Linker

Abstract: The three-dimensional structure of a 70-kilodalton amino terminally truncated form of human topoisomerase I in complex with a 22–base pair duplex oligonucleotide, determined to a resolution of 2.8 angstroms, reveals all of the structural elements of the enzyme that contact DNA. The linker region that connects the central core of the enzyme to the carboxyl-terminal domain assumes a coiled-coil configuration and protrudes away from the remainder of the enzyme. The positively charged DNA-proximal surface of the linker makes only a few contacts with the DNA downstream of the cleavage site. In combination with the crystal structures of the reconstituted human topoisomerase I before and after DNA cleavage, this information suggests which amino acid residues are involved in catalyzing phosphodiester bond breakage and religation. The structures also lead to the proposal that the topoisomerization step occurs by a mechanism termed “controlled rotation.”

Abstract: Compounds having structure (1) wherein R 1 is —H a protecting group, a linker or a binding partner; and R 2 and R 34 are as defined in the specification. The invention also provides intermediates and methods make the structure (1) compounds, as well as methods to use the compounds as labels in diagnostic assays and to enhance binding to complementary bases.

Abstract: Antibody-drug conjugates are targeted anticancer agents consisting of a cytotoxic drug covalently linked to a monoclonal antibody for tumor antigen-specific activity. Once bound to the target cell-surface antigen, the conjugate must be processed to release an active form of the drug, which can reach its intracellular target. Here, we used both biological and biochemical methods to better define this process for antibody-maytansinoid conjugates. In particular, we examined the metabolic fate in cells of huC242-maytansinoid conjugates containing either a disulfide linker (huC242-SPDB-DM4) or a thioether linker (huC242-SMCC-DM1). Using cell cycle analysis combined with lysosomal inhibitors, we showed that lysosomal processing is required for the activity of antibody-maytansinoid conjugates, irrespective of the linker. We also identified and characterized the released maytansinoid molecules from these conjugates, and measured their rate of release compared with the kinetics of cell cycle arrest. Both conjugates are efficiently degraded in lysosomes to yield metabolites consisting of the intact maytansinoid drug and linker attached to lysine. The lysine adduct is the sole metabolite from the thioether-linked conjugate. However, the lysine metabolite generated from the disulfide-linked conjugate is reduced and S-methylated to yield the lipophilic and potently cytotoxic metabolite, S-methyl-DM4. These findings provide insight into the mechanism of action of antibody-maytansinoid conjugates in general, and more specifically, identify a biochemical mechanism that may account for the significantly enhanced antitumor efficacy observed with disulfide-linked conjugates.

Abstract: A novel linker for the synthesis of oligonucleotides on a glass support is described. Oligonucleotides synthesised on the support remain tethered to the support after ammonia treatment and are shown to take part in sequence specific hybridisation reactions. These hybridizations were carried out with oligonucleotides synthesised on 'ballotini' solid sphere glass beads and microscope slides. The linker has a hexaethylene glycol spacer, bound to the glass via a glycidoxypropyl silane, terminating in a primary hydroxyl group that serves as starting point for automated or manual oligonucleotide synthesis.