Daniel L. Rathbone

Synthesis of potent water-soluble tissue transglutaminase inhibitors

Dipeptide-based sulfonium peptidylmethylketones derived from 6-diazo-5-oxo-L-norleucine (DON) have been investigated as potential water-soluble inhibitors of extracellular transglutaminase. The lead compounds were prepared in four steps and exhibited potent activity against tissue transglutaminase.

New solid phase Knoevenagel catalyst

Abstract A new solid phase Knoevenagel catalyst has been prepared and evaluated. The new resin based catalyst reduces by-product formation and has the advantage over conventional Knoevenagel catalysts of simple removal by filtration.

Molecular recognition by fluorescent imprinted polymers

Abstract New fluorescent imprinted polymers have been prepared for guest–host selectivity studies. Significant differences were found in the fluorescence response between the bound and unbound states.

Characterization of Heparin-binding Site of Tissue Transglutaminase ITS IMPORTANCE IN CELL SURFACE TARGETING, MATRIX DEPOSITION, AND CELL SIGNALING

Background: TG2 is a multifunctional matrix protein and cross-linking enzyme. Results: Identification and mutation of its heparan sulfate (HS)-binding site blocks matrix deposition of TG2 as do inhibitors of syndecan shedding. Conclusion: Coordinated binding to cell surface heparan sulfates facilitates TG2 cell surface trafficking and deposition into the ECM. Significance: Blocking heparan sulfate binding provides an avenue for regulating the pathological roles of the enzyme. Tissue transglutaminase (TG2) is a multifunctional Ca2+-activated protein cross-linking enzyme secreted into the extracellular matrix (ECM), where it is involved in wound healing and scarring, tissue fibrosis, celiac disease, and metastatic cancer. Extracellular TG2 can also facilitate cell adhesion important in wound healing through a nontransamidating mechanism via its association with fibronectin, heparan sulfates (HS), and integrins. Regulating the mechanism how TG2 is translocated into the ECM therefore provides a strategy for modulating these physiological and pathological functions of the enzyme. Here, through molecular modeling and mutagenesis, we have identified the HS-binding site of TG2 202KFLKNAGRDCSRRSSPVYVGR222. We demonstrate the requirement of this binding site for translocation of TG2 into the ECM through a mechanism involving cell surface shedding of HS. By synthesizing a peptide NPKFLKNAGRDCSRRSS corresponding to the HS-binding site within TG2, we also demonstrate how this mimicking peptide can in isolation compensate for the RGD-induced loss of cell adhesion on fibronectin via binding to syndecan-4, leading to activation of PKCα, pFAK-397, and ERK1/2 and the subsequent formation of focal adhesions and actin cytoskeleton organization. A novel regulatory mechanism for TG2 translocation into the extracellular compartment that depends upon TG2 conformation and the binding of HS is proposed.

Synthesis of water-soluble prodrugs of the cytotoxic agent Combretastatin A4

Water-soluble phosphate and glycine carbamate prodrugs of the cytotoxic agent Combretastatin A4 (1) have been prepared. The phosphate prodrug was degraded slowly in plasma at 37°C. The degradation was accelerated by the addition of alkaline phosphatase.

Metal complexes of carboxamidrazone analogs as antitubercular agents: 1. Synthesis, X-ray crystal-structures, spectroscopic properties and antimycobacterial activity against Mycobacterium tuberculosis H37Rv

N(1)-Benzylidene-pyridine carboxamidrazones and their metal conjugates have emerged as a new class of potential antimycobacterial agents. Nine such carboxamidrazone analogs (L(1)-L(9)) along with their Cu(II) (MC(1)-MC(9)) and Fe(III) (MC(10)-MC(18)) complexes were synthesized. Single crystal X-ray structures of copper complexes MC(1) and MC(5) were determined which suggest slightly distorted square planer geometries for copper complexes and octahedral geometries for ferric compounds. All compounds were evaluated for their in vitro antimycobacterial activity against Mycobacterium tuberculosis H(37)Rv. The results show 32-64-fold enhancement in antitubercular activity upon copper complexation.

