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
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[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 both,products, light—leading reaction pathways. to the energy-rich Given the product possibilities and dark—leading of modern to competitivequantum chemistry products, methods, reaction we pathways. focused Givenour stud they possibilitieson the ground of modernstate reaction quantum profiles chemistry and methods,formation we focusedof the our electronically study on the groundexcited state 10 reaction-methyl-9-acridinone. profiles and formation By understanding of the electronically the excited 10-methyl-9-acridinone. By understanding the chemiluminescence mechanism, new potential chemiluminogens with specificity and susceptibility could be rationally designed. Int. J. Mol. Sci. 2020, 21, 4417 3 of 16 chemiluminescence mechanism, new potential chemiluminogens with specificity and susceptibility couldInt. J. Mol. be Sci.rationally2020, 21, designed. 4417 3 of 17
2. Results and Discussion 2. Results and Discussion 2.1. Selection of Investigated Models 2.1. Selection of Investigated Models Our previous investigations [12,13,17] on the mechanism of the chemiluminescence of acridiniumOur previous derivatives investigations indicate that [12,13 the,17 ]type on theof mechanismthe leaving ofgroup the chemiluminescence is affected the efficiency of acridinium of the formationderivatives of indicate the electronically that the type excited of the products. leaving For group this is reason, affected we the began efficiency to analyze of the the formation molecules, of inthe which electronically elimination excited of the products. leaving group For this could reason, be much we began easier tothan analyze in acridinium the molecules, esters inand which thus transformationelimination of the into leaving cyclic group intermediate, could be leading much easier to the than electronically in acridinium excited esters product, and thus much transformation efficient. Weinto decided cyclic intermediate, to replace only leading one atom to the in electronicallythe leaving group excited (Scheme product, 2) and much check efficient. how such We decideda small differenceto replace onlyin the one molecular atom in the structure leaving groupof acridini (Schemeum 2)esters and checkwill howaffect such the aefficiency small difference of the chemiluminescencein the molecular structure reaction. of The acridinium selection esters of the will replacing affect theatom efficiency was based of on the the chemiluminescence electronegativity ofreaction. chemical The elements selection (χ of in the the replacing Pauling atom scale was is equal based to on 3.44 the for electronegativity oxygen atom and of chemical 2.58 for elementssulphur atom(χ in the[44]) Pauling and structural scale is equal proper toties 3.44 within for oxygen the leaving atom and group 2.58 of for the sulphur investigated atom [44 compounds.]) and structural The bondproperties lengths within in the the thiocarboxyl leaving group group of the are investigated much longer compounds. than in the The carboxyl bond lengths group in (the the thiocarboxylC–S bond is equalgroup to are 1.8 much Å, the longer C–O than bond in is the ca. carboxyl 1.3 Å) and group the (the C–C C–S bond bond between is equal thiocarbox to 1.8 Å, theyl C–O group bond and is ca.acridine 1.3 Å) moietyand the in C–C acridinium bond between thioester thiocarboxyl (Scheme group 2, ATE) and increases acridine moietyslightly in compared acridinium to thioester the same (Scheme bond in2, acridiniumATE) increases ester slightly (Scheme compared 2, AE). The to the reduction same bond of electronegativity in acridinium ester and (Scheme increasing2, AE). the The bond reduction lengths withinof electronegativity the leaving andgroup increasing suggest the easier bond elimination lengths within of the the leaving groupgroup suggestand thus easier more elimination efficient transformationof the leaving group to the and electronically thus more efficient excited transformation product. Other to aspects the electronically to consider excited are feasible product. for Other the synthesisaspects to of consider new chemiluminogens are feasible for the after synthesis the oxygen-by of new chemiluminogens-sulphur exchange after and thetheir oxygen-by-sulphur stability of using inexchange applications. and their Based stability on the of usingliterature in applications. [1,5,12,13,17], Based it can on thebe predicted literature [that1,5,12 the,13 ,17synthesis], it can beof acridiniumpredicted that thioesters the synthesis will be of similar acridinium to those thioesters of acridinium will be similar esters toand those will of proceed acridinium in three esters steps: and willthe conversionproceed in threeof acridine-9-carboxylic steps: the conversion acid of acridine-9-carboxylic into 9-chlorocarbonyl acid intoacridine 9-chlorocarbonyl hydrochloride, acridine the esterificationhydrochloride, of the9-chlorocarbonyl esterification of acridine 9-chlorocarbonyl to acridine acridine thioester to and acridine the methylation thioester and of the the methylation endocyclic nitrogenof the endocyclic atom to obtain nitrogen acridinium atom to obtain salt. Considering acridinium salt.the physical Considering or/and the chemical physical properties or/and chemical of the sulphurproperties atom, of theit can sulphur be assumed atom, that it can also be acridinium assumed thioesters that also will acridinium be stable thioesters in aqueous will and be organic stable environments.in aqueous and The organic chemiluminogens environments. Thecontaining chemiluminogens sulphur atom containing are known sulphur [13], atom so arewe knownexpect [the13], possibilityso we expect of the application possibility ofof application acridinium of acridiniumthioesters thioestersas a chemiluminogenic as a chemiluminogenic fragment fragment in chemiluminescentin chemiluminescent labels. labels.
Scheme 2. 2. ComparisonComparison of of structural structural properties properties within within the the leaving leaving group group of acridinium of acridinium ester ester (AE) (AE)and investigatedand investigated acridinium acridinium thioester thioester (ATE). (ATE).
The models selected to investigation are 10-methyl-9-(thiophenoxycarbonyl)acridinium cations substituted with various alkyl alkyl groups groups or an elec electronegativetronegative methoxy group, nitro group or halogen in the thiophenyl fragment (Scheme 33,, a–h)a–h) andand alsoalso inin thethe acridineacridine moietymoiety (Scheme(Scheme3 ,3, i–l). i–l). These These compounds were selected for investigations since th thee electron-attracting features of the substituents in the thiophenyl fragmentfragment maymay substantiallysubstantially a affectffect their their a ffiaffinitynity for for nucleophiles nucleophiles and and susceptibility susceptibility to tooxidation oxidation (chemiluminescence (chemiluminescence effi efficiency),ciency), and and consequently consequently their their applicability. applicability.
