Active N-hydroxysuccinimide esters

Cefpiramide (64) is a third generation cephalosporin with a l-methyl-[lH)-tetra2ol-5-ylthio-methyl moiety at C-3 and an acylated -hydroxyphenylglycine moiety at C-7. It includes in its activity spectrum reasonable potency in vitro against many strains of Pseudomonas. It can be synthesized in a variety of ways including condensation of cephalosporin antibiotic 63 with 6-methyl-4-(l-H)-pyridone-3-carboxylic acid in the form of its active N-hydroxysuccinimide ester (62) to produce cefpiramide (64) [20,21],... 

The Active Esters Method. Selected amino-protecting groups and conditions required for their deprotection in organic solvents are shown in Table III. Active N-hydroxysuccinimide esters of f-butyloxycarbonyl-L-... 

Currently, a common form of activated mPEG used for preparation of therapeutic enzymes is mPEG-succinate-N-hydroxysuccinimide ester (SS-PEG) (11). It reacts with proteins in short periods of time under mild conditions, producing extensively modified conjugates with well preserved biological activity. However, the ester linkage between the polymer and the succinic acid residue has limited stability in aqueous media (5,12). 

The avidin-biotin interaction has also been used to immobilize antibodies and proteins, especially in commercial systems based on surface plasmon resonance (SPR) measurements (e.g., the BIAcore). The extraordinary affinity (Kl 10-15 M) of avidin (or its bacterial relative, streptavidin) for the vitamin biotin is the basis of this immobilization procedure. A solid support (e.g., glass beads, sensor chip, optical fiber) covered with avidin can be used as an activated carrier for a very sturdy immobilization of previously biotinylated antibodies. In spite of the many methods for biotinylating proteins described in the literature, the use of biotinyl N-hydroxysuccinimide ester (BNHS) and similar derivatives, remains the most useful [65]. 

Limiting essential amino acids covalently attached to proteins by using activated amino acid derivatives can improve the nutritional quality and change the functional properties of proteins. The best chemical methods for incorporating amino acids into water-soluble proteins involve using car-bodiimides, N-hydroxysuccinimide esters of acylated amino acids, or N-carboxy-a-amino acid anhydrides. The last two methods can give up to 75% incorporation of the amount of amino acid derivative used. With the anhydride method, as many as 50 residues of methionine have been linked to the 12 lysine residues of casein. The newly formed peptide and isopeptide bonds are hydrolyzed readily by intestinal aminopeptidase, making the added amino acids and the lysine from the protein available nutritionally. 

Proteins may be modified covalently via the e-amino group of lysyl residues using two types of activated amino acids N-hydroxysuccinimide esters and N-carboxyanhydrides. 

Having established the selectivity of the microspheres for 2,4-D, the template was then conjugated to tobacco peroxidase (TOP). Briefly, TOP was activated using NaI04 then coupled with diaminopropane. 2,4-D was converted to its N-hydroxysuccinimide ester and allowed to react with the derivatised TOP. After purification, the final 2,4-D/TOP ratio was estimated to be 8/1. 

The synthesis of the protein conjugates is shown in Figure 3. Commercially available carboxylic acids were converted to activated esters by direct DCC (N,N-dicyclohexylcarbodiimide) coupling with N-hydroxysuccinimide or by conversion to the corresponding acid chloride derivative followed by reaction with N-hydroxysuccinimide. Reactiion of the resulting N-hydroxysuccinimide esters with either BSA or OVA led to the desired lysine bonded nitroaromatic hapten-protein conjugates. 

Commercially available preactivated matrices [N-hydroxysuccinimide ester or hydrazide, Affi-Gel (BioRad) or a variety of different activated Sepharose derivatives (Amersham Pharmacia Biotech) I have been primarily developed for affinity chromatography and are generally too expensive for use as a biocatalyst matrix. 

Dimyristoylphosphatidylethanolamine (DMPE), biotinyl-N-hydroxysuccinimide ester (BNHS), triethylamine, myoglobin, bovine serum albumin (BSA), lysozyme, guanidinium chloride, dimethylamino-cinnamaldehyde, acrylamide, ammonium persulfate, sodium dodecyl sulfate (SDS), and molybdenum blue reagent were all obtained from Sigma Chemicals and used without further purification as received. The compounds N, N, N, N -tetramethylethylenediamine (TEMED), N, N -methylene-bis-acrylamide, and Coomassie Brilliant Blue R-250 were from BioRad Laboratories. Avidin was obtained from Vector Laboratories with a quoted activity of 14 units/mg. All solvents were from Fisher Scientific. Water was passed through a Barnstead Nanopure system. 

