NHS ester reagents

With bifunctional NHS ester reagents, one of three modification products can occur with proteins (1) a dead-end linkage wherein one end of the crosslinker has attached to an amine group within a protein and the other end has hydrolyzed and not formed an attachment, (2) an intra-protein crosslink wherein the PIR reagent has been coupled at both ends to amines within the same protein, or (3) an inter-protein crosslink wherein both ends of the PIR reagent have been coupled with amines on two different protein molecules (Figure 28.7). 

NHS ester reagent in DMSO (50 mM for biotin or TAMRA 70 mil for fluorescein)... 

SMPT, succinimidyloxycarbonyl-a-methyl-a-(2-pyridyldithio)toluene, contains an NHS ester end and a pyridyl disulfide end similar to SPDP, but its hindered disulfide makes conjugates formed with this reagent more stable (Thorpe et al., 1987) (Chapter 5, Section 1.2). The reagent is especially useful in forming immunotoxin conjugates for in vivo administration (Chapter 21, Section 2.1). A water-soluble analog of this crosslinker containing an extended spacer arm is also commercially available as sulfo-LC-SMPT (Thermo Fisher). 

Figure 2.1 Three types of fluorophenyl esters have been used for coupling to amine-containing molecules. The PFP and TFP esters are relatively hydrophobic and typically have better stability toward hydrolysis in aqueous solution than NHS esters. The STP ester is water-soluble due to the negatively charged sulfonate group, and it provides better solubility to associated crosslinkers or bioconjugation reagents similar to that of a sulfo-NHS ester group.

CMC should be able to participate in the two-step reaction using a sulfo-NHS ester intermediate similar to EDC, however there are no reports in the literature to this effect. Protocols for the use of this reagent in biological crosslinking applications should be essentially the same as those given previously for EDC, except substituting a molar equivalent quantity of CMC. See Sections 1.1 and 1.2 in this chapter for additional information concerning carbodiimide reactions. 

Since SIAB is water-insoluble, it must be dissolved first in organic solvent prior to addition to an aqueous reaction medium. The most commonly used solvents for this purpose include DMSO and DMF. Typically, a concentrated stock solution is prepared in one of these solvents and an aliquot added to the protein conjugation solution. Long-term storage of the reagent in these solvents is not recommended, however, due to slow uptake of water and breakdown of the NHS ester end. 

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