1. Syntheses of T(N) building blocks Nalpha-(9-fluorenylmethoxycarbonyl)-O-(3,4,6-tri-O-acetyl-2-azido-2-deoxy-alpha-D-galactopyranosyl)-L-serine/L-threonine pentafluorophenyl esters: comparison of protocols and elucidation of side reactions
Mian Liu, Victor G Young Jr, Sachin Lohani, David Live, George Barany Carbohydr Res. 2005 May 23;340(7):1273-85. doi: 10.1016/j.carres.2005.02.029.
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.
2. A 'conovenomic' analysis of the milked venom from the mollusk-hunting cone snail Conus textile--the pharmacological importance of post-translational modifications
Zachary L Bergeron, et al. Peptides. 2013 Nov;49:145-58. doi: 10.1016/j.peptides.2013.09.004. Epub 2013 Sep 18.
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.
3. The synthesis and application of Fmoc-Lys(5-Fam) building blocks
Michal Tokmina-Roszyk, Dorota Tokmina-Roszyk, Gregg B Fields Biopolymers. 2013 Jul;100(4):347-55. doi: 10.1002/bip.22222.
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.