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SchemeScheme 3.3. StructureStructure of of investigated investigated acridinium acridinium thioesters thioes (ATEs)ters (ATEs) with the with numbering the numbering of atoms indicated.of atoms indicated.Scheme 3. Structure of investigated acridinium thioesters (ATEs) with the numbering of atoms 2.2. Orbitalsindicated. 2.2. Orbitals As is known from previous studies [12,13,17], acridinium derivatives may react with oxidants 2.2. Orbitals in alkalineAs is known media from relatively previous easily, studies yielding [12,13,17], electronically acridinium excited derivatives products may that react are with able oxidants to emit inlight. alkaline In theAs media is environment known relatively from ofprevious easily, the reaction yieldingstudies (alkaline [12,13,17], electronic media), acridiniumally excited an oxidantderivatives products dissociates may that react are to withable the tooxidants anionic emit light. form in alkaline media relatively easily, yielding electronically excited products that are able to emit light. Inand the then environment reacts with of acridinium the reaction cation, (alkaline which media) initiates, an the oxidant chemiluminescence dissociates to reaction.the anionic The form anion and of In the environment of the reaction (alkaline media), an oxidant dissociates to the anionic form and thenthe oxidant reacts with is the acridinium nucleophile cation, and it willwhich attack initiate the electrophilics the chemiluminescence center in the acridinium reaction. The cation. anion To of better the then reacts with acridinium cation, which initiates the chemiluminescence reaction. The anion of the oxidantunderstandoxidant is the whereis nucleophilethe nucleophile the electrophilic and and it willit will center attack attack of the the electrophilicmoleculeelectrophilic is located,center center in itinthe canthe acridinium referacridinium to the cation. frontier cation. To better molecularTo better understandorbitalunderstand theory where [ 45where]. the According the electrophilic electrophilic to this center theory,center ofof which thethe molecule describes is is located, thelocated, electronic it canit can refer structure refer to theto offrontierthe molecules, frontier molecularthe bondingmolecular orbital between orbital theory theory the [45]. molecules [45]. According According approximates to to thisthis theory, the molecular whichwhich describes describes orbitals the the aselectronic linearelectronic combinationsstructure structure of of of molecules,atomicmolecules, orbitals the (LCAO).thebonding bonding Therefore, between between thethe the Lowest moleculesmolecules Unoccupied approximates Molecular the the molecular Orbitalmolecular (LUMO) orbitals orbitals distributionas linearas linear of combinationsan electrophilecombinations of determines atomicof atomic orbitals theorbitals molecular (LCAO). (LCAO). center TherefTheref sensitiveore, thethe to Lowest nucleophilicLowest Unoccu Unoccu attack.piedpied Molecular The Molecular computationally Orbital Orbital (LUMO)predicted(LUMO) distribution LUMO distribution orbitals of ofan ofanelectrophile acridiniumelectrophile determine determine thioesters withthethe molecularmolecular indicating center center potential sensitive sensitive electrophilic to nucleophilicto nucleophilic centers: attack. The computationally predicted LUMO orbitals of acridinium thioester with indicating attack.the endocyclic The computationally C9 and the carbonyl predicted C15 atom, LUMO and orbita the valuesls of ofacridinium the LCAO coethioesterfficient with of the indicating pz atomic potential electrophilic centers: the endocyclic C9 and the carbonyl C15 atom, and the values of the potentialorbital in electrophilic LUMO orbitals centers: at these the centersendocyclic are shownC9 and in the Figure carbonyl1. The C15 average atom, value and the of the values calculated of the LCAO coefficient of the pz atomic orbital in LUMO orbitals at these centers are shown in Figure 1. LCAOLCAO coefficient coefficient of the ppzz LUMOatomic orbital ofin theLUMO investigated orbitals at ATEs these is equalcenters to are 0.317 shown at the in endocyclic Figure 1. The average value of the calculated LCAO coefficient of the pz LUMO orbital of the investigated ATEs TheC9 atomaverageis equal and valueto 0.0140.317 of at the the the calculated endocyclic carbonyl LCAO C9 C15 atom atom coefficient and (for 0.014 more ofat thethe details pcarbonylz LUMO see C15 orbital Table atom S1 of (for inthe more the investigated Supplementary details see ATEs isMaterials). equalTable to S10.317 The in the ca.at Supplementary the 20 timesendocyclic higher Materi C9 LCAO atomals). The coeand ffica. 0.014cient 20 times at at the C9 higher carbonyl than LCAO at C15 C15 coefficient atom atom implies (forat C9 more than that atdetails C9 C15 rather see Tablethanatom C15S1 in implies makesthe Supplementary that the C9 site rather of the than Materi primary C15als). makes The nucleophilic the ca. site 20 oftimes the attack primary higher of LCAOnucleophilic the anionic coefficient attack form ofat of theC9 the anionicthan oxidants. at C15 atomThe Mullikenform implies of the that partial oxidants. C9 rather charges The thanMulliken at C15 the samepartialmakes potentialcharges the site at of electrophilicthe the same primary potential center nucleophilic electrophilic are also attack showncenter of are the in alsoTable anionic S1 formin the shownof Supplementary the inoxidants. Table S1 The in Materials. the Mulliken Supplementary The partial computational Materials.charges at The the result computational same of potential Mulliken result electrophilic charge of Mulliken at the center charge C9 isare at lower also shownthanthe at in theC9 Table is C15 lower S1 (ca. thanin 2 the times). at theSupplementary C15 These (ca. 2 results times). Materials. suggestThese results thatThe suggest thecomputational population that the population result analysis of Mulliken showsanalysis which shows charge atom at thehas C9 thewhich is largest lower atom deficitthan has theat of the largest the C15 negative deficit (ca. 2 times).of charge the negative These and does resultscharge not indicatesuggestand does wellthat not indicatethe the population location well the of the analysislocation electrophilic ofshows the electrophilic center. In this subject, the more appropriate approach is to use the frontier molecular whichcenter. atom In this has subject, the largest the more deficit appropriate of the negative approach charge is to and use does the frontier not indicate molecular well orbitalthe location theory. of orbital theory. the electrophilic center. In this subject, the more appropriate approach is to use the frontier molecular orbital theory.
FigureFigure 1. LUMO 1. LUMO molecular molecular orbital orbital of of acridinium acridinium thioester thioester (a) (a) with with the the values values of LCAO of LCAO coefficient coeffi cientof of the pz LUMO orbital at C9 and C15 atoms. the pz LUMO orbital at C9 and C15 atoms.
Figure 1. LUMO molecular orbital of acridinium thioester (a) with the values of LCAO coefficient of
the pz LUMO orbital at C9 and C15 atoms.
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2.3. Mechanism of Chemiluminescence
Considering the reaction of acridinium derivatives with oxidants (e.g., OOH−) in alkaline environment (e.g., in presence of OH−), two processes should be considered: the reaction leading to the light emission (chemiluminescence) and other competitive processes (so-called dark processes or non-CL processes). Schemes4 and5 present the processes that account for CL and non-CL processes, respectively (the summary of all investigated processes is shown in Scheme S1 in the Supplementary Materials). The theoretically predicted thermodynamic data for all reaction steps are given in Table1, whileInt. J. Table Mol. Sci.2 contains 2020, 21, 4417 kinetic characteristics of the selected steps. Additionally, the density functional8 of 16 theory (DFT)-optimized geometries originating from the acridine molecules produced by the reaction results of computational study on excited states of 1,2-dioxetane suggest that in the first step the O−O of ATEs with OOH− or OH− are shown in Table S3 (lowest energy and transition state structures) bond is broken and the molecule enters in a region of biradical character, and next the chemiexcitation in the Supplementary Materials. occurs concomitantly with C–C bond breaking [52,53].