The crystal structure of first copper(II) complex of a pyridine-2-carboxamidrazone – a potential antitumor agent

Abstract The crystal structure of the neutral, distorted square planar copper(II) complex, viz. cis-[dichloro (N1-(2-benzyloxybenzylidene) pyridine-2-carboxamidrazone) copper(II)] (1) is reported which is the first well-characterized copper(II) complex of the series (space group C2/c; a=18.414(5) A ; b=8.6510(10) A ; c=27.091(4) A ; α=90°; β=109.800(17)°; γ=90°; R1=0.0331 wR2=0.0852). The distortion from square planar geometry is thought to arise from the non-equivalent ligand donor set. The Cu–Cl bonds are equivalent with one of the Cl atoms making intermolecular contact at 2.6070 A with adjacent copper rendering a pseudo-square pyramidal geometry around copper. The complex is further characterized by UV/VIS spectroscopy, electrochemistry and magnetic susceptibility measurements. The in vitro antitumor activity of this complex and the parent ligand is determined against the human breast cancer cell-line MCF-7 which revealed that copper complexation renders a highly antiproliferative compound with IC50 value of 3 μM.

Selectivity of response in fluorescent polymers imprinted with N1-benzylidene pyridine-2-carboxamidrazones

Abstract Fluorescent polymers imprinted with various N 1 -benzylidene pyridine-2-carboxamidrazones were evaluated for their recognition of the original template and cross-reactivity to similar molecules. Dramatic quenching of fluorescence approaching background levels was observed for most cases where the “empty” MIP was re-exposed to its template. Molecules too large to enter the imprinted cavities gave no reduction of fluorescence. Other compounds were found to quench the fluorescence and are assumed to have entered the imprinted cavities. There is also evidence for partial responses which may give some measure of partial binding. The fluorescence response profiles of substrates containing polycyclic aromatics were found to be quite different from those containing flexible substituents. In order to make this approach more suitable for high-throughput screening a method has been validated wherein the extent of substrate-induced fluorescence quenching may be obtained without having to know how much polymer is present.

Structure–activity relationships for α‐calcitonin gene‐related peptide

Calcitonin gene‐related peptide (CGRP) is a member of the calcitonin (CT) family of peptides. It is a widely distributed neuropeptide implicated in conditions such as neurogenic inflammation. With other members of the CT family, it shares an N‐terminal disulphide‐bonded ring which is essential for biological activity, an area of potential α‐helix, and a C‐terminal amide. CGRP binds to the calcitonin receptor‐like receptor (CLR) in complex with receptor activity‐modifying protein 1 (RAMP1), a member of the family B (or secretin‐like) GPCRs. It can also activate other CLR or calcitonin‐receptor/RAMP complexes. This 37 amino acid peptide comprises the N‐terminal ring that is required for receptor activation (residues 1–7); an α‐helix (residues 8–18), a region incorporating a β‐bend (residues 19–26) and the C‐terminal portion (residues 27–37), that is characterized by bends between residues 28–30 and 33–34. A few residues have been identified that seem to make major contributions to receptor binding and activation, with a larger number contributing either to minor interactions (which collectively may be significant), or to maintaining the conformation of the bound peptide. It is not clear if CGRP follows the pattern of other family B GPCRs in binding largely as an α‐helix.

Synthesis and characterization of copper(II) complexes of pyridine-2-carboxamidrazones as potent antimalarial agents

Copper(II) complexes of some pyridine-2-carboxamidrazones have been prepared and characterized. The crystal structures of the copper complex cis-[dichloro(N1-2-acetylthiophene-pyridine-2-carboxamidrazone) copper(II)] 8a and one of the free ligands, viz. {(p-chloro-2-thioloxy-benzylidine-pyridine-2-carboxamidrazone)} 6, have been determined. The former shows a highly distorted square planar geometry around copper, with weak intermolecular coordination from the thiophenyl sulfur resulting in a stacking arrangement in the crystal lattice. The in vitro activities of the synthesized compounds against the malarial parasite Plasmodium falciparum are reported for the first time, which clearly shows the advantage of copper complexation and the requirement of four coordinate geometry around copper as some of the key structural features for designing such metal-based antimalarials.