A Non-Conventional Stereogenic Element for Designing Inherently Chiral Ionic Liquids for Electrochemical Enantiodifferentiation

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Article Helicity: A Non-Conventional Stereogenic Element for Designing Inherently Chiral Ionic Liquids for Electrochemical Enantiodifferentiation

Francesca Fontana 1,2,*, Greta Carminati 1, Benedetta Bertolotti 1, Patrizia Romana Mussini 3, Serena Arnaboldi 3, Sara Grecchi 3, Roberto Cirilli 4, Laura Micheli 5 and Simona Rizzo 6,*

1 Dipartimento di Ingegneria e Scienze Applicate, Università di Bergamo, Viale Marconi 5, 24044 Dalmine, Italy; [email protected] (G.C.); [email protected] (B.B.) 2 CSGI Bergamo R.U., Viale Marconi 5, 24044 Dalmine, Italy 3 Dipartimento di Chimica, Università Degli Studi di Milano, Via Golgi 19, 20133 Milano, Italy; [email protected] (P.R.M.); [email protected] (S.A.); [email protected] (S.G.) 4 Centro Nazionale per Il Controllo e la Valutazione dei Farmaci, Istituto Superiore di Sanità, Viale Regina Elena 299, 00161 Rome, Italy; [email protected] 5 Dipartimento di Scienze e Tecnologie Chimiche, Università Degli Studi di Roma Tor Vergata, Via della Ricerca Scientiﬁca, 1, 00133 Roma, Italy; [email protected] 6 CNR Istituto di Scienze e Tecnologie Chimiche “Giulio Natta”, Via C. Golgi 19, 20133 Milano, Italy * Correspondence: [email protected] (F.F.); [email protected] (S.R.)

Abstract: Configurationally stable 5-aza[6]helicene (1) was envisaged as a promising scaffold for non-conventional ionic liquids (IL)s. It was prepared, purified, and separated into enantiomers by preparative HPLC on a chiral stationary phase. Enantiomerically pure quaternary salts of 1 with appropriate counterions were prepared and fully characterized. N-octyl-5-aza[6]helicenium Citation: Fontana, F.; Carminati, G.; bis triflimidate (2) was tested in very small quantities as a selector in achiral IL media to perform Bertolotti, B.; Mussini, P.R.; preliminary electrochemical enantiodifferentiation experiments on the antipodes of two different Arnaboldi, S.; Grecchi, S.; Cirilli, R.; chiral probes. The new organic salt exhibited outstanding enantioselection performance with respect Micheli, L.; Rizzo, S. Helicity: A to these probes, thus opening the way to applications in the enantioselective electroanalysis of Non-Conventional Stereogenic Element for Designing Inherently relevant bioactive molecules. Chiral Ionic Liquids for Electrochemical Enantiodifferentiation. Keywords: azahelicenes; ionic liquids; enantiodifferentiation; chiral additives; inherent chirality; Molecules 2021, 26, 311. https:// chiral voltammetry doi.org/10.3390/molecules26020311

Academic Editor: Jacek Nycz Received: 23 December 2020 1. Introduction Accepted: 3 January 2021 Some of us recently unveiled the amazing potential of chiral electroanalysis [1–3] (in Published: 9 January 2021 remarkable analogy with chiroptics [4,5]) for “inherently chiral” functional compounds, in which the stereogenic scaffold responsible for chirality and the molecular group responsible Publisher’s Note: MDPI stays neu- for their specific properties coincide. This structural combination invariably results in tral with regard to jurisdictional clai- outstanding enantioselection properties that are much greater than those exhibited by ms in published maps and institutional affiliations. compounds in which the stereogenic unit and functional group are independent molecular portions. This strategy was at first implemented in terms of inherently chiral thiophene- based oligomer electrode surfaces. They were prepared using the electrochemical or chemical oxidation of suitable inherently chiral monomers. In most cases these presented Copyright: © 2021 by the authors. Li- with an atropisomeric biheteroaromatic system as the stereogenic element, as in the BT2T4 censee MDPI, Basel, Switzerland. (Figure1) proof-of-concept case [3], or a helix in a recent case [6]. This article is an open access article More recently, the inherent chirality strategy was once again proven to be a winning distributed under the terms and con- approach when implemented in ionic liquid media. The proof-of-concept case was based ditions of the Creative Commons At- on a family of 3,30-bicollidinium salts featuring an atropisomeric biheteroaromatic system tribution (CC BY) license (https:// as a stereogenic element (Alk2BicX2, Figure1)[ 1]. Impressive potential differences were creativecommons.org/licenses/by/ observed for the enantiomers of chiral probes in voltammetry experiments on unmodified 4.0/).

Molecules 2021, 26, 311. https://doi.org/10.3390/molecules26020311 https://www.mdpi.com/journal/molecules Molecules 2021, 26, x FOR PEER REVIEW 2 of 14

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observed for the enantiomers of chiral probes in voltammetry experiments on unmodified electrodes, performed in achiral ionic liquids with inherently chiral molecular salts as chi- electrodes, performed in achiral ionic liquids with inherently chiral molecular salts as ral additives. The strategy worked with different family members, including those solid chiral additives. The strategy worked with different family members, including those solidat room at room temperature temperature [1,2], [1,2 and], and those those with with long long alkyl alkyl chains and and bistriﬂimide bistriflimide anions anions which whichwere liquid were liquid at room at room temperature temperature [1] [ 1and] and can can be be regarded regarded as inherently chiral chiral ionic ionic liquids liquids(ICILs). (ICILs).

0 0 0 0 0 Figure 1.1. ((RR)-2,2)-2,2′-bis[2-(5,2-bithienyl)]-3,3′-bithienyl)]-3,3-bithianaphthene′-bithianaphthene ((R )-((BTR)-2TBT4),2T (R4),)- N(R,N)-N-dialkyl-3,3,N′-dialkyl-3,3- ′-bicol- 0 bicollidiniumlidinium salts salts ((R (()-AlkR)-Alk2BicX2BicX2)2 and) and (R (R)-)-NN-alkyl-3,3-alkyl-3,3 -bicollidinium′-bicollidinium salts salts ((R )-Alk((R)-AlkBicXBicX). ).

