As a whole, the results described herein, based on molecular orbital calculations and the matrix-isolation technique coupled to FTIR and EPR spectroscopies, pave the way towards a better elucidation of the mechanistic pathways followed by 5-aminotetrazoles upon UV-irradiation and of the effect of the ring substitution pattern on the photoreactivity of these compounds.
Here we report the UV-induced photochemistry of monomeric 3-(2-methyl-(2H)-tetrazole-5-amino)-1,2-benzisothiazole 1,1-dioxide 4 (herein termed as 2-methyl-(2H)-tetrazole-5-amino-saccharinate, 2MTS ) isolated in solid argon. Compound 4 is one of our most promising ligands, since it proved to be non-toxic and selective towards Cu (II) and could be developed in the context of a new anti-tumor therapeutic approach based on selective copper chelation [ 11 ]. The matrix photochemistry of 4 is compared with that of other 5-aminotetrazole derivatives ( Scheme 2 ), including 2-methyl-5-aminotetrazole 2 , used as building block for the preparation of the ligand 4 , and its isomer, 1-methyl-5-aminotetrazole 5 . It is worth noting that 5-aminotetrazoles are of interest as gas-generating compositions [ 22 ], [ 23 ], as high energy materials (e.g. as air-bag insufflators and as propellants components for missiles) [ 24 ], [ 25 ], [ 26 ], [ 27 ] and, accordingly to recent findings, are being developed as powerful tools for proteome profiling in living cells [ 28 ], [ 29 ], [ 30 ], [ 31 ], [ 32 ]. Thus, a deeper investigation of the photochemistry of 5-aminotetrazoles and their derivatives is also especially relevant and timely.
Despite the promising properties observed for these new tetrazole-saccharinate ligands, the tetrazole ring is known to undergo easy cleavage, induced thermally or photochemically [ 19 ], [ 20 ], [ 21 ]. Thus, when considering the applications of tetrazole-saccharinates, the low stability of this heterocycle steams as a concern. In this context we considered relevant to investigate the photochemistry of tetrazole-saccharinates, as the electron withdrawing saccharyl system, known to be relatively photostable, will probably affect the photostability of the tetrazole moiety [ 10 ].
An investigation on the chelating ability of the new compounds revealed that selected N-linked tetrazole-saccharinates ( Scheme 1 ; X=&#;NH&#; or &#;N=) exhibit strong binding selectivity to copper ions [ 11 ]. This property was considered of interest for therapeutic applications [ 12 ], mainly because of the recent findings that link elevated levels of copper to cancer progression [ 13 ], [ 14 ], boosting the interest in selective copper chelators [ 12 ], [ 15 ], [ 16 ]. The copper complexes prepared from selected N-linked tetrazole-saccharinates were tested in vitro against cancer cell lines and have shown a considerable increase in cytotoxicity against tumoral cells, compared to the corresponding nontoxic ligands [ 11 ]. Also, in a different line of investigation, two mononuclear copper (II) and cobalt (II) complexes based on the tetrazole-saccharinate ligand 4 ( Scheme 2 ) were used as effective catalysts for selective oxidation of diverse secondary alcohols [ 17 ], while the ligand 4 alone proved effective as organo-catalyst for the oxidation of benzyl alcohols [ 18 ].
Tetrazoles exhibit excellent coordination abilities through their four nitrogen atoms, acting either as multidentate ligands or as bridging building blocks in supramolecular assemblies [ 1 ], [ 2 ], [ 3 ], [ 4 ]. Additionally, the structure of the tetrazolyl-based coordination complexes can be tailored by employing functionalysed tetrazoles in the assembly process [ 5 ], increasing the versatility and widening the applications of the tetrazole chemotype in coordination and supramolecular chemistry. Likewise, benzisothiazoles (known as saccharins) bear interesting coordination abilities and are thermally and photochemically much more stable than tetrazoles, although the saccharyl system is comparatively more difficult to functionalise [ 6 ], [ 7 ]. Taking advantage of the properties of both classes of heterocycles, we designed new scaffolds, termed as tetrazole-saccharinates ( Scheme 1 ), with the aim of exploring their applications as multidentate nitrogen ligands. In recent years we prepared a representative library of tetrazole-saccharinates and investigated their structure in detail, prior to exploring their reactivity and applications [ 8 ], [ 9 ], [ 10 ].
