NHS esters chemistry

Analogously, the d5SICS-dNaM unnatural base pair developed by Romesberg et al. [107, 142] tolerates amino-modifications at both components of this unnatural base pair with sufficient efficiency in in vitro transcription reactions to allow postsynthetic RNA labeling using NHS-ester chemistry [111, 143]. As a first example of post-transcriptional functionalization of a long RNA, a site-specifically labeled 77 nucleotide amber suppressor tyrosyl tRNA from Methanocaldococcus jannaschii was synthesized via in vitro transcription and post-transcriptionally reacted with NHS-biotin [143]. 

Most immobilizahon chemistries for microarrays currently rely upon derivatization of the substrate with amine-reactive functional groups such as aldehydes, epoxides, or NHS esters. While we can choose from many available surface-reactive chemistries, it is important to keep in mind that they must be compatible with a printing process. Ideally, the biomolecule should react completely and rapidly with the substrate in order to achieve good spot formation. It is also critical that the probe remain or be recoverable in its active state following printing. If too reactive a chemistry is employed there is the possibility for excessive crosslinking that can hinder performance by reducing the number of rotatable bonds in the probe. 

A surface coated with a SAM of 3-mercaptoproponic-acid, was used for the covalent attachment of Alexa-Fluor 647 labeled streptavidin (AF-SA) by means of NHS-active ester chemistry [38]. This reaction yielded an SPR minimum shift of A0 O.27° and contributed a fluorescence intensity of A/A 350 eps (layer b ). Subsequently, the alternate binding of biotinylated IgG and streptavidin from 20 pg ml-1 solutions was monitored. 

Soper et al. [211] presented the fabrication of DNA microarrays onto PMMA surfaces using a UV modification protocol as shown in Fig. 11. Briefly, the PMMA surface was first activated via exposure to UV irradiation, which produced carboxylic acid functional groups onto its surface. EDC/NHS coupling chemistry was then used to facilitate the formation of succinimidyl ester intermediates on the surface, which allowed for the covalent attachment of amine-terminated... 

In addition to the design principles just mentioned, peptides, proteins, and peptide-based supramolecular structures can be cross-linked into 3D networks via conventional synthetic chemistry (e.g., active ester chemistry or short synthetic cross-linkers) or via bioorthogonal click chemistry (e.g., Michael-type addition), utilizing their inherent multitude of functional groups (—NH, —COOH, —SH) and/or other additionally introduced functionalities. 

FIGURE 11.6 Covalent enzyme immobilization via reaction of (a) lysine residues with NHS esters or aldehydes, (b) cysteine residues with maleimide groups, and (c) carhoxyl residue of glutamic acid or aspartic add with amine-functionalized materials via NHS-mediated ester chemistry. 

Unlike the succinimidyl succinate or succinimidyl glutarate activation methods, SC chemistry does not suffer from the presence of a labile ester bond. The intermediate reactive NHS carbonate may hydrolyze in aqueous solution to release NHS and CO2, essentially regenerating... 

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