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such as –OCH3, into the acridine moiety reduces the activation barrier with similar or slightly lower Scheme 4. CL pathway of acridinium thioester (ATE). values of thermodynamic Schemecharacteristics 4. CL pathway of individual of acridinium steps thioesterof CL pathway. (ATE). 2.3.2. Non-CL Processes The values of quantum yield of chemiluminescence of acridinium derivatives (2–7% in aqueous environments [54,55]) indicate such a small portion of chemiluminogen is transformed into electronically excited product, which leads to the light emission. Unfortunately, most of the molecules undergo competitive reactions (the dark processes), that do not lead to the formation of electronically excited 10-methyl-9-acridinone, but 10-methyl-9-acridinone in the ground state. The first group of transformation of acridinium derivatives, which not leading to the electronically excited products, is all reactions, which transform of the acridinium cation to the non-emitting product (Scheme 5, pathway starting with step VI), and the second group is the hydrolysis of the acridinium molecule (Scheme 5, pathway XI). Both above-mentioned groups significantly reduce the chemiluminescence quantum yield of acridinium derivatives. The first group of the dark processes starts from the nucleophilic attack of OH– at the endocyclic carbon atom (C9) of acridine moiety, creating of so-called ‘pseudobase’. The computationally predicted thermodynamic data (Table 1) indicated that the two – stepsScheme of competitive 5. Non-CLNon-CL processes pathways pathways— ofleading of ATEs: ATEs: (toa, ( atheblue), blue) CL the (step the transformation transformation I, addition ofof thethe of the OOHacridinium acridinium) and cationthe cation formation to the to – of thenon-emittingthe non-emitting ‘pseudobase’ product, product, (step and and VI,(b, orange) (baddition, orange) the of thehydrolysis the hydrolysis OH of) theare of the acridiniumpossible acridinium for molecule. molecule.all of investigated ATEs. Considering the values of Gibbs’ free energies in aqueous environment, one can be noted that the addition of OH– into position 9 of the acridine moiety is thermodynamically more preferred than the OOH–. In this case, the kinetic factors will be determined the preference for the addition of ions. All proposed non-CL steps (VI–X) are possible for all investigated compounds in gaseous and aqueous phases. Our calculations reveal that the activation barriers (Table 2) predicted for step VIII are relatively small (in the range of 0.3–2.8 kcal mol–1). On the other hand, the thermodynamic data show that the energy released in this step exceeds that which is necessary to cause the electronic excitation of product of CL reaction. Therefore, steps VI–VIII will not lead to the formation of electronically excited 10-methyl-9-acridinone. The thermodynamic data suggest that step X can lead to the formation of product in electronically excited state (Table 1), but the activation barriers (Table 2) predicted for this step are very high (43.3 and 36.1 kcal mol–1 for unsubstituted (compounds a–h) and substituted (compounds i–l), respectively), so the step X will not occur. In this situation, both the above pathways may account for the dark transformation of ATEs. Comparing the thermodynamic and kinetic results of non-CL pathway, starting with the formation of ‘pseudobase’, of unsubstituted (compounds a–h) and substituted (compounds i–l) in the acridine moiety derivatives (Tables 1 and 2, stepsFigure VI 3.– RelativeX), no significant Gibbs free differencesenergies of thein thereaction values steps of ofenthalpies two possibilities and Gibbs of non-CL free energies pathways can of be seen.acridinium The Gibbs thioesters: free energy (a, black) profiles unsubstituted of the full andnon-CL (b, blue) pathway substituted (Figure in 3)the for acridine acridinium moiety. thioesters without any substituent in the acridine moiety (derivative a) and with the methoxy group in the 2 positionAmong in thethe acridine compounds moiety (derivativeinvestigated, i), show f (electron-acceptor that the addition of ansubstituent electron-donor in 4 substituent, position of thiophenoxy group), g (two electron-acceptor substituents in 2 and 6 positions of thiophenoxy group) and h (two electron-donor substituents in 2 and 6 positions and electron-acceptor substituent in 4 position of thiophenoxy group) pay attention. The thermodynamic results indicate decrease of Gibbs free energy values compared to the same values for other compounds investigated. The highest decrease can be seen in the case of II (formation of cyclic intermediate 3) and IX (hydrolysis of acridinium cation) steps. It can be assumed, that the addition of an electron-acceptor substituents in 2 and 6 positions or an electron-donor substituents in 2 and 6 position while an electron-acceptor substituent in 4 position, will affect the more efficient transformation into electronically excited product, but also sensitizes to the hydrolysis of ATEs. Considering the utilitarian perspectives of the studied compounds, we compared the energetical values of the main steps (the chemiluminescence pathway and the formation of the pseudobase as affecting the whole process) between acridinium thioesters and currently used in the analysis – acridinium esters. Figure S2 in the Supplementary Materials presents the Gibbs free energy profiles of the CL pathway and the first step of the non-CL pathway (formation of the pseudobase) of acridinium thioesters and acridinium esters. The results show slight differences of the values of Gibbs free energies for step I (addition of OOH– at the C9 atom on acridinium moiety; –44.8 and –43.1 kcal mol–1 for AE and ATE, respectively) and VI (formation of the pseudobase; –62.8 and –61.9 kcal mol–1