As described in the Experimental Section, a sample of crystalline 2-methyl-(2H)-tetrazole-5-amino-saccharinate 4 was sublimated under reduced pressure (at &#;150°C) and the vapors of the compound were co-deposited with argon (ca. 1: molar ratio) onto a CsI substrate kept at 15 K. In previous studies, 4 was found to undergo a complete amino&#;imino tautomerization under the conditions described, where the amino-bridged tautomeric form existing in the crystalline phase of the compound was completely converted into the theoretically predicted most stable imino-bridged tautomer [9]. In this most stable tautomeric form, observed in the matrix isolation experiments, the labile hydrogen atom is connected to the saccharyl nitrogen and the two heterocyclic fragments are linked by an imino moiety in which the double-bond is established with the carbon atom belonging to the saccharyl fragment (see structure 4 in Scheme 3).
Scheme 3:
To investigate the photochemistry of 4, the deposited matrix was irradiated with a tunable UV-laser source, starting at λ=330 nm and gradually decreasing until λ=222 nm (the shortest wavelength available in our experimental setup), with the sample being followed after each irradiation by recording its infrared spectrum. It was observed that irradiations at around λ~290 nm and λ~250 nm were the most effective in inducing changes in the spectrum of 4.
Irradiations at λ=290 nm, during up to 100 min., resulted in a decrease in the intensity of the bands due to 4 (indicating that the compound was being consumed) and, simultaneously, in a continuous increase of a distinctive absorption band at around cm&#;1. Further irradiations of this same matrix at λ=250 nm resulted in several new bands, with complete consumption of the reagent 4 after 10 min. of irradiation. Figure 1 presents the spectral changes in the range &#; cm&#;1: (i) after 100 min. of irradiation at λ=290 nm, when around 60% of 4 was consumed and a chemical species, with a characteristic absorption at cm&#;1, was produced, which could be identified as the 1H-diazirene 7 (see Scheme 3); (ii) after 5 and 10 min. of irradiation at λ=250 nm, subsequent to the irradiation at λ=290 nm, showing bands due to the different photoproducts generated at this wavelength. As reported for the photochemistry of an S-linked 1-methyltetrazole-saccharinate [10], the presence of the saccharyl ring, which seems to be unaffected by irradiation under the experimental conditions used, results in extensive overlap of the bands of 4 with those of the photoproducts, especially in the low-frequency spectral range (below cm&#;1), hampering the interpretation of the data based on the low frequency spectral region. However, the most characteristic bands of these photoproducts are expected to appear in the clean &#; cm&#;1 spectroscopic window.
Fig. 1:
As it can be observed in Fig. 1, it is clear that in the &#; cm&#;1 spectral range no other bands increased besides the distinctive cm&#;1 absorption due to the diazirene 7, even after 100 min. of irradiation at λ=290 nm. Subsequent irradiation of the same matrix at λ=250 nm resulted in a fast consumption of both the reactant 4 (see absorption at cm&#;1) and the diazirene 7 while, simultaneously, several new bands (, , , cm&#;1), due to other photoproduced species, appeared in the spectrum. The identification of the photoproducts corresponding to these characteristic absorption bands could be easily achieved by comparison with the reported results obtained for the photolysis of an S-linked tetrazole-saccharinate and the parent 2-methyl-5-aminotetrazole [10], [33]. The band at cm&#;1 was ascribed to the νNCN antisymmetric stretching of carbodiimide 8 (see Scheme 3), the band at cm&#;1 was ascribed to the νC&#;N stretching of nitrile 9 and the band at cm&#;1 was ascribed to the νCN stretching of CNH 12. The distinctive absorption at cm&#;1, which increased during the first 5 min. of irradiation and then started decreasing with further irradiations at λ=250 nm, was assigned to the νC=N stretching mode of the nitrile imine 6, calculated at cm&#;1. The spectral changes observed in the range &#; cm&#;1, after 5 and 10 min of irradiation at λ=250 nm, are shown in detail in Fig. 2. The calculated IR spectra for the proposed photoproducts are also shown in this figure, for comparison.
