General Considerations and Problems with Interpretation

Hydrogen bromides have been used extensively to completely remove blocking groups such as the carbobenzoxy (CBZ) moiety from amines. Consequently, contamination of this particular hydrogen halide can also effectively disrupt attempts to synthesize several of the exemplary vitaletheine modulators, especially those with free carbamate/carbonimidate moieties. Hydrogen halides can be artifactually introduced by i) using the hydrogen halide salts of cystamine instead of subliming the free base as outlined in the synthesis of bis-CBZ-ß-alethine, ii) by using impure reagents in equilibrating the ion-exhange columns, or iii) through an incomplete chromatographic removal of HBr from ß-alethine by this chromatographic technique prior to synthesizing vitalethine, most commonly caused by applying too large of a sample volume to the column. In addition, acetic acid, halides, and added amines such as triethylamine may alter the products of the reaction mixtures and decrease the efficiency of bromide removal in the DEAE ion exchange chromatography step. Since ammonium carbamate appears to be less stable to thermal decomposition than other carbamate salts, there also is some evidence that incomplete phosgenation of the free amines of ß-alethine may lead to catalytic hydrolysis of any carbamates and carbonimidates that may be formed.

The vitaletheine modulators also tend to rearrange in different organic solvents, so organic reference standards were generally avoided in the initial NMR spectra of these compounds to ensure sample integrity. For example, triturating one of the vitaletheine modulators with ethyl ether caused obvious and complete rearrangement into two other products having radically different chemical properties and solubilities. Unfortunately, these precautions of generally avoiding unproven organic exposures in the interest of sample integrity are not without consequence in the accurate scaling of the data. For example, because of the indicated tenacious binding of water to vitaletheine V4, this proton spectra in DMSO was not shifted 0.18 ppm downfield when originally reported, even though an argument for doing so could have been made using the signal in this original spectra that might be contaminating DMSO; water's proton NMR shift is too inconsistent in the presence of hydrogen-bonding solvents and solutes to be definitive in confirming these assignments for the vitaletheine modulators. The legitimacy for making such a correction has since been established in a repeat of this synthesis by others using TMS as a spectral reference standard for proton NMR. Similarly, in the original report proton assignments for the paired methylenes in the cysteamine and ß-alethine moieties may have been inverted from what they actually are. These assignments were originally based upon changes observed when ß-alethine is reduced to its thiol. More recent 2-D NMR indicates that assignments for the two sets of coupled methylenes are juxtaposed, possible discrepancies not affecting the chemical identity of the substance. These discrepancies in assignment resulted from the then unappreciated effects of hydrogen bonding in these structures.

There are also indications that several of the vitaletheine modulators rearrange when reduced to their thiol forms, so this reductive probe of structure must be subjected to considerable scrutiny before definitive assignments can be made. These rearrangements are most likely facilitated by five- and six-membered nucleophilic attack of the thiolate upon carbonyl carbons or the various amides, imidates, carbamates, and carbonimidates present in this series of compounds. Consequently, thiol reagents may or may not detect these compounds when reduced to thiols. Careful chemists are still studying these assignments with shift reagents and other probes of structure, including 2-D NMR.

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