Sulfa antibiotics (sulfonamides) are derivatives of p-aminobenzenesulfonamide that are widely used in veterinary medicine. Foods derived from treated animals may be contaminated with these drugs. However, current immunobased sulfonamide detection methods are unfit for screening of products because they are either too insensitive or specific for a few compounds only. An immunoassay capable of detecting all sulfas in a single reaction would be ideal for screening. For development of a binder capable of binding all sulfas, a protein engineering approach was chosen and the properties of monoclonal antibody 27G3 were improved with mutagenesis followed by selection with phage display. Several different mutant antibodies were isolated. The cross-reaction profile of the best mutant antibody was significantly improved over that of the wild-type antibody: it was capable of binding 9 of the tested 13 sulfonamides within a narrow concentration range and also bound the rest of the sulfas, albeit within a wider concentration range.

In the adrenergic receptor subfamily, the seven ␣-helical transmembrane (TM) 1 domains form a crevice for the recognition and binding of ligands (1). The amino acid sequences are highly conserved within the seven hydrophobic TM domains of the three human ␣ 2 -adrenoreceptor (␣ 2 -AR) subtypes (ϳ 75% amino acid identity) (2-5). The ␣ 2 -AR subtypes also share significant structural identity within the TM domains with other members of the adrenoreceptor family (2, 6). For example, of the 182 amino acids comprising the TM domains of the human ␣ 2A -AR, about 40% are identical with the human ␤ 2 -AR. The conserved regions are known to contain structural determinants responsible for recognizing and binding the endogenous hormones/neurotransmitters adrenaline and noradrenaline and other receptor ligands (2, 7-9).Phenoxybenzamine (PB) is an irreversible, subtype-nonselective ␣-AR antagonist. PB has been used as a pharmacological tool to study ␣-AR subpopulations in tissue preparations. PB was also the first ␣-AR antagonist to be therapeutically evaluated in humans. It produces long lasting ␣-AR blockade and reduces blood pressure, but its clinical use was limited by severe side effects (10 -12). Although the pharmacology of PB has been studied quite extensively, the molecular basis of its interaction with ␣-ARs has not been examined in detail. It is known that ␤-haloalkylamines, such as PB, cyclize in aqueous solution to form an unstable aziridinium ion, which can bind to target proteins with a strong ionic bond. The aziridinium ion then opens to create a reactive intermediate, with the consequence that a covalent bond between the drug molecule and the binding site can be formed. Side chains of amino acid residues that can be alkylated by haloalkylamines include -SH, -OH, ϭNH, and -COOH (13). Of the susceptible amino acid residues, cysteine (-SH) is the most reactive (14).The helical arrangement of the TM domains indicated by receptor modeling (15,16), in conjunction with analysis of sequence alignments (see Fig. 1), suggested to us an interaction between PB and the TM3 region of the ␣-ARs. The amino acid sequence alignment of TM3 of adrenergic receptors presented in Fig. 1 is validated by the position of the conserved aspartate residue (D 3.32 according to the nomenclature of Ballesteros and Weinstein (17) or position 113 in the ␣ 2A -AR), known to be crucial for binding the charged nitrogen present in adrenergic phenethylamine ligands (7, 9). According to our hypothesis, the reactive aziridinium derivative of PB forms a covalent bond with C 3.36 in TM3 of the ␣ 2A -, ␣ 2B -and ␣ 2C -AR (Fig. 2) (corresponding to amino acid residues 117, 96 and 135, respectively). Also the three ␣ 1 -AR subtypes have a cysteine in this position, but the three ␤-AR subtypes have a valine residue in its place (Fig. 1). To test our hypothesis, we determined the irreversible binding of PB to the three human ␣ 2 -AR subtypes and constructed and tested an ␣ 2A -AR mutant lacking the Cys 117 (Cys 117 was substituted with valine; ␣ 2A -C117V...

Synthetic glycoclusters are extensively used as mimetics of naturally occurring, multivalent carbohydrate ligands in various glycobiological applications. Their preparation, however, is far from trivial, and it still is a limiting factor in the study of carbohydrate binding. We herein report the synthesis of an orthogonally protected building block, N-Alloc-N'-Boc-N' '-Fmoc-alpha,alpha-bis(aminomethyl)-beta-alanine (1), and its use in the preparation of triantennary peptide glycoclusters (21-24) on a solid support. The assembly of the clusters involves removal of the amino protections of the solid-supported branching unit 1 in the order Fmoc, Boc, and Alloc, and subsequent coupling of peracetylated O-(glycopyranosyl)-N-Fmoc-L-serine pentafluorophenyl esters (galactose, glucose, mannose, and ribose) to each amino group exposed.

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