MALDI Mass Spectra of Authentic Sulfenate-linked Benzyl Carbamate Dimer

MASS SPECTRAL STRUCTURAL PROOF? JUDGE FOR YOURSELF!

UNM and its licensee through the testimony of Drs. Wallace and Murray have alleged that the authentic Sulfenate-Linked Benzyl Carbamate Dimer as originally prepared by Drs. Knight and Scallen are taurine derivatives, or essentially sulfonates. However, there is little, if any, scientific evidence that the authentic compounds are sulfonates. Drs. Murray’s and Wallace’s attempts to make the authentic compounds when allegedly using their corruption of the authentic procedures, failed to yield “taurine derivatives” in any appreciable amount. The true identity of the authentic compounds is graphically illustrated by the following careful analyses of MALDI mass spectra of authentic sulfenate-linked benzyl carbamate dimer (SLBCD), as obtained from Los Alamos National Laboratories and Bruker Instruments, respectively. Although these are not high resolution spectra, the LANL data being ± 1, the fairly good resolution of the Bruker instrument makes the true identity of the SLBCD obvious:

Fragmentation patterns of the authentic sulfenate-linked benzyl carbamate dimer, especially the calcium salt that is resistant to loss of the counterions in the high vacuum of the mass spectrometer, indicate several signature, mass ion fragments, following, such as 548.144 (547+1) and 563.116, the former being observed at Los Alamos (supra) and the latter being observed at Bruker whether KI is present in the analyses or not:

Another key, definitive mass ion was observed at Los Alamos National Laboratories. Despite allegations of UNM’s attorneys that the dihydrate of the sulfenate-linked benzyl carbamate dimer cannot exist, the structure of the authentic dihydrate is actually stabilized by hydrogen bonds and is observed as its mass ion in in the LANL spectrum as a finely split peak at about 726-728:

POTASSIUM IODIDE AS SHIFT REAGENT

Potassium iodide is an extremely valuable shift reagent for analyzing MALDI (Matrix Assisted Laser Desorption and Ionization) mass spectra of sulfenates, sulfinates, and sulfonates. Note that in KI's presence, another signature mass is observed at 777.712 for an authentic preparation that was contaminated with HCl, oxidized by ICl to the bis-sulfinate-linked artifact, and then reacted with iodide to form a characteristic adduct in the MALDI mass spectrometer.

KI's presence in the MALDI analyses also causes reduction of sulfenates and sulfenate-linked compounds to thiols. It has long been known that sulfenates react with iodide to form sulfenyl iodides , which are then reduced to thiol compounds by ultraviolet light, such as that emitted by the UV laser of the MALDI mass spectrometer. (Thiols and unreacted sulfenates or sulfenyl iodides form disulfides.) Thus, sulfenates and sulfenate-linked compounds tend to be reduced by iodide to smaller sulfhydryl products in the MALDI mass spectrometer, whereas sulfinates and sulfinate-linked compounds tend to form large complexes with iodide that apparently do NOT reduce into the smaller thiol compounds. A second signature peak for the artifactually oxidized authentic compound is observed at 739.622 when KI is present under these acidic conditions:

Presumably, sulfonates and sulfonate-linked compounds, due to their high oxidation states, are even more resistant to reduction to thiols under these conditions, and may even be resistant to adduct formation with iodide. This last suspicion remains to be tested, since there is absolutely no evidence for sulfonates or sulfonate-linked compounds in the authentic preparations, even when the authentic synthetic process using iodine was contaminated with HCl (ICl) to control a pH overshoot. It is highly unlikely that iodide and UV light could completely reduce a sulfonate to a thiol in the split second between its desorption with a UV laser and its analysis by the detector of the mass spectrometer, especially when even the sulfinate-linked, -S(O)-O-, compounds appear resistant to such reduction. Full reduction of the sulfonic acid to sulfide or thiol would require the highly unlikely chance meeting of the sulfonic acid with several iodide ions in the UV-illuminated flight path of the mass spectrometer.

Having chloride ions from HCl present in the iodine reaction constitutes a serious departure from the authentic procedure, since the iodine monochloride ( ICl ) so generated is known to be extremely reactive, and capable of artifactually oxidizing sulfur compounds to higher oxidation states. Similar polarity problems exist for reactive mixtures of bromine and chloride .

Additional proof that the authentic sulfenate-linked benzyl carbamate dimer, -S-O-, is not the bis-sulfinate-linked structure, -S(O)-O-, is provided by mass ion adducts that are intermediary between the two, especially in preparations known to be contaminated with HCl. While a mechanism can be drawn for the sulfenate-linkage being reduced by iodide, presumably the sulfinate-linkage is less likely to be reduced to thiol and, therefore, more likely to form a stable adduct with iodide, the doubly charged mass ion of which is observed in the MALDI mass spectrometer when KI is present:

Some of the more compelling evidence that the authentic compound is sulfenate-linked, and NOT the alleged sulfonates or taurine derivatives, stems from the fragmentation of the HCl-contaminated preparation to the free sulfenates of vitaletheine, presumably as derived from the substantial amount of authentic sulfenate-linked compound remaining in this preparation that was only partially artifactually oxidized:

Still, the most compelling evidence for the authentic sulfenate-linked structure is the actual appearance, upon reduction, of mass ions for the sulfide or thiol forms, vitaletheine. These reduced forms are among the more abundant masses in the spectrum of the authentic, albeit somewhat artifactually oxidized preparation, but only when analyzed in the presence of KI. The prevalence of the mass ions observed at 231.25 and 193.19, and the somewhat surprising lack of the smaller mass ions expected for beta-aletheine and its adducts, indicate that the authentic structure is as assigned by the true inventors, i.e., the sulfenate-linked benzyl carbamate dimer.

