Biomedical subjects
Publications and source records attributed to M Quibell.
Prototype irreversible inhibitors of the cysteinyl protease Der p I were designed, synthesised and evaluated in vitro. Candidates were designed using a modular approach, whereby a peptide sequence was appended with known thiophilic moieties. This hinged on utilizing peptide sequences from substrate specificity data compiled using proprietary RAPiD technology.
Malignant melanoma is increasing in incidence, and, though early lesions are readily treatable, systemic therapy for metastatic disease remains disappointing. Integrins are a family of cell-surface molecules that mediate adhesion between the cell and the extracellular matrix. One member of the integrin family, the alpha v beta 3 integrin, is associated with progression of melanomas, in that the most malignant cells express the highest levels of alpha v beta 3. Like many members of the integrin family, alpha v beta 3 recognizes the sequence Arg-Gly-Asp (RGD) in its ligands, and other molecules that contain this sequence will complete with the natural ligands (such as vitronectin) for binding. There is growing evidence that integrins function as receptors for signal transduction, and that integrin-mediated signalling can affect cell behaviour and even cell survival. Under certain circumstances, loss of integrin-mediated signalling will induce apoptosis, or programmed cell death, and we have demonstrated that melanoma cells treated with a cyclic peptide with high affinity for the alpha v beta 3 integrin will undergo apoptosis within three days. This mechanism might be exploited therapeutically.
Two monoclonal antibodies, AXO 49 and TAP 952, probed with carboxy-terminal peptides from Paramecium axonemal tubulin and with polyglycylated synthetic peptides, are found to recognize differently tubulin polyglycylation, the most recently identified posttranslational modification discovered in Paramecium axonemal tubulin. With these antibodies, we show that tubulin polyglycylation is widely distributed in organisms ranging from ciliated protozoa to mammals; it arose early in the course of evolution, but seems to be absent in primitive protozoa such as the Euglenozoa. Tubulin polyglycylation is the last posttranslational modification which takes place in the course of Drosophila spermatogenesis and its occurrence corresponds to the end of spermatozoan maturation. An involvement of polyglycylated tubulin in axoneme motility is suggested since AXO 49 and TAP 952 specifically inhibit the reactivated motility of sea urchin spermatozoa.
Case studies of the solid-phase synthesis of some 'difficult sequences' using continuous-flow Fmoc-polyamide techniques are presented. Solvent change and peptide side chain and backbone modification procedures recently found to reduce association effects are applied, and the results compared to syntheses under standard conditions.
The solid (gel)-phase peptide synthesis of peptides, each containing an azaglutamine residue, has been examined. Procedures using various mono-, di- and tripeptide and carbazate fragments containing or relating to an azaglutamine (1) residue have been evaluated. N-Activation of the amino-terminus of a resin-bound peptide with bis-(2,4-dinitrophenyl)carbonate (2) yielded the terminal isocyanate species, which reacted with protected carbazates to give resin-bound protected peptides containing the aza-residue. By contrast, coupling of activated amino-acid derivates to the free amino-group of a resin-bound peptide with an aza-residue at the N-terminus was a slow and unsatisfactory process. It is concluded that the route yielding the best results involves the reaction of a protected amino-acyl carbazate to a resin-bound isocyanate-activated peptide.
2-Hydroxy-4-methoxybenzyl-amino acid residues inhibit interchain association in solid phase peptide synthesis. They are easily introduced through their N,O-bisFmoc derivatives. Preparation of a range of these derivatives is described.
A scheme combining the preparation of building blocks in solution followed by solid-phase combinatorial chemistry has been developed to side-chain diversify 5-(hydroxymethyl)oxazole scaffold (1) into aryl ethers, thioethers, sulfones, sulfonamides, and carboxamides. Protected heterocyclic scaffolds 2 were linked to the solid phase and N-terminal derivatized using active ester chemistry, providing chemset 4¿1-4,1-4¿. The free side-chain hydroxyl of 4 was smoothly converted to aryl ethers 6 under Mitsunobu conditions, with a broad range of substituted phenols. Alternatively, quantitative conversion of hydroxyl to bromide followed by displacement with alkyl and aryl thiols gave thioethers 8. Thioethers were optionally oxidized to sulfones 9. Bromide displacement by azide, followed by reduction to amine and acylation with a range of carboxylic acids and sulfonyl chlorides gave carboxamides 11 and sulfonamides 13, respectively. Crude purity at typically >90% was observed for each of the five modifications detailed. A series of 20 compounds, exemplifying each modification, was reprepared, purified, and fully characterized.
Deprotection peak profiles have been determined as a measure of internal aggregation during Fmoc-polyamide continuous flow solid phase synthesis. The results have been correlated with amino-acid structure and discussed in terms of minimising aggregation during synthesis.
The synthesis of a 36-residue fragment of HIV integrase by standard Fmoc-polyamide chemistry was unsuccessful (Trt-protected Cys) or difficult (Acm-protected Cys); after 10-12 couplings, deprotection became hindered and a major portion (30%-50%) of the growing peptide chains became inaccessible to reagents. The synthesis was repeated using N-(2-hydroxy-4-methoxybenzyl) (Hmb) backbone protection at two strategic positions in the sequence, and Acm protection of Cys. The synthesis was trouble-free and progressed smoothly to completion. Electrospray mass spectrometric analysis at each step of the cleavage and purification showed that Hmb groups become O-acetylated during treatment of the peptide with acetic anhydride in the presence of tertiary base. The acetylated Hmb was refractory to cleavage with TFA/scavenger mixtures but could be readily removed following deacetylation with piperidine. The target peptide was purified to > 98% purity and an overall yield of 15%.
Analogues of beta-amyloid (32-42) peptide, containing N-(2-hydroxy-4-methoxybenzyl) (Hmb) amide backbone substitutions at various positions have been prepared using fluoren-9-ylmethoxycarbonyl (Fmoc)-polyamide based solid phase peptide synthesis. On-line N alpha-Fmoc deprotection monitoring during assembly exhibited hindered release in the native and beta A(34-42, (Hmb)Gly38) analogue syntheses. No such hindrance was observed during the synthesis of beta A(34-42, (Hmb)Gly37) nor beta A(34-42, (Hmb)Val36). However, the latter contained an exceptionally slow coupling reaction. Cleaved peptides were analysed for solubility in a variety of solvents and insoluble pellets tested for congophilic staining. X-ray analysis of Fmoc (and H-) beta A(34-42) and the corresponding (Hmb)Gly38 analogues as dimethylformamide swollen gels gave very similar structures. Secondary structure prediction and model-building of ordered arrays, compatible with our results, suggest that beta A(34-42) forms a beta-hairpin structure, with the reverse turn at Val36-Gly37-Gly38-Val39 both in solution and on the resin during synthesis.