Hydrochloric Acid → 1-Cyano-4-(dimethylamino)pyridin-1-ium tetrafluoroborate

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Reaction Steps

This example describes a generalized method for making insulin-glycogen conjugates using cyanodimethylamino-pyridinium tetrafluoroborate (CDAP) as a coupling agent. This synthesis was used to prepare various insulin-glycogen conjugates that are described in later Examples. The inventors have found that this method produces increased insulin loading as compared to the CNBr method of Example 2. Briefly, 8.0 g of glycogen is dissolved in 160 ml of 25 mM HEPES, 0.15 M NaCl, pH 9.0. 2.4-12.0 mL of a 1.0M CDAP solution in DMSO is added dropwise to the glycogen solution at 0° C. After stirring for 1 minute, a volume equal to that of the CDAP solution consisting of 0.2M triethylamine (TEA) is added dropwise over one minute. At this time, the pH of the reaction solution is adjusted to 9.0 using 1.2N HCl. Then, 80-2000 ml of a 10 mg/ml insulin solution in a 20 mM HEPES (pH 9.0) solution is added over the next three minutes and the pH adjusted again to 9.0 using 0.3N NaOH. Finally, in some cases 1.6 ml of a 100 mg/ml glycine solution in 100 mM HEPES buffer containing 0.15M NaCl (pH 9.0) can be added. In other cases, amine-functionalized sugars such as mannosamine, glucosamine, and other glucose or mannose-containing sugars. The inventors have found that the addition of the glycine step increases the solubility of the conjugate. The entire reaction mixture is slowly stirred at room temperature overnight.

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