Carbodiimide technique

Various preparations of antisera to atropine have been reported. A racemic hemisuccinate ester was prepared and conjugated to bovine serum albumin by the carbodiimide technique. Antisera formed to the original immunogen selectively bound the R isomer (34), but a later antiserum prepared by this approach was reported to bind both R and S forms with "equal efficiency" (35). R, S-atropine was treated with diazotized p-amino-benzoic acid, and the resulting compound (which was not further characterized) was used for conjugation to bovine serum albumin by means of a carbodiimide-mediated reaction. Antisera resulting from use of this material were quite selective for the R isomer, with a cross-reaction of only about 2% for the S isomer (36). Virtanen et al. followed this procedure with S-atropine. Their antiserum bound equally to S- and R,S-atropine, as measured by displacement of tritium-labeled R,S-atropine (37). In another study (31), both racemic atropine and the S isomer were coupled to human serum albumin by the technique of Wurtzburger et al. (36), Antisera were obtained that were selective for both the R and S isomers (33). 

In conclusion, IR analysis of polymer/additive extracts before chromatographic separation takes advantage mainly of straightforward transmission measurements. Without separation it is often possible to make class assignments (e.g. in the reported examples on plasticisers and carbodiimide hydrolysis stabilisers) it may eventually be necessary to use multivariate techniques. Infrared detection of chromatographic effluents is dealt with in Chapter 7. 

AMCA may be coupled to amine-containing molecules through the use of the carbodiimide reaction using EDC (Chapter 3, Section 1.1). EDC will activate the carboxylate on AMCA to a highly reactive o-acylisourea intermediate. Attack by a nucleophilic primary amine group results in the formation of an amide bond (Figure 9.22). Derivatization of AMCA off its carboxylate group causes no major effects on its fluorescent properties. Thus, proteins and other macromolecules may be labeled with this intensely blue probe and easily detected by fluorescence microscopy and other techniques. 

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). 

For high-performance lAC, the preferred solid support is a glass bead solid support coated wilh either protein-A or protein-A covalently linked with the antibody through a carbodiimide bond (165, 166). In either case, protein-A binds to the Fc portion of the antibody so that the combining sites are oriented to the mobile phase. Once the protein is attached, the lAC matrix is packed into the column either as a slurry or dry. Pump-slurry techniques use buffers with a low salt content, such as Tris or 0.01 M phosphate buffer to minimize friction and denaturation of the immobilized antibody (16). If the solid support consists of glass beads, the packing can be freeze-dried after antibody attachment and packed dry. 

Since depsipeptides, in contrast to classical peptides, contain units constructed from amino and hydroxy acid residues, the various methods for their preparation are generally pathways involving the formation of ester bonds. The novel achievements in this area discussed (vide infra) are associated with further developments in the mixed anhydride technique, the application of effective catalysts in the carbodiimide procedure, and adaptation of the known Mitsunobu reaction to the depsipeptide case. A number of significant and efficient esterification procedures utilized for the preparation of depsipeptides are considered. 

In 1959 Shchukina et a I. 56 showed that the addition of pyridine to DCC increases the yield (65-85%) of depsipeptides constructed from Z-protected amino acids and serine, threonine, or salicylic acid derivatives. The ferf-butyl ester of Af-(benzyloxycarbonyl)-leucylleucic acid (Table 2) was prepared in 60% yield under the same conditions of addition using a 2 molar excess of pyridine to carbodiimide. 57 The DCC/pyridine technique was successfully utilized in the ring closure between ()-alanine and leucic acid residues during the synthesis of the cyclic hexadepsipeptide destruxin B.[58 Under this modified approach the... 

In summary, among the carbodiimide-based methods only the carbodiimide/dialkyl-aminopyridine technique has been widely used in the synthesis of depsipeptides. It usually affords the desired products in moderate or high yield and has good potential for routine use in the esterification of urethane-protected amino acids or peptide fragments even in the case... 

A simple, one-step immobilization technique is bulk cross-linking of the functional protein with bovine serum albumin (BSA) using glutaraldehyde as the cross-linking agent (Fig. 2.19). It is popular because of its simplicity, but it usually leads to reduction of the biological activity of the biomolecule. A cleaner and preferable approach is the two-step carbodiimide route. A partial summary of the various immobilization options was shown in Table 2.3. 

Andres and coworkers demonstrated the iCP of densely packed alkanethiolate-functionalized Au nanoparticle arrays in monolayer and multilayer structures.81,82 Dense and hexagonally packed monolayers of nanoparticles were first assembled on a water surface. By using the Langmuir-Schafer technique, the Au nanoparticle monolayer was transferred to a PDMS stamp, and printed onto a substrate. Multilayers were prepared by repeating the printing process in an LbL scheme, in which subsequent particle layers may be made up of the same or different types of particles. Similarly, the assembly of irregular, densely packed monolayers of polystyrene nanoparticles on iCP substrates via carbodiimide coupling was reported.83 The conformal contact of the carbodiimide-functionalized polystyrene particles resulted in the covalent attachment of the nanoparticles at a carboxylate-functionalized surface. 

Eledroosmotic flow (EOF) conditions were applied and yielded only 10% conversion with constant reactant movement [9]. The use of stopped-flow techniques, which periodically push and mix the flow, led to a 50% increase in yield. A change in the coupling agent from l-(3-dimethylaminopropyl)-3-ethyl-carbodiimide hydrochloride (EDCI) to dicyclohexylcarbodiimide (DCC) for reasons of limited solubility resulted in a 93% yield of the dipeptide. Batch P-dipeptide synthesis using EDCI gave a yield of 50% [6]. 

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