Heparin carboxylic derivatization

After determining the control heparin-APTT response curve, derivatized heparin was added to unheparinized bovine plasma (the same plasma used for preparing the control response curve) at known weight concentrations, and APTT was determined. Based on the interpolated heparin activity (units/mL plasma) of the derivatized heparin at known weight concentration (mg/mL plasma), the anticoagulant activity (units/mg) of the N-acetylated and hydroxyl- and carboxylic-derivatized heparins was determined. 

Based on comparison of NMR spectra with carboxylic derivatized heparins where the % derivatization was measured. 

Functional Group Derivatization. Carboxylic Derivatization. Two grams of N -acetylated heparin and 2 mL of n-butylamine were dissolved in 40 mL of water, and the pH was adjusted to 4.75. A total of 0.8 g of l-ethyl-3-(3-dimethylaminopro-pyl)carbodiimide hydrochloride was added to the heparin/n-butylamine solution in approximately 10-mg portions over a 6-h period while the reaction was maintained at 4°C and pH 4.75. Periodic samples were withdrawn from the reaction vessel, dialyzed, and freeze-dried. In a separate reaction, 0.5 g of N-acetylated heparin and 1.0 g of 2-aminoethyl hydrogen sulfate were dissolved in 10 mL of water and adjusted to pH 4. 75. A total of 0.2 g of l-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride was added to the reaction vessel in approximately 10-mg portions over a 4-h... 

Nuclear Magnetic Resonance (NMR) Spectrometry. Spectral verification of derivatization was provided by proton NMR of N-acetylated and carboxylic- and hydroxyl-derivatized heparins using a 300-MHz Varian SC 300 NMR spectrometer. Forty milligrams/milliliter of the respective heparin was dissolved in deuterium oxide with 1% 2,2-dimethyl-2-silapentane-5-sulfonate(DSS) and transferred to 5-mm NMR tubes. Spectra were obtained at room temperature with a spin rate of 20 rps and a gas flow rate of 13 ft3/h. 

This chapter focuses on the effects of anticoagulant activity caused by specific functional group derivatization of heparin, and on preliminary immobilization spacer group evaluations. The functional groups selected for immobilization are hydroxyl and carboxylic heparin groups. 

Free amine groups on porcine intestinal heparin (Sigma) were first blocked with acetic anhydride to form IV-acetylated heparin (13). This blocking reaction was performed to insure against intermolecular or intramolecular cross-linking during heparin carboxylic activation reactions. The N -acetylated heparin was then dialyzed extensively and freeze-dried. This heparin preparation was used in all further derivatization and immobilization reactions. 

Figure 1. The l-ethyl-3-(3-dimethylaminopropyl)carbodiimide-mediated N-acetylated heparin carboxylic group derivatization reaction scheme.

These results indicate that, under partial derivatization, the chemical nature of the derivatized end group is not critical to anticoagulant activity within the limited number of derivatizing agents tested however, the degree of derivatization is critical. Furthermore, both carboxylic and hydroxyl heparin groups can be utilized in heparin surface coupling reactions. 

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