N-α-(9-Fluorenylmethoxycarbonyl)-DL-α-aminolauric acid

We describe an unprecedented solid phase peptide synthesis (SPPS) of short peptide-based multimetal tags designated as elemental tags for the quantification of biomolecules via inductively coupled plasma mass spectrometry (ICP-MS). The macrocyclic chelator 1,4,7,10-tetraazacyclododecane N,N',N″,N‴-tetra acetic acid (DOTA) was attached to the side chain of N-α-(9-fluorenylmethoxycarbonyl)-l-lysine (Fmoc-Lys-OH) and metalated with a lanthanide to provide a building block for Fmoc-based SPPS. Thereby, in contrast to existing strategies for the synthesis of DOTA-peptide conjugates, an already metalated DOTA-amino acid was used as a building block for SPPS. The DOTA-lanthanide complex was stable throughout the whole SPPS, even during the final cleavage in concentrated trifluoroacetic acid. This indicates that the strategy to first metalate the Fmoc-Lys(DOTA)-OH and to utilize the metal coordination to protect the carboxyl groups of DOTA offers an alternative to conventional synthetic routes using tert-butyl protected DOTA. Several small peptides containing up to four metal ions were synthesized, among them peptides carrying defined metal sequences consisting of two different lanthanides. The peptides were N-terminally maleimide-functionalized, thus introducing a moiety for conjugation to thiol-bearing biomolecules. The final objective of this work was the signal enhancement in ICP-MS-based DNA quantification assays. To evaluate the performance of the multimetal peptide tags in assay, they were applied to label thiol-modified 15mer DNA oligonucleotide probes. These served as reporter probes in a model sandwich-type hybridization assay. Thereby, we found that the ICP-MS signal increased linearly with the number of lanthanide ions attached to the reporter probe.

Cone snail venoms provide a largely untapped source of novel peptide drug leads. To enhance the discovery phase, a detailed comparative proteomic analysis was undertaken on milked venom from the mollusk-hunting cone snail, Conus textile, from three different geographic locations (Hawai'i, American Samoa and Australia's Great Barrier Reef). A novel milked venom conopeptide rich in post-translational modifications was discovered, characterized and named α-conotoxin TxIC. We assign this conopeptide to the 4/7 α-conotoxin family based on the peptide's sequence homology and cDNA pre-propeptide alignment. Pharmacologically, α-conotoxin TxIC demonstrates minimal activity on human acetylcholine receptor models (100 μM, <5% inhibition), compared to its high paralytic potency in invertebrates, PD50 = 34.2 nMol kg(-1). The non-post-translationally modified form, [Pro](2,8)[Glu](16)α-conotoxin TxIC, demonstrates differential selectivity for the α3β2 isoform of the nicotinic acetylcholine receptor with maximal inhibition of 96% and an observed IC50 of 5.4 ± 0.5 μM. Interestingly its comparative PD50 (3.6 μMol kg(-1)) in invertebrates was ~100 fold more than that of the native peptide. Differentiating α-conotoxin TxIC from other α-conotoxins is the high degree of post-translational modification (44% of residues). This includes the incorporation of γ-carboxyglutamic acid, two moieties of 4-trans hydroxyproline, two disulfide bond linkages, and C-terminal amidation. These findings expand upon the known chemical diversity of α-conotoxins and illustrate a potential driver of toxin phyla-selectivity within Conus.

Native chemical ligation has proven to be a powerful method for the synthesis of small proteins and the semisynthesis of larger ones. The essential synthetic intermediates, which are C-terminal peptide thioesters, cannot survive the repetitive piperidine deprotection steps of N(alpha)-9-fluorenylmethoxycarbonyl (Fmoc) chemistry. Therefore, peptide scientists who prefer to not use N(alpha)-t-butyloxycarbonyl (Boc) chemistry need to adopt more esoteric strategies and tactics in order to integrate ligation approaches with Fmoc chemistry. In the present work, side-chain and backbone anchoring strategies have been used to prepare the required suitably (partially) protected and/or activated peptide intermediates spanning the length of bovine pancreatic trypsin inhibitor (BPTI). Three separate strategies for managing the critical N-terminal cysteine residue have been developed: (i) incorporation of N(alpha)-9-fluorenylmethoxycarbonyl-S-(N-methyl-N-phenylcarbamoyl)sulfenylcysteine [Fmoc-Cys(Snm)-OH], allowing creation of an otherwise fully protected resin-bound intermediate with N-terminal free Cys; (ii) incorporation of N(alpha)-9-fluorenylmethoxycarbonyl-S-triphenylmethylcysteine [Fmoc-Cys(Trt)-OH], generating a stable Fmoc-Cys(H)-peptide upon acidolytic cleavage; and (iii) incorporation of N(alpha)-t-butyloxycarbonyl-S-fluorenylmethylcysteine [Boc-Cys(Fm)-OH], generating a stable H-Cys(Fm)-peptide upon cleavage. In separate stages of these strategies, thioesters are established at the C-termini by selective deprotection and coupling steps carried out while peptides remain bound to the supports. Pilot native chemical ligations were pursued directly on-resin, as well as in solution after cleavage/purification.