Within this framework, we decided to implement the inherent chirality concept in Within this framework, we decided to implement the inherent chirality concept in a a new family of ICILs based on the helix as an ideal stereogenic element in an inherent chiralitynew family design, of ICILs and to based investigate on the whether helix as the an great ideal enantiodiscrimination stereogenic element ability in an of inherent the chi- ILsrality based design, on a stereogenicand to investigate axis was alsowhether exhibited the bygreat helical enantiodiscrimination organic salts. ability of the ILs basedAza[ onn a]helicenes stereogenic are a axis class was of chiral also multinuclearexhibited by molecules helical organic possessing salts. peculiar elec- tronicAza[ andn chiroptical]helicenes characteristics are a class of due chiral to theirmultinuc extendedlear conjugatedmolecules aromaticpossessing system peculiar elec- associatedtronic and with chiroptical a central distortioncharacteristics from planarity. due to Theytheir are extended configurationally conjugated stable aromatic above system roomassociated temperature with a only central when distortion the number from of condensed planarity. six-membered They are configurationally aromatic rings stable is ≥6 [7]. above room temperature only when the number of condensed six-membered aromatic In contrast to carbohelicenes, azahelicenes possess one or more nitrogen atoms in their molecularrings is ≥6 framework [7]. which can be exploited to direct the reactivity of these molecules. Furthermore,In contrast since to the carbohelicenes, nitrogen atom of azahelicenes azahelicenes canpossess react withone alkylatingor more nitrogen agents to atoms in affordtheir molecular quaternary framework salts, we envisaged which thecan possibility be exploited of obtaining to direct a completelythe reactivity new of class these mole- ofcules. CILs. Furthermore, If their melting since point couldthe nitrogen be lowered atom below of 100azahelicenes◦C, aza-[6]helicenium can react cationswith alkylating wouldagents clearly to afford fulfil quaternary the requirements salts, towe be envisa definedged as ICILs,the possibility since the pyridiniumof obtaining unit a iscompletely essentialnew class part of ofCILs. the helicalIf their scaffold. melting These point saltscould are be reasonably lowered solublebelow 100 in polar °C, aza-[6]helicenium solvents and exhibit physical properties that could be modulated by varying the length and shape of cations would clearly fulfil the requirements to be defined as ICILs, since the pyridinium the alkyl moiety as well as the counterion [8]. This latter point is known to be particularly importantunit is essential for determining part of the meltinghelical rangescaffold. of the These organic salts salts: are the reasonably use of appropriate soluble in polar counteranions,solvents and likeexhibit bistriflimidate, physical properties can lower the that melting could point be bymodulated many dozens by ofvarying degrees the length comparedand shape to of halide the alkyl salts [ 1moiety,9]. as well as the counterion [8]. This latter point is known to be particularlyAs mentioned important above, in thefor casedetermining of solid salts, the evenmelting those range with a of high the melting organic point, salts: the use theyof appropriate could be employed counteranions, as chiral dopants like bistriflimidate, of achiral ILs, in can which lower they the should melting be perfectly point by many solubledozens consideringof degrees theircompared structural to halide affinity. salts We [1,9]. have already found that bicollidinium ICILs, when used as low-concentration additives to commercial achiral ILs, can impart high As mentioned above, in the case of solid salts, even those with a high melting point, enantioselection properties towards the antipodes of chiral probes differing in structure, functionalthey could groups, be employed and stereogenic as chiral elementsdopants [of1, 10achiral]. This ILs, project in which could they open should the way be perfectly towardssoluble newconsidering tools for enantioselectivetheir structural electroanalysis affinity. We of have bioactive already molecules. found that bicollidinium ICILs, when used as low-concentration additives to commercial achiral ILs, can impart 2.high Results enantioselection and Discussion properties towards the antipodes of chiral probes differing in structure,The functional molecules groups, chosen toand verify stereogenic the viability elemen of thets approach [1,10]. This were project the quaternary could saltsopen the way oftowards 5-aza[6]helicene new tools (1 ),for a configurationallyenantioselective stable electroanalysis azahelicene. of bioactive molecules.

2. Results and Discussion The molecules chosen to verify the viability of the approach were the quaternary salts of 5-aza[6]helicene (1), a configurationally stable azahelicene.

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Molecules 2021, 26, 311 The synthesis of 1 was realized following the procedure reported in the3 of literature 13 [11]. It began with Wittig condensation between benzyl-triphenylphosphonium bromide and 4-tolualdehyde,The synthesis of followed 1 was realized by photochemica following thel ring procedure closure toreported yield 2-methylphenan-in the literature threne;[11]. It the beganThe latter synthesis with was Wittig ofbrominated1 was condensation realized with following N-br betweenomosuccinimide the procedure benzyl-triphenylphosphonium reported (NBS) in theand literature the resulting [11 bromide]. bro- momethylandIt 4-tolualdehyde, began derivative with Wittig convertedfollowed condensation by into photochemica between the corre benzyl-triphenylphosphoniumspondingl ring closure triphenylphosphonium to yield bromide2-methylphenan- and salt. This, 4-tolualdehyde, followed by photochemical ring closure to yield 2-methylphenanthrene; inthrene; turn, underwent the latter was Wittig brominated condensation with N-br withomosuccinimide commercially available (NBS) and quinoline-3-carbox- the resulting bro- the latter was brominated with N-bromosuccinimide (NBS) and the resulting bromomethyl aldehydemomethyl to derivativeyield 1-(2-phenanthrenyl)-2- converted into the corre(3-quinolyspondingl)ethane triphenylphosphonium 3, mainly as E isomer, salt. in This, 71% in turn,derivative underwent converted Wittig into condensation the corresponding with triphenylphosphonium commercially available salt. quinoline-3-carbox- This, in turn, yieldunderwent (Scheme Wittig1). condensation with commercially available quinoline-3-carboxaldehyde aldehyde to yield 1-(2-phenanthrenyl)-2-(3-quinolyl)ethane 3, mainly as E isomer, in 71% to yield 1-(2-phenanthrenyl)-2-(3-quinolyl)ethane 3, mainly as E isomer, in 71% yield yield(Scheme (Scheme1). 1).

CH 3OH, CH 3ONa CH OH, CH ONa reflux, 3 2h 3 reflux, 2h 3 3

Scheme 1. Wittig condensation to yield precursor 1-(2-phenanthrenyl)-2-(3-quinolyl)ethane (3). Scheme 1. 3 Scheme 1. WittigWittig condensation condensation to to yield yield precursor precursor 1-(2-phenanthrenyl)-2-(3-quinolyl)ethane1-(2-phenanthrenyl)-2-(3-quinolyl)ethane ( ). (3). The final step was again a photochemical ring closure, which, however, unexpectedly The ﬁnal step was again a photochemical ring closure, which, however, unexpectedly led toled Thetwo to two finalconstitutional constitutional step was again isomers, isomers, a photochemical namely 1 1and and ring4 (Scheme4 (Scheme closure,2). which,2). however, unexpectedly led to two constitutional isomers, namely 1 and 4 (Scheme 2).