Scheme 7.36 Synthesis of medium-ring lactams and N-hydroxysuccinimide esters employing P-HOBt-derived active esters.

Additives such as HOBt or DMAP can be used while attached to a polymer. Thus, the polymeric //-benzyl-1-hydroxybenzotriazole-6-sulfona-mide (19) [47] and the polymeric 1-hydroxybenzotriazole (20) [48] have been shown to be highly efficient for the solution synthesis of amides. The efficiency of 19 could be attributed to its high acidity, conferred by the sulfonyl moiety. The procedure for amide construction involves the formation of an activated ester on the derivatized polymer followed, in a second step, by treatment with an amine to generate the amide in solution. This HOBt-supported polymer has also been applied for the preparation of N-hydroxysuccinimide esters, useful for the modification of proteins [49]. Polymeric DMAP is a less basic compound and generally gives very low racemization [50]. 

Figure 5.7. Coupling of ligands to matrices. (A) Via its primary amino group, a ligand couples to a matrix activated with N-hydroxysuccinimide ester. (B) Carbodiimide coupling of the carboxyl group of a ligand to matrix-bound primary amino groups. In the process, the carbodiimide changes into the corresponding urea derivative.

Activated CH-Sepharose Pharmacia Agarose (Sepharose 4B) N-hydroxysuccinimide ester (6-10 pMol/ml gel) 6 C Hydrophobe Primary amino group... 

The active ferrocenylethanoic N-hydroxysuccinimide ester was prepared in situ from ferrocenylethanoic acid (1.0 mmolj, HSU (1.1 mmol), and DCC (1.2 mmol) in THF (2 ml) at -5 C (Ih) and ambient temperature (5h). The ester solution, filtered from the urea by-product, combined with the THF washings of the residue, and reduced in volume to 7 ml, was added dropwise to the precooled (0 ) solution of copolyamide 5 (x/y = 3.0 0.7 mmol) in H2O (5 ml) with rapid stirring. Following the addition of more THF (1 ml) to dissolve some precipitated active ester, the mixture was stirred for 18h at ambient temperature. Upon the addition of NEts (1.0 mmol), stirring was continued for another 6h, the THF component removed under reduced pressure, and the mixture diluted with H2O (8 ml). The filtered solution was dialyzed and freeze-dried as before to give 12 as a w ater-soluble, brown solid. Yield, 78% 77inh(H20) 12 ml g l. Anal, found Fe, 2.6%. Calcd. for 12 (x/y = 3.0 45% substitution) Fe, 2.7%. 

Carbodi-imide-mediated reaction with epichlorohydrin-activated agarose previously treated with glycine Reaction with a syw-trichlorotriazine-activated polystyrene anion exchanger reaction with 1,4-benzoqumone-activated aminopropyl silica reaction with an N-hydroxysuccinimide ester derivative of 3-succinimidopropyl silica Carbodi-imide- or 2,4-toluenedi-isocyanate-mediated reaction with amino-or carboxy-aerosU... 

Better reaction rates were observed with the already inherently more reactive o-nitrophenyl esters, in part because they are less sensitive to steric hindrance than their para-substituted analogs. Bulkyness of the activating groups renders N-hydroxysuccinimide esters and pentachlorophenyl esters less well suited for acylation inside the matrix of a polymer than in solution. Only the introduction of the powerful pentafluorophenyl esters led to a revival of the active ester idea in the praxis of solid phase peptide synthesis. Pentafluorophenyl esters of Fmoc amino acids... 

Preformed polymers bearing a reactive or activated group that can react with a functional group of an enzyme have been reported. Schuhmann described the synthesis of polymers 130 and 131 derived from dithienylpyrrole containing a pendant amino group that can fix a glucose oxidase [244]. Bauerle and coworkers have developed poly(bithiophene) 132 and the related poly(terthio-phene) substituted with easily replaceable N-hydroxysuccinimide ester [245] for the immobilization of glucose oxidase [246]. 

---