N-α-(9-Fluorenylmethoxycarbonyl)-2,4-dinitro-D-phenylalanine

T(N) antigen building blocks Nalpha-(9-fluorenylmethoxycarbonyl)-O-(3,4,6-tri-O-acetyl-2-azido-2-deoxy-alpha-D-galactopyranosyl)-L-serine/L-threonine pentafluorophenyl ester [Fmoc-L-Ser/L-Thr(Ac3-alpha-D-GalN3)-OPfp, 13/14] have been synthesized by two different routes, which have been compared. Overall isolated yields [three or four chemical steps, and minimal intermediary purification steps] of enantiopure 13 and 14 were 5-18% and 6-10%, respectively, based on 3,4,6-tri-O-acetyl-D-galactal (1). A byproduct of the initial azidonitration reaction of the synthetic sequence, that is, N-acetyl-3,4,6-tri-O-acetyl-2-azido-2-deoxy-alpha-D-galactopyranosylamine (5), has been characterized by X-ray crystallography, and shown by 1H NMR spectroscopy to form complexes with lithium bromide, lithium iodide, or sodium iodide in acetonitrile-d3. Intermediates 3,4,6-tri-O-acetyl-2-azido-2-deoxy-alpha-D-galactopyranosyl bromide (6) and 3,4,6-tri-O-acetyl-2-azido-2-deoxy-beta-D-galactopyranosyl chloride (7) were used to glycosylate Nalpha-(9-fluorenylmethoxycarbonyl)-L-serine/L-threonine pentafluorophenyl esters [Fmoc-L-Ser/L-Thr-OPfp, 11/12]. Previously undescribed low-level dehydration side reactions were observed at this stage; the unwanted byproducts were easily removed by column chromatography.

Fluorescence resonance energy transfer (FRET) peptide substrates are often utilized for protease activity assays. This study has examined the preparation of FRET triple-helical peptide (THP) substrates using 5-carboxyfluorescein (5-Fam) as the fluorophore and 4,4-dimethylamino-azobenzene-4'-carboxylic acid (Dabcyl) as the quencher. The N(α)-(9-fluorenylmethoxycarbonyl)-N(ε)-(5-carboxyfluorescein)-L-lysine [Fmoc-Lys(5-Fam)] building block was synthesized utilizing two distinct synthetic routes. The first involved copper complexation of Lys while the second utilized Fmoc-Lys with microwave irradiation. Both approaches allowed convenient production of a very pure final product at a reasonable cost. Fmoc-Lys(5-Fam) and Fmoc-Lys(Dabcyl) were incorporated into the sequence of a THP substrate utilizing automated solid-phase peptide synthesis protocols. A second substrate was assembled where (7-methoxycoumarin-4-yl)-acetyl (Mca) was the fluorophore and 2,4-dinitrophenyl (Dnp) was the quencher. Circular dichroism spectroscopy was used to determine the influence of the fluorophore/quencher pair on the stability of the triple-helix. The activity of the two substrates was examined with three matrix metalloproteinases (MMPs), MMP-1, MMP-13, and MT1-MMP. The combination of 5-Fam as fluorophore and Dabcyl as quencher resulted in a triple-helical substrate that, compared with the fluorophore/quencher pair of Mca/Dnp, had a slightly destabilized triple-helix but was hydrolyzed more rapidly by MMP-1 and MMP-13 and had greater sensitivity.

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.