Int. J. Mol. Sci. 2020, 21, 4417 6 of 17
1 Table 1. Thermodynamic data of the elementary steps of the reaction of ATE with OOH− and OH−.
Gaseous Phase Aqueous Phase Gaseous Phase Aqueous Phase Step No. Compound Step No. Compound 0 0 0 0 0 0 ∆r,298H ∆r,298G ∆r,298G ∆r,298H ∆r,298G ∆r,298G I a 166.2 153.0 43.1 II a 80.1 93.2 69.0 − − − − − − b 166.0 153.1 43.2 b 86.3 99.5 73.3 − − − − − − c 167.1 152.7 44.9 c 80.3 93.9 67.7 − − − − − − d 165.8 152.0 43.8 d 83.3 97.2 67.5 − − − − − − e 163.6 150.0 42.3 e 75.8 89.7 67.8 − − − − − − f 171.1 157.4 44.0 f 101.7 115.0 79.3 − − − − − − g 166.0 151.6 44.1 g 92.1 105.1 76.2 − − − − − − h 171.0 158.0 44.6 h 102.2 115.1 78.9 − − − − − − i 162.3 149.4 41.3 i 80.1 93.1 68.7 − − − − − − j 162.5 149.5 41.2 j 85.9 99.6 73.3 − − − − − − k 162.6 148.9 41.6 k 80.9 94.5 68.9 − − − − − − l 161.5 148.2 41.2 l 83.7 97.3 68.2 − − − − − − III a–h 16.5 27.2 33.2 IV a–h 78.1 77.6 73.3 − − − − − − i–l 23.0 32.6 38.7 i–l 72.3 71.8 68.2 − − − − − − V a–h 94.6 103.9 106.5 VII a 64.7 63.9 37.4 − − − − − − i–l 95.3 104.5 107.0 b 76.2 75.5 38.9 − − − − − − VI a 197.1 187.5 61.9 c 72.1 72.6 37.9 − − − − − − b 188.9 179.5 61.7 d 73.6 74.3 37.4 − − − − − − c 189.9 178.6 62.3 e 64.1 63.8 37.1 − − − − − − d 188.8 178.5 62.3 f 83.0 83.0 41.2 − − − − − − e 186.4 176.0 60.7 g 80.9 81.3 44.7 − − − − − − f 194.3 183.7 62.6 h 82.4 83.4 40.4 − − − − − − g 188.9 177.6 62.1 i 73.6 73.2 38.0 − − − − − − h 194.3 183.6 62.9 j 77.3 77.0 39.3 − − − − − − i 185.1 175.4 59.1 k 73.2 74.0 37.7 − − − − − − j 184.7 175.3 59.5 l 74.4 74.7 37.5 − − − − − − k 185.7 174.9 60.3 IX a 92.0 93.2 68.9 − − − − − − l 184.8 174.9 60.5 b 97.9 99.6 73.3 − − − − − − VIII a 13.2 37.1 48.6 c 93.0 93.6 69.4 − − − − − − b 15.9 39.8 51.7 d 94.8 96.1 68.0 − − − − − − c 14.0 37.6 48.2 e 85.0 86.7 66.8 − − − − − − d 15.4 38.7 47.3 f 118.6 119.4 80.1 − − − − − − e 11.7 35.5 48.0 g 103.7 103.7 77.1 − − − − − − f 24.3 48.1 55.3 h 118.9 120.2 80.3 − − − − − − g 16.9 40.0 49.3 i 92.3 93.4 68.9 − − − − − − h 25.2 48.4 55.9 j 98.3 100.1 73.4 − − − − − − i 13.0 36.6 49.2 k 93.3 94.4 69.4 − − − − − − j 15.8 39.6 52.0 l 95.1 96.5 68.3 − − − − − − k 13.9 37.3 48.8 X a 60.3 58.8 46.9 − − − − − − l 15.3 38.7 47.6 b 74.5 73.2 51.4 − − − − − − XI a–h 83.5 103.8 109.9 c 68.5 68.0 46.9 − − − − − − i–l 84.6 104.8 110.6 d 71.4 70.8 45.5 − − − − − − e 64.1 63.8 45.9 − − − f 89.7 88.9 57.3 − − − g 80.2 79.2 54.7 − − − h 90.0 89.6 57.1 − − − i 68.0 66.7 47.2 − − − j 74.5 73.5 51.4 − − − k 68.4 68.2 46.6 − − − l 71.4 70.3 45.2 − − − 1 0 0 1 ∆r,298H and ∆r,298G (both in kcal mol− ), respectively, represent the enthalpy and Gibbs’ free energy (gaseous phase) or free energy (aqueous phase) of the reaction corresponding to a given step number at standard temperature and pressure.
Table 2. Kinetic characteristics 1 of the selected steps of the CL reaction of ATEs.
Gaseous Phase Aqueous Phase Step No. Compound 0 0 0 0 ∆r,298H ∆r,298G 298k (298τ99) ∆r,298G TS-III a–h 12.9 14.2 2.5 102 (1.9 10 2) 15.6 × × − i–l 12.8 14.2 2.4 102 (1.9 10 2) 14.5 × × − TS-VIII a 2.1 0.3 4.1 1012 (1.1 10 12) 2.1 × × − b 2.3 1.2 8.9 1011 (5.2 10 12) 1.6 × × − c 1.9 0.9 1.3 1012 (3.4 10 12) 2.4 × × − d 2.4 1.7 3.8 1011 (1.2 10 11) 0.5 × × − Int. J. Mol. Sci. 2020, 21, 4417 7 of 17
Table 2. Cont.
Gaseous Phase Aqueous Phase Step No. Compound 0 0 0 0 ∆r,298H ∆r,298G 298k (298τ99) ∆r,298G e 2.4 1.1 1.0 1012 (4.5 10 12) 2.1 × × − f 1.1 0.4 3.2 1012 (1.5 10 12) 1.3 × × − g 3.1 2.2 1.5 1011 (3.1 10 10) 2.8 × × − h 0.5 0.4 3.4 1012 (1.3 10 12) 0.3 × × − i 2.7 1.5 4.8 1011 (9.5 10 12) 2.4 × × − j 2.3 1.2 8.6 1011 (5.4 10 12) 2.0 × × − k 2.5 1.5 4.9 1011 (9.3 10 12) 2.0 × × − l 2.5 0.8 1.7 1012 (2.7 10 12) 2.7 × × − TS-XI a–h 43.6 45.3 4.1 10 21(1.1 1021) 43.3 × − × i–l 35.1 36.9 6.0 10 15(7.7 1014) 36.1 × − × 1 0 0 1 ∆a,298H and ∆a,298G (both in kcal mol− ), respectively, represent the enthalpy and Gibbs’ free energy (gaseous 0 1 phase) or free energy (aqueous phase) of activation at standard temperature and pressure; 298k (in s− ) and 298τ99, respectively, denote the rate constant and the time after which the reaction is 99% complete.
