Mieczysław Sajewicz

Experimental and Model Investigation of the Oscillatory Transenantiomerization of L-α-Phenylalanine

Abstract In an earlier study, we obtained experimental evidence of the oscillatory transenantiomerization of selected profen drugs (e.g., S-(+)-ibuprofen, S-(+)-naproxen, and S-(+) and R-(−)-flurbiprofen) dissolved in aqueous, aqueous-organic, and purely organic liquid media. This process was apparently catalyzed by basic or amphiprotic environments and involved keto-enol tautomerism, and the self-organization of molecules in the solution via association of the carboxylic functional group of profens through hydrogen bonding to form mixed H-bonded associates with the remaining constituents of the solution. A model of the oscillatory transenantiomerization of profens was also developed by adapting an earlier oscillatory model, the Templator. Our new model comprises two linked Templators. The essence of the Templator model adapted to the oscillatory transenantiomerization of profens is the assumption that the H-bonded profen homodimer acts as a template, able to generate new dimers having the same steric configuration as their respective monomeric units. As profens belong to the class of 2-arylpropionic acids (2-APAs), we concluded that the phenomenon of oscillatory transenantiomerization may occur in other 2-APAs as well, among them those amino acids whose molecular structure can formally be derived from propionic acid. Thus, in this study, we focus our attention on L-α-phenylalanine (LPA; one of the nine amino acids essential for humans). Using thin layer chromatography (TLC) and polarimetry, we demonstrate the ability of LPA to undergo oscillatory transenantiomerization analogous to that observed with profens. The self-organization of molecules in a 70% ethanol solution of LPA is confirmed with photographs taken in UV light (λ = 254 nm). Finally, we propose a skeleton molecular mechanism for the transenantiomerization of LPA and simulate the oscillatory interconversion of its L and D forms with two linked Templators.

Enantioseparation and Oscillatory Transenantiomerization of S,R‐(±)‐Ketoprofen, as Investigated by Means of Thin Layer Chromatography with Densitometric Detection

Abstract In the course of our earlier and rather extensive investigations, we discovered a striking ability of the two non‐steroidal anti‐inflammatory drugs (NSAIDs) from the group of 2‐arylpropionic acids (2‐APAs), i.e., ibuprofen and naproxen, and also of 2‐phenylpropionic acid (which is not a drug) to, in vitro, undergo a repeated structural conversion from one chiral configuration to the opposite one. We labelled the discovered phenomenon ‘oscillatory transenantiomerization’ and formulated a hypothesis that all the 2‐APAs can behave in a similar manner when dissolved in certain low‐molecular‐weight (aqueous or non‐aqueous) solvents. Furthermore, we assumed that structural differences among the various 2‐APAs can result in differentiated dynamics of oscillatory transenantiomerization, which is omnipresent with this class of compounds. In this paper,we present the results of an analogous study, this time devoted to still another 2‐APA, S,R‐(±)‐ketoprofen. The chemical structure of ketoprofen is, in a sense, unique as a keto‐enol tautomer (a transition form between S‐(+)‐ketoprofen and its R‐(‐) antimer) that contains a long assembly of eight conjugated π‐electron pairs. Such a peculiar electron structure seems to particularly well stabilize the keto‐enol tautomer derived from ketoprofen and, consequently, to efficiently promote oscillatory transenantiomerization of this compound. The results of our investigations fully confirmed this intuition, and in our thin‐layer chromatographic experiment, we managed to demonstrate the ability of S,R‐(±)‐ketoprofen to undergo oscillatory transenantiomerization, in a manner similar to that of the other already examined 2‐APAs. Moreover, we performed a successful densitometric scrutiny of the chromatograms of S,R‐(±)‐ketoprofen, proved to have separated three different species, to run, in situ, their respective UV spectra (all three of them practically identical), and to speculate about their chemical nature. In the HPLC experiment we produced evidence of a considerable viscosity of S,R‐(±)‐ketoprofen when chromatographed with pure acetonitrile and with the acetonitrile+water mixtures (containing very low amounts of water). This viscosity seems to be the main factor that contributes to the oscillatory (i.e., repeated) nature of the observed transenantiomerization.

TLC in a search for structural limitations of spontaneous oscillatory in-vitro chiral conversion. α-hydroxybutyric and mandelic acids

As a result of our earlier studies, we were the first research group to report the spontaneous oscillatory in-vitro chiral conversion of a considerable number of α-substituted propionic acids (i.e., selected profens, amino acids, and l-lactic acid). It is noteworthy that TLC proved highly instrumental in collecting relevant confirmatory evidence on this issue. Besides, in two papers we introduced a theoretical model and with its aid, we could simulate basic physical and physicochemical features of the discussed process. Reflecting on the oscillatory chiral conversion, we realized that it seems to be a rather general phenomenon and we started contemplating its scope and limitations. It became our primary concern to check whether it is confined to a-substituted propionic acids only or can occur with other classes of chiral compounds also. To this effect, in this study we present empirical (thin-layer chromatographic and polarimetric) evidence on an ability of R and S-α-hydroxybutyric acids, and R and S-mandelic acids to undergo spontaneous oscillatory in-vitro chiral conversion when dissolved in 70% aqueous ethanol. From the obtained results, general conclusion can be drawn that the process of interest occurs not only with chiral carboxylic acids having three carbon atoms per molecule, but also with those having two and four carbon atoms.