Fig. 2:
The mechanisms of fragmentation of tetrazoles remain under debate and the formation of nitrile imines from thermal and photochemical decomposition of tetrazoles has been subject of intense investigation over the last decades [19]. From the very first studies [32], it was postulated that the parent tetrazole in its gas phase most stable isomeric 2H-form, as well as 2-, 5- and 2,5-substituted tetrazoles, undergo fragmentation through formation of a nitrile imine intermediate that cyclizes to a 1H-diazirene, then leading to a final carbodiimide through rearrangement. However, this pattern of reactivity was demonstrated experimentally only very recently, during studies on the photochemistry of tetrazoles under low-temperature matrix isolation conditions [5], [13], [15], [21], [33], [35]. The observed outline of photo-fragmentation of 4 upon irradiation at λ=250 nm follows this general pattern, which, as also found in other cases [10], [33], is accompanied by an additional reaction path involving as reactant the 1H-diazirene (see Scheme 3).
The initial step of the photochemistry of matrix-isolated 4 corresponds to selective photoinduced cleavage of the C5&#;N4 and N2&#;N3 bonds of the tetrazole ring, leading to extrusion of molecular nitrogen and production of nitrile imine 6, which reached its maximum amount after the first 5 min. of irradiation at λ=250 nm, and then was gradually consumed with increased irradiation times (see band cm&#;1 in Fig. 2; blue and red lines represent 5 min and 10 min of irradiation at λ=250 nm, respectively), generating the 1H-diazirene 7 through a ring closing process.
It shall be noticed that, as mentioned above, upon irradiation of 4 at λ=290 nm, the diazirene 7 was observed as the sole photoproduct. Although around 40% of 4 remained after 100 min. of irradiation, suggesting that the efficiency of the reaction is rather low at this wavelength, it should be noticed that the capture of antiaromatic (i.e. 4π systems) structures such as 7 proved to be quite challenging, and has only been observed in rare cases, mostly upon isolation in cryogenic matrices [10], [33], [35], [36], [37]. Nevertheless, under the experimental conditions used, the antiaromatic three membered ring 7 could indeed be generated as the sole photoproduct and was found to be photostable. This result is even more remarkable because, contrarily to what is observed in this case, the diazirene derivative of 2-methyl-5-aminotetrazole was found to react upon irradiation at 325 nm [33]. On the other hand, the observed photostability of the antiaromatic three membered diazirene 7 follows the trend observed for a previously studied S-linked tetrazole-saccharinate [10], and appears to be a common phenomenon on this type of conjugates, probably induced by the stabilizing effect of the electron-withdrawing saccharyl moiety.
It is also interesting to note that during the irradiation experiments performed at 290 nm no spectroscopic evidence of any intermediate from 4 to the diazirene 7 was found, in particular no bands ascribable to nitrile imine 6, which is observed upon irradiation at λ=250 nm. This may be explained considering that the longer wavelength of excitation (λ=290 nm), being closer to the absorbance maximum of the expected preceding nitrile imine 6 [38], facilitates its fast photoconversion into the diazirene 7.
Upon irradiation at λ=250 nm, the diazirene 7 undergoes subsequent photoconversion into carbodiimide 8 (pathway a in Scheme 3), in a process that will be discussed in more detail below. A second pathway was also perceived, pathway b, involving concomitant decomposition of the diazirene 7 and formation of nitrile 9 and CNH 12.