2.3.1. Chemiluminescence Pathway The chemiluminescence reaction begins from nucleophilic attack of anionic form of oxidant (e.g., OOH−) at the C9 atom on acridinium moiety (step I). In the next steps, the addition product 2 reacts with an OH− ion and after elimination of the thiophenyl anion, forms cyclic intermediate 3 (step II), which after the elimination of carbon dioxide leads to the energy-rich 10-methyl-9-acridinone (5 *, step III), and then followed by the relaxation of electronically excited 10-methyl-9-acridinone to the ground state with the emission of light (step IV). The computationally predicted thermodynamic data (Table1) indicated that all proposed CL steps (I–V) are possible for all investigated compounds in gaseous and aqueous phases. Our calculations reveal that the activation barriers (Table2) predicted for step III (formation of electronically excited product) are similar to the activation barriers of the same step predicted for acridinium esters 1 (ca. 15.4 kcal mol− )[12]. Bou˙zyket al. have shown that the necessary value of energy to cause 1 the electronic excitation of 10-methyl-9-acridinone is 88.2 kcal mol− [46]. Our calculation reveal that the energy released in step V (Table1) exceeds that which is necessary to cause the electronic excitation of product of CL reaction. Comparing the thermodynamic and kinetic results of CL pathway (Tables1 and2, steps I–V) of unsubstituted (compounds a–h) and substituted (compounds i–l) in the acridine moiety derivatives, no significant differences in the values of enthalpies and Gibbs free energies can be seen. However, after analyzing the Gibbs free energy profiles of the full reaction leading to the generation of light (Figure2) for acridinium thioesters without any substituent in the acridine moiety (derivative a) and with the methoxy group in the 2 position in the acridine moiety (derivative i), one can conclude that the addition of an electron-donor substituent, such as –OCH3, into the acridine moiety reduces the activation barrier with similar or slightly lower values of thermodynamic characteristics of individual steps of CL pathway. Int. J. Mol. Sci. 2020, 21, 4417 7 of 16
Int. J. Mol. Sci. 2020 21 substituent, such, ,as 4417 –OCH3, into the acridine moiety reduces the activation barrier with similar8 of 17or slightly lower values of thermodynamic characteristics of individual steps of CL pathway.
FigureFigure 2. 2.Relative Relative GibbsGibbs freefree energies energies of of the the chemiluminescence chemiluminescence (CL) (CL) reaction reaction steps steps (CL (CL pathway) pathway) of of acridiniumacridinium thioesters: thioesters: ( a(a,, black) black) unsubstituted unsubstituted and and ( b(b,, green) green) substituted substituted in in the the acridine acridine moiety. moiety.
ComparingComparing thethe chemiluminescent properties properties of of acridinium acridinium thioesters, thioesters, the the nature nature of the of thedecomposition decomposition of cyclic of cyclic intermediates intermediates (structure (structure 3, Scheme 3, Scheme 4) 4should) should be beconsidered. considered. This This entity entity has hastwo two important important bonds bonds (O (O–O–O and and C– C–C),C), which which have have an an effect effect on on the the tr transformationansformation in thethe electronicallyelectronically excitedexcited product—10-methyl-9-acridinone. product—10-methyl-9-acridinone. In In geometrical geometrical terms, terms, the the bond bond lengths lengths of C–C of C and–C and O–O O are–O 1.532are 1.532 Å and Å 1.491and 1.491 Å, respectively Å, respectively and both and appear both appe to bear covalent. to be covalent. The VDD The population VDD population analysis analysis results alsoresults present also apresent typical a distributiontypical distribution of charges of ch (seearges Table (see S2 Table in the S2 Supplementary in the Supplementary Materials)—oxygen Materials)— atomsoxygen have atoms a negative have a negative charges charges and carbon and atomscarbon haveatoms positive have positive charges. charges. It can It be can concluded be concluded that thethat combination the combination of negative of negative charges charges of both of oxygen both oxygen atoms andatoms the and small the di smallfference difference between between charge indicatescharge indicates a repulsive a repulsive electrostatic electrostatic interaction interactio betweenn between oxygen atoms.oxygen Inatoms. the case In the of carboncase of atoms,carbon theatoms, charge the di chargefferences differences are significantly are significantly higher, which higher, also indicates which also a repulsive indicates eff ecta repulsive of interaction, effect but of attenuatedinteraction, by but the polarizationattenuated effbyect. the To betterpolarization understand effect. the natureTo better of intramolecular understand interactionsthe nature in of a dioxetanoneintramolecular entity, interactions the calculations in a dioxetanone were performed entity, using the the calculations quantum theory were ofperformed atoms in moleculesusing the (QTAIM)quantum [47theory]. The of topology atoms in of molecules the electron (QTAIM) localization [47]. functionThe topology using aof bond the electron critical pointlocalization (BCP) (seefunction Table using S2 in thea bond Supplementary critical point Materials) (BCP) (see indicates Table S2 that in thethe C–C Supplementary bond is a typical Materials) covalent indicates bond (combinationthat the C–C bond of negative is a typical value covalent of Laplacian bond ((combi2ρ(r))nation with highof negative value of value electron of Laplacian density distribution(∇2ρ(r)) with ∇ (ρhigh(r)) andvalue the of O–Oelectron bond density is charge-shifted distribution (combination (ρ(r)) and the ofO– positiveO bond valueis charge-shifted of Laplacian (combination ( 2ρ(r)) with of ∇ highpositive value value of electron of Laplacian density (∇ distribution2ρ(r)) with (highρ(r)) value [48,49 ].of Toelectron determine densityσ or distributionπ bond character, (ρ(r)) the[48,49]. values To ofdetermine the ellipticity σ or (πε) bond were analysedcharacter, (see the Table values S2 of in the the ellipticity Supplementary (ε) were Materials). analysed The (see analysis Table S2 of thesein the parametersSupplementary demonstrates Materials). that The both analysis O–O and of C–C these bonds parameters represent demonstrates a sigma bond that character both O (larger–O and values C–C (>bonds0.1) indicate representπ bond a sigma character bond andcharacter lower values(larger indicatevalues (>0.1)σ bond indicate character π bond [50]). character However, and the valuelower ofvalues ellipticity indicate for O–O σ bond bond character is very close[50]).to However, the value the that value indicates of ellipticityπ bond character.for O–O bond Finally, is very the ratioclose of to –(G(rthe valueb)/V(r bthat)) was indicates used as π a criterionbond character. of the nature Finally, of bondsthe ratio (Table of – S2,(G(r Supplementaryb)/V(rb)) was used Material). as a criterion For O–O of bond,the nature value of thesebonds parameters (Table S2, demonstrateSupplementary covalent Material). bond, For and O for–O C–C bond, bond, value the of –(G(r theseb)/ V(rparametersb)) ratio isdemonstrate lower than thecovalent range indicatingbond, and partially for C–C covalent bond, bondthe –(G(r (largerb)/V(r valuesb)) ratio (–(G(r isb )lower/V(rb)) than> 1.0) the indicate range noncovalentindicating partially bond character covalent and bond values (larger in thevalues range (–(G(r (0.5b)/V(r< –(G(rb)) b>) /1.0)V(r bindicate)) < 1.0) noncovalent indicate partially bond covalentcharacter bond and charactervalues in [the51]). range The (0.5 results < –(G(r of calculationb)/V(rb)) < 1.0) using indicate quantum partially theory covalent of atoms bond in molecules character does[51]). not The clearly results indicate of calculation the covalent using quantum nature of theory the O–O of andatoms C–C in molecules bonds in the does cyclic not clearly intermediates. indicate Onthe the covalent other hand,nature the of resultsthe O– ofO computationaland C–C bonds study in the on cyclic excited intermediates. states of 1,2-dioxetane On the other suggest hand, that the
Int. J. Mol. Sci. 2020, 21, 4417 9 of 17 in the first step the O O bond is broken and the molecule enters in a region of biradical character, − and next the chemiexcitation occurs concomitantly with C–C bond breaking [52,53].