1 1 3a 3b 4 3a 3b 4 SchemeScheme 2. Photochemical 2. Photochemical ring ring closure closure of of the twotwo conformers conformers of precursorof precursor3. 3. Scheme 2. Photochemical ring closure of the two conformers of precursor 3. In fact, precursor 3, which was formed as an E/Z mixture, during photolysis isomerized toIn the fact,Z stereoisomer, precursor 3 for, which which was two conformationalformed as ans -cisE/Z ( 3amixture,) and s-trans during (3b) photolysis isomers were isomerized expected.toIn the fact, Z stereoisomer, Oxidativeprecursor photocyclization 3, which for which was twoformed of the conformational former as an afforded E/Z mixture, 5-aza[6]helicene s-cis (3a during) and s (photolysis-trans1), while (3b ring) isomer- isomers wereizedclosure expected.to theof Z thestereoisomer, Oxidative latter led to thephotocyclizationfor formationwhich two of conformational the achiralof the phenanthreno[2,3-k]phenanthridineformer s-cis afforded (3a) and 5-aza[6]helicenes-trans (3b) isomers (1), whilewere(4 )ring expected. (Scheme closure2). Oxidative of the latter photocyclization led to the formation of the of former the achiral afforded phenanthreno[2,3-k]phe- 5-aza[6]helicene (1), When performing the analytical HPLC separation of the racemate of 1 on chiral nanthridinewhile ring closure (4) (Scheme of the 2). latter led to the formation of the achiral phenanthreno[2,3-k]phe- stationary phase (see below), we observed the presence of 39% of 4, previously undetected nanthridineWhen performing (4) (Scheme the 2). analytical HPLC separation of the racemate of 1 on chiral sta- dueWhen to its performing very high gas-chromatographic the analytical HPLC retention separation time. At of this thepoint, racemate while of proceeding 1 on chiral sta- tionarywith phase the experiments (see below), with we the observed isolated enantiomers, the presence we undertookof 39% of the 4, taskpreviously of improving undetected tionary phase (see below), we observed the presence of 39% of 4, previously undetected due tothe its synthesis very high of compound gas-chromatographic1 by reducing retention or eliminating time. the At formation this point, of byproductwhile proceeding4. due to its very high gas-chromatographic retention time. At this point, while proceeding withWe the found experiments rather difﬁcult with the to separateisolated4 enantifrom racemicomers,1 weby undertook column chromatography the task of improving on with the experiments with the isolated enantiomers, we undertook the task of improving the synthesissilica gel and of compound minimize its 1 formation by reducing by varying or eliminating the reaction the conditions. formation We of attempted byproduct to 4. We the performsynthesis photochemical of compound ring 1 closureby reducing of 3 in differentor eliminating solvents the as previousformation investigations of byproduct on 4. We found rather difficult to separate 4 from racemic 1 by column chromatography on silica founda diazapentahelicene rather difficult hadto separate evidenced 4 howfrom this racemic step could 1 by be column preferentially chromatography directed towards on silica gel and minimize its formation by varying the reaction conditions. We attempted to per- gel theand formation minimize of its the formation azahelicene by by varying the use ofthe polar reaction solvents conditions. [12]. However, We attempted in this case, to per- formthe photochemical use of acetonitrile ring instead closure of ethyl of 3 acetatein different resulted solvents in the almost as previous exclusive investigations formation of on a form photochemical ring closure of 3 in different solvents as previous investigations on a diazapentahelicene had evidenced how this step could be preferentially directed towards diazapentahelicene had evidenced how this step could be preferentially directed towards the formation of the azahelicene by the use of polar solvents [12]. However, in this case, the formation of the azahelicene by the use of polar solvents [12]. However, in this case, thethe use use of of acetonitrile acetonitrile instead instead ofof ethylethyl acetatacetate resulted in the almoalmostst exclusiveexclusive formation formation ofof 4 ,4 while, while using using dichloromethane dichloromethane or or toluenetoluene wewe obtained bothboth productsproducts with with a a prevalence prevalence

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of 4. We eventually managed to obtain an almost complete prevalence of 1 by using a 9:1 4, while using dichloromethane or toluene we obtained both products with a prevalence hexane/ethylof 4. We eventually acetate mixture, managed where to obtain ethyl an ac almostetate completewas at just prevalence the minimum of 1 by quantity using a nec- essary9:1 hexane/ethylfor the solubilization acetate mixture, of 3. whereHowever, ethyl the acetate yield was of at 1 just in thethese minimum conditions quantity was 34%, withnecessary just traces for of the 4 solubilization, while the remaining of 3. However, starting the yieldmaterial of 1 in underwent these conditions degradation was 34%, during the withprocess. just traces of 4, while the remaining starting material underwent degradation during theIn process. order to avoid this drawback, we explored the possibility of approaching the synthesis ofIn 1 orderthrough to avoid the alternative this drawback, pathway we explored depicted the in possibility Scheme 3 ofbased approaching on the Heck the con- synthesis of 1 through the alternative pathway depicted in Scheme3 based on the Heck densation. 11-[(E)-styryl]benzo[k]phenanthridine 5 was obtained as an E/Z isomeric mix- condensation. 11-[(E)-styryl]benzo[k]phenanthridine 5 was obtained as an E/Z isomeric turemixture and its and photocyclization its photocyclization exclusively exclusively provided provided the the desired desired product product 11.. It It is is to to be be noted thatnoted in this that case inthis two case conformational two conformational isomer isomerss would would also also be possible, be possible, one one of of which which would bringwould the bringformation the formation of 4. of 4.

CH OH, t-BuOK 3 hν reflux, 2h, 79 % 366 nm, 3h cat. I , 86% 2

Pd(PPh ) Cl , AcONa 3 2 2 DMA, 140°C 2 days, 94%

Scheme 3. Alternative synthetic approach to azahelicene (1). Scheme 3. Alternative synthetic approach to azahelicene (1). Product 5 was also prepared by using the classical Wittig approach between the four- ringsProduct phosphonium 5 was also ylide prepared moiety andby using benzaldehyde, the classical but overall Wittig yields approach were considerablybetween the four- ringslower phosphonium than with the ylide Heck moiety approach. and The benzaldehyde, yield of the photocyclization but overall yields of 5 wereat 366 considerably nm to lowerproduct than1 withwas 90%.the Heck approach. The yield of the photocyclization of 5 at 366 nm to productThe 1 was racemate 90%. of 1 obtained by photolysis of intermediate 3 in ethyl acetate solution wasThe resolved racemate into of enantiomers 1 obtained by by semipreparative photolysis of HPLCintermediate on Chiralpak 3 in ethyl IA chiral acetate station- solution ary phase; the process also allowed the total removal of the achiral product 4 from the was resolved into enantiomers by semipreparative HPLC on Chiralpak IA chiral station- enantiopure antipodes of 1 (Figure2) and its isolation in a high-purity state. ary phase;In accordance the process with also the circularallowed dicroism the total (CD) removal data in theof the literature, achiral [11 product], the absolute 4 from the enantiopureP configuration antipodes was assigned of 1 (Figure to the 2) first and eluted its isolation enantiomer. in a high-purity state. Both enantiomers were alkylated with octyl iodide excess at 80 ◦C, followed by anion metathesis with silver bis(trifluoromethanesulfonyl)amide AgNTf2 to provide enantiomerically pure quaternary salts as waxy solids (Scheme4). The starting material configuration is supposed to be retained in the corresponding salts on account of the high racemization barrier of 1 (the enantiomeric excess of 1 remains unchanged after 24 h heating in dimethylformamide solution at 100 ◦C).

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)-1 P )-1 ( M 4(39%) (

Molecules 2021, 26, x FOR PEER REVIEW 5 of 14 Molecules 2021, 26, 311 5 of 13 )-1 P )-1 ( M 4(39%) ( 0 3 6 9 12 15 18 Elution time (min)

Figure 2. HPLC resolution of (±)-5-aza[6]helicene (1) and simultaneous purification from by-product 4. Column: Chiralpak IA 250 mm × 4.6 mm, mobile phase: n-hexane-isopropanol-ethyl acetate- diethylamine 100/5/5/0.2, flow rate: 1 mL/min, detector: UV/CD at 325 nm.

In accordance with the circular dicroism (CD) data in the literature, [11], the absolute P configuration was assigned to the first eluted enantiomer. Both enantiomers were alkylated with octyl iodide excess at 80 °C, followed by anion metathesis with silver bis(trifluoromethanesulfonyl)amide AgNTf2 to provide enantio- merically0 3pure quaternary 6 salts 9 as waxy 12 solids (Scheme 15 4). 18 The starting material configu- Elution time (min) ration is supposed to be retained in the corresponding salts on account of the high race- Figuremization 2. HPLC barrier resolution of of1 (±(the)-5-aza[6]helicene enantiomeric (1) and excess simultaneous of 1 puriﬁcationremains fromunchanged by-product after 24 h heating Figure4. Column: 2. HPLC Chiralpak resolution IA 250 mmof (±×)-5-aza[6]helicene4.6 mm, mobile phase: (1) andn-hexane-isopropanol-ethyl simultaneous purification acetate- from by-prod- uctdiethylaminein 4dimethylformamide. Column: 100/5/5/0.2, Chiralpak ﬂow IA rate:solution 250 1mm mL/min, × at 4.6 100 detector:mm, °C). mobile UV/CD phase: at 325 n nm.-hexane-isopropanol-ethyl acetate- diethylamine 100/5/5/0.2, flow rate: 1 mL/min, detector: UV/CD at 325 nm.

