S S symmetry

Article A General Phenomenon of Spontaneous Amplification of Optical Purity under Achiral Chromatographic Conditions

K. Michał Pietrusiewicz 1 , Mariusz Borkowski 1,2 , Dorota Strzelecka 1, Katarzyna Kielar 1, Wioleta Kici ´nska 1, Sergei Karevych 1, Radomir Jasi ´nski 3 and Oleg M. Demchuk 1,4,*

1 Faculty of Chemistry, Maria Curie-Skłodowska University, 2 M. Sklodowskiej-Curie Square, 20-031 Lublin, Poland; [email protected] (K.M.P.); [email protected] (M.B.); [email protected] (D.S.); [email protected] (K.K.); [email protected] (W.K.); [email protected] (S.K.) 2 Institute of Catalysis and Surface Chemistry, Polish Academy of Sciences, 8 Niezapominajek Str., 31-155 Cracow, Poland 3 Institute of Organic Chemistry and Technology, Cracow University of Technology, 24 Warszawska Str., 31-155 Cracow, Poland; [email protected] 4 Pharmaceutical Research Institute, 8 Rydygiera Str., 01-793 Warsaw, Poland * Correspondence: [email protected]



Received: 2 April 2019; Accepted: 15 May 2019; Published: 17 May 2019


Abstract: This work explores the behavior of chiral compound mixtures enriched in one of the enantiomers whilst a typical chromatography on the achiral stationary phase is employed. The influence of several factors, such as the eluent composition, ratio of the compound to the stationary phase, and the initial enatiomeric purity of the compound used on the distribution of the enantiomers in the collected chromatographic fraction, was studied. The obtained results indicate that the phenomenon of Self Disproportionation of Enantiomer (SDE) occurred in all cases, and some of the collected fractions got higher optical purities than the initial one. Thus, achiral column chromatography could be applied in some cases as the simplest approach for chiral purification. Based on the experimental results and DFT calculations, an alternative concept explaining the SDE phenomenon was proposed. Due to its generality and simplicity, SDE may also be responsible for the formation of the first chiral non-racemic compounds on the early Earth.

Keywords: enantiomer self-disproportionation; SDE; achiral stationary phase; homochiral and heterochiral aggregates; chiral separation; chirality; genesis of life chirality

1. Introduction The mystery of life on Earth is closely connected with the phenomenon of the chiral nature of all living organisms (human beings, animals, plants, insects, etc.). Chirality, as a fundamental property of 3-dimensional objects (including many molecules and ions) to be non-superinposable on their mirror images, is imprinted in such basic building blocks of life as amino acids and sugars; it is further reflected by the chirality of more complex biochemical objects such as DNA and eventually by the chirality of entire bodies [1,2]. Interestingly, amino acids [3] and sugars [4] derived from natural sources are non-racemic and are constituted of single stereoisomers. At the same time, according to the fundamental chemical principles the formation of non-racemic chemical compounds is impossible when the chiral component of the chemical reaction (substrate, catalyst, medium) is absent [5]. Despite its crucial value for the understanding of the origin of life, the origins of the chirality of molecules of life are still unknown [6]. Apart from the creationist approach stating that “the only possible way

