Dextran ethylene diamine

Another approach uses reactive alkyl halogen compounds containing a terminal carboxylate group on the other end to form spacer arms off the dextran polymer from each available hydroxyl. In this manner, Brunswick et al. (1988) used chloroacetic acid to modify the hydroxyl groups to form the carboxymethyl derivative. The carboxylates then were aminated with ethylene diamine to create an amine-terminal derivative (Inman, 1985). Finally, the amines were modified with iodoacetate to form a sulfhydryl-reactive polymer (Figure 25.14). 

In a somewhat similar scheme, Noguchi et al. (1992) prepared a carboxylate spacer arm by reacting 6-bromohexanoic acid with a dextran polymer. The carboxylate then was aminated with ethylene diamine to form an amine-terminal spacer (Figure 25.15). This dextran derivative finally was reacted with N-Succinimidyl 3-(2-pyridyldithio)propionate (SPDP) (Chapter 5, Section 1.1) to create the desired sulfhydryl-reactive polymer (Section 2.4, this chapter). The SPDP-activated polymer then could be used to prepare an immunoconjugate composed of an antibody against human colon cancer conjugated with the drug mitomycin-C. 

Figure 25.14 An amine derivative of dextran may be prepared through a two-step process involving the reac-tion of chloroacetic acid with the hydroxyl groups of the polymer to create carboxylates. Next, ethylene diamine is coupled in excess using a carbodiimide-mediated reaction to give the primary amine functional groups.

Figure 25.15 Amino-dextran derivatives may be prepared by the reaction of 6-bromohexanoic acid with the hydroxyl groups of the polymer followed by coupling of ethylene diamine using EDC.

To make an amine derivative of dextran, dissolve ethylene diamine (or another suitable diamine) in 0.1 M sodium phosphate, 0.15 M NaCl, pH 7.2, at a concentration of 3 M. Note Use of the hydrochloride form of ethylene diamine is more convenient, since it avoids having to adjust the pH of the highly alkaline free-base form of the molecule. Alternatively, to prepare a hydrazide-dextran derivative, dissolve adipic acid dihydrazide (Chapter 4, Section 8.1) in the coupling buffer at a concentration of 30 mg/ml (heating under a hot water tap may be necessary to completely dissolve the hydrazide compound). Adjust the pH to 7.2 with HC1 and cool to room temperature. 

Dissolve polyaldehyde dextran in the ethylene diamine (or adipic dihydrazide) solution at a concentration of 25 mg/ml. 

The ethylene diamine-dextran derivative may be used for the coupling of carboxylate-contain-ing molecules by the carbodiimide reaction, for the coupling of amine-reactive probes, or to modify further using heterobifunctional crosslinkers. The hydrazide-dextran derivative may be used to crosslink aldehyde-containing molecules, such as oxidized carbohydrates or glycoproteins. 

Several chemical approaches may be used to form the amine- or carboxyl-terminal dextran derivative. The simplest procedure may be to prepare polyaldehyde dextran according to the procedure of Section 2.1, and then make the spacer arm derivative by reductively animating an amine-containing organic compound onto it. For instance, short diamine compounds such as ethylene diamine or diaminodipropylamine (3,3 -iminotepropylamine) can be coupled in excess to polyaldehyde dextran to create an amine-terminal derivative. Carboxyl-terminal derivatives may be prepared similarly by coupling molecules such as 6-aminocaproic acid or p-alanine to polyaldehyde... 

Sihca impregnated with saturated and unsaturated hydrocarbons (squalene, paraffin oil) silicone and plant oils complexing agents (silver ions, boric acid, and borates carbohydrates unsaturated and aromatic compounds) chelating compounds [ethylene diamine tetra-acetic add (EDTA), digitonin] transition metal salt synthetic peptides 18-crown-6 and ammonium sulfate silanized sdica gel impregnated with anionic and cationic surfactants Cross-hnked, polymeric dextran gels (Sephadex)... 

Sodium poly(styrene sulfonate) (PSS, MW = 70-000) and poly(allylamine hydrochloride) (PAH, MW = 70-000), calcinm chloride dehydrate, sodium carbonate, sodium chloride, ethylene diamine tetra acetic acid (EDTA), TRIS, maleic anhydride, sodium Itydroxide (NaOH) and Bromocresol purple were purchased from Sigma-Aldrich (Munich, Germary). Urease (Jack bean, Canavalia ensiformis) was purchased from Fluka SNARF-1 dextran (MW = 70,000) was obtained from Invit-rogen GmbH (Molecular Probes D3304, Karlsrahe, Germarty). All chemicals were used as received. The bi-distillated water was used in all experiments. 

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