Dextran derivative

Many covalentiy modified derivatives of dextran have been described. Of these, the most important are dextran sulfate [9042-14-2] and cross-linked dextran. The uses and properties of these and other dextran derivatives have been reviewed (66,69,71—73,203). 

The above chemicals can be obtained by fermentation (qv) of other sugars. However, some compounds require sucrose as a unique feedstock. Examples are the polysaccharides dextran, alteman, andlevan, which are produced by specific strains of bacteria (48,54—56). Dextrans are used to make chromatographic separation media, and sulfated dextran derivatives are used as plasma extenders (41). Levans show promise as sweetness potentiators and, along with alteman, have potential as food thickeners and bulking agents in reduced-caloric foods (55,56) (see Carbohydrates). 

The following protocol for creating the polyaldehyde dextran derivative is based on the method of Bernstein et al. (1978). 

Dextran derivatives containing carboxyl- or amine-terminal spacer arms may be prepared by a number of techniques. These derivatives are useful for coupling amine- or carboxylate-containing molecules through a carbodiimide-mediated reaction to form an amide bond (Chapter 3, Section 1). Amine-terminal spacers also can be used to create secondary reactive groups by modification with a heterobifunctional crosslinking agent (Chapter 5). 

This type of modification process has been used to form sulfhydryl-reactive dextran polymers by coupling amine spacers with crosslinkers containing an amine reactive end and a thiol-reactive end (Brunswick et al., 1988 Noguchi et al., 1992). The result was a multivalent sulfhydryl-reactive dextran derivative that could couple numerous sulfhydryl-containing molecules per polymer chain. 

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. 

Hydrazide derivatives also may be prepared from a periodate-oxidized dextran polymer or from a carboxyl-containing dextran derivative by reaction with te-hydrazidc compounds (Chapter 4, Section 8). A hydrazide terminal spacer provides reactivity toward aldehyde- or ketone-containing molecules. Thus, the hydrazide-dextran polymer can be used to conjugate specifically glycoproteins or other polysaccharide-containing molecules after they have been oxidized with periodate to form aldehydes (Chapter 1, Section 4.4). 

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. 

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. 

The carboxymethyl-dextran derivative may be used to couple amine-containing molecules by the carbodiimide reaction. Heindel et al. (1994) prepared the lactone derivative of carboxymethyl-dextran by refluxing for 5 hours in toluene or other anhydrous solvents. The lactone derivative is highly reactive toward amine-containing molecules, thus creating a preactivated polymer for conjugation purposes. 

Figure 25.17 An amine-functionalized dextran derivative may be further reacted with SPDP to create a sulfhydryl-reactive product.

CDDP was obtained from Nihon Kayaku Co. Ltd. Dextran M = 6.0 X 10" ) was purchased from Wako Pure Chemical Industry and converted to two kinds of dextran derivatives, OX-Dex and DCM-Dex. The organic solvents were of commercial grades and used without further purification. 

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... 

A remarkable new method for the conversion of dextran and dextran derivatives, mainly towards mimetics of heparan sulfate uses comparable sulfation but applies 2-methyl-2-butene (2M2B) as an acid scavenger of neutral character [110]. This procedure shows a more efficient reaction combined with diminished chain degradation, as can be seen in Table 5. The method was used for the sulfation of carboxymethylated dextran. 

Subsequent conversion of dextran derivatives with acetic acid or propionic acid anhydride is an effective method for revealing structural features on the molecular level. This is illustrated on a dextran propionate (Mw 5430 gmol x) which can be completely acetylated with acetic anhydride/pyridine in a separate step yielding a peracetylated sample (dextran propionate acetate, DPA). The assignment of the chemical shifts of DPA is carried out via 2D NMR (Fig. 17). 

Interestingly, CDI can also be utilised for the introduction of substituents by inter- or intramolecular coupling of OH moieties of the polysaccharide via a carbonate function. This synthesis was used to obtain dextran with 2-hydroxyethyl methacrylate moieties (dex-HEMA) and dex-HEMA with lactate spacer functions (Fig. 32). A new class of dextran derivatives (DS < 0.2) that can be polymerised containing hydrolysable groups is accessible [177]. 

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