The final partial structure consists of a thiazoline ring with a terminal alkene substituent, as determined by electron ionization mass spectrometry (EI-MS) and 13C NMR.

[4] The chemical shifts of ring carbons adjacent to the sulfur and nitrogen heteroatoms were compared to 13C NMR data from model compounds.

[8] With these partial structures established, their connectivity was evaluated via HMBC spectroscopy,[4] a 2D NMR technique which allows for the determination of heteronuclear J-coupling values for nonadjacent carbons and protons.

These methods use NMR to evaluate the spin-spin coupling constants which directly relate to the dihedral angle of the atoms being analyzed, allowing for the determination of chirality.

Because C7 and C8 are adjacent stereocenters, these techniques allowed for immediate determination of their relative stereochemistry, however C10 is separated from C8 by C9, which carries two diastereotopic protons.

[6] Stereochemistry at C3 was determined by Marfey's analysis, wherein the compound was ozonized and subsequently hydrolyzed to obtain cysteic acid from the thiazoline ring and attached terminal alkene.

[9] Kalkitoxin analogs lacking the complete thiazoline ring exhibit on the order of 1000-fold decreased toxicity to solid tumor cell lines.

[7] Furthermore, the removal of the C10 methyl group has a smaller impact on potency than does epimerization of C7, supporting the trend of decreased SAR correlation at core chiral centers on the aliphatic chain.

This group is lost in asymmetric conjugate addition of an (R)-amino alcohol, which, through two cyclodehydration steps using Wipf's oxazoline-thiazoline interconversion protocol, produces the thiazoline ring.

This method is advantageous because it allows for stereoselectivity of the resulting 1,3-dimethyl configuration during the larger sequential introduction of the methyl substituents at the C7, C8 and C10 chiral centers.

This group was separated by one carbon from C7 in the first total synthesis, so the keto-auxiliary moiety could be converted to a carboxylic acid, in anticipation of addition of the amino alcohol immediately thereafter.

[6] In this synthesis, this keto-auxiliary group is directly adjacent to C7, necessitating a one carbon homologation, before construction of the thiazoline ring.

This was achieved through reductive bond cleavage of the auxiliary group to a primary alcohol and oxidation to the corresponding aldehyde, Wittig reaction using an ylide carrying a methoxy group to produce an enol ether, hydrolysis to the aldehyde and finally oxidation to produce the carboxylic acid.

Furthermore, this avoids repetitive interconversion and purification steps normally required for repeat chain extensions, which increases yield and efficiency and decreases labor.

[12] Published on Nature : Burns, M., Essafi, S., Bame, J. et al. Assembly-line synthesis of organic molecules with tailored shapes.

Kalkitoxin is ichthyotoxic to goldfish (Carassius auratus, LC50: 700nM) and to aquatic crustacean brine shrimp (Artemia salina, LC50: 150-180nM [7]).

[2] Many efforts to discover cancer therapeutic drugs focus on the screening of novel biomolecules produced and isolated from various plants and animals.

Kalkitoxin was originally isolated from Lyngbya majuscula as an effort to collect new molecules for testing as antitumor or antifungal agents.

At concentrations comparable to those required for tumor-selective cytotoxicity, kalkitoxin induces cell death when applied to rat cerebellar granule neurons (CGN) in culture.

[2] Kalkitoxin acts as an N-methyl-D-aspartate (NMDA) receptor agonist, and induces cytotoxicity in cultured rat CGNs at delayed time points.