Covalent character of hydrogen bonding
As part of our investigations to study the nature and characteristics of hydrogen bonding, we have, some years ago, synthesized and studied the NMR spectra of 15N-labeled formazans as model compounds to differentiate between hybridization and tautomerism [15]. To conclude this study, we also synthesized and investigated the NMR spectrum of 15N-labeled compound VII. The combined results of our earlier and present study now unequivocally support our conclusions of the differentiation between the covalent nature and electrostatic character of the hydrogen bond, notwithstanding other assumptions.
Formazans provide a typical example of intramolecular -NH–N = hydrogen bonding, such as between the nitrogenous bases of RNA, responsible for its helical structure. In addition, formazans are of particular biological interest because they are the precursors of the tetrazolium salts used in histochemistry and for assessing cell viability. The process involves reduction by mitochondrial dehydrogenase enzymes in living cells [16–19].
In formazans (Fig. 1), the hydrogen proton can either be shared by hybridization between N1 and N5 or can wander between these two sites due to tautomerism. In the case of compound IA, a slightly broad singlet proton signal was observed at 15.62 ppm. In compound II, the singlet was replaced by a doublet with a coupling constant of 45 Hz, exactly half of what is to be expected based on the known spin-spin coupling constant of 90 Hz between 15N and H-1. Based on this finding, the hydrogen bonding could either be due to the hybridization or the rapidly mutating tautomerism of the contributing structures Ia and Ib.
However, in the NMR spectrum of nitro formazan IV, no spin-spin coupling 15N-H was observed, confirming the residence of H exclusively on 14N5 and therefore excluding any postulation of hybridization. To confirm this conclusion, we synthesized 15N5 nitrofarmazan compound VII. The NMR spectrum indeed showed the expected spin-spin coupling 15N-H of 90 Hz, expected of covalency.
On the other hand, the p-methoxy formazan VI exists almost exclusively in form VIb with almost excusive positioning of the H at 15N1, as indicated by a spin-spin coupling of 86.5 Hz between 15N-H. A slight deviation of 3.5 Hz from the expected value of 90 Hz is perhaps due to the 4 percent contribution of structure VIa to the rapidly mutating tautomerism.
Chemical shifts of the chelated proton clearly indicate the strength of the hydrogen bond. In compounds I & II, they appear at 15.68 ppm; in compounds III, IV & VII, they appear at 14.68 ppm (weaker bonding due to withdrawal of electrons by the directly conjugated nitro group); and in compounds V & VI, they appear at 15.33 ppm (methoxy group being far away from the location of N-H to only marginally influence the chemical shift). These conclusions are further supported by the chemical shifts of the R2 aromatic protons.
The absence of any difference in chemical shifts between 14N and 15N compounds also indicates that there is no influence of the 15N isotope on hydrogen bonding.
Furthermore, we have also confirmed the covalency of NH–N = and NH–O = in many such systems, either by 15N isotope labeling or by studying the nature and chemical shifts of the proton [20].
15N isotope labelling can provide new opportunities of the development of novel contrast agents for Magnetic Resonance Imaging (MRI). The state-of art contrast materials are mostly organo-inorganic 14N-gadolinium(III) based complexes. Due to the alleged concerns of their long-term safety and the requirement of more accuracy in imaging, research into alternatives has recently “spurred” [21]. In NMR 14N signals are sometimes usually significantly broad due to quadrupolar interactions, and in some cases they are even unobservable. Whereas 15N yields sharp signals but is very insensitive to NMR. For safety and to improve the resolution of MRI, therefore, 15N-H containing functionalities could be an alternative to gadolinium(III) based chelates.
Moreover, such non-chelates could even be readily metabolized after their actual use for diagnostics.
Autocatalysis in chemical reactions due to hydrogen bonding
In 2021, Benjamin List and David MacMillan were awarded the Nobel Prize in chemistry for their development of a precise new tool for molecular construction: organocatalysis.
“Many research areas and industries are dependent on chemists’ ability to construct molecules that can form elastic and durable materials, store energy in batteries or inhibit the progression of diseases. This work requires catalysts, which are substances that control and accelerate chemical reactions without becoming part of the final product. For example, catalysis in cars transforms toxic substances in exhaust fumes to harmless molecules. Our bodies also contain thousands of catalysts and are thus fundamental tools for chemists, but researchers long believed that there were, in principle, just two types of catalysts available: metals and enzymes. Benjamin List and David MacMillan are awarded the Nobel Prize in Chemistry 2021 because in 2000, they, independent of each other, developed a third type of
Catalysis. It is called asymmetric organocatalysis and builds upon small organic molecules” [22].
