Maleimide reactive crosslinkers

The first two compounds discussed in this section are truly sulfhydryl-reactive, using the common iodoacetyl and maleimide functionalities, respectively. The third derivative, however, is not reactive directly with sulfhydryl groups, but contains a protected sulfhydryl which, after deprotection, can be used to react with other sulfhydryl-reactive crosslinkers. 

The most common type of heterobifunctional reagent used for the activation of lipid components includes the amine- and sulfhydryl-reactive crosslinkers containing an N-hydroxysuccinim-ide (NHS) ester group on one end and either a maleimide, iodoacetyl, or pyridyl disulfide group on the other end (Chapter 5, Section 1). Principle reagents used to effect this activation process include SMCC (Chapter 5, Section 1.3), MBS (Chapter 5, Section 1.4), SMPB (Chapter 5, Section 1.6), SIAB (Chapter 5, Section 1.5), and SPDP (Chapter 5, Section 1.1). Other... 

Other heterobifunctional crosslinkers with NHS and maleimide reactive groups (e.g., BMPS, MBS, SMCC) can be used in place of GMBS. The final concentration of these other linkers in the ethanolic mix should be 1 mM. 

BMPA is N-(3-maleimidopropionic acid (or 3-maleimidopropionic acid), which contains a thiol-reactive maleimide group at one end and a carboxylate group on the other end (Rich et al., 1975 Moroder, 1983, 1987). The compound is the acid precursor to the short, heterobifunctional crosslinker 3-maleimidopropionic acid N-hydroxysuccinimide ester (BMPS). 

A common choice of crosslinker for this type of reaction is sulfo-SMCC, which has been used extensively for antibody conjugation (Chapter 20, Section 1.1). A better option for dendrimer conjugation is to use a similar crosslinker design, but one that contains a hydrophilic PEG spacer arm to promote dendrimer hydrophilicity after modification. Derivatization of an amine-dendrimer with a NHS-PEG-maleimide can create an intermediate that is coated with water-soluble PEG spacers. This modification helps to mask any potential for nonspecific interactions that the PAMAM surface may have, while providing terminal thiol-reactive maleimides for coupling ligands (Figure 7.10). 

Coupling of affinity molecules to surfaces also can be enhanced by the use of discrete PEG linkers. Nishimura et al. (2005) modified an amino surface with a NHS-PEG -maleimide crosslinker to create a hydrophilic self-assembled monolayer (SAM) surface that was thiol reactive for the conjugation of sulfhydryl-modified RNAs. This array then was used to investigate the binding specificity of synthetic kanamycins with selected RNA sequences to prove the specific interaction of ribosomal RNA with this molecule. The PEG linkers on surfaces provide lower nonspecific binding character than alkyl linkers, when preparing SAM surfaces for affinity interactions. 

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 19.18 Carrier proteins may be activated with sulfo-SMCC to produce maleimide derivatives reactive with sulfhydryl-containing molecules. The graphs show the gel filtration separation on Sephadex G-25 of male-imide-activated BSA (A) and OVA (B) after reaction with sulfo-SMCC. The first peak is the protein and the second peak is excess crosslinker. The maleimide groups create increased absorbance at 280 nm in the activated proteins.

Succinimidyl-4-(p-maleimidophenyl)butyrate (SMPB), is a heterobifunctional analog of MBS containing an extended cross-bridge (Chapter 5, Section 1.6). The crosslinker has an amine-reactive NHS ester on one end and a sulfhydryl-reactive maleimide group on the other. Conjugates formed using SMPB thus are linked by stable amide and thioether bonds. 

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