Hydroxyl-derivatized heparin

Hydroxyl Group Derivatization. Epichlorohydrin Activation. One-half gram of N-acetylated heparin was dissolved in 10 mL of 1.0M Na2C03 solution. One milliliter of epichlorohydrin and either 1 mL of n-butylamine or 1 g of glycine or 2-aminoethyl hydrogen sulfate were added to the heparin solutions and allowed to react at 40°C for 5 h. Reaction schemes are illustrated in Figure 2. The hydroxyl-derivatized heparins were then dialyzed and freeze-dried. 

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. 

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. 

The hydroxyl group at the C6 position is more active than that at the C3 position and is, therefore, derivatized favorably. Figure 4 shows the sulfation as a common example of this type of reaction. When this type of reaction (Figure 4) is conducted on chitin, the end products behave like heparin, an anti-blood-clotting agent (Wolform and Shen-Han, 1959). 

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. 

Figure 2. Epichlorohydrin-mediated N-acetylated heparin hydroxyl 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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