Chemical syntheses and biological activities of lactam analogues of alpha-conotoxin SI.

Bicyclization represents an effective method for the introduction of conformational constraints into small, biologically important peptides. Several strategies have been developed for the preparation of bicyclic lactam analogues of alpha-conotoxin SI, a 13-residue peptide neurotoxin found in cone snail venom. Four analogues of the natural regioisomer of alpha-conotoxin SI were designed and synthesized, each with one of the two paired cysteines of the parent peptide being replaced by a side-chain lactam bridged glutamic acid/lysine pair. Solid-phase lactamization was studied to determine rates of formation of the two possible loops and to document the extent of dimerization and higher oligomerization. Radioligand binding assays were carried out on all synthesized peptides, including the naturally occurring two-disulfide form, in order to determine their affinities for nicotinic acetylcholine receptors (nAChRs). Replacement of the Cys(2)-Cys(7) loop of alpha-conotoxin SI with a lactam bridge resulted in complete loss of activity, whereas replacement of the Cys(3)-Cys(13) disulfide loop resulted in a approximately 60-fold reduction in affinity for one orientation and a approximately 70-fold increase in affinity for the other. The two active lactam analogues retain the selectivity exhibited by the naturally occurring peptide for the alpha/delta subunit of nAChRs, as judged by competition experiments with the curariform antagonist metocurine.

Photoactive porphyrin derivative with broad-spectrum activity against oral pathogens In vitro.

Photodynamic therapy (PDT) has historically been used as a means to treat cancerous tumors but has recently been used to kill bacterial cells through the use of targeted photosensitizers. PDT is a potential adjunct to scaling and root planing in the treatment of periodontal disease. However, the effectiveness of porphyrin derivatives against microorganisms has been limited because some gram-negative bacteria are refractory to photodynamic treatment with these agents. We have designed a porphyrin derivative conjugated to a pentalysine moeity that endows the molecule with activity against gram-positive and gram-negative bacteria. Whereas the porphyrin, chlorin e6, showed in vitro activity against a limited spectrum of bacteria, chlorin e6 conjugated to pentalysine showed in vitro activity against all oral microorganisms tested, including Porphyromonas gingivalis, Actinobacillus actinomycetemcomitans, Bacteroides forsythus, Campylobacter rectus, Eikenella corrodens, Fusobacterium nucleatum subsp. polymorphum, Actinomyces viscosus, and the streptococci. Potent antimicrobial activity (>/=5-log-unit reduction in the numbers of CFU per milliliter) was retained in the presence of up to 25% whole sheep blood. The use of potent, selective agents such as this chlorin e6-pentalysine conjugate to more effectively reduce the pathogenic bacteria in the periodontal pocket may be a significant tool for the treatment of periodontal disease.

"High-load" polyethylene glycol-polystyrene (PEG-PS) graft supports for solid-phase synthesis.

The choice of a polymeric support is a key factor for the success of solid-phase methods for syntheses of organic compounds and biomolecules such as peptides and oligonucleotides. Classical Merrifield solid-phase peptide synthesis (SPPS), performed on low cross-linked hydrophobic polystyrene (PS) beads, sometimes suffers from sequence-dependent coupling difficulties. The concept of incorporating polyethylene glycol (PEG) into supports for solid-phase synthesis represents a successful approach to alleviating such problems. Previous reports from our laboratories have shown the advantages of "low-load" PEG-PS (0.15-0.25 mmol/g) for SPPS. Herein, we demonstrate that the beneficial aspects of the PEG-PS concept can be extended with resins that have higher loadings (0.3-0.5 mmol/g).

Novel N omega-xanthenyl-protecting groups for asparagine and glutamine, and applications to N alpha-9-fluorenylmethyloxycarbonyl (Fmoc) solid-phase peptide synthesis.

The N alpha-9-fluorenylmethyloxycarbonyl (Fmoc), N omega-9H-xanthen-9-yl (Xan), N omega-2-methoxy-9H-xanthen-9-yl (2-Moxan) or N omega-3-methoxy-9H-xanthen-9-yl (3-Moxan) derivatives of asparagine and glutamine were prepared conveniently by acid-catalyzed reactions of appropriate xanthydrols with Fmoc-Asn-OH and Fmoc-Gln-OH. The Xan and 2-Moxan protected derivatives have been used in Fmoc solid-phase syntheses of several challenging peptides: a modified Riniker's peptide to probe tryptophanalkylation side reactions, Briand's peptide to assess deblocking, at the N-terminus and Marshall's ACP (65-74) to test difficult couplings. Removal of the Asn and Gln side-chain protection occurred concomitantly with release of peptide from the support, under the conditions for acidolytic cleavage of the tris(alkoxy)benzylamide (PAL) anchoring linkage by use of trifluoroacetic acid/scavenger mixtures. For each of the model peptides, the products obtained by the new protection schemes were purer than those obtained with N omega-2,4,6-trimethoxybenzyl (Tmob) or N omega-triphenylmethyl (Trt) protection for Asn and Gln.

Optimized preparation of deca(L-alanyl)-L-valinamide by 9-fluorenylmethyloxycarbonyl (Fmoc) solid-phase synthesis on polyethylene glycol-polystyrene (PEG-PS) graft supports, with 1,8-diazobicyclo [5.4.0]-undec-7-ene (DBU) deprotection.

Deca(L-alanyl)-L-valinamide is known to be a challenging model target for solidphase peptide synthesis, due to its hydrophobicity and its tendency to form secondary structures which inhibit acylation and deprotection. Here we report systematic studies on the synthesis of this peptide on an automated continuous-flow instrument, using the 9-fluorenylmethyloxycarbonyl (Fmoc) group for N alpha-amino protection and a polyethylene-glycol polystyrene (PEGPS) graft support. The optimal deprotection reagent proved to be DBU-piperidine-DMF (1:1:48, vol/vol/vol). The synthetic peptides were analyzed and characterized by high-performance liquid chromatography and several mass spectrometric techniques.