Concerning pathway b, it should be noticed that the formation of nitrile 9 could in principle result either from (i) the cleavage of the C5&#;N4 and N1&#;N2 bonds of the tetrazole ring of 4, generating the nitrile 9 and methyl azide, or from (ii) cleavage of the diazirene ring 7, generating nitrile 9 and methyl nitrene 10, the latter undergoing subsequent isomerization to methylenimine 11, from which isocyanic acid (CNH) can be produced [39], [40]. However, formation of nitrile 9 via cleavage of the tetrazole ring can be ruled out, since no evidence of the intense νNNN antisymmetric mode of methyl azide could be found [40]. On the other hand, isocyanic acid was identified beyond doubt [39] and its formation from methylenimine 11 is expected based on the available knowledge regarding the photochemistry of this last compound [39], [40]. This mechanistic proposal is also supported by the results gathered from the photochemical investigation of the matrix-isolated parent 2-methyl-5-aminotetrazole 2, which included EPR measurements, enabling the identification of methyl nitrene 10 (as described in detail below).
The present study revealed that photolysis of 4 results in a selective fragmentation of the tetrazole ring, while the saccharyl system seems to be completely photostable under the conditions used. As such, comparison of the photochemistry of 4 with that reported for the parent 2-methyl-5-aminotetrazole 2 [33] may be viewed as an efficient approach to support the interpretation of the present spectroscopic data, and to evaluate the photochemical stability induced by the electron withdrawing saccharyl system into the photolabile tetrazole.
The photochemistry of 4, isolated in solid argon at 15 K, was investigated under similar conditions to those used for the parent tetrazole 2. Irradiation was performed with a tunable laser at λ=250 nm, with an output power of ~40 mW. It should be noted that both 5-aminotetrazole derivatives 2 and 4 show an absorption maximum at ~250 nm.
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Comparison of the photochemistry of the conjugate 4 with that of the parent 2-methyl-5-aminotetrazole 2, in the same experimental conditions, revealed that the reaction pathways and obtained photoproducts are equivalent for both compounds (Scheme 4). However, kinetic studies on the photodegradation of both compounds unfolded interesting differences in the kinetic profiles. Photolysis of matrix-isolated 2 at λ=250 nm (~40 mW) generated the corresponding nitrile imine 13 in a maximal amount after 2 s of irradiation, which was totally consumed after 4 min. of irradiation. Also, ca. 50% of the initial compound 2 was consumed after 4 s of irradiation [33]. On the other hand, photolysis of 4 under the same conditions (λ=250 nm, ~40 mW) generated the corresponding nitrile imine 6 in a maximal amount after 5 min. of irradiation, and only after 60 min. of irradiation the nitrile imine was completely consumed. Moreover, ca. 50% of the initial 4 was consumed only after 2 min. of irradiation. These results suggest that the saccharyl system increases the photostability of the tetrazole ring and also of the nitrile imine intermediate by more than 20×, compared to parent tetrazole 2. These results are in keeping with the above noticed stabilization of the diazirene formed from 4 in result of the presence of the saccharyl substituent.
Scheme 4:
The general pattern of photoreactivity of 2-, 5- and 2,5-substituted tetrazoles has been described above. For 1,5-disubstituted tetrazoles, earlier studies [34], [38] postulated direct formation of the carbodiimide through an imidoylnitrene intermediate (see Fig. 3). In addition, more recent investigations demonstrated that 1H-diazirenes are common intermediates on the photochemistry of both 1,5- and 2,5-disubstituted tetrazoles [33].