Symmetry 2019, 11, 680; doi:10.3390/sym11050680 www.mdpi.com/journal/symmetry Symmetry 2019, 11, 680 2 of 20 for unique homochirality to exist in the chiral biochemical molecules found in living organisms is for those biomolecules to have been created with unique homochirality when that organism was first created. Just as a fingerprint identifies its creator” [7], there are several other theories trying to address this issue [8]: partial photochemical decomposition of the racemic mixtures of chiral compounds in the interstellar clouds caused by polarized cosmic irradiation [9,10]; photochemical reactions in the primordial soup influenced by the Sun’s light polarized by passing though the Earth atmosphere [11,12]; the effect of the parity-symmetry violation of the weak force [13]; the stereochemical outcome of the chemical reactions influenced by a magnetic field [14]; asymmetric catalysis mediated with chiral minerals e.g., single quartz crystals [15,16]; stereoselective crystallization of a single enantiomer from the racemic mixture [17,18]. Since the formation of both enantiomers is statistically equal, all the variety of the chiral compounds on Earth should have originated from almost a single act of creation. Moreover, in all those rare cases, mixtures with only an extremely low excess of one of the enantiomers could be formed. Thus, the major question of the chirality genesis is how—from an almost racemic mixture of chiral probiotic compounds formed accidentally, once or few times only—an optically pure substance could have been spontaneously created. The process of the spontaneous enrichment of the enantiomer mixture and the generality of this phenomenon could be studied on the example of several typical benchmark asymmetric syntheses where some enantio-enriched mixture of the chiral compounds is formed. As a result of the significant progress made in the last few decades, the asymmetric catalysis has become the basic tool of synthetic organic chemistry used to obtain modern medicines [19], and cosmetics [20], plant protecting substances [21], and variety of functional materials [22,23]. Valuable features of those kinds of transformations include: the highest atom economy (thus, only one of two possible enantiomers are usually desirable); a short synthetic pathway because of the elimination of chiral derivatisation and racemic mixture separation steps; mild reaction conditions; wide substitution pattern toleration; and many others—all of the above made these transformations highly attractive economically. Therefore, they draw the attention of both academics and the industry. The importance of this field has also entailed the development of methods for the determination of the enantiomeric composition of the obtained products directly or after derivatisation by chiral reagents. There are the classical measurement of optical rotation power [24,25], chiral chromatographic approaches [26,27], NMR methodologies utilising chiral shifting reagents [28–31] and chiral derivatisation reagents [32–34]. In the majority of cases, the determination of the enantiomeric composition of the mixtures of isomeric products obtained in the reaction is performed after preliminary purification of the sample studied. Distillation is only marginally useful in the case of polysubstituted organic materials. Such routine purification techniques as crystallisation and sublimation cannot ensure the invariability of the enantiomeric composition in a rough reaction mixture and isolated substance because of a widely observed enantiomer self-disproportionation phenomenon (SDE). SDE was defined in 2006 as a transformation of an enantiomerically enriched system resulting in the formation of fractions with different, in comparison to the initial enantiomeric distribution [35]. The SDE phenomenon is attributed to two distinct models of the intermolecular interaction between the enantiomers of a chiral compound. These are homochiral (R,R or S,S) and heterochiral (R,S)-associations (Figure1) which are present in a not completely racemic mixture and are largely responsible for the nonlinear behavior of the optical rotation [35,36]. Symmetry 2019, 11, 680 3 of 20 Symmetry 2018, 10, x FOR PEER REVIEW 3 of 20

