International Journal of Molecular Sciences Article Computational Insights on the Mechanism of the Chemiluminescence Reaction of New Group of Chemiluminogens—10-Methyl- 9-thiophenoxycarbonylacridinium Cations Milena Pie ´nkosand Beata Zadykowicz * Faculty of Chemistry, University of Gda´nsk,Wita Stwosza 63, 80-308 Gda´nsk,Poland; [email protected] * Correspondence: [email protected] Received: 1 June 2020; Accepted: 20 June 2020; Published: 21 June 2020 Abstract: Immunodiagnostics, in which one of the promising procedures is the chemiluminescent labelling, is essential to facilitate the detection of infections in a human organism. One of the standards commonly used in luminometric assays is luminol, which characterized by low quantum yield in aqueous environments. Acridinium esters have better characteristics in this topic. Therefore, the search for new derivatives, especially those characterized by the higher quantum yield of chemiluminescence, is one of the aims of the research undertaken. Using the proposed mechanism of chemiluminescence, we examined the effect of replacing a single atom within a center of reaction on the efficient transformation of substrates into electronically excited products. The density functional theory (DFT) and time dependent (TD) DFT calculated thermodynamic and kinetic data concerning the chemiluminescence and competitive dark pathways suggests that some of the scrutinized derivatives have better characteristics than the chemiluminogens used so far. Synthesis of these candidates for efficient chemiluminogens, followed by studies of their chemiluminescent properties, and ultimately in chemiluminescent labelling, are further steps to confirm their potential applicability in immunodiagnostics. Keywords: mechanism of chemiluminescence; DFT and TD DFT calculations; acridinium derivatives; chemiluminogens 1. Introduction Chemical demonstrations are very popular and allow the promotion of science, especially among children and youth. Spectacular chemical experiments, especially those that are unexpected, explosive and colorful, attract the full auditoria. One of the most amazing chemical experiments successfully presented at chemical demonstrations is chemiluminescence (CL) [1], in which by mixing several substances a luminous product is formed [2]. In fact, chemiluminescence is a phenomenon of the emission of light as a result of a chemical reaction [3]. In this reaction, a product is formed in an electronically excited state [4,5]. When an electronically excited electron returns to a ground state, an energy is released in a form of light (as a photon) [5]. In addition to cognitive features, chemiluminescence has also found many utilitarian applications in clinical [6,7], pharmaceutical [8], chemical and biochemical analysis [9], and also in environmental [10] and food analysis [11]. One of the most efficient chemiluminogens are compounds based on quaternary acridine derivatives substituted in the central ring at the endocyclic nitrogen atom and the opposing carbon atom (in position 9), especially 10-methyl-9-phenoxycarbonylacridnium cations (acridinium esters) [12–16]. These acridinium salts exhibit chemiluminogenic features as a result of reaction Int. J. Mol. Sci. 2020, 21, 4417; doi:10.3390/ijms21124417 www.mdpi.com/journal/ijms Int.Int. J. Mol. J. Mol. Sci. Sci.2020 2020, 21, ,21 4417, 4417 2 2of of 16 17 [12–16]. These acridinium salts exhibit chemiluminogenic features as a result of reaction with withoxidants oxidants in alkaline in alkaline environments environments [12,17]. [12 Acridinium,17]. Acridinium esters are esters used arein clinical used in[18,19], clinical chemical [18,19 ], chemical[5,20], [or5, 20environmental], or environmental analysis analysis [10,21] [ 10as ,fragme21] as fragmentsnts of chemiluminescent of chemiluminescent labels labelsin commercial in commercial tests tests[22] [22 in] inwhich which the the determination determination of concentration of concentration of macromolecules, of macromolecules, e.g., α e.g.,-fetoproteinsα-fetoproteins [22], β [-22D-], β-dgalactosidase-galactosidase [23,24], [23,24 ],glucose-6-phosphate glucose-6-phosphate dehydrogenase dehydrogenase [22], [22 ],TSH TSH [24,25], [24,25 ],FT4 FT4 [22], [22 ],or or anti-HIV anti-HIV antibodiesantibodies [26 [26,27],,27], are are very very precise precis (ite (it is is possible possible to to determine determine thethe concentrationsconcentrations of of macromolecules macromolecules at theat the femtomole femtomole level level [ 6[6,11,26,28,11,26,28––3333]).]). Considering the the utilitarian utilitarian ro rolele of ofacridinium acridinium derivatives, derivatives, it it isis importantimportant toto understandunderstand howhow electronicallyelectronically excitedexcited products are formed. The The mechanism mechanism of of chemiluminescencechemiluminescence of of acridinium acridinium esters esters was was suggested suggested byby McCapraMcCapra in the late 1980s 1980s [1,34], [1,34], and and then then waswas studied studied using using quantum quantum chemistry chemistry methods methods (first (first at at the the semiempiricalsemiempirical [[35]35] and then then at at the the density density functionalfunctional theory theory level level [17 ]).