mization barrier of 1 (the enantiomeric excess of 1 remains unchanged after 24 h heating inSchemeScheme dimethylformamide 4. Synthesis4. Synthesis of (P)-5-octyl-5-aza[6]helicenium of (solutionP)-5-octyl-5-aza[6]helicenium at 100 °C). bistriflimidate bistriflimidate (2). (2). Then, 1 and 2 were electrochemically characterized by cyclic voltammetry (CV, Figure3 ). As a firstThen, general 1 and consideration, 2 were electrochemically in the parent azahelicene characterize the nitrogend by lone cyclic pair involtammetry the (CV, Fig- pyridineure 3). As ring a providesfirst general a preferential consideration, site for first in oxidation, the parent resulting azahelicene in radical the cation nitrogen lone pair in formation.the pyridine However, ring provides upon alkylation a preferential the situation site is for reversed, first oxidation, since the nitrogen resulting be- in radical cation comes cationic and hence much more electron-poor, thus providing preferential sites of firstformation. reduction However, (similarly e.g., upon to the alkylation effect of alkylation the situ onation the electrochemicalis reversed, since behavior the nitrogen becomes ofcationic other pyridine and hence and benzimidazole much more scaffolds electron-poor, [1,13]). Cation thus reduction providing leads preferential to radical sites of first re- formation,duction (similarly with possible e.g., coupling to the follow-ups, effect of resulting alkylation e.g., inon dimerization, the electrochemical as reported behavior of other forpyridine the 1-methyl-1-aza[6]helicenium and benzimidazole scaffolds cation [14]. [1,13]). Cation reduction leads to radical formation, Comparing the CV features of 1 and 2, the alkylation of the aza site resulted in: with possible coupling follow-ups, resulting e.g., in dimerization, as reported for the 1- Scheme(1) The 4. disappearance Synthesis of ( ofP)-5-octyl-5-aza[6]helicenium the first irreversible oxidation peakbistriflimidate at 1.07 V; (2). (2)methyl-1-aza[6]heliceniumThe disappearance of most of cation the complex [14]. reduction peak system between −2.2 and Then,−2.7 V,leaving 1 and a2 singlewere nearly electrochemically chemically and electrochemically characterize reversibled by cyclic peak voltammetry at −2.58 V; (CV, Fig- (3) The appearance of a nearly irreversible and nearly splitting first reduction peak at −1.19 V. ure 3). As a first general consideration, in the parent azahelicene the nitrogen lone pair in Both the reduction peak systems of 2 were at potentials very close to those reported the pyridine ring provides a preferential site for first oxidation, resulting in radical cation for the N-methyl-1-aza[6]helicenium cation [14]. Furthermore, a peak, surely associated formation.with reoxidation However, of reduced upon products alkylation on the electrodethe situation surface, is couldreversed, be seen since for 2 theat about nitrogen becomes cationic−0.45 V and in a scanhence starting much from more reduction electron-poor, and also including thus providing oxidations preferential (Figure4a) (a sites of first re- ductionsecond oxidative (similarly return e.g., peak to the at about effect 0 Vof was alkylation instead associated on the electrochemical with the only partially behavior of other pyridinereversible and second benzimidazole reduction peak, Figurescaffolds4b). Thus,[1,13]). reversible Cation dimer reduction formation leads as in to [ 14 radical], at formation, with possible coupling follow-ups, resulting e.g., in dimerization, as reported for the 1- methyl-1-aza[6]helicenium cation [14].

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least as partial follow-up of the first reduction process, cannot be ruled out. An additional similarity with a case in the literature [14] is that the first reduction peak of 2 tended to split or to feature a preceding shoulder, as confirmed in two experiments at different times (see Supplementary Materials). Notably, the same feature also appeared in the CV patterns of the azahelicenium compound described in [14], although the authors do not specifically mention it. Since we cannot assume any conformational isomerism for the single molecule, Molecules 2021, 26, x FOR PEER REVIEW 6 of 14 the above features might be linked to two pathways with different preceding or following chemical steps (for example, partial coupling as in [10]), besides solid state adsorption or aggregation effects.

60.0 25.0

50.0 20.0

40.0 15.0 s) s) 1 1 30.0 − − 10.0 V V 3 3 20.0

dm 5.0 dm 1 1 −

− 10.0 0.0 mol 1 mol −

1 0.0 − −5.0 −10.0 (Acm (Acm / −10.0 / −20.0

−30.0 −15.0

−40.0 cv^0.5 j / −20.0 j / cv^0.5 −50.0 −25.0 −3 −2.5 −2 −1.5 −1 −0.5 0 0.5 1 1.5 2 −3.0 −2.5 −2.0 −1.5 −1.0 −0.5 0.0 0.5 1.0 1.5 2.0 E vs Fc+|Fc / (V) E vs Fc+|Fc / (V)

−4 MoleculesFigureFigure 2021 3., Normalized 3.26,Normalized x FOR PEER CV REVIEW CV patterns patterns obtained obtained on on glassy glassy carbon carbon electrode electrode at a 0.2 V/s scan scan rate for 1 andand 22 withwith7.5 7.5× × 1010−4 M7M of 14

in CHin CH3CN3CN + 0.1 + 0.1M tetrabutylammonium M tetrabutylammonium hexafluorophosphate, hexaﬂuorophosphate, TBAPF TBAPF6 (starting6 (starting oxidative oxidative and and re reductiveductive half-cycles, half-cycles, red red andand blue, blue, respectively). respectively).

Comparing the CV features of 1 and 2, the alkylation of the aza site resulted in: (1) The disappearance of the first irreversible oxidation peak at 1.07 V; (2) The disappearance of most of the complex reduction peak system between −2.2 and −2.7 V, leaving a single nearly chemically and electrochemically reversible peak at −2.58 V; (3) The appearance of a nearly irreversible and nearly splitting first reduction peak at

−1.19 V. Both the reduction peak systems of 2 were at potentials very close to those reported for the N-methyl-1-aza[6]helicenium cation [14]. Furthermore, a peak, surely associated with reoxidation of reduced products on the electrode surface, could be seen for 2 at about −0.45 V in a scan starting from reduction and also including oxidations (Figure 4a) (a second oxidative return peak at about 0 V was instead associated with the only partially reversible second reduction peak, Figure 4b). Thus, reversible dimer formation as in [14], at

