride (Fmoc-EDOBEA, Iris Biotech GmbH) was added to an anhydrous DMSO solution (5 mg/mL) of HA-TBA synthesized using HA-Na (99 kDa) as a starting material in Example 1-2 in an amount of 0.1 molar equivalents relative to the HA unit. Next, DMT-MM was added in an amount of 0.2 molar equivalents relative to the HA unit, and stirring was performed at room temperature for 6 hours. Then, L-asparagine methyl ester hydrochloride and DMT-MM were added in this order in amounts of 5 and 3-6 molar equivalents, respectively, relative to the HA unit, and stirring was performed at room temperature overnight. The reaction solution was dialyzed for purification against DMSO. Piperidine (Wako Pure Chemical Industries, Ltd.) was added to the resulting dialyzate to g a concentration of 20%, and stirring was performed for 2 hours to remove the Fmoc group. Thereafter, the mixture was dialyzed for purification against DMSO, an aqueous solution of 0.3 M NaCl, and distilled water in this order. 5N NaOH solution was added to the resulting dialyzate to adjust the pH to at least 12.5, and stirring was performed for an hour to perform ester hydrolysis and carboxy deprotection. Thereafter, the mixture was neutralized with 5N HCl solution and further dialyzed for purification against distilled water and ultrapure water in this order, and then the dialyzate was freeze-dried to give HA-Asn/EDOBEA. synthesized respectively, relative to the HA unit, and stirring was performed at room temperature overnight The reaction solution was dialyzed for purification against DMSO Piperidine (Wako Pure Chemical Industries, Ltd.) was added to the resulting dialyzate stirring was performed for 2 hours to remove the Fmoc group Thereafter, the mixture was dialyzed for purification against DMSO an aqueous solution of 0.3 M NaCl, and distilled water in this order 5N NaOH solution was added to the resulting dialyzate stirring was performed for an hour ester hydrolysis and carboxy deprotection further dialyzed for purification against distilled water and ultrapure water in this order the dialyzate was freeze-dried

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

Procedure: 1-(9-fluorenylmethyloxycarbonyl-amino)-3,6-dioxa-8-octanamine hydrochloride (Fmoc-EDOBEA, Iris Biotech GmbH) was added to an anhydrous DMSO solution (5 mg/mL) of HA-TBA synthesized using HA-Na (99 kDa) as a starting material in Example 1-2 in an amount of 0.1 molar equivalents relative to the HA unit. Next, DMT-MM was added in an amount of 0.2 molar equivalents relative to the HA unit, and stirring was performed at room temperature for 6 hours. Then, L-asparagine methyl ester hydrochloride and DMT-MM were added in this order in amounts of 5 and 3-6 molar equivalents, respectively, relative to the HA unit, and stirring was performed at room temperature overnight. The reaction solution was dialyzed for purification against DMSO. Piperidine (Wako Pure Chemical Industries, Ltd.) was added to the resulting dialyzate to g a concentration of 20%, and stirring was performed for 2 hours to remove the Fmoc group. Thereafter, the mixture was dialyzed for purification against DMSO, an aqueous solution of 0.3 M NaCl, and distilled water in this order. 5N NaOH solution was added to the resulting dialyzate to adjust the pH to at least 12.5, and stirring was performed for an hour to perform ester hydrolysis and carboxy deprotection. Thereafter, the mixture was neutralized with 5N HCl solution and further dialyzed for purification against distilled water and ultrapure water in this order, and then the dialyzate was freeze-dried to give HA-Asn/EDOBEA. synthesized respectively, relative to the HA unit, and stirring was performed at room temperature overnight The reaction solution was dialyzed for purification against DMSO Piperidine (Wako Pure Chemical Industries, Ltd.) was added to the resulting dialyzate stirring was performed for 2 hours to remove the Fmoc group Thereafter, the mixture was dialyzed for purification against DMSO an aqueous solution of 0.3 M NaCl, and distilled water in this order 5N NaOH solution was added to the resulting dialyzate stirring was performed for an hour ester hydrolysis and carboxy deprotection further dialyzed for purification against distilled water and ultrapure water in this order the dialyzate was freeze-dried