Formation of NHS esters

P-coupling occurs in the formation of azophosphonic esters [ArN2PO(OCH3)2] from diazonium salts and dimethyl phosphite [HPO(OCH3)2] (Suckfull and Hau-brich, 1958). P-coupled intermediates are formed in the reaction between diazonium salts and tertiary phosphines, studied by Horner and Stohr (1953), and by Horner and Hoffmann (1956). The P-azo compound is hydrolyzed to triphenylphosphine oxide, but if a second equivalent of the tertiary phosphine is available, phenyl-hydrazine is finally obtained along with the phosphine oxide (Scheme 6-26 Horner and Hoffmann, 1958). It is likely that an aryldiazene (ArN = NH) is an intermediate in the hydrolysis step of the P-azo compounds. 

HOSu or NHS) or pentafluorophenol to give water-soluble activated esters, which can not be purified by extraction [30]. It has also been employed with sulfonamides to afford acylsulfonamide libraries, this last case requiring the addition of DMAP as additive [31]. The formation of thiol esters [32] and also the synthesis of ben-zoxazinone and benzoxatine scaffolds, via cyclodehydration, has been performed using PS-EDC (11) [33],... 

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... 

Benzoyl Chloride.—The formation of esters by ibe action of benzoyl chloride or othei acid chloride on an alcohol or ]ohcnol in presence of caustic soda is known as the Schotten- 13riumann reaction. The reaction may also be employed in the preparation of deri ati es of the aromatic amines containing i.n acid radical, like benzanilide, CjjH NH.CO... 

Figure 16 Conjugation of an amine and a carboxylic acid via the (V-hydroxysuccinimide (NHS)-activated ester method. NHS esters may be isolated and characterized and are stable to long term storage as the powder. Alternatively, the NHS esters may be used immediately upon formation without isolation. Details of the reaction are given in Table 4...

Partly saturated pyrazino[l,2-r-]pyrimidines were prepared by formation of the pyrazine ring. 2-Substituted-8-hydroxy-3,4-dihydro-177,277-pyrazino[l,2-r-]pyrimidin-l-ones were prepared by a [6+0] synthesis involving cyclization of 6-hydroxy-pyrimidine-4-(fV-hydroxyethyl)carboxamides <2005W02005/087766>. The 2/7-pyra-zino[l,2-c]pyrimidine-3-carboxamide 164 (Y = NH) was formed from [5+1] atom fragments via the uracil derivative 163 (Y = NH) and DMF-dimethyl acetal. Compounds 163 were prepared from 6-chloromethyluracil and glycine methyl ester 162 (Y = NH) (Scheme 20) <2004W02004/014354>. 

Figure 1.113 The NHS ester-suberate derivative of lactose can be used to add lactose groups to amine-containing molecules. The reaction results in the formation of amide bonds containing terminal lactose groups.

Figure 3.2 The efficiency of an EDC-mediated reaction may be increased through the formation of a sulfo-NHS ester intermediate. The sulfo-NHS ester is more effective at reacting with amine-containing molecules. Thus, higher yields of amide bond formation may be realized using this two-step process as opposed to using a single-step EDC reaction.

Carbodiimide coupling to carboxylate-containing QDs usually involves the use of EDC in a single-step or two-step process to form an amide bond. If a one-step reaction is done, the QD is activated with EDC in the presence of an amine-containing molecule, such as a protein. Many protocols use this method, but it can result in protein polymerization in addition to coupling, because proteins contain both carboxylates and amines. A two-step protocol results in better control of the reaction (Figure 9.61). In the first step, EDC is used in the presence of sulfo-NHS to activate the carboxylates on the particles to intermediate sulfo-NHS esters. After a quick separation step to remove excess reactants, the activated QDs are added to the protein solution to be coupled. This then results in amide bond formation without polymerization of the protein in solution. See Chapter 3, Section 1 and Chapter 14, Section 1 for additional information on this process. 

Biotinylated liposomes usually are created by modification of PE components with an amine-reactive biotin derivative, for example NHS-LC-Biotin (Chapter 11, Section 1). The NHS ester reacts with the primary amine of PE residues, forming an amide bond linkage (Figure 22.19). A better choice of biotinylation agent may be to use the NHS-PEG -biotin compounds (Chapter 18), because the hydrophilic PEG spacer provides better accessibility in the aqueous environment than a hydrophobic biotin spacer. Since the modification occurs at the hydrophilic end of the phospholipid molecule, after vesicle formation the biotin component protrudes out from the liposomal surface. In this configuration, the surface-immobilized biotins are able to bind (strept)avidin molecules present in the outer aqueous medium. 

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