Figure 4. Highlighted returnleast oxidation as partial processes follow-up related of tothe the first first reductio (a) and secondn process, (b) reduction cannot processesbe ruled forout. compound An additional2. FigureArrows 4. ofHighlighted same colour return indicatesimilarity oxidation correlated with processes peaks.a case related in the to literaturethe first (a) [14]and issecond that (theb) reduction first reduction processes peak for compound of 2 tended to 2. Arrows of same colour indicate correlated peaks. split or Theto feature irreversible a preceding oxidation shoulder, peaks at as 1.46 confirmed V in 1 (the in two second experime one afternts azaat different oxidation) times and (see Supplementary Materials). Notably, the same feature also appeared in the CV pat- 1.47The V in irreversible2 (only one) oxidation could instead peaks be at ascribed 1.46 V in to 1 the (the carbahelicene second one terminal,after aza consideringoxidation) ternsits consistencyof the azahelicenium with the reported compound first oxidationdescribed potential in [14], although of 1.405 V the for authors 6-carbahelicene do not spe- [15]. cificallyand 1.47 mention V in 2 (only it. Since one) we could cannot instead assume be ascribed any conformation to the carbaheliceneal isomerism terminal, for the consid- single eringThe smallits consistency positive shiftwith couldthe reported be justified first foroxidation1 considering potential that of 1.405 in that V casefor 6-carbaheli- the electron molecule,transfer the process above takes features place might after be a linked former to one, two that pathways is, with with a positive different charge preceding already or cene [15]. The small positive shift could be justified for 1 considering that in that case the followingpresent onchemical the molecule, steps (for and example, for 2 since partial the molecule coupling is as electron-poorer in [10]), besides on solid account state of ad- the electron transfer process takes place after a former one, that is, with a positive charge al- sorptionalkylated or aggregation aza site. effects. ready present on the molecule, and for 2 since the molecule is electron-poorer on account The nearly reversible (both chemically and electrochemically) second reduction peak of the alkylated aza site. of 2 at −2.6 V is more likely to be related to first reduction (to radical anion) of the The nearly reversible (both chemically and electrochemically) second reduction peak carbahelicene terminal than to a second reduction on the aza site, since in 6-carbahelicene of 2 at −2.6 V is more likely to be related to first reduction (to radical anion) of the carba- the first reduction is at an even more positive potential [15]. Consistently, the superimposed helicene terminal than to a second reduction on the aza site, since in 6-carbahelicene the first reduction is at an even more positive potential [15]. Consistently, the superimposed peak system appearing in the case of 1 should correspond to the reduction of the opposite helicene side, the one with the pyridyl ring adjacent to the phenyl terminal. To check the enantioselection ability of the (P)-2 and (M)-2 salts, we first tested them as 0.01 M additives in achiral ionic liquid (BMIM)NTf2, recording in small volumes of the resulting chiral media the CV patterns of the (R)-(+)- and (S)-(−)-N,N′-dimethyl-1-ferrocenylethylamine chiral probe enantiomers on screen-printed electrode (SPE) cells with an Au working electrode (Figure 5).

(S)- FcA (R)- FcA

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Figure 4. Highlighted return oxidation processes related to the first (a) and second (b) reduction processes for compound 2. Arrows of same colour indicate correlated peaks.

The irreversible oxidation peaks at 1.46 V in 1 (the second one after aza oxidation) and 1.47 V in 2 (only one) could instead be ascribed to the carbahelicene terminal, considering its consistency with the reported first oxidation potential of 1.405 V for 6-carbahelicene [15]. The small positive shift could be justified for 1 considering that in that case the electron transfer process takes place after a former one, that is, with a positive charge already present on the molecule, and for 2 since the molecule is electron-poorer on account of the alkylated aza site. The nearly reversible (both chemically and electrochemically) second reduction peak of 2 at −2.6 V is more likely to be related to first reduction (to radical anion) of the carba- Molecules 2021, 26, 311 helicene terminal than to a second reduction on7 ofthe 13 aza site, since in 6-carbahelicene the first reduction is at an even more positive potential [15]. Consistently, the superimposed peak system appearing in the case of 1 should correspond to the reduction of the opposite helicene side, the one with the pyridyl ring adjacent to the phenyl terminal. peak system appearing in the case of 1 should correspond to the reduction of the opposite helicene side, the one with the pyridylTo ring check adjacent the enantioselection to the phenyl terminal. ability of the (P)-2 and (M)-2 salts, we first tested them To check the enantioselectionas ability0.01 M of additives the (P)-2 and in achiral (M)-2 salts, ionic we liquid ﬁrst tested(BMIM)NTf them 2, recording in small volumes of the as 0.01 M additives in achiral ionicresulting liquid chiral (BMIM)NTf media2, the recording CV patterns in small of volumes the (R)-(+)- of and (S)-(−)-N,N′-dimethyl-1-ferro- the resulting chiral media the CVcenylethylamine patterns of the chiral (R)-(+)- probe and (Senantiomers)-(−)-N,N0-dimethyl-1- on screen-printed electrode (SPE) cells with an ferrocenylethylamine chiral probeAu enantiomers working electrode on screen-printed (Figure electrode 5). (SPE) cells with an Au working electrode (Figure5).

Molecules 2021, 26, x FOR PEER REVIEW 8 of 14

(S)- FcA (R)- FcA

Figure 5. CV patterns of (S)- or (R)-ferrocenyl chiral probes (2.0 × 10−3 M, blue and yellow, respectively) on screen- printed electrode (SPE) cells with an Au working electrode, in (BMIM)NTf2 with either (P)-5-n-octyl-5-aza[6]helicenium bistriflimidate (1.6 × 10−2 M, green frame) or (M)-5-n-octyl-5-aza[6]helicenium bistriflimidate (1.6 × 10−2 M, red frame) as Figure additives.5. CV patterns Grey voltammograms of (S)- or (R)-ferrocenyl refer to experiments chiral probes in absence (2.0 × 10 of-3 azahelicenium M, blue and saltsyellow, as chiral respectively) inducers. on screen-printed electrode (SPE) cells with an Au working electrode, in (BMIM)NTf2 with either (P)-5-n-octyl-5-aza[6]helicenium bistriflimidate (1.6 × 10-2 M, green frame) or (M)-Despite5-n-octyl-5-aza[6]helicenium the medium viscosity, neatbistriflimidate CV peaks (chemically(1.6 × 10-2 M, and red electrochemicallyframe) as additives. re- Grey voltammograms refer to experimentsversible or quasi-reversible)in absence of azahelicenium were obtained salts for as allchiral probe+selector inducers. combinations (Figure5 ). At the same time, a remarkable difference was observed for the enantiomer peak poten- tialsDespite (~140 the mV) medium together viscosity, with slight neat differences CV peaks in (chemically the peak shape and (for electrochemically example, there re- was a greater difference between forward and backward peak potentials for the peak at versible or quasi-reversible) were obtained for all probe+selector combinations (Figure 5). higher potential). In particular, the oxidation peak potential of one enantiomer remained At theapproximately same time, ina remarkable the same position, difference while was the observed other shifted for tothe much enantiomer higher potentials, peak poten- tialswhile (~140 maintaining mV) together its reversiblewith slight peak differences shape, a featurein the peak often shape linked (for to preferential example, there stabi- was a greaterlization difference of the reactant between (or destabilizationforward and backward of the product). peak Neatlypotentials specular for the features peak at were higher potential).obtained In invertingparticular, either the oxidation selector or peak probe potential configuration, of one while enantiomer the (R)- remained and (S)-probe approx- imatelyenantiomers in the same gave practicallyposition, coincidentwhile the CVother peaks shifted when to performing much higher the same potentials, protocol inwhile maintainingachiral (BMIM)NTf its reversible2 in the peak absence shape, of the a feat chiralure additive. often linked Repetitions to preferential on new SPE stabilization supports of thewere reactant performed (or destabilization in all cases in order of the to checkproduct). the result Neatly repeatability specular (Figurefeatures5). were obtained A second series of enantioselection experiments was performed using differential inverting either selector or probe configuration, while the (R)- and (S)-probe enantiomers pulse voltammetry (DPV) on SPE cells with working graphite electrodes using the (P)- gave practically coincident CV peaks when performing the same protocol in achiral 2 salt as 0.01 M additive in achiral (BMIM)NTf2 to test the (R)- and (S)-enantiomers of (BMIM)NTftyrosine methyl2 in the ester absence hydrochloride of the chiral at 0.002 additive M concentration.. Repetitions Once on more,new SPE a neat supports and fairly were performedreproducible in all peakcases potential in order difference to check the of ~130 result mV repeatability was observed (Figure for the 5). first oxidation peaksA second in the series CV patterns of enantioselection of each enantiopure experiments probe, with was a constantperformed working using protocol, differential pulse voltammetry (DPV) on SPE cells with working graphite electrodes using the (P)-2 salt as 0.01 M additive in achiral (BMIM)NTf2 to test the (R)- and (S)-enantiomers of tyrosine methyl ester hydrochloride at 0.002 M concentration. Once more, a neat and fairly reproducible peak potential difference of ~130 mV was observed for the first oxidation peaks in the CV patterns of each enantiopure probe, with a constant working protocol, while the same probe enantiomers gave practically the same peak in the absence of the chiral additive in the achiral IL medium (Figure 6).