Figure 1. Preferential formation of homochiral or heterochiral aggregates in the solution of optically Figure 1. Preferential formation of homochiral or heterochiral aggregates in the solution of optically enriched compounds. enriched compounds. Thus, column chromatography is usually the method of choice for the purification of reaction mixturesThus, in column laboratory chromatography practice and it isis extensivelyusually the used method for the of preparationchoice for the of analyticalpurification samples of reaction from dimixturesfferent asymmetricin laboratory reactions. practice Forand ait long is extensively time, the chemicalused for communitythe preparation accepted of analytical the assumption samples that,from withdifferent very fewasymmetric exceptions reactions. [37], the For ratio a oflong enantiomers time, the wouldchemical not community be significantly accepted changed the duringassumption chromatographic that, with very purification, few exceptions unless [37], some the otherratio of chiral enantiomers players suchwould as not chiral be significantly solvents or additiveschanged orduring a chiral chromatographic stationary phase purification, are used. Recent unless studies some indicate other chiral that this players rule could such beas broken chiral insolvents the case or additives of chiral fluorinatedor a chiral stationary compounds phase because are used. of notableRecent studies chiral self-aggregationindicate that this promotedrule could bybe abroken strong in dispersive the case of interaction chiral fluorinated of fluorous compounds tails [35,38 because]. Rare of literature notable exampleschiral self-aggregation also suggest thatpromoted a partial by separationa strong dispersive of the racemate interaction from of thefluo enantiomerrous tails [35,38]. could Rare be observed literature for examples some other also enantiomericalysuggest that a partial enriched separation compounds of the subjected racemate to from achiral the chromatographic enantiomer could purification be observed via for regular some gravityother enantiomericaly driven chromatography enriched oncompounds silica gel [subjecte39–42],d or to HPLC achiral [43 chromatographic], MPLC [37], flash purification [44], or other via chromatographicregular gravity driven purification chromatography techniques on [45 silica]. However, gel [39–42], this or possibility HPLC [43], is stillMPLC usually [37], neglectedflash [44], inor routineother chromatographic chromatographic purificationpurification ontechniques regular achiral [45]. stationaryHowever, phases.this possibility is still usually neglectedHerein, in routine we would chromatographic like to demonstrate purificati thaton SDE on regular is a common achiral phenomenonstationary phases. observed during the achiralHerein, low-pressure we would like column to demonstrate chromatography that SDE of is the a common chiral compounds phenomenon obtained observed in benchmark during the asymmetricachiral low-pressure reactions suchcolumn as allylicchromatography substitution, of [46 the,47 ]chiral cross-coupling compounds [48 –obtained52] and organocatalytic in benchmark aldolasymmetric condensation reactions [53 ,54such] as as well allylic as other substitution important, [46,47] asymmetric cross-coupling transformations. [48–52] Thus,and organocatalytic such processes mayaldol play condensation a significant [53,54] role in as the well course as ofother the chiral,important non-racemic, asymmetric pro-biotic transformations. compound formation.Thus, such processes may play a significant role in the course of the chiral, non-racemic, pro-biotic compound 2.formation. Materials and Methods All the chemicals used in this research were purchased from Sigma-Aldrich, Avantor Performance 2. Materials and Methods Materials Poland S.A. (formerly POCH S.A) and Merck. Analytical thin-layer chromatography (TLC) was performedAll the chemicals using silica used gel 60in Fthis254 precoatedresearch plateswere (0.25purchased mm thickness). from Sigma-Aldrich, Visualization Avantor of TLC platesPerformance was performed Materials by Poland means ofS.A. UV (formerly light. NMR PO spectraCH S.A) were and recorded Merck. onAnalytical Bruker Avance thin-layer 500 MHzchromatography spectrometers, (TLC) and was chemical performed shifts using are silica reported gel 60 in F ppm,254 precoated and calibrated plates (0.25 to residual mm thickness). solvent 1 13 peaksVisualization at 7.27 ppm of TLC and plates 77.00 ppmwas performed for H and byC mean in CDCls of 3UVor TMSlight. as NMR internal spectra reference were compounds.recorded on TheBruker enantiomeric Avance 500 compositions MHz spectrometers, of the obtained and chemical fractions shifts were are determinedreported in ppm, by a chromatographicand calibrated to methodresidual (Shimadzusolvent peaks LCMS at 7.27 IT-TOF ppm spectrometer)and 77.00 ppm usingfor 1H chiraland 13 chromatographicC in CDCl3 or TMS columns as internal Chiralpak reference® andcompounds. Chiralcel ®Thewith enantiomeric the following compositions dimensions: 150 of the2.1 mm,obtained or by 1fractionsH NMR andwere13 Cdetermined NMR techniques by a × usingchromatographic an appropriate method chiral (Shimadzu differentiating LCMS factor IT-TOF and spectrometer) solvents. The using purification chiral chromatographic process and the studycolumns of theChiralpak SDE phenomenon® and Chiralcel were® with conducted the following on a flash dimensions: low pressure 150 BUCHI × 2.1 mm, chromatograph or by 1H NMR using and 1213C NMR150 mm techniquesPE columns using packed an appropriate by authors chiral with diffe 10 grentiating spherical factor silica geland 60 solvents. with the The particle purification size of × 230–400process meshand the as the study stationary of the phase.SDE phenomenon The studied compounds were conducted were dissolved on a flash in 5low mL ofpressure an appropriate BUCHI solventchromatograph and mixed using with 12 1g × of 150 silica mm gel. PE Next, columns the solvent packed was by evaporatedauthors with and 10 the g spherical sample supported silica gel on60 silicawith wasthe placedparticle at size the topof of230–400 the column mesh prepacked as the stationary with silica. phase. After The that, studied the column compounds was connected were todissolved the MPLC in to5 mL be subjectedof an appropriate to SDE studies. solvent In and the mixed case of with compounds 1g of silica1 and gel.6, Next, instead the of solvent supporting was evaporated and the sample supported on silica was placed at the top of the column prepacked with silica. After that, the column was connected to the MPLC to be subjected to SDE studies. In the case