Fig. 3:
During a study on the photochemistry of 5-methyl-tetrazole, Nunes et al. [35] have shown that 1H-diazirenes exhibit a close structural relation to imidoylnitrenes. Indeed, geometry optimization at the B3LYP/cc-pVTZ level on the triplet state of 5-methyl-1H-diazirene revealed convergence to the respective imidoylnitrene structure [35]. However, these imidoylnitrenes were only observed using internal or external traps [36], [37], since in the absence of these traps the reactive singlet state imidoylnitrene shall promptly cyclize to the 1H-diazirene or undergo a Wolff-type rearrangement into carbodiimide [41]. Recent studies on the photolysis of matrix-isolated tetrazoles allowed to identify several isomers formed from elimination of molecular nitrogen, but attempts to trap the putative imidoylnitrene intermediates were unsuccessful [35], [42].
Following the reactivity patterns described above, two important observations result from recent studies regarding the effect of the ring substitution outline on the photofragmentation pathways of 5-aminotetrazoles [33]: (i) the 1H-diazirene was observed as a common intermediate from photolysis of both 1-methyl- and 2-methyl-5-aminotetrazole, subsequently isomerizing to carbodiimide as final photoproduct; (ii) upon irradiation at short wavelengths (222 nm), an amino cyanamide was obtained, together with the 1H-diazirene and carbodiimide, from photolysis of 1-methyl-5-aminotetrazole, unraveling a new reaction pathway; this cyanamide isomerizes to the carbodiimide upon irradiation at longer wavelengths (325 nm).
In order to deepen our understanding of the effect of the ring substitution pattern on the mechanistic pathways followed by 5-aminotetrazole derivatives, further studies were undertaken. The C2H5N3 isomers were calculated at the B3LYP/6-311++G(d,p) level and the structures are represented in Fig. 3. The calculated energies indicate that 1H-diazirene (14 in Fig. 3) is the most energetic species in the singlet state; ~22 and ~11 kJ mol&#;1 below the imidoylnitrene triplet states TN1 and TN2, respectively. The high energetic character of 14, 94.1 kJ mol&#;1 above the amino cyanamide 17 (calculated as the most stable isomer in the singlet state potential energy surface), is conceivably due to both ring strain and antiaromatic destabilization. The observed trends for these isomeric species are similar to the trends found in calculations for H4C2N2 and H2CN2 isomers, which can be formed from the photolysis of 5-methyl-tetrazole and of the parent unsubstituted tetrazole, respectively [35], [42]. The optimized isomeric form E of triplet imidoylnitrene (TN2) is more stable than form Z (TN1) by ~10 kJ mol&#;1.
In Scheme 5, a mechanistic proposal for the photochemical transformations of 5 and 2 isomers is presented, which is also in agreement with the observed photochemistry of 4, described above. This mechanism considers as pivotal intermediate the highly reactive open-shell singlet state imidoylnitrene species, sN. Structurally, sN can be visualized as delocalized resonance structures with some biradical character at the two nitrogen atoms, and a central CN bond with appreciable double bond character [43], [44], [45].
Scheme 5:
As reported before [33], the λ=250 nm photolysis of 2 results in the cleavage of the tetrazole ring, with initial formation of nitrile imine 13 that develops to diazirene 14. This last one is expected to generate the imidoylnitrene in the geometrically most accessible Z isomeric form SN1, through ring opening. The reactive Z-sN1 species can then rearrange to the observed carbodiimide 15, via a direct R-group 1,2-shift (R=amino group) from the carbon of the imidoylnitrene to the sterically accessible nitrene moiety (in this isomeric form of the imidoylnitrene, the methyl substituent precludes the 1,2-shift to the methyl-substituted nitrogen). Several thermal and photochemical reactions of tetrazoles involving imidoylnitrenes&#; rearrangements were postulated based on this 1,2-shift [19]. Indeed, imidoylnitrenes are aza analogs of vinylnitrenes and acylnitrenes, thus these reactions can be expected to follow the same Wolff-type rearrangement to carbodiimides [41].
