Biotin side chain

Figure 6.1 The Wedekind trifunctional crosslinker can react with amine groups via its p-nitrophenyl ester to form amide bond linkages. The phenyl azide group then can be photoactivated with UV light to generate covalent bond formation with a second molecule. The biotin side chain provides binding capability with avidin or streptavidin probes.

Activation of imines A recent synthesis of (+ )-biotin (6) is based on activation of the imine group of a 3-thiazoline (1) by BF, etherate to nucleophilic attack. Thus reaction of 1, substituted by the biotin side chain, with the ester enolate 2 in the presence of 1 equiv. of the Lewis acid results in the thiazolidine 3 as the major product. The stereochemistry at the future C7-center is determined to some extent by the ester group of 2. Selective reduction of the Chester group furnishes the alcohol 4. Reaction of 4 with... 

Biotin is involved in carboxylation and decarboxylation reactions. It is covalently bound to its enzyme. In the carboxylase reaction, C02 is first attached to biotin at the ureido nitrogen, opposite the side chain in an ATP-dependent reaction. The activated C02 is then transferred from carboxybiotin to the substrate. The four enzymes of the intermediary metabolism requiring biotin as a prosthetic group are pyruvate carboxylase (pyruvate oxaloacetate), propionyl-CoA-carboxylase (propionyl-CoA methylmalonyl-CoA), 3-methylcroto-nyl-CoA-carboxylase (metabolism of leucine), and actyl-CoA-carboxylase (acetyl-CoA malonyl-CoA) [1]. 

Figure 6.2 The trifunctional reagent sulfo-SBED reacts with amine-containing bait proteins via its NHS ester side chain. Subsequent interaction with a protein sample and exposure to UV light can cause crosslink formation with a second interacting protein. The biotin portion provides purification or labeling capability using avidin or streptavidin reagents. The disulfide bond on the NHS ester arm provides cleavability using disulfide reductants, which effectively transfers the biotin label to an unknown interacting protein.

Figure 11.1 The basic design of a biotinylation reagent includes the bicyclic rings and valeric acid side chain of D-biotin at one end and a reactive group to couple with target groups at the other end. Spacer groups may be included in the design to extend the biotin group away from modified molecules, thus ensuring better interaction capability with avidin or streptavidin probes.

Amine-reactive biotinylation reagents contain reactive groups off biotin s valeric acid side chain that are able to form covalent bonds with primary amines in proteins and other molecules. Two basic types are commonly available N-hydroxysuccinimide (NHS) esters and carboxylates. NHS esters spontaneously react with amines to form amide linkages (Chapter 2, Section 1.4),... 

Biocytin is e-N-biotinyl-L-lysine, a derivative of D-biotin containing a lysine group coupled at its e-amino side chain to the valeric acid carboxylate. It is a naturally occurring complex of biotin that is typically found in serum and urine, and probably represents breakdown products of recycling biotinylated proteins. The enzyme biotinidase specifically cleaves the lysine residue and releases the biotin component from biocytin (Ebrahim and Dakshinamurti, 1986, 1987). 

The only disadvantage to the use of NHS-biotin or sulfo-NHS-biotin is the lack of a long spacer group off the valeric acid side chain. Since the binding site for biotin on avidin and streptavidin is somewhat below the surface of the proteins, some biotinylated molecules may not interact as efficiently with (strept)avidin as when longer cross-bridges are used (Green et al., 1971 Bonnard et al., 1984). 

The Derivative, 5-(biotinamido)pentylamine, contains a 5-carbon cadaverine spacer group attached to the valeric acid side chain of biotin (Thermo Fisher). The compound can be used in a carbodi-imide reaction process to label carboxylate groups in proteins and other molecules, forming amide bond linkages (Chapter 3, Section 1). However, the main use of this biotinylation reagent is in the determination of factor XHIa or transglutaminase enzymes in plasma, cell, or tissue extracts. 

Strepavidin A 60 kD extracellular protein of Streptomyces avidinii with four high-affinity biotin binding sites. Unlike avidin, streptavidin has a near neutral isoelectric point and is free of carbohydrate side chains. 

The way biotin participates in carbon dioxide fixation was established in the early 1960s. In 1961 Kaziro and Ochoa using propionyl CoA carboxylase provided evidence for 14C02 binding in an enzyme-biotin complex. With excess propionyl CoA the 14C label moved into a stable position in methyl malonyl CoA. In the same year Lynen found biotin itself could act as a C02 acceptor in a fixation reaction catalyzed by B-methylcrotonyl CoA carboxylase. The labile C02 adduct was stabilized by esterification with diazomethane and the dimethyl ester shown to be identical with the chemically synthesized molecule. X-ray analysis of the bis-p-bromanilide confirmed the carbon dioxide had been incorporated into the N opposite to the point of attachment of the side chain. Proteolytic digestion and the isolation of biocytin established the biotin was bound to the e-NH2 of lysine. 

Vitamin H (biotin) is present in liver, egg yolk, and other foods it is also synthesized by the intestinal flora. In the body, biotin is covalently attached via a lysine side chain to enzymes that catalyze carboxylation reactions. Biotin-dependent carboxylases include pyruvate carboxylase (see p. 154) and acetyl-CoA carboxylase (see p. 162). CO2 binds, using up ATP, to one of the two N atoms of biotin, from which it is transferred to the acceptor (see p. 108). 

Biotin is composed of an imidazolidone ring joined to a tetrahydrothiophene ring (Fignre 19.17). The latter possesses a valeric acid side chain. The unique stereoisomer occurring in natnre is d(+)-biotin. 

Biotin is solnble in water and insolnble in organic solvents. It is stable at pH 5-8 and to heating in strong acid. However it can be oxidized in the sulfur atom and the shortening of the valeric acid side chain results in the loss of vitamin activity. 

Because at a given pH the reactivity and/or accessibility of NH2 groups of amino acid side chains of a protein are not the same, the number of conjugated biotin residues may be altered by variation of the buffer pH between pH 6 and 9 (the higher pH the larger the degree of conjugation). 

Biotin (6.24) consists of an imidazole ring fused to a tetrahydrothiophene ring with a valeric acid side chain. Biotin acts as a co-enzyme for carboxylases involved in the synthesis and catabolism of fatty acids and for branched-chain amino acids and gluconeogenesis. 

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