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Molecules 2021, 26, x FOR PEER REVIEW 9 of 14 while the same probe enantiomers gave practically the same peak in the absence of the chiral additive in the achiral IL medium (Figure6).

Figure 6. Enantiodiscrimination differential pulse voltammetry (DPV) tests with L-and D- tyrosine methyl ester, in achiral Figure 6. Enantiodiscrimination differential pulse voltammetry (DPV) tests with L-and D- tyrosine IL 1-butyl-3-methylimidazolium bistriflimide (BMIM)NTf2 with (P)-2 or (M)-2 chiral additives. Repetitions are shown as dashed lines. IL: ionic liquid.methyl ester, in achiral IL 1-butyl-3-methylimidazolium bistriflimide (BMIM)NTf2 with (P)-2 or (M)-2 chiral additives. Repetitions are shown as dashed lines. IL: ionic liquid. It is interesting to compare the effects produced on enantioselectivity by ICILs charac- terizedIt is interesting by different to stereogenic compare elements, the effects namely prod auced stereogenic on enantioselectivity axis and a helix, when by ICILs used char- as chirality inducers in low concentration in achiral ILs. The terms of comparison were acterized by different stereogenic elements,0 namely a stereogenic axis and a helix, when usedthe asN -monoalkyl-chirality inducers and the inN,N’ low-dialkyl-3,3 concentration-bicollidinium in achiral salts ILs. (Figure The1 ),terms C 2 symmetric of comparison when the alkyl chains are identical and asymmetric when the alkyl groups are different. wereUnder the comparableN-monoalkyl- experimental and the N,N’ conditions-dialkyl-3,3 and using′-bicollidinium the same ferrocenylamine salts (Figure probes, 1), C2 sym- metricthe bicollidiniumwhen the alkyl salt representedchains are identical in Figure1 ,an characterizedd asymmetric by two when identical the alkyln-octyl groups groups, are different.at a 0.01Under M concentrationcomparable developedexperimental a potential conditions difference and higherusing thanthe same 170 mV ferrocenylamine [1], while probes,the asymmetric the bicollidinium bicollidinium salt compound represented with in the Figure nitrogen 1, atomscharacterized differently by quaternarized two identical n- octylwith groups, a methyl at anda 0.01 a n-octyl M concentration group respectively developed afforded a a potential 120-mV peak difference potential higher separation than 170 mVat [1], a 0.016 while M concentration.the asymmetric Therefore, bicollidinium in this first compound attempt to with verify the the nitrogen enantioselection atoms differ- entlyobtainable quaternarized by azahelicenium with a methyl salts, we and observed a n-octyl a potential group differencerespectively comparable afforded to thata 120-mV attainable with other inherently chiral additives, such as bicollidinium compounds. peak potential separation at a 0.016 M concentration. Therefore, in this first attempt to verify3. Materials the enantioselection and Methods obtainable by azahelicenium salts, we observed a potential dif- ference3.1. General comparable Procedures to that attainable with other inherently chiral additives, such as bicol- lidiniumThe compounds. starting materials and solvents for azahelicene synthesis were purchased from Carlo Erba (Milan, Italy) and used without further purification; photolysis reactions were 3. Materialsperformed and using Methods a Multirays instrument equipped with sets of 10 lamps of different wave- lengths. NMR spectra were recorded on Bruker AV400 and Bruker AC300 spectrometers. 3.1. General Procedures Chemical shifts (δ) are expressed in parts per million (ppm), and coupling constants are givenThe instarting Hz. Splitting materials patterns and are solvents indicated for as az follows:ahelicene s = singlet, synthesis d = doublet, were purchased t = triplet, from Carloq = Erba quartet, (Milan, quint Italy) = quintet, and used m = multiplet.without fu Purificationsrther purification; by column phot chromatographyolysis reactions were performedwere performed using a usingMultirays Merck instrument silica gel 60 equipped (230–400 mesh with forsets flash-chromatography of 10 lamps of different and wave- lengths. NMR spectra were recorded on Bruker AV400 and Bruker AC300 spectrometers. Chemical shifts (δ) are expressed in parts per million (ppm), and coupling constants are given in Hz. Splitting patterns are indicated as follows: s = singlet, d = doublet, t = triplet, q = quartet, quint = quintet, m = multiplet. Purifications by column chromatography were performed using Merck silica gel 60 (230–400 mesh for flash-chromatography and 70–230 mesh for gravimetric chromatography) and aluminium oxide 90 neutral. Melting points were determined on a Büchi B-540 instrument. GC-MS analyses were performed on an Agilent 6850 chromatograph equipped with an Agilent 5975N mass spectrometer.

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70–230 mesh for gravimetric chromatography) and aluminium oxide 90 neutral. Melting points were determined on a Büchi B-540 instrument. GC-MS analyses were performed on an Agilent 6850 chromatograph equipped with an Agilent 5975N mass spectrometer. HPLC-grade solvents were purchased from Sigma-Aldrich (Milan, Italy). HPLC enantioseparations were performed by using stainless-steel Chiralpak IA (250 mm × 4.6 mm, 5 µm and 250 mm × 10 mm, 5 µm) columns (Chiral Technologies Europe, Illkirch, France). The HPLC apparatus used for analytical enantioseparations consisted of a PerkinElmer (Norwalk, CT, USA) 200 LC pump equipped with a Rheodyne (Cotati, CA, USA) injector, a 50-µL sample loop, an HPLC PerkinElmer oven, and a Jasco (Jasco, Tokyo, Japan) Model CD2095 Plus UV/CD detector. The signal was acquired and processed by Clarity software (DataApex, Prague, Czech Republic). For semipreparative separation, a PerkinElmer 200 LC pump equipped with a Rheodyne injector, a 5000-µL sample loop, a PerkinElmer LC 101 oven, and a Waters 484 detector (Waters Corporation, Milford, MA, USA) were used.

3.2. Electrochemistry 3.2.1. Helicene Characterization Cyclic voltammetry (CV) was performed at scan rates in the range of 0.05–2 V/s using an AutoLab PGStat potentiostat and a classical three-electrode glass minicell (with a working volume of about 3 cm3). The latter included as a working electrode a glassy carbon (GC) disk embedded in glass (Metrohm) polished by diamond powder (1 µm Aldrich) on a wet cloth (Struers DP-NAP), as counter electrode a platinum disk, and as a reference electrode a saturated aqueous calomel electrode (SCE) inserted into a compartment with the working medium ending in a porous frit to avoid contamination of the working solution by water and KCl traces. Experiments were run with 0.00075 M azahelicene solutions in acetonitrile (ACN, Aldrich, HPLC grade) + 0.1 M tetrabutylammonium hexaﬂuorophosphate TBAPF6 (Fluka, ≥98 %) as the supporting electrolyte, previously deaerated by nitrogen bubbling. (S)-L-Tyr Me Ester was purchased from Sigma-Aldrich and (R)-D-Tyr Me Ester from Alfa Aesar.