TLC-MS VERSUS TLC-LC-MS FINGERPRINTS OF HERBAL EXTRACTS. PART II. PHENOLIC ACIDS AND FLAVONOIDS

In the previous paper from this series, we proposed mass spectrometric fingerprinting of a complex and volatile botanical sample upon an example of the essential oil derived from Salvia lavandulifolia. In that paper, we compared two variants of fractionation of such a mixture. A simpler one-dimensional variant consisted of the low-temperature thin-layer chromatographic fractionation coupled with mass spectrometric fingerprinting of each separated fraction (1D LT TLC-MS). A more sophisticated variant was the two-dimensional liquid chromatographic system composed of the low-temperature thin-layer chromatography, high-performance liquid chromatography, and mass spectrometric detection (2D LT TLC-LC-MS). In this study, we present an analogous approach to the non-volatile botanical mixtures upon an example of the pharmacologically important phenolic acids and flavonoids selectively extracted from Salvia lavandulifolia. With these non-volatile fractions, the thin-layer chromatographic separations were carried out at ambient temperature (21 ± 0.5°C). Once again, we compared two variants of fractionation. A simpler one-dimensional variant consisted of the thin-layer chromatographic mode coupled with mass spectrometric fingerprinting of each separated fraction (1D TLC-MS). A more sophisticated variant was the two-dimensional liquid chromatographic system composed of the thin-layer chromatography and high-performance liquid chromatography, with mass spectrometric detection (2D TLC-LC-MS). As expected, the two-dimensional mode proved better performing than the one-dimensional mode (1D TLC-MS). It was concluded that thin-layer chromatography directly or indirectly coupled with mass spectrometric detection can prove very useful in the analysis of the phenolic acid and flavonoid fraction selectively extracted from botanical material.

Application of thin-layer chromatography to the investigation of oscillatory instability of selected profen enantiomers in physiological salt

Abstract In earlier investigations, we evaluated the performance of thin‐layer chromatography as an analytical tool to study oscillatory instability of selected profens dissolved in 70% ethanol and dichloromethane. In this study, the solvent used to dissolve profens is changed to physiological salt. The purpose was to assess if the selected test profens [i.e., S‐(+)‐ibuprofen, S‐(+)‐naproxen, and R,S‐(±)‐2‐phenylpropionic acid] can undergo oscillatory transenantiomerization if dissolved in physiological salt. It is a commonly known fact that physiological salt is often drip‐infused into the human (or animal) body with the addition of a drug. Therefore, the question arises whether profen drugs dispensed as physiological salt solutions can undergo oscillatory transenantiomerization in this particular medium. The results of our study confirm that such a transformation is in fact possible.

Experimental Investigation of the Oscillatory Transenantiomerization of L-Tyrosine

Abstract In our earlier studies, we demonstrated an ability of selected enantiomeric profen drugs (e.g., S-(+)-ibuprofen, S-(+)-naproxen, and S-(+)- and R-(−)-flurbiprofen) and one amino acid (i.e., L-α-phenylalanine) to undergo oscillatory transenantiomerization when dissolved in simple, low molecular weight solvents (e.g., water, ethanol, dichloromethane, acetonitrile, etc.) and stored for a longer period of time at ambient temperature or in a refrigerator. Experimental evidence of this process originates from a number of analytical techniques, with thin layer chromatography (TLC) and polarimetry among the best performing ones. There are two common structural features of all these compounds, namely: (i) they are 2-arylpropionic acids (2-APAs), and (ii) their chirality center is located on the α-carbon atom of the respective molecules. It has also been established that the basic and the amphiprotic environment catalyzes the oscillatory transenantiomerization of the investigated compounds, while the acidic environment tends to hamper this process. Moreover, it has been established that all the aforementioned compounds can organize molecules present in the solution in such a manner as to produce the density anisotropy of the liquids considered. Model explanation of the oscillatory transenantiomerization of profens and L-α -phenylalanine was also developed as a starting point, adapting an earlier established oscillator known as Templator. The new model comprises two linked Templators. The quintessence of the Templator model adapted to the demands of the oscillatory transenantiomerization of profens and amino acids was based on an assumption that the H-bonded 2-APA dimer is a template, able to generate the new dimers having the same steric configuration of their respective monomeric units. From our earlier studies, it clearly comes out that in spite of common traits of the oscillatory transenantiomerization of the selected profens and L-α-phenylalanine, the dynamics of this process can significantly differ from one compound to another, due to their differentiated molecular structure and, hence, to the different electron density distribution. Thus, in this study, we investigated the ability of L-tyrosine (another 2-APA and the amino acid regarded as essential for the humans) to undergo oscillatory transenantiomerization. Solubility of L-tyrosine in the amphiprotic binary mixture (70{%} aqueous ethanol solution) widely used in our earlier studies proved too low to use it as a solvent in the present investigation. Instead, we traced the behavior of L-tyrosine when stored for over one week in the following mixed solvents: ethanol–1M NaOH (7:3, v/v) and ethanol-1M HCl (7:3, v/v). The results of our experiments clearly confirm the ability of L-tyrosine to undergo the oscillatory transenantiomerization, similar to that of the previously studied profens and L-α-phenylalanine, although the individual dynamics of the oscillatory transenantiomerization with this particular enantiomer is also evident and discussed. It is apparent that the model of the two linked Templators applies to L-tyrosine as well, as an adequate explanation of the mechanism of its oscillatory transenantiomerization.