This authentic sulfenate-linked structure is the only one that can account for the many definitive mass ions observed in the authentic and partially oxidized preparations. Similar analyses of pristine, pharmaceutical grade material, prepared by Dr. Knight, is expected to confirm these conclusions, the oxidative artifacts being expected to drop out of the MALDI mass spectrum of this pristine material, as the sulfenate and thiol mass ions and adducts of vitaletheine increase in relative abundance when analyzed in the presence of KI. The following table summarizes the compelling mass spectral evidence for the authentic sulfenate-linked benzyl carbamate dimer, and for oxidative artifacts generated, therefrom, when the otherwise authentic preparation was contaminated with chloride ions:

*Asterisk indicates good to excellent agreement of observed values with calculated ones.

UNM’s licensee and Dr. Murray, and UNM, for some reason, seem doggedly fixated upon the taurine derivatives when there is absolutely NO discriminating scientific evidence that these were the compounds used by Drs. Knight and Scallen to perform the enabling biological experiments with the authentic vitaletheine modulators. Scientists at Los Alamos's mass spectra group have stated “(sic., Some) Scientists lie. Mass spectra tells the truth. ” Furthermore, Dr. Knight has been presented with NO credible evidence that decarboxylated taurine derivatives have any biological activity, whereas the authentic compounds were phenomenally active. As illustrated in the following table, even when Dr. Murray allegedly used the authentic procedure, he did NOT make product having any of the above mass spectral signatures observed by the true inventors for the authentic compounds. There is absolutely NO mass spectral NOR any IR evidence for the alleged sulfonates in the authentic preparations, even when artifactually oxidized by exposing them to contaminating HCl, and there is virtually NO evidence for the sulfonate mass at 723 in Dr. Murray’s alleged attempt to make authentic compound when using his corrupted version of the authentic procedure:

**Attempts by Dr. Murray allegedly using UNM’s method produced an oxidized artifact of the authentic vitaletheine modulator (755.89), but this product from Hauser Chemical clearly lacks the mass spectra fingerprint of the authentic compound. His attempt produced only tiny traces of the 723 mass expected for the alleged sulfonates. The artifactually oxidized authentic preparation, contaminated with HCl and discussed previously, apparently did not fully reduce, and instead produced some large, artifactually oxidized adducts with iodide. Similar resistance to KI/UV reduction is expected for the sulfonates alleged by Drs. Wallace and Murray. In contrast, the unoxidized SLBCD remaining in the partially oxidized preparation appears to be readily reduced by KI and the MALDI's UV laser to vitaletheine and other small fragments and adducts.
One possible explanation for Dr. Murray’s artifact, produced through his admitted departures from the authentic procedures, including much longer reaction times with iodine, is the much higher oxidation state at 755.621 than observed for the authentic compound. The calculated value, following, is in good agreement with Dr. Murray’s observed mass ion adduct at 755.89, the 723 mass for the alleged sulfonate being much smaller and virtually absent from the mass spectrum of Dr. Murray’s alleged attempt:

The striking fit of the many chemically defined masses for the authentic compound and its oxidized artifacts (previous analyses, supra, and when contaminated with HCl) is in stark contrast to the products generated by the significant departures from the authentic procedures in Dr. Murray's alleged attempts. Dr. Knight could deduce few, if any, mass ions for the alleged CBZ-beta-alanyl-taurines (sulfonates), or iodine adducts thereof, following, that could substitute for the fit observed with the authentic assignments:

This failure of the alleged sulfonates to fit the observed data for the authentic compounds is further illustrated by calculations performed for the alleged sulfonates, the UV laser of the MALDI instument conceivably capable of removing the benzyl groups from the alleged sulfonates, but NOT capable of reducing the sulfonates to thiols, especially in the absence of iodide. As before, the following table provides few, if any, alternative "sulfonic" assignments that provide better "fits" than those graphically illustrated, supra, for the authentic, and for the somewhat artifactually oxidized preparations:

In conclusion, UNM, its licensee, and their so-called "experts" appear to be simply wrong about the structure of the authentic sulfenate-linked benzyl carbamate dimer. The masses assigned in the first table, supra, for the authentic sulfenate-linked benzyl carbamate dimer, and for oxidative artifacts thereof, appear to produce an accurate "mass signature" for the authentic sulfenate-linked compound. This hasn’t stopped UNM and its licensee from promoting the alleged sulfonate or taurine derivatives for human use, as if they were the same as the authentic compounds, and apparently without much testing of these alleged sulfonate and taurine derivatives in animals for biological efficacy and safety.

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