3.2.2. Enantiodiscrimination Experiments CV enantiodiscrimination tests were performed by cyclic voltammetry at a 0.05 V/s scan rate on screen-printed electrode (SPE) supports (Dropsens, custom made without paint, with Au working and counter electrodes and an Ag pseudoreference electrode, resulting in good reproducibility at constant conditions with the present working protocol). The experiments were performed using (P)-2 or (M)-2 salts as 0.01 M chiral additives in achiral commercial IL 1-butyl-3-methylimidazolium bis(triﬂuoromethanesulfonyl)imidate (BMIM)NTf2 (CAS 174899-83-3; Aldrich 98%) with the same counteranions. CVs were recorded in open air conditions, depositing on the working electrode a drop of one of the above chiral media with 0.002 M (R)- or (S)-antipodes of N,N0- dimethyl-1- ferrocenylethylamine (Aldrich, submitted to a further chromatographic puriﬁcation step), usually employed as model chiral probe by some of us when testing “inherently chiral” electrode surfaces and media on account of its chemical and electrochemical reversibility. A second series of enantioselection experiments was performed using differential pulse voltammetry (DPV) on laboratory screen-printed electrode cells on a plastic (polyester) sheet with an insulating layer including graphite working and counter electrodes and an Ag pseudoreference, resulting in good reproducibility at constant conditions with the present working protocol. Again, the experiments were performed in open air conditions, using the (P)-2 salt as the 0.01 M chiral additive in achiral commercial IL (BMIM)NTf2 to test (R)- and (S)-enantiomers of tyrosine methyl ester hydrochloride ((S)-L-Tyr Me Ester from Aldrich; (R)-D-Tyr Me Ester from Alfa Aesar) at 0.002 M concentration.

3.3. Synthesis of 5-aza[6]helicene The synthesis was realized as described in [11]. Precursor 3, which is formed as a mixture of Z and E isomers, was photolysed in various solvents or solvent mixtures in Molecules 2021, 26, 311 10 of 13

Pyrex vessels with visible light for 24 h. When the photolysis took place in a hexane - ethyl acetate 9:1 a mixture of the desired product (1) was obtained in 34% yield, with traces of its isomer (4) (Scheme2), The byproduct proved difficult to separate completely from the azahelicene by column chromatography; after many attempts, separation was obtained by using alumina as stationary phase and by eluting with hexane/ethyl ether 4:1. (±) phenanthro[3,2-k]phenanthridine (4). 1H NMR (300.14 MHz, HZ/pPT(Hz) = 0.15, 3 3 CDCl3) δ 9.69 (s, 1H), 9.39 (s, 1H), 9.30 (s, 1H), 9.28 (d, J = 2.7 Hz, 1H), 8.91 (d, J = 8.1 Hz, 1H), 8.44–8.36 (m, 1H), 8.24 (d, 3J = 8.7 Hz, 1H), 8.07–7.92 (m, 3H), 7.91–7.66 (m, 5H); 13C NMR (75.48 MHz, HZ/pPT(Hz) = 0.13, CDCl3) δ 151.94 (s), 146.43 (s), 133.30 (s), 132.42 (s), 131.69 (s), 131.13 (s), 130.10 (s), 129.95 (s), 129.48 (s), 128.77 (s), 128.48 (s), 128.35 (s), 127.97 (s), 127.76 (s), 127.45 (s), 127.31 (s), 127.14 (s), 126.77 (s), 125.05 (s),124.82 (s), 124.75 (s), 123.25 (s), 122.58 (s). The alternative synthesis of the same product (Scheme3) was realized by refluxing p- bromobenzylphosphonium bromide (3.9 mmol) and 3-quinolinecarboxaldehyde (3.9 mmol) with 11.7 mmol (3 eq) of t-BuOK in 50 mL of methanol overnight. Methanol was evaporated and the crude dissolved in ethyl acetate and washed with water. The organic phase was dried on anhydrous Na2SO4, evaporated and purified by column chromatography on silica gel, eluting with hexane/ethyl acetate 1:1. The product, p-bromophenyl-3-quinolyl-ethane, was obtained in 79% yield. It was then photolyzed in ethyl acetate in quartz vessels with 366-nm lamps for 2.5 h, in the presence of catalytic I2-. The photolyzed solutions were united, evaporated and chromatographed on silica gel with hexane/ethyl acetate 1:1. The yield of 11-bromobenzo[k]phenanthridine was 86%. This latter product was then reacted with styrene (4 eq) in dimethylacetamide (DMA, 22 mL) in the presence of 0.01 eq of ◦ (Ph3P)2PdCl2 and 3 eq of sodium acetate trihydrate, under nitrogen atmosphere at 140 C for 2 days. The solution was cooled at room temperature, diluted with AcOEt, washed with water (three times) to eliminate the DMA, evaporated, and chromatographed on silica gel with hexane/ethyl acetate 1:1. The yield of product 5 was 94%. Product 5 was then dissolved in ethyl acetate (0.5 mg/mL) and photolyzed at 366 nm for 2 h. The solution was then evaporated and product 1 purified by column chromatography over silica gel, eluting with hexane-acetate 1:1. Yield 90%

3.4. Synthesis of (P)-5-octyl-5-aza[6]helicenium Iodide The iodooctane (0.4 mL) was added to (P)-5-aza[6]helicene (3.31 mg, 0.01 mmol). The reaction mixture was heated at 80 ◦C for 56 h, then the crude was washed with hexane. The 1 product was obtained (5.3 mg, 93%). H NMR (300.14 MHz, HZ/pPT(Hz) = 0.09, CDCl3) δ 11.41 (s, 1H), 8.88 (d, 3J = 8.1 Hz, 1H), 8.39 (d, 3J = 8.1 Hz, 1H), 8.32 (d, 3J = 8.1 Hz, 1H), 8.18 (d, 3J = 9.0 Hz, 1H), 8.15 (d, 3J = 8.4 Hz, 1H), 8.10 (d, 3J = 8.7 Hz, 1H), 8.03 (d, 3J = 9.0 Hz, 1H), 7.95 (d, 3J = 7.8 Hz, 1H), 7.79 (d, 3J = 8.4 Hz, 1H), 7.70 (t, 3J = 7.8 Hz, 1H), 7.48 (d, 3J = 8.4 Hz, 1H), 7.37 (t, 3J = 7.5 Hz, 1H), 7.04 (t, 3J = 7.8 Hz, 1H), 6.87 (t, 3J = 7.7 Hz, 1H), 5.47 (t, 3J = 7.5 Hz, 2H), 2.33 (quint, 3J = 7.5 Hz, 2H), 1.80-1.19 (m, 10H), 0.89 (t, 3J = 6.6 Hz, 3H); APT NMR (75.48 MHz, HZ/pPT(Hz) = 0.14, CDCl3) δ 152.82 (s), 138.55 (s), 135.38 (s), 133.67 (s), 132.78 (s), 132.42 (s), 132.34 (s), 131.35 (s), 131.00 (s), 129.90 (s), 128.94 (s), 128.40 (s), 127.84 (s), 127.76 (s), 127.54 (s), 127.09 (s), 126.95 (s), 126.12 (s), 125.82 (s), 125.40 (s), 124.97 (s), 123,25 (s), 121,49 (s), 117.69 (s), 57.51 (s), 31.64 (s), 31.53 (s), 30.56 (s), 29.14 (s), 29.05 (s), 26.59 (s), 22.52 (s), 14.00 (s).