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Symmetryof compounds2019, 11, 6801 and 6, instead of supporting them on silica, the solutions of the studied compounds4 of 20 in 0.6 mL of mobile phase were injected onto the column prepacked with 11 g of silica and prewashed themwith onthe silica,mobile the phase. solutions The offractions the studied were compounds automatically in 0.6 collected mL of mobile by 18 phasemL volume. were injected The elution onto theprogress column was prepacked monitored with by 11 TLC. g of silicaAll DFT and calculations prewashed withwere theperformed mobile phase. using Thethe fractions“Prometheus” were automaticallycluster in the collected“Cyfronet” by computational 18 mL volume. center The elution in Cracow. progress A new was monitoredgeneration byM062x TLC. [55]; All DFT[56] calculationsfunctional, implemented were performed in the using Gau thessian “Prometheus” 09 package cluster[57], was in used. the “Cyfronet” This functional computational has been centerrecently in Cracow.used by Aus newto solve generation several M062x similar [ 55problems]; [56] functional, [58–60]. All implemented stationary instructures the Gaussian have 09been package optimized [57], wasusing used. the advanced This functional 6-311++G(d,p) has been basis recently set and used were by characterized us to solve several by only similar positive problems eigenvalues [58–60 in]. Alltheir stationary diagonalized structures Hessian have matrices. been optimized For the using optimized the advanced structures, 6-311 thermochemical++G(d,p) basis setdata and for were the characterizedtemperature T by = 298 only K positive and pressure eigenvalues p = 1 atm in theirwere diagonalizedcomputed using Hessian vibrational matrices. analysis For the data. optimized For the structures,simulation thermochemicalof the solvent presence, data for the the PCM temperature model ha T s= been298 Kapplied, and pressure similar p to= in1 atmprevious were computedour works using[59,60]. vibrational analysis data. For the simulation of the solvent presence, the PCM model has been applied, similar to in previous our works [59,60]. 3. Results and Discussion. 3. ResultsIn order and to Discussion. investigate the impact of a routine chromatographic purification process on the distributionIn order of to enantiomers investigate in the collected impact offraction a routines, non-racemic chromatographic mixtures purification of the studied process compounds on the distributionwere obtained of in enantiomers the usual benchmark in collected reactions. fractions, non-racemic mixtures of the studied compounds were obtained in the usual benchmark reactions. 3.1. Study on the SDE of Dicarboxylic Acid Esters: (R)-Dimethyl [(2E)-1,3-Diphenyl-2-Propen-1-yl] 3.1.MalonateStudy (1) on the SDE of Dicarboxylic Acid Esters: (R)-Dimethyl [(2E)-1,3-Diphenyl-2-Propen-1-yl] Malonate (1) TheThe asymmetricasymmetric allylicallylic substitutionsubstitution reactionreaction leadingleading toto ((RR)-dimethyl)-dimethyl [(2[(2E)-1,3-diphenyl-2-)-1,3-diphenyl-2- propen-1-yl]propen-1-yl] malonatemalonate (1)) isis aa standardstandard processprocess usedused toto assessassess thethe eefficiencyfficiency andand comparecompare thethe chiralchiral transitiontransition metalmetal complexcomplex basedbased catalystscatalysts [[59].59]. The product of this benchmark reaction was evaluated first.first. The reaction wa wass carried out using (R)-(-)-)-(-)-BisNap-Phos [48] [48] as aa ligandligand andand furnishedfurnished crudecrude productproduct 11(Scheme (Scheme1 ),1), which which was was subsequently subsequently subjected subjected to theto the enantiomeric enantiomeric purity purity determination determination by anby HPLCan HPLC technique. technique. The The enantiomeric enantiomeric composition composition and and (R)-absolute (R)- absolute configuration configuration of the of product the product were determinedwere determined by the by peak the peak integration integration and elutionand elution order order from from chiral chiral HPLC HPLC using using a Chiralcel a Chiralcel® OD-H® OD- columnH column [61 [61].]. The The enantiomeric enantiomeric excess excess of theof the crude crude product product was was 49.0%. 49.0%.