[17]). The The chemiluminescence chemiluminescence reaction reaction of acridinium of acridinium salts salts (Scheme (Scheme1) begins 1) begins with – thewith attack the of attack oxidant, of e.g.,oxidant, OOH e.g.,− on OOH carbon on atom carbon in position atom in 9 position of acridine 9 of moiety, acridine followed moiety, by followed the reaction by the reaction of the addition product with OH− [12,17], either the cyclization of the anion to cyclic of the addition product with OH− [12,17], either the cyclization of the anion to cyclic intermediate [17], andintermediate the elimination [17],of and phenyl the elimination carbonateanion, of phenyl leading carbonate to the energy-richanion, leading 10-methyl-9-acridinone, to the energy-rich 10- or themethyl-9-acridinone, elimination of the adequate or the elimination phenoxy anion of the and adequate formed thephenoxy cyclic intermediate—four-memberedanion and formed the cyclic highlyintermediate strained dioxetane—four-membered structure highly [12,36 –strained41], and dioxetane the subsequent structure elimination [12,36–41], of carbon and the dioxide subsequent leading elimination of carbon dioxide leading to the energy-rich 10-methyl-9-acridinone. The theoretically to the energy-rich 10-methyl-9-acridinone. The theoretically predicted thermodynamic data indicated predicted thermodynamic data indicated that these two pathways are possible for most compounds that these two pathways are possible for most compounds [12,17]. The relaxation of electronically [12,17]. The relaxation of electronically excited product is accompanied by the emission of light, i.e., excited product is accompanied by the emission of light, i.e., chemiluminescence [4,42]. chemiluminescence [4,42]. SchemeScheme 1. 1.Proposed Proposed chemiluminescence chemiluminescence mechanism mechanism ofof acridiniumacridinium derivatives. TheThe acridinium acridinium derivativesderivatives are are one one of of the the most most important important groups groups of compounds of compounds used in usedthe in thechemiluminometric chemiluminometric analysis. analysis. Their Their biggest biggest advantage advantage is the is the relatively relatively high high quantum quantum yield yield of of chemiluminescence—upchemiluminescence—up to to 7% 7% [34 [34].]. It It is is also also important important thatthat acridiniumacridinium esters are are easy easy to to synthesize synthesize andand oxidize oxidize in in an an aqueous aqueous alkaline alkaline environment environment [12[12,43].,43]. However,However, the sear searchch for for new new derivatives, derivatives, especiallyespecially those those characterized characterized by by the the higher higher quantum quantum yield yield ofof chemiluminescence,chemiluminescence, is is one one of of the the aims aims ofof the the research research undertaken. undertaken. TakingTaking intointo accountaccount previousprevious investigationsinvestigations on on the the mechanism mechanism of of chemiluminescence of acridinium derivatives, it is known that the type of the leaving group is chemiluminescence of acridinium derivatives, it is known that the type of the leaving group is affected by the efficiency of the formation of the electronically excited products [12,17,35]. Therefore, affected by the efficiency of the formation of the electronically excited products [12,17,35]. Therefore, the studies on new chemiluminogens, in which a phenoxy group was replaced by a thiophenoxy the studies on new chemiluminogens, in which a phenoxy group was replaced by a thiophenoxy group, group, were initiated. were initiated. In the current work, we computationally investigate a series of 10-methyl-9- In the current work, we computationally investigate a series of 10-methyl-9- thiophenoxycarbonylacridinium derivatives, comparing their susceptibility to oxidation in an thiophenoxycarbonylacridiniumalkaline media. We suggest the derivatives, mechanism comparing of the chemiluminescence their susceptibility reaction to oxidation of new in angroup alkaline of media.compounds We suggest based the on mechanism acridine moiety, of the considering chemiluminescence both, light reaction—leading of to new the groupenergy-rich of compounds product basedand on dark acridine—leading moiety, to competitive considering