Fmoc-β-HomoCys(Trt)-OH

Cyclodepsipeptides are cyclic peptides in which at least one amide link on the backbone is replaced with an ester link. These natural products present a high structural diversity that corresponds to a broad range of biological activities. Therefore, they are very promising pharmaceutical candidates. Most of the cyclodepsipeptides have been isolated from marine organisms, but they can also originate from terrestrial sources. Within the family of cyclodepsipeptides, 'head-to-side-chain' cyclodepsipeptides have, in addition to the macrocyclic core closed by the ester bond, an arm terminated with a polyketide moiety or a branched amino acid, which makes their synthesis a challenge. This protocol provides guidelines for the synthesis of 'head-to-side-chain cyclodepsipeptides' and includes-as an example-a detailed procedure for preparing pipecolidepsin A. Pipecolidepsin was chosen because it is a very complex 'head-to-side-chain cyclodepsipeptide' of marine origin that shows cytotoxicity in several human cancer cell lines. The procedure begins with the synthesis of the noncommercial protected amino acids (2R,3R,4R)-2-{[(9H-fluoren-9-yl)methoxy]carbonylamino}-3-hydroxy-4,5-dimethylhexanoic acid (Fmoc-AHDMHA-OH), Alloc-pipecolic-OH, (4R,5R)-5-((((9H-fluoren-9-yl)methoxy)carbonylamino)-4-oxo-4-(tritylamino)butyl)-2,2-dimethyl-1,3-dioxolane-4-carboxylic acid (Fmoc-DADHOHA(acetonide, Trt))-OH and the pseudodipeptide (2R,3R,4R)-3-hydroxy-2,4,6-trimethylheptanoic acid ((HTMHA)-D-Asp(OtBu)-OH). It details the assembly of the depsipeptidic skeleton using a fully solid-phase approach (typically on an amino polystyrene resin coupled to 3-(4-hydroxymethylphenoxy)propionic acid (AB linker)), including the key ester formation step. It concludes by describing the macrocyclization step performed on solid phase, and the global deprotection and cleavage of the cyclodepsipeptide from the resin using a trifluoroacetic acid-H2O-triisopropylsilane (TFA-H2O-TIS; 95:2.5:2.5) cocktail, as well as the final purification by semipreparative HPLC. The entire procedure takes ~2 months to complete.

Tritylation using trityl alcohol (Trt-OH) in 1,1,1,3,3,3-hexafluoro-2-propanol (HFIP) is a convenient and efficient procedure that can offer S-protection of the Cys located in fully unprotected peptides. The procedure simply requires Trt-OH and HFIP to selectively promote S-tritylation in the presence of peptide nucleophilic functionalities.

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.