Esterification, Transesterification, Acylation >. This half-life decreases to 10 mins at pH 8.6 and 4°C. 0000342553 00000 n Another example is conjugation of peptide antigens to KLH or other carrier protein to prepare an effective immunogen. acidic aqueous workup procedure. Amine-reactive chemical groups in biomolecular probes for labeling and crosslinking primary amines include NHS esters (N-hydroxysuccinimide esters) and imidoesters. efficiency. during the Yamaguchi esterification reaction, a symmetric aliphatic 234 0 obj <> endobj 2006, 8, 47-50. The isothiocyanate group is familiar to researchers who have used the traditional fluorescent labeling reagent called FITC (fluorescein isothiocyanate). 309 0 obj <>stream 0000020662 00000 n 0000344063 00000 n Structures of DSS and BS3 (Sulfo-DSS) amine-to-amine crosslinkers. 0000004508 00000 n 0000342428 00000 n Activation levels (moles of maleimide per gram of carrier protein) were determined with a cysteine coupling assay. recovery and reuse of commercially available polystyrene-supported DMAP was 0000343307 00000 n A macroporous polymeric acid catalyst enables a direct esterification of This In these experiments, large molar excesses of crosslinker (100- to 1000-fold) and low concentrations of protein (less than 1mg/mL) are used to favor intramolecular over intermolecular crosslinking. The most popular and effective biotinylation and fluorescent labeling reagents for antibodies and proteins are NHS-ester compounds. The catalyst was reused up Hydrolysis of the NHS ester competes with the primary amine reaction. 0000013858 00000 n Soc., 2007, Imidoester crosslinkers react rapidly with amines at alkaline pH but have short half-lives. The half-life of hydrolysis for NHS-ester compounds is 4 to 5 h at pH 7.0 and 0°C. NHS vs. Sulfo-NHS crosslinkers. Left lane: Infrared molecular weight marker. 0000199155 00000 n 0000342926 00000 n and MeSO3H gave monoesterified products in excellent yields The functional groups are frequently used in protein biology methods. of carboxylic acids to ester or amide analogues in high yields. Crosslinking chemistry is addressed in an easy-to-follow format designed to convey the important information you need without getting lost in details. hydroxamates, peptides, and esters more environmentally friendly and coupling reagents TBTU, TATU, or COMU, in the presence of organic bases. The simplest, most common and versatile techniques for crosslinking or labeling peptides and proteins such as antibodies involve the use of chemical groups that react with primary amines (–NH2). 0000009673 00000 n As the pH becomes more alkaline, the half life and reactivity with amines increases; therefore, crosslinking is more efficient when performed at pH 10 than at pH 8. For more information on this related amine-immobilization method (AminoLink Products), see the page on Carbonyl-reactive Crosslinker Chemistry. Furthermore, the 2479-2482. Commonly needed proteins, such as HRP or KLH, are commercially available in pre-activated form (i.e., in which the NHS ester reaction with SMCC is already complete). Imidoester crosslinkers also have been examined as a substitute for glutaraldehyde for tissue fixation. chromatography. 0000143179 00000 n The solubility of NHS-ester reagents varies with buffer composition and the physical properties of the remainder of the molecular structure (e.g., spacer arm). 0000199271 00000 n Anal Biochem 156:220-2. Molecular Biology Reports (1993) 17:167-183. 1,2 Propylphosphonic anhydride (T3P ® ) 3 is a very efficient, nontoxic coupling reagent especially suited for connecting sterically hindered amino acids. 0000143210 00000 n 14, 4910-4913. Chem. 0000343232 00000 n 0000005181 00000 n Surprisingly, only a 0.05−2 mol % of DMAP can efficiently promote acylation of DSS is not directly water-soluble but once dissolved can permeate across cell membranes to crosslink inside cells. The reaction between carboxylic acids and dialkyl dicarbonates, in the presence Synlett, 2017, 28, 981-985. By comparing results using crosslinkers with different spacer arm lengths or different cleavability or solubility features, different characteristics of an interaction can be elucidated. Lett., 2012, from an acid and either a phenol or an aliphatic alcohol using the peptide Hydrolysis of the NHS ester competes with the primary amine reaction. Y.-H. Kim, J. Han, B. Y. Jung, H. Baek, Y. M. A. Yamada, Y. Uozumi, Y.-S. Lee, The reaction releases N-hydroxysuccinimide (NHS). Silica chloride is an efficient catalyst for esterification of carboxylic Enhancement by N-hydroxysulfosuccinimide of water-soluble carbodiimide-mediated coupling reactions. particular oligoethylene glycols - has been developed. and CO2. with choice of base and coupling agent. Reactions using TBTU and TATU are faster but do not occur with tertiary Synlett, 2016, 27, 29-32. A final yield of 25 mg of protein was obtained. Not for use in diagnostic procedures. 0000268130 00000 n