Heparin derivatization

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. 

Anticoagulant Activity Assay. The anticoagulant activities of IV-acetylated heparin and all further derivatized heparins were determined based on activated partial thromboplastin time (APTT) assay methods (15). Bovine blood was collected in 3.8% sodium-citrated (9 parts blood to 1 part citrate solution) and centrifuged at 5000 x g for 15 min. The supernatant plasma was collected and pooled for subsequent APTT testing. Prior to testing plasma was kept refrigerated no longer than 6 h after... 

Figure 6. APTT heparin activity assay curve. Key 9, control heparinized plasma (units mL plasma) calibration APTT and O, derivatized heparin (at known weight concentrations, mg/mL plasma) APTT.

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. 

Another major site of peptide metabolism is the blood and especially blood serum and plasma. From an extensive compilation of peptide tm values (over 100 peptides, plus derivatized and cyclized analogues, D-amino acid stereoisomers, peptide bond isosteres, etc.), it appears that the differences between serum, heparinized plasma, and whole blood are fairly limited [161]. Interspecies differences are larger, particularly between humans and rats, with most human/rat t1/2 ratios ranging from 1 1 to 25 1 ... 

Covalent immobilization of chemically synthesized carbohydrates onto derivatized surfaces by Seeberger group, (a) Attachment of thiol containing carbohydrates to maleimide-functionalized glass slides (b) attachment of amine-terminated heparin oligosaccharides onto amine-reactive glass slides... 

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). 

To elucidate heparin anticoagulant effects caused by covalent coupling via specific functional groups, those heparin groups involved in immobilization reactions are first derivatized, and the effect of derivatization on anticoagulant activity is determined. 

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. 

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... 

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

Not taking into account the weight increase above the initial amount of heparin due to derivatization. 

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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