Homobifunctional compounds

DSC also is used to activate hydroxylic particles for coupling to amine-containing ligands (Miron and Wilchek, 1993). For methods involving particle conjugation using this homobifunctional compound, see Chapter 14. 

Compounds having the same functionality on both ends are homobifunctional in nature and can be conjugated with the same target functionality on biomolecules, surfaces, or other molecules. Chapter 4 describes traditional homobifunctional compounds in detail, but the discrete PEG-based reagents are described here, because of their unique hydrophilic properties. 

Fluorobenzene-type compounds have been used as functional groups in homobifunctional crosslinking agents (Chapter 4, Section 4). Their reaction with amines involves nucleophilic displacement of the fluorine atom with the amine derivative, creating a substituted aryl amine bond (Reaction 9). Detection reagents incorporating reactive aryl chemistry include 2,4-dinitrofluorobenzene and trinitrobenzenesulfonate (Eisen et al., 1953). These compounds form... 

The following list of homobifunctional imidoesters represent compounds that are commonly used for protein crosslinking and are currently available from commercial sources. 

Dimethyl suberimidate, DMS, is a homobifunctional crosslinking agent-containing amine-reactive imidoester groups on both ends. The compound is reactive toward the s-amine groups... 

Difluorobenzene derivatives are small homobifunctional crosslinkers that react with amine groups. Conjugation using these compounds results in bridges of only about 3 A in length, potentially providing information concerning very close interactions between macromolecules. 

Reported structures for homobifunctional aryl azides include a biphenyl derivative and a naphthalene derivative (Mikkelsen and Wallach, 1976), a biphenyl derivative containing a central, cleavable disulfide group (Guire, 1976), and a compound containing a central l,3-diamino-2-propanol bridge between phenyl azide rings that are nitrated (Guire, 1976). The only commercially available homobifunctional photoreactive crosslinker is BASED. 

The homobifunctional photoreactive BASED (Chapter 4, Section 5.1) has two photoreactive phenyl azide groups, each of which contains an activating hydroxyl. Radioiodination of this crosslinker can yield one or two iodine atoms on each ring, creating an intensely radioactive compound. Crosslinks formed between two interacting molecules are reversible by disulfide reduction, thus allowing traceability of both components of the conjugate. 

A homobifunctional amine-reactive compound can be used initially to modify the amine groups on particles, while leaving the remaining amine-reactive groups available to couple with ligands. This type of reaction must be done with the crosslinker in great excess to prevent polymerization of the amine particles themselves. There must be enough crosslinker present... 

Unlike the use of homobifunctional crosslinkers, heterobifunctional compounds usually don t have to be used in large excess with amine particles to prevent aggregation. This is due to the fact that only one of the ends of the crosslinker can react with the amines on the particles. 

Hydrophilic short biotin-PEG tags also have found their way into the design of multifunctional crosslinkers to study protein structures by mass spec. Fujii et al. (2004) developed a homobifunctional NHS ester crosslinker that in addition has a PEG-biotin handle (Figure 18.1). The reagent actually is a trifunctional compound similar to the biotinylated PIR compound... 

Figure 18.3 Homobifunctional NHS ester compounds containing PEG spacers for water solubility.

The following protocol describes a general method for using bis-NHS ester PEG compounds. Optimization of concentrations should be done for each application to assure the best possible results. See also the protocol in Chapter 28, Section 1, which describes the use of homobifunctional... 

Homobifunctional crosslinkers containing thiol-reactive maleimides on each end of a PEG spacer are available in several sizes. These compounds are hydrophilic and react with sulfhydr-yls to produce thioether linkages, which are stable under most conditions. The following compounds can be obtained from Thermo Fisher or Quanta BioDesign. 

Figure 25.10 PEG-amine compounds may be reacted with this heterobifunctional crosslinker to form amide bond derivatives terminating in maleimide groups. This results in a homobifunctional reagent capable of crosslinking thiol molecules. Subsequent reaction with sulfhydryl-containing molecules yields thioether linkages.

The use of PIR compounds to study protein interactions is a significant advance over the use of standard homobifunctional crosslinkers. The unique design of the PIR reagent facilitates deconvolution of putative protein interaction complexes through a simplified mass spec analysis. The software can ignore all irrelevant peak data and just focus analysis on the two labeled peptide peaks, which accompany the reporter signal of appropriate mass. This greatly simplifies the bioinformatics of data analysis and provides definitive conformation of protein-protein crosslinks. 

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