2.3.2. Non-CL Processes The values of quantum yield of chemiluminescence of acridinium derivatives (2–7% in aqueous environments [54,55]) indicate such a small portion of chemiluminogen is transformed into electronically excited product, which leads to the light emission. Unfortunately, most of the molecules undergo competitive reactions (the dark processes), that do not lead to the formation of electronically excited 10-methyl-9-acridinone, but 10-methyl-9-acridinone in the ground state. The first group of transformation of acridinium derivatives, which not leading to the electronically excited products, is all reactions, which transform of the acridinium cation to the non-emitting product (Scheme5, pathway starting with step VI), and the second group is the hydrolysis of the acridinium molecule (Scheme5, pathway XI). Both above-mentioned groups significantly reduce the chemiluminescence quantum yield of acridinium derivatives. The first group of the dark processes starts from the nucleophilic attack of OH− at the endocyclic carbon atom (C9) of acridine moiety, creating of so-called ‘pseudobase’. The computationally predicted thermodynamic data (Table1) indicated that the two steps of competitive processes—leading to the CL (step I, addition of the OOH−) and the formation of the ‘pseudobase’ (step VI, addition of the OH−) are possible for all of investigated ATEs. Considering the values of Gibbs’ free energies in aqueous environment, one can be noted that the addition of OH− into position 9 of the acridine moiety is thermodynamically more preferred than the OOH−. In this case, the kinetic factors will be determined the preference for the addition of ions. All proposed non-CL steps (VI–X) are possible for all investigated compounds in gaseous and aqueous phases. Our calculations reveal that the activation barriers (Table2) predicted for step VIII are relatively small (in the range of 1 0.3–2.8 kcal mol− ). On the other hand, the thermodynamic data show that the energy released in this step exceeds that which is necessary to cause the electronic excitation of product of CL reaction. Therefore, steps VI–VIII will not lead to the formation of electronically excited 10-methyl-9-acridinone. The thermodynamic data suggest that step X can lead to the formation of product in electronically excited state (Table1), but the activation barriers (Table2) predicted for this step are very high (43.3 1 and 36.1 kcal mol− for unsubstituted (compounds a–h) and substituted (compounds i–l), respectively), so the step X will not occur. In this situation, both the above pathways may account for the dark transformation of ATEs. Comparing the thermodynamic and kinetic results of non-CL pathway, starting with the formation of ‘pseudobase’, of unsubstituted (compounds a–h) and substituted (compounds i–l) in the acridine moiety derivatives (Tables1 and2, steps VI–X), no significant di fferences in the values of enthalpies and Gibbs free energies can be seen. The Gibbs free energy profiles of the full non-CL pathway (Figure3) for acridinium thioesters without any substituent in the acridine moiety (derivative a) and with the methoxy group in the 2 position in the acridine moiety (derivative i), show that the addition of an electron-donor substituent, such as –OCH3, into the acridine moiety reduces the activation barrier with similar or slightly lower values of thermodynamic characteristics of individual steps of CL pathway. Int. J. Mol. Sci. 2020, 21, 4417 9 of 16
such as –OCH3, into the acridine moiety reduces the activation barrier with similar or slightly lower values of thermodynamic characteristics of individual steps of CL pathway.
Int. J. Mol.Scheme Sci. 2020 5., 21 Non-CL, 4417 pathways of ATEs: (a, blue) the transformation of the acridinium cation to the 10 of 17 non-emitting product, and (b, orange) the hydrolysis of the acridinium molecule.
FigureFigure 3. Relative3. Relative Gibbs Gibbs free free energies energies of of the the reactionreaction stepssteps of two possibilities of of non-CL non-CL pathways pathways of of acridiniumacridinium thioesters: thioesters: (a (,a black), black) unsubstituted unsubstituted and and ((bb,, blue)blue) substituted in in the the acridine acridine moiety. moiety.
AmongAmong the the compounds compounds investigated, investigated, f (electron-acceptor f (electron-acceptor substituent substituent in 4 position in 4 ofposition thiophenoxy of group),thiophenoxy g (two electron-acceptorgroup), g (two electron-acceptor substituentsin substituents 2 and 6 positions in 2 and of6 positions thiophenoxy of thiophenoxy group) and group) h (two electron-donorand h (two electron-donor substituents insubstituents 2 and 6 positions in 2 and and6 positions electron-acceptor and electron-acceptor substituent substituent in 4 position in 4 of thiophenoxyposition of group)thiophenoxy pay attention. group) pa They attention. thermodynamic The thermodynamic results indicate results decrease indicate of decrease Gibbs free of Gibbs energy valuesfree comparedenergy values to the compared same values to the for same other values compounds for other investigated. compounds The investigated. highest decrease The highest can be seendecrease in the casecan ofbe II seen (formation in the case of cyclic of II intermediate (formation of 3) cyclic and IX intermediate (hydrolysis of3) acridiniumand IX (hydrolysis cation) steps. of acridinium cation) steps. It can be assumed, that the addition of an electron-acceptor substituents in It can be assumed, that the addition of an electron-acceptor substituents in 2 and 6 positions or an 2 and 6 positions or an electron-donor substituents in 2 and 6 position while an electron-acceptor electron-donor substituents in 2 and 6 position while an electron-acceptor substituent in 4 position, substituent in 4 position, will affect the more efficient transformation into electronically excited will affect the more efficient transformation into electronically excited product, but also sensitizes to product, but also sensitizes to the hydrolysis of ATEs. the hydrolysis of ATEs. Considering the utilitarian perspectives of the studied compounds, we compared the energetical valuesConsidering of the main the steps utilitarian (the chemiluminescence perspectives pathway of the and studied the formation compounds, of the pseudobase we compared as theaffecting energetical the valueswhole process) of the main between steps acridinium (the chemiluminescence thioesters and currently pathway used and in the the formation analysis – of theacridinium pseudobase esters. as a ffFigureecting S2 the in wholethe Supplementary process) between Materials acridinium presents the thioesters Gibbs free and energy currently profiles used in theof the analysis–acridinium CL pathway and the esters. first step Figure of S2the inno then-CL Supplementary pathway (formation Materials of the presents pseudobase) the Gibbs of freeacridinium energy profiles thioesters of and the acridinium CL pathway esters. and The the resu firstlts stepshow ofslight the differences non-CL pathway of the values (formation of Gibbs of thefree pseudobase) energies for of step acridinium I (addition thioesters of OOH and– at acridiniumthe C9 atom esters. on acridinium The results moiety; show –44.8 slight and di –ff43.1erences kcal of themol values–1 for of AE Gibbs and ATE, free energies respectively) for step and IVI (addition (formation of OOHof the− pseudobase;at the C9 atom –62.8 on and acridinium –61.9 kcal moiety;mol–1 1 44.8 and 43.1 kcal mol− for AE and ATE, respectively) and VI (formation of the pseudobase; − − 62.8 and 61.9 kcal mol 1 for AE and ATE, respectively) and significant differences of the values − − − of Gibbs free energies for step II (formation of the cyclic intermediate; 47.6 and 69.0 kcal mol 1 − − − for AE and ATE, respectively) between acridinium esters and acridinium thioesters. A comparison of these values suggests the more thermodynamically preferred formation of the cyclic intermediate for acridinium thioesters, and thus its decomposition to the electronically excited product. It can be concluded that acridinium thioesters will be more efficient chemiluminogens than acridinium esters.