Compared to 2, the first step of the photolysis of 5 leads to a different product, though in both cases cleavage of the tetrazole ring, with extrusion of molecular nitrogen, takes place. In the case of 5, this process directly generates the postulated imidoylnitrene intermediate Z-sN1. Once formed, Z-sN1 can undergo the above mentioned Wolff-type rearrangement to carbodiimide 15, or collapse to give the 1H-diazirene 14, which are then common photoproducts from photolysis of both 5-aminotetrazole isomers 2 and 5. The observation of the cyanamide 17 photoproduct during the short wavelength irradiation (222 nm) [33] of 5 can be explained by considering that at this irradiation wavelength the channel for photoisomerization between Z and E forms of the imidoylnitrene is accessible. In the E imidoylnitrene isomeric form sN2, the methyl substituent is no longer blocking the 1,2-shift from the amino group to the methyl-substituted nitrogen, thus allowing generation of the cyanamide 17 by a mechanism similar to that discussed above leading to rearrangement of the imidoylnitrene into the carbodiimide 15.
As mentioned above, it was also shown [33] that upon subsequent irradiation at 325 nm of a photolysed argon matrix of 5, the formed cyanamide 17 (and also the diazirene 14) converts into the carbodiimide 15, a result that is also explained by the mechanism shown in Scheme 4 and involves participation of the imidoylnitrene intermediate. Recently, Abe et al. [46] reported the formation of an imidoyl nitrene from photolysis of 1-methyl-5-phenyl-tetrazole, which was observed by EPR (electron paramagnetic resonance) spectroscopy.
We performed EPR (electron paramagnetic resonance) spectroscopy for the photolysis of 5 and 2 in cryogenic conditions, aimed at trapping and identifying putative nitrene species. According to the reported data [47], we can expect to observe different EPR signatures for nitrenes and diradicals and they can be distinguished by EPR, where nitrenes give rise to transitions at high field (typically ~ G) and zero-field splitting parameters D of around 1 cm&#;1, whereas diradicals resonate at much lower field (typically ~ G) and show much smaller D values.
In these EPR studies, ca. 50 mM 2-methyltetrahydrofuran (MTHF) solution of 2 and the suspension of 5 were degassed under high vacuum at 1.0×10&#;2 Pa and sealed under the vacuum conditions, respectively. The solubility of tetrazole 5 was quite low. The EPR sample of 2 was placed in the EPR cavity, cooled to 5 K, and then irradiated at 266 nm (~10 mJ). After irradiation of compound 2 under these conditions for 60 min., the EPR spectrum of photolyzed 2 evidenced a signal at around G, at the resonance frequency of 9.39 GHz (Fig. 4a), which was identified as methyl nitrene 10, with |D/hc|=1.61 cm&#;1 and |E/hc|=0. cm&#;1, which are consistent with reported values in organic matrix [48]. This observation brings further support to our proposal that nitriles 9 and 16 result from photolysis of diazirenes 7 and 14, respectively (see Schemes 2 and 3), with concomitant formation of methyl nitrene 10 that rearranges to methylene imine 11, then affording isocyanic acid 12.
Fig. 4:
For compound 5, the irradiation with a broadband Xe-light source had to proceed for 11 h until a very weak signal could be observed, at ~ G, also ascribed to a triplet nitrene (Fig. 4b). It should be noted that the irradiation wavelength (λ=266 nm) is far from the maximum absorption (~222 nm) observed for 5, and even the Xe-lamp has a very weak light intensity below ~250 nm. In addition, the solubility of tetrazole 5 proved to be quite low, as mentioned above.
In consonance with the reported information, these EPR results seem to be the first direct observation of the postulated methylnitrene intermediate from the photolysis of diazirenes. Unfortunately, we could not observe evidence of the postulated diradical imidoyl nitrene SN1 which, according to the reported data, can be expected to be mixed with the strong signals at ~ G, which were derived from doublet impurities.
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