We have demonstrated in the past how hydrogen bonding can even override (Fig. 2b) the well-established influence of the Cl group (Fig. 2a) on the orientation of an aromatic electrophilic reaction by aligning the reactive sites, involving intramolecular autocatalysis, yet another form of catalysis, as shown in Fig. 2 [23].
Later, we industrially exploited such principles along with the principle of “Seeding” (underlining one of the principles of organocatalysis) for the synthesis & “tailoring" of organic pigments in predetermined homogenous microcrystalline, particulate size, shape and distribution, the most important functionality of pigments for their actual multipurpose uses, such as for the coloration of plastics, coatings, printing inks, cosmetics and now in electronics for LCDs (Liquid Crystal Displays) [24–26].
This paper specifically discusses the intramolecular autocatalysis mechanism in the synthesis of THI (thiazine indigo) pigments, a novel class of environmentally benign synthetic colorants [27] related to naturally occurring trichosiderins contained in human and animal keratinic materials [28]. Trichosiderins formed biochemically from sulfur-containing amino acids are now attracting increasing interest in medicinal research due to melanoma studies [29, 30]
The paper also describes the structural study of cis-THI (Thiazine Indigo) and trans-THI (Thiazine Indigo). Although the proof of the structure of trans-THI is well established on the basis of X-ray crystal structure studies [31, 32], evidence of the structure and characteristics of cis-THI is provided herein for the first time. This characterization is of particular importance because in our opinion, the recent revision of the structure of “The Δ2,2′-Bi (2H-1,4-benzothiazine)”, the basic chromophoric system of THI, needs to be rerevised [33].
Multistep synthesis of THI pigments involving intramolecular catalysis
This totally novel chromophoric system synthesized for the first time in 1974 is obtained in an apparently one-step synthesis but inharently occurs in multiple stages by the reaction of o-aminothiophenols with 2,3-dichloromaleic anhydride. Depending upon the reaction conditions, the THI molecule can, however, be obtained either in the cis- or the trans-form (Fig. 3) [34, 35].
Moreover, the procedure provides an approach to the synthesis of unsymmetrical trichosiderins found in nature.
Mechanism of the synthesis of cis-THI
In water or ethanol, the reaction between dichloromaleic anhydride and an o-aminothiophenol is the first stage of the stepwise nucleophilic substitution of the chlorine atoms by mercapto groups, which is intramolecularly catalysed by the proximate amino group. The liberated acid is intermittently captured by the neighbouring NH2 group but later liberated after cyclization to form the first thiazine ring. This reaction is repeated for the second time with the second mole of an o-aminophenol, which can be the same or different, to form the cis-THI molecule. We believe that the nucleophilic substitution reaction of both Cl atoms occurs by the SN2 principle catalysed by intramolecular hydrogen bonding of NH2 with C = O.
`-
Mechanism of the synthesis of trans-THI
Under acidic conditions, however, after the addition of the mercapto group but before the elimination of the halogen atom in the second step, the intermediate now undergoes rotation, followed by ring closure, leading to the formation of trans-THI.
Structural study of cis-THI and trans-THI
Although proof of the structure of trans-THI is well documented, the structure of cis THI is still not well understood. To differentiate between the cis and the trans forms of THI, in addition to their colour appearance (cis: yellow powder; trans: red powder), we determined the FTIR (Fig. 6) and NMR (Fig. 7) spectra of 7,7′-dichloro-THI.
In the FTIR spectrum (Fig. 6) of the cis form, the C = O absorption appears at 1689 (cm− 1), and the C = C band appears at 1645 (cm− 1), whereas in the trans form, the corresponding bands appear at 1625 (cm− 1) and 1589 (cm− 1). These distinct differentiations can be interpreted as the restricted conjugation of C = O across the C = C double bond in the cis form due to its nonplanar structure.
In the NMR spectrum (in D6-DMSO + NaOD) of cis-7,7′ dichloro THI, all aromatic protons are shifted downfield compared to the trans-7,7′ dichloro THI. However, the most prominent is the downfield shift of the protons at the C8 and C8′ positions due to their proximity to the peri-sulfur atoms. Reduced conjugation within the chromophoric system due to the nonplanarity of cis-THI increases the electronegativity of the sulfur atoms and hence the downfield shifts of the H8 &H8` protons.