2.4. Electronically Excitation The mechanism of chemiluminescence reaction of ATEs indicates that CL pathway leads to energy-rich 10-methyl-9-acridinone, which returning to the ground state emits light. In our previous studies on acridinium esters [12,17], we reported formation of electronically excited product after elimination of phenyl carbonate anion [17] or firstly, phenoxy anion and secondly, carbon dioxide from dioxetane intermediate [12]. As mentioned in Section 2.3.1., the formation of energy-rich 10-methyl-9-acridinone in the CL reaction of acridinium thioesters follows by the transformation to the dioxetane intermediate after the elimination of the thiophenyl anion, and then the elimination Int. J. Mol. Sci. 2020, 21, 4417 11 of 17 of the carbon dioxide (Scheme4, step III and IV). The formation of cyclic intermediate 3 (Scheme4) and then its decomposition is a rate determining step of the chemiluminescence. To better understand the nature of an electronically excited product, an investigation of excited states was carried out using the time dependent (TD) DFT calculation. As is known, the intersystem crossing (ISC) mechanism, which is the nonradiative transition between two electronic states of different multiplicity, plays an important role in photochemistry and explains well the properties of organic chromophores [56,57]. Taking into account the previous reports about the decomposition mechanism of 1,2-dioxetane [52], 1,2-dioxetanedione [58] and acridinium esters [5], the spin-orbit coupling between the S0 and the T1 states occurs along the chemiluminescence reaction pathway. The computed energy profiles on the singlet (S1), triplet (T1) and ground (S0) states started from the transition state of decomposition of cyclic intermediate 3 (TS-III) show that the potential energy curve of the triplet state (T1) lays closer than the potential energy curve of the singlet state (S1) to the potential energy curve of the ground state (S0). Figure4 and Figure S3 in the Supplementary Materials show the energy diagram of CL pathway in aqueous and gaseous phases, respectively, including thermal decomposition (S0) and formation of excited states (S1 and T1). The vertical excitation energies in aqueous phase of 10-methyl-9-acridinone in equilibrium geometry are 2.51 and 2.26 eV for T1 state and 3.29 and 3.33 eV for S1 state for unsubstituted and substituted in the acridine moiety, respectively. Therefore, the emission energy from T1 corresponds well to experimental results that show the maximum emission speak at 451 nm (2.75 eV) for 10-methyl-9-acridinone [46]. It should be noted that the emission energy from S1 state in gaseous phase for unsubstituted in the acridine moiety product of the chemiluminescence reaction are higher than that for the product substituted in position 2 in acridine moiety (4.82 and 3.23 eV, respectively). This difference decreases in the aqueous phase as indicated above. According to this, the photoemission of the mixed state (from T1 and S1 states) of 10-methyl-9-acridinone in water solution is expected. On the other hand, the phosphorescence will be quenched in solution, and we expect that it may not play a significant role in the chemiluminescence of acridiniumInt. J. Mol. derivatives.Sci. 2020, 21, 4417 11 of 16
FigureFigure 4. Energy4. Energy diagram diagram of of the the chemiluminescencechemiluminescence reaction reaction inin the the aqueous aqueous phase phase with with energy energy of the of lowest triplet (T1) and singlet (S1) states. the lowest triplet (T1) and singlet (S1) states.
3. Materials3. Materials and and Methods Methods UnconstrainedUnconstrained geometry geometry optimizations optimizations of of isolated isolated moleculesmolecules were carried carried out out at at the the DFT DFT [59] [59 ] andand TD DFTTD DFT [60] level[60] oflevel theory of theory to determine to determine ground andground excited and states, excited respectively. states, respectively. All the calculations All the calculations were conducted using the popular Becke’s three-parameter hybrid functional B3LYP [61,62] and the 6-31G** basis set [63,64] with the Gaussian09 package [65]. B3LYP functional is known to produce reliable results [5,12,17] for the thermodynamic data and have been proven to provide accurate qualitative results for the mechanism of chemiluminescence reaction of investigated molecules. After completion of each optimization, the Hessian (second derivatives of the energy as a function of the nuclear coordinates) was calculated and checked for positive definiteness to assess whether the structures were true minima. The solvent (water) effect included in the DFT calculations at the level of the polarizable continuum model (PCM) was used to mimic water environment (UAHF radii were used to obtain the molecular cavity) [66,67]. The transition energies for the electronically excited product of chemiluminescence reaction of acridinium thioesters—10-methyl-9-acridinone— were calculated from an excited state optimized structure using the state-specific approach. The Voronoi deformation density (VDD) population analysis was performed by using the Multiwfn code [68] with the same level of theory and basis set as geometry optimization. The electron density at the bond critical point (BCP) were analysed using the quantum theory of atoms in molecules (QTAIM) methodology [47]. Visualization of the results of the calculations of equilibrium structures of all molecules was made using the ChemCraft software [69]. The enthalpies (Δr,298H0) and Gibbs free energies (free energies in the case of DFT(PCM)) (Δr,298G0) of the reactions (r), as well as the enthalpies (Δa,298H0) and Gibbs free energies (free energies in the case of DFT(PCM)) (Δa,298G0) of activation (a) were calculated by following the basic rules of thermodynamics [44]. The rate constants (298k0) for the gaseous phase reactions were obtained by applying the equation:
o = RT −Δ o 298 k exp[ a,298G /(RT )] (1) Nh
Resulting from the transition state theory, and reaction completion time (298τ99) from the formula: τ = o 298 99 ln 100 / 298 k (2)
Int. J. Mol. Sci. 2020, 21, 4417 12 of 17 were conducted using the popular Becke’s three-parameter hybrid functional B3LYP [61,62] and the 6-31G** basis set [63,64] with the Gaussian09 package [65]. B3LYP functional is known to produce reliable results [5,12,17] for the thermodynamic data and have been proven to provide accurate qualitative results for the mechanism of chemiluminescence reaction of investigated molecules. After completion of each optimization, the Hessian (second derivatives of the energy as a function of the nuclear coordinates) was calculated and checked for positive definiteness to assess whether the structures were true minima. The solvent (water) effect included in the DFT calculations at the level of the polarizable continuum model (PCM) was used to mimic water environment (UAHF radii were used to obtain the molecular cavity) [66,67]. The transition energies for the electronically excited product of chemiluminescence reaction of acridinium thioesters—10-methyl-9-acridinone—were calculated from an excited state optimized structure using the state-specific approach. The Voronoi deformation density (VDD) population analysis was performed by using the Multiwfn code [68] with the same level of theory and basis set as geometry optimization. The electron density at the bond critical point (BCP) were analysed using the quantum theory of atoms in molecules (QTAIM) methodology [47]. Visualization of the results of the calculations of equilibrium structures of all molecules was made using the ChemCraft software [69]. 0 The enthalpies (∆r,298H ) and Gibbs free energies (free energies in the case of DFT(PCM)) 0 0 (∆r,298G ) of the reactions (r), as well as the enthalpies (∆a,298H ) and Gibbs free energies (free energies 0 in the case of DFT(PCM)) (∆a,298G ) of activation (a) were calculated by following the basic rules of 0 thermodynamics [44]. The rate constants (298k ) for the gaseous phase reactions were obtained by applying the equation: 0 RT 0 k = exp[ ∆a G /(RT)] (1) 298 Nh − ,298
Resulting from the transition state theory, and reaction completion time (298τ99) from the formula:
298τ99 = ln100/298k0 (2) where R, T, N and h denote the gas constant, temperature (298.15 K), Avogadro number and Planck’s constant, respectively [44].
