Hydrazide Particles

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. 

The following protocol describes the oxidation of carbohydrate (glycans) on antibody molecules to form aldehydes and the subsequent coupling to hydrazide particles. 

Dissolve the antibody to be coupled in 10 mM sodium phosphate, 0.15 M NaCl, pH 7.5, at a concentration of 10 mg/ml. The antibody must be glycosylated to work in this procedure. 

Prepare a solution of 0.1 M sodium periodate in water. Protect from light. 

With mixing, add 0.1 ml of the periodate solution to each ml of the antibody solution. 

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Figure 14.18 Carboxylate-particles or aldehyde-particles can be modified with the carbohydrazide in excess to create a hydrazide-particle that can be used to couple with aldehyde-containing molecules.

Figure 14.19 Aldehyde-containing molecules, such as periodate-oxidized carbohydrates or glycoproteins, can be coupled to hydrazide-particles to form a hydrazone bond. This bond can be further stabilized by reduction...

After the final wash, resuspend the particles at a concentration of 10 mg/ml in coupling buffer and add an appropriate amount of the solution from step 6, which contains the purified, oxidized antibody. The amount of oxidized antibody to add to the particles should be about 1-10 X over the amount of the calculated monolayer for the particle type used. (Note For 100 mg of 1 pm hydrazide particles, a monolayer equivalent of antibody will be about 1.5 mg, so the total amount added should be in the range of 1.5-15 mg for a 1-10 X excess). 

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). 

Aldehyde particles are spontaneously reactive with hydrazine or hydrazide derivatives, forming hydrazone linkages upon Schiff base formation. Reactions with amine-containing molecules, such as proteins, can be done through a reductive amination process using sodium cyanoborohydride (Figure 14.21). 

Miwa and Yamamoto (31) described a simple and rapid method with high accuracy and reliability for the determination of C8 0-C22 6 fatty acids, which occur in esterified forms in dietary fats and oils and in living cells [the biological effects of routinely consumed fats and oils are of wide interest because of their impact on human health and nutrition (28,29), in particular, the ratio of cu-3 polyunsaturated fatty acid to w-6 polyunsaturated fatty acids (w-3/cu-6) seems to be associated with atherosclerosis and breast and colon cancers (30)]. They report improved separation of 29 saturated and mono- and polyunsaturated fatty acids (C8-C22), including cis-trans isomers and double-bond positional isomers, as hydrazides formed by direct derivatization with 2-nitrophenylhydrazine hydrochloride (2-NPH HC1) of saponified samples without extraction. The column consisted of a J sphere ODS-M 80 column (particle size 4 /xm, 250 X 4.6-mm ID), packed closely with spherical silica encapsulated to reach a carbon content of about 14% with end-capped octadecyl-bonded-spherical silica (ODS), maintained at 50°C. The solvent system was acetonitrile-water (86 14, v/v) maintained at pH 4-5 by adding 0.1 M hydrochloric acid with a flow rate of 2.0 ml/min. Separation was performed within only 22 min by a simple isocratic elution (Fig. 6). The resolution of double-bond positional isomers, such as y-linolenic ( >-6) and a-linolenic acid ( >-3) hydrazides and w-9, >-12, and >-15 eicosenoic acid hydrazides was achieved by use of this column. 

Green 488 carboxylic acid, Cascade Blue hydrazide, FQ-labeled aspartic acid, CBQCA-labeled serine and tyrosine, GFP, TAMRA-labeled 20-mer oligonucleotide, and fluorescently labeled PS particles see Figure 5.20). This focusing effect leads to a pre-concentration factor of 10,000 or greater (from 8 nM to 90 dM for Oregon Green) [597]. 

Fig. 1 Dependence of k on adrenaline (squares) and L-tyrosine hydrazide (circles), on mobile-phase concentration of 1-hexane-sulfonate. Column Synergi Hydro-RP (Phenomenex) 150 x 4.6 mm ID, particle size 4 pm, and bonded phase coverage 4.05 pmol/m. Eluent phosphate buffer 37.10 mM KH2PO4 and 4.29 mM Na2HP04 calculated to provide a pH of 6.0. After addition of the desired amount of sodium 1-hexanesulfonate, NaCl was added so that the total sodium concentration was 50 mM (constant ionic strength). Experimental data were fitted by Eq. 8.

This technique has also been used to add a cyano group to an aromatic ring. l-Methoxy-4-methoxycarbonyl pyridinium methyl sulphate was absorbed on silica gel particles. After these particles had been dried under vacuum, they were placed in a column and H CN was passed through the column. Then the column was sealed and heated at 40 °C for 5 minutes. After the methyl-2[ C]cyano-isonicotinate had been purified by HPLC chromatography, it was converted into 2-[ C]-isonicotinic acid hydrazide in a 30% radiochemical yield by treatment with hydrazine in anhydrous ethanol (equation 78). The synthesis took 30 minutes to complete. 

Microgels can be produced by the mixing and emulsification of two different polymer solutions that can react to form cross-linked microgel particles that are monodisperse and have predictable swelling properties [8]. An example of this is the use of aldehyde and hydrazide-functionaUzed carbohydrates where heating or UV irradiation can be used for gelation. 

Hybrid dynamic proteoids, containing alternating imine and acylhydrazone linkages, have been obtained by polycondensation of amphiphilic dialdehydes with amino acid hydrazides. The polymerization displays nucleation-elongation behavior driven by hydrophobic effects, resulting in the formation of globular particles reminiscent of folded proteins (Fig. 6) [89]. 

The polymer particles contain co-polymerized carbonyl groups which, on film formation, react with hydrazide groups of the water-soluble acid dihydrazide to form hydrazone. Increased cohesion strength results due to both inter-particle and intraparticle crosslinking reactions. A similar process also occurs between corona-pre-treated polypropylene film and the emulsion adhesive thereby significantly increas-... 

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