Bis- -hydrazide

Most proteins contain an abundance of carboxylic acid groups from C-terminal functionalities and aspartic and glutamic acid side chains. These groups are readily modified with bis-hydrazide compounds to yield useful hydrazide-activated derivatives. Both carbohydrazide and adipic acid dihydrazide have been employed in forming these modifications using the carbodi-imide reaction (Wilchek and Bayer, 1987). 

Bis-hydrazide-containing molecules also can be used to activate soluble polymeric sub-stances-containing aldehyde groups. For instance, dextran may be periodate oxidized to create numerous formyl functionalities on each molecule. Subsequent reaction with a homobifunctional hydrazide in large excess results in a hydrazide-activated polymer having multivalent-binding capability toward aldehydes or ketones (Chapter 25, Section 2.2). Insoluble support matrices suitable for affinity chromatography have been activated in a similar fashion to create the hydrazide derivative (O Shannessy and Wilchek, 1990). 

Perhaps a better design for a bis-hydrazide compound to modify carboxylate particles would include a short PEG spacer arm between the two hydrazide groups. This type of linker would result in a hydrophilic surface due to the presence of the PEG spacers, while providing the terminal hydrazide functionality necessary for coupling to carbonyl compounds. Unfortunately, this type of compound is not currently available, so the aliphatic bis-hydrazides are the only choice. 

Another route to the formation of a hydrazide on a surface is to use an aldehyde-containing particle (such as HEMA/acrolein copolymers) and subsequently modify the aldehydes to form hydrazone linkages with bis-hydrazide compounds, which then can be stabilized by reduction with sodium cyanoborohydride (Chapter 2, Section 5). The resulting derivative contains terminal hydrazides for immobilization of carbonyl ligands (see Figure 14.18). 

The following protocol describes the modification of DNA or RNA probes at their 5 -phosphate ends with a bis-hydrazide compound, such as adipic acid dihydrazide or carbohydrazide. A similar procedure for coupling the diamine compound cystamine can be found in Section 2.2 (this chapter). 

Add to the tube 7.5 pi of RNA or DNA containing a 5 phosphate group. The concentration of the oligonucleotide should be 7.5-15nmol or total of about 57-115.5 pg. Also immediately add 5 pi of 0.25 M bis-hydrazide compound dissolved in 0.1M imidazole, pH 6.0. Because EDC is labile in aqueous solutions, the addition of the oligo and bis-hydrazide/imidazole solutions should be done quickly. 

Conjugation of SFB-Modified Alkaline Phosphatase to Bis-Hydrazide-Modified Oligonucleotides... 

The following protocol assumes the prior derivatization of an oligonucleotide at the 5 end using a bis-hydrazide compound according to the protocol of Section 2.1 (this chapter) using a carbodiimide-mediated reaction. 

Figure 27.17 SFB may be used to create aldehyde groups on enzyme molecules for subsequent conjugation to a 5 -bis-hydrazide-modified oligonucleotide, forming hydrazone bonds.

Bei dieser Reaktion konnen allerdings auch Kohlensaure-bis-hydrazide entstehen. So cyclisiert l-Amino-2-oxo-4,6-diphenyl-l,2-dihydro-pyridin mit Kohlensaure-bis-imidazolid zu 5,7-Di-phenyl-(pyridio[2,l-b]-l,3,4-oxadiazolium )-2-olat innvA 37%iger AusbeutelS0(vgl. S. 533, 535). AuBerdem wird 1,3-Bis-[4,6-diphenyl-2-oxo-l, 2-dihydro-pyrido -harnstoff (45%) gebildet. 

Eine interessante Variante der Umsetzung von Carbonsaure-hydraziden mit Alkalimetall-Salzen von Dithiokohlensiiure-O-estern (Xanthogenaten) ist die Reaktion von Kohlensaure-bis-hydrazid mit (Ethoxy-thiocarbonyl)-thioessigsaure316. Man erhalt in maBigen Ausbeuten 2-Ethoxy-5-[2-(ethoxy-thiocarbonyl)-hydrazino]-l,3,4-oxadiazol (25-30% Schmp. 149-1510)316. Die im Reaktionsschema angegebenen Zwischenstufen wurden dabei nicht isoliert. 

Von Interesse ist, daB Oxalsaure-bis-hydrazid mit Triethoxy-methan unter Cyclisierung nur einer Hydrazid-Gruppe zu 2-(2-Ethoxymethylen-hydrazinocarbonyl)-l,3,4-oxadiazol (22% Schmp. 192-1940)288 reagiert ... 

The key intermediate chloromethyloxadiazole 18 was prepared in two steps from inexpensive, commercially available materials as shown in Scheme 5.10. Bishydrazide 19 was prepared in a one-pot procedure by reaction of 35% aqueous hydrazine with ethyl trifluoroacetate in acetonitrile and subsequent addition of chloroacetyl chloride and base. This procedure affords the unsymmetrical bis(hydrazide) 19 in higher than 95% assay yield. While a number of dehydrating agents were found to be effective in the dehydration to prepare 18, phosphorus oxychloride was chosen because of its low cost and relatively benign waste stream. Sub-stoichiometric (0.3 equiv.) amounts were found to be as effective as full equivalents in the reachon when used in conjunction with catalyhc amounts of DMAP as a nucleophilic catalyst. The entire sequence was transformed into a one-pot through process in order to improve efficiency. Following the cyclization, an aqueous work-up was performed and the organic extracts carried directly into the next step. 

Amine functional groups also may be created on polysaccharides (Section 4.3). The reducing ends of carbohydrate molecules (or generated aldehydes) may be reacted with small diamine compounds to yield short alkylamine spacers that can be used for subsequent conjugation reactions. Hydrazide groups may be similarly created using bis-hydrazide compounds (Section 4.5). 

The creation of hydrazide probes most often is based on the derivatization of a detectable molecule with a bis-hydrazide compound. Although hydrazine itself (in the form of hydrazine hydrate) can be used in a methanolic solution to modify activated carboxylate molecules forming hydrazides, the availability of the bifunctional hy-drazides provides a built-in spacer to accommodate greater steric accessibility. 

The reaction of an excess of adipic acid dihydrazide with aldehyde groups present on proteins or other molecules will result in modified proteins containing alkyl-hydrazide groups (Fig. 96). Another bis-hydrazide compound, carbohydrazide, also may be employed with similar results, except that the spacer afforded through its use is considerably shorter. Target aldehydes may be created on macromolecules according... 

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