4. Conclusions Due to the rapid growth of population and factors which negatively affect health, the increasing number of diseases motivates the intensive search for new effective diagnostic methods. One of the promising immunological diagnostic procedures is chemiluminescent labelling, in which efficient chemiluminogens are used. Searching for new chemiluminogens with better features, especially higher quantum yields, is the main aim of this work. As acridinium esters are an efficient chemiluminogens, we modelled new group of compounds based on acridinium moiety—10-methyl-9-thiophenoxycarbonylacridinium derivatives. The proposed chemiluminescence reaction mechanism of these compounds was based on our previous computational and experimental studies [12,13,17]. The CL reaction begins with the nucleophilic attack of hydroperoxide anion, then forms highly strained dioxetanone ring after addition of hydroxyl anion, and finally energy-rich 10-methyl-9-acridinone as a result of decomposition of cyclic intermediate. The step, in which the decomposition of the cyclic dioxetanone ring is observed, is the rate-determining step in the whole process. All the considered compounds were computationally proven to easily transform into electronically excited products as a result of the chemiluminescence reaction. However, all investigated derivatives could be divided into two groups: unsubstituted and substituted in the acridine moiety. The addition of an electron-donor substituent, e.g., methoxy group, into the acridine moiety reduces the activation barrier with almost similar values of thermodynamic characteristics of individual steps of CL pathway. Additionally, the studied acridinium derivatives are susceptible to two reactions competing with chemiluminescence: formation of ‘pseudobase’ and hydrolysis. The computational Int. J. Mol. Sci. 2020, 21, 4417 13 of 17 results show that the possible pathway of the reaction, CL or one of the competitive pathways, seems to be exclusively dependent on kinetic factors. The most promising are the addition of an electron-acceptor substituents in the 2 and 6 positions or an electron-donor substituent in the 2 and 6 position while an electron-acceptor substituent in the 4 position moves into the thiophenoxy group. These substitutions will affect the more efficient transformation into an electronically excited product, but also sensitize it to the hydrolysis of investigated compounds. The computationally predicted emission energy from T1 for electronically excited products of CL reaction—10-methyl-9-acridinone corresponds well to experimental results. The theoretical studies of the excited state indicate that the photoemission of the mixed state (from T1 and S1 states) of 10-methyl-9-acridinone in water solution is expected. To sum up, with the use of a simple computational approach, we were able to identify potential chemiluminogens for further experimental studies. Synthesis, followed by studies of their chemiluminescent properties, and ultimately in chemiluminescent labelling, are further steps to confirm their potential applicability in immunodiagnostics.
Supplementary Materials: The following are available online at http://www.mdpi.com/1422-0067/21/12/4417/s1. Figure S1. The VDD population analysis results of the investigated dioxetanone molecules (a) unsubstituted and (b) substituted in the acridine moiety. Figure S2. Relative Gibbs free energies of the CL pathway (reaction steps: I–IV) and non-CL step VI (formation of pseudobase) of acridinium thioesters: (a, black) and acridinium esters (c, violet). Figure S3. Energy diagram of chemiluminescence reaction in the gaseous phase with energy of the lowest triplet (T1) and singlet (S1) states. Scheme S1. Pathways of the reactions of ATE with OOH and OH (CL pathway (green arrows), non-CL pathways: transformation of ATE to the non-emitting product (blue− arrows)− and hydrolysis of ATE (orange arrows)). Table S1. The calculated Mulliken atomic charges and LCAO coefficient of the pZ LUMO orbital at the endocyclic C9 and the carbonyl C15 atoms. Table S2. The VDD population analysis results of the investigated dioxetanone molecules (a) unsubstituted and (b) substituted in the acridine moiety. Table S3. Cartesian coordinates and structures of the lowest energy and transition state structures of investigated molecules. Author Contributions: Conceptualization: B.Z.; methodology: B.Z.; investigation: M.P. and B.Z.; writing—original draft preparation: M.P. and B.Z.; writing—review and editing: B.Z.; visualization: M.P.; supervision: B.Z.; project administration: B.Z.; funding acquisition: M.P. and B.Z. All authors have read and agreed to the published version of the manuscript. Funding: This research was funded by: (1) Polish Ministry of Science and Higher Education within grants BMN 538-8220-B776-17/18 and 539-T080-B490-20 (M.P.); (2) Wroclaw Center for Networking and Supercomputing (wcss.pl) within grant No. 215 (B.Z.). Conflicts of Interest: The authors declare no conflict of interest.
Abbreviations
AE Acridinium ester ATE Acridinium thioester BCP Bond critical point CL Chemiluminescence DFT Density functional theory FT4 Thyroxine hormone HIV Human immunodeficiency virus ISC Intersystem crossing LCAO Linear combination of atomic orbitals LUMO Lowest Unoccupied Molecular Orbital PCM Polarizable Continuum Model QTAIM Quantum theory of atoms in molecules TD DFT Time dependent density functional theory TS Transition State TSH Thyroid-stimulating hormone VDD Voronoi deformation density Int. J. Mol. Sci. 2020, 21, 4417 14 of 17
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