3.5. Synthesis of (P)-5-octyl-5-aza[6]helicenium bis(trifluoromethanesulfonyl)imidate A solution of silver bis(trifluoromethanesulfonyl)imide (3.06 mg, 7.9 × 10−3 mmol) in EtOH (0.2 mL) was dropped into a stirred solution of (P)-5-octyl-5-aza[6]helicenium iodide (4.5 mg, 7.9 × 10−3 mmol) in EtOH (0.2 mL). Stirring was continued for 72 h at room temperature to complete the reaction. During this time the precipitate was formed, and this was removed by filtration. Finally, the filtrate was evaporated under reduced pressure and the corresponding product was obtained as a waxy solid (4.8 mg, 84%). 1 3 H-NMR (300.14 MHz, HZpPT(Hz)=0.15, CDCl3) δ 10.14 (s, 1H), 8.55 (d, J = 8.4 Hz, Molecules 2021, 26, 311 11 of 13

1H), 8.39 (d, 3J = 8.4 Hz, 1H), 8.33 (d, 3J = 8.4 Hz, 1H), 8.17 (d, 3J = 8.4 Hz, 1H), 8.15 (d, 3J = 8.1 Hz, 1H), 8.11 (d, 3J = 8.7 Hz, 1H), 8.03 (d, 3J = 8.7 Hz, 1H), 7.95 (d, 3J = 7.8 Hz, 1H), 7.80 (d, 3J = 8.7 Hz, 1H), 7.72 (t, 3J = 7.4 Hz, 1H), 7.44 (d, 3J = 8.7 Hz, 1H), 7.37 (d, 3J = 7.5 Hz, 1H), 7.04 (d, 3J = 7.7 Hz, 1H), 6.85 (d, 3J = 7.8 Hz, 1H), 5.32-5.12 (m, 2H), 2.27 (quint, 3J = 7.6 Hz, 2H), 1.70-1.52 (m, 2H),1.52-1.20 (m, 8H), 0.89 (t, 2J = 6.6 Hz, 3H); 19 F NMR (300.14 MHz, HZ/pPT(Hz) = 0.87, CDCl3) δ −78.0 (s); APT NMR (100.62 MHz, HZ/PT(Hz)=1.47, CDCl3); δ 152.46 (s), 138.82 (s), 135.68 (s), 133.55 (s), 132.84 (s), 132.62 (s), 132.47 (s), 131.54 (s), 131.47 (s), 130.11 (s), 129.15 (s), 128.51 (s), 128.07 (s), 127.90 (s), 127.54 (s), 127.50 (s), 127.12 (s), 126.14 (s), 125.86 (s), 125.58 (s), 123.22 (s), 121.60 (s), 119.82 (q, 2J(C,F) = 322.0 Hz), 117.61 (s), 58.26 (s), 31.62 (s), 30.20 (s), 29.03 (s), 26.53 (s), 22.55 (s), 14.02 (s). APT NMR Only Quaternary Carbon (100.62 MHz, HZ/PT(Hz)=1.47, CDCl3); δ 138.84 (s), 135.68 (s), 132.86 (s), 132.62 (s), 132.49 (s), 128.54 (s), 127.92 (s), 125.60 (s), 123.27 (s), 121.62 (s), 119.87 (q, 2J(C,F) = 321.0 Hz).

3.6. Enantioselective HPLC Column: Chiralpak IA 250 mm × 4.6 mm, mobile phase: n-hexane-IPA-ethyl acetate- DEA 100/5/5/0.2, ﬂow rate: 1 mL/min, detector: UV/CD at 325 nm. The CD spectra of the enantiomers collected on a semipreparative scale were recorded in chloroform at 25 ◦C by using a Jasco Model J-700 spectropolarimeter. The optical path was 0.1 cm. The spectra are average computed over three instrumental scans and the intensities are presented in terms of ellipticity values (mdeg).

3.7. Off-Column Racemization Study A solution of (P)-1 in dimethylformamide (concentration about 0.2 mg/mL) was held at 100 ◦C in a closed vessel. The temperature was monitored by a thermostat Julabo HE-4. Samples were withdrawn at ﬁxed time intervals and the ee decay over time was monitored by HPLC on the Chiralpak IA (250 mm × 4.6 mm i.d.) column under normal-phase mode.

4. Conclusions A new ICIL was synthesized, characterized, and successfully tested as additive of a commercial IL for electrochemical enantiodiscrimination purposes. In fact, significant peak potential differences were reproducibly observed in CV or DPV experiments for the enantiomers of two different chiral electroactive probes. The conclusions that we can draw from this very preliminary experiment are twofold. The first consideration is that inherent chirality extraordinarily enhances the enantioselection ability in comparison with chiral ILs designed according to more traditional schemes involving the separation between chiral and onium moieties [16]. In fact, peak potential separations jump here from a few dozen mV to more than 100. The second point is that helicity induces enantioselectivities comparable to those produced by stereogenic axes. The helix can be regarded as an ideal stereogenic element in the design of inherently chiral selectors, even though the access to enantiopure helical systems is definitely more troublesome than the synthesis of chiral compounds endowed with axial stereogenicity. We plan to extend the study to diazahelicenes quaternarized on one or both nitrogen atoms in order to compare them to the mono- and dialkylated bicollidinium compound.

Supplementary Materials: The following are available online. Figure S1: Normalized CV patterns of compound 1 (0.00075 M) at scan rates in the 0.02–2 V/s range, in acetonitrile, with 0.1 M TBAPF6 as the supporting electrolyte, applying ohmic drop compensation by the positive feedback method and referring the potentials to the Fc+|Fc redox couple (the intersolvental standard recommended by IUPAC) measured in the same conditions (~0.39 V vs. SCE). Figure S2: Normalized CV pattern of compound 2 (0.00075 M) at scan rates in the 0.02–2 V/s range, in acetonitrile, with 0.1 M TBAPF6 as the supporting electrolyte, applying ohmic drop compensation by the positive feedback method and referring the potentials to the Fc+|Fc redox couple (the intersolvental standard recommended by IUPAC) measured in the same conditions (~0.39 V vs. SCE). Figure S3. The ﬁrst reduction peak of 2 Molecules 2021, 26, 311 12 of 13

tending to split or to feature a preceding shoulder, confirmed in two experiments at different times and different scan rates. Author Contributions: Conceptualization, S.R. and F.F.; Investigation, G.C., B.B., R.C., S.A., S.G., and S.R.; Resources, L.M.; Supervision, F.F. and P.R.M.; Writing—original draft preparation, S.R., P.R.M., and F.F. All authors have read and agreed to the published version of the manuscript. Funding: B.B. acknowledges funding from Università di Bergamo, Program STaRs Supporting Talented Researchers 2017–2018. P.R.M., S.A., and S.G. acknowledge financial support from the Fondazione Cariplo and Regione Lombardia (2016-0923 RST—Avviso congiunto FC-RL Sottomisura B) rafforzamento (Enhancing VINCE (Versatile INherently Chiral Electrochemistry)) and from the Università degli Studi di Milano. Institutional Review Board Statement: Not applicable. Informed Consent Statement: Not applicable. Data Availability Statement: Data sharing not applicable. Acknowledgments: The authors gratefully acknowledge Francesco Sannicolò for helpful discussion and Pasquale Illiano for his contributions on NMR characterization. Conflicts of Interest: The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results. Sample Availability: Samples of compound 1 are available from the authors.

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