Condensation oscillations in the peptidization of phenylglycine

In earlier studies, we showed that certain low-molecular-weight carboxylic acids (profens, amino acids, hydroxy acids) can undergo spontaneous in vitro chiral conversion accompanied by condensation to from oligomers, and we proposed two simple models to describe these processes. Here, we present the results of investigations using non-chiral high-performance liquid chromatography with diode array detector (HPLC-DAD) and mass spectrometry (MS) on the dynamics of peptidization of S-, R-, and rac-phenylglycine dissolved in 70% aqueous ethanol and stored for times up to one year. The experimental results demonstrate that peptidization of phenylglycine can occur in an oscillatory fashion. We also describe, and carry out simulations with, three models that capture key aspects of the oscillatory condensation and chiral conversion processes.

Chiral separation of S -(+)- and R -(−)-ibuprofen by thin-layer chromatography. An improved analytical procedure

Chiral separations by gas and liquid chromatography (thin-layer chromatography included) remain an analytical challenge, hence even moderate success in this field is very likely to be regarded as a valuable separation achievement. There has been only one literature report of chiral separation of S-(+)- and R-(-)-ibuprofen by TLC. The original procedure was performed with laboratory-coated glass plates and resolution of the two enantiomers in one-dimensional mode was incomplete. In an attempt to enhance the resolution the authors made use of less convenient and considerably more time-consuming two-dimensional TLC and the final result was not very impressive (ΔRF = 0.03). These chromatograms were visualized by exposure of the developed plates to iodine vapor and no direct confirmation of the identity of the two chromatographic bands was produced. The goals of this study were: (i) to adapt the experimental conditions used for this separation to commercial chromatographic glass plates; (ii) to enhance the resolution of the two antipodes; and (iii) to produce up-to-date UV spectroscopic evidence of successful separation.

Condensation dynamics of L-proline and L- hydroxyproline in solution

We employ high-performance liquid chromatography with evaporative light scattering and mass spectrometric detection (HPLC/ELSD and LC/MS) to monitor the dynamic behavior of L-Pro, L-Hyp, and L-Pro–L-Hyp in 70% aqueous methanol. The individual amino acid solutions show evidence of oscillatory oligomerization. In the binary solution, the behavior is controlled by the dynamics of L-Pro oligomerization. A simple model involving oligomerization, formation of catalytic oligomer aggregates and cross-catalysis provides qualitative insight into the process.

Exploring the Anti-Cancer Activity of Novel Thiosemicarbazones Generated through the Combination of Retro-Fragments: Dissection of Critical Structure-Activity Relationships

Thiosemicarbazones (TSCs) are an interesting class of ligands that show a diverse range of biological activity, including anti-fungal, anti-viral and anti-cancer effects. Our previous studies have demonstrated the potent in vivo anti-tumor activity of novel TSCs and their ability to overcome resistance to clinically used chemotherapeutics. In the current study, 35 novel TSCs of 6 different classes were designed using a combination of retro-fragments that appear in other TSCs. Additionally, di-substitution at the terminal N4 atom, which was previously identified to be critical for potent anti-cancer activity, was preserved through the incorporation of an N4-based piperazine or morpholine ring. The anti-proliferative activity of the novel TSCs were examined in a variety of cancer and normal cell-types. In particular, compounds 1d and 3c demonstrated the greatest promise as anti-cancer agents with potent and selective anti-proliferative activity. Structure-activity relationship studies revealed that the chelators that utilized “soft” donor atoms, such as nitrogen and sulfur, resulted in potent anti-cancer activity. Indeed, the N,N,S donor atom set was crucial for the formation of redox active iron complexes that were able to mediate the oxidation of ascorbate. This further highlights the important role of reactive oxygen species generation in mediating potent anti-cancer activity. Significantly, this study identified the potent and selective anti-cancer activity of 1d and 3c that warrants further examination.