Using covalent dimers of human carbonic anhydrase II to model bivalency in immunoglobulins.

This paper describes the development of a new bivalent system comprising synthetic dimers of carbonic anhydrase linked chemically through thiol groups of cysteine residues introduced by site-directed mutagenesis. These compounds serve as models with which to study the interaction of bivalent proteins with ligands presented at the surface of mixed self-assembled monolayers (SAMs). Monovalent carbonic anhydrase (CA) binds to benzenesulfonamide ligands presented on the surface of the SAM with K(d)(surf) = 89 nM. The synthetic bivalent proteins--inspired by the structure of immunoglobulins--bind bivalently to the sulfonamide-functionalized SAMs with low nanomolar avidities (K(d)(avidity,surf) = 1-3 nM); this difference represents a ~50-fold enhancement of bivalent over monovalent association. The paper describes dimers of CA having (i) different lengths of the covalent linker that joined the two proteins and (ii) different points of attachment of the linker to the protein (either near the active site (C133) or distal to the active site (C185)). Comparison of the thermodynamics of their interactions with SAMs presenting arylsulfonamide groups demonstrated that varying the length of the linker between the molecules of CA had virtually no effect on the rate of association, or on the avidity of these dimers with ligand-presenting surfaces. Varying the point of attachment of the linker between monomeric CA's also had almost no effect on the avidity of the dimers, although changing the point of attachment affected the rates of binding and unbinding. These observations indicate that the avidities of these bivalent proteins, and by inference the avidities of structurally similar bivalent proteins such as IgG, are unexpectedly insensitive to the structure of the linker connecting them.

Photoaffinity labeling with 8-azidoadenosine and its derivatives: chemistry of closed and opened adenosine diazaquinodimethanes.

The reactive intermediate produced upon photolysis of 8-azidoadenosine was studied by chemical trapping studies, laser flash photolysis with UV-vis and IR detection, and modern computational chemistry. It is concluded that photolysis of 8-azidoadenosine in aqueous solution releases the corresponding singlet nitrene which rapidly tautomerizes to form a closed adenosine diazaquinodimethane in less than 400 fs. A perbenzoylated derivative of 8-azidoadenosine cannot undergo this tautomerization, and instead, it fragments upon photolysis to form an opened adenosine diazaquinodimethane. The singlet nitrene is too short-lived to be observed and, thus, to relax to the lowest triplet state or to become covalently attached to targeted biological macromolecules. The pivotal closed adenosine diazaquinodimethane, the product of nitrene tautomerization, has a lifetime of ca. 1 min or longer in water and in HEPES buffer at ambient temperature. However, this intermediate reacts rapidly with good nucleophiles such as amines, thiols, and phenolates, and significantly more slowly with weak nucleophiles such as alcohols and water. On the basis of these studies, it is clear that the closed adenosine diazaquinodimethane, and not the singlet or triplet nitrene, is the pivotal reactive intermediate involved in photolabeling and cross-linking studies using the 8-azidoadenosine family of photoaffinity labeling reagents.

DNA photocleavage and biological activity of a pyrene dihydrodioxin.

A pyrene dihydrodioxin has been synthesized, shown to bind to duplex DNA by intercalation, and cleave the phiX 174 supercoiled plasmid upon irradiation with UV light. This compound also exhibits cytotoxic activity at the micromolar range in a number of human cancer cell lines.