Scheme 1.1. Synthesis of (R)-dimethyl [(2E)-1,3-diphenyl-2-propen-1-yl][(2E)-1,3-diphenyl-2-propen-1-yl] malonatemalonate ((11).).

Next,Next, thethe productproduct waswas chromatographicallychromatographically purifiedpurified onon thethe achiralachiral stationarystationary phase.phase. TheThe eluenteluent system selection was based on the Rf values (~0.2) and hence the mixtures of hexane/acetone in the system selection was based on the Rf values (~0.2) and hence the mixtures of hexane/acetone in the ratiosratios ofof (99(99/1)/1) andand (99.7 (99.7/0.3)/0.3) were were selected selected as as mobile mobile phases. phases. 50.0 50.0 mg mg of of compound compound1 was 1 was subjected subjected to the purification process on an achiral 12 150 mm column filled with 11.0 g silica gel. The separations to the purification process on an achiral× 12 × 150 mm column filled with 11.0 g silica gel. The wereseparations run at were the eluent run at flowthe eluent rate of flow 18 mLrate/min of 18 and mL/min the collected and the collected fractions fractions had the volumehad the volume of 18 mL. of The18 mL. fractions The fractions were collectedwere collected from from the moment the moment when when the the eluted eluted compound compound was was detected detected byby TLC. TheThe enantiomericenantiomeric compositionscompositions of consecutiveconsecutive fractionsfractions were determined by means ofof chiralchiral HPLCHPLC with the application of a Chiralpak® AS-RH column and the mobile phase H O/CH CN = 55/45, and with the application of a Chiralpak® AS-RH column and the mobile phase H2O/CH2 3CN3 = 55/45, and 0.4 ® ® 0.4mL/min mL/min flow flow (Figure (Figure 1(ESI)). S1). TheThe ChiralpakChiralpak® OJ-RH and ChiralcelChiralcel® OD-H columns could also be usedused forfor this purpose. The The elution elution profiles profiles for for both both eluent eluent systems systems are are depicted depicted in in Figure Figure 22 and and Table Table 1(ESI). S1.

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FigureFigure 2. SDE 2. SDE of compound of compound1 in 1 achiralin achiral MPLC MPLC conditions: conditions: the the elution elution profiles.profiles. The The fitting fitting equation equation for 2 hexanefor/acetone hexane/acetone= 99/1 is = y99/1= 0.018x is y = 0.018x+ 49.429 + 49.429 (R = (R0.851)2 = 0.851) and forandhexane for hexane/acetone/acetone = 99.7 = 99.7/0.3/0.3 is yis =y 2.099x= + 40.42.099x (R2 = +0.768). 40.4 (R2 = 0.768).

The mobileThe mobile phases phases were were selected selected by by the the optimizationoptimization process process using using the the TLC TLC method. method. Several Several possiblepossible solvent solvent mixtures mixtures and and pure pure solvents solvents were were testedtested in this this process. process. In Inthe the next next step, step, those those mobile mobile phases,phases, for whichfor which the the separation separation on on the the plates plates gavegave promising results results were were selected; selected; these these mobile mobile phasesphases were were then testedthen tested in the in column the column separation separation process. process. In those In those experiments, experiments, several several mobile mobile phases werephases examined were and examined finally thoseand finally for which those thefor separationwhich the separation result was result good was enough good were enough obtained were and obtained and results obtained in the context of the SDE phenomenon. This procedure of mobile phase results obtained in the context of the SDE phenomenon. This procedure of mobile phase optimization optimization was used in a course of all further separations. In this case, in the optimization process, was usedhexane/acetone in a course with of all ratios further of 99:1 separations. and 99.7:0.3 In was this selected case, in as the mobile optimization phase. It can process, be seen hexane that in /theacetone with ratioscourse ofof 99:1the purification, and 99.7:0.3 the was ee values selected of the as mobilecollected phase. fractions It canincreased be seen slightly that infrom the the course initial of the purification,level of 49.0% the ee forvalues hexane/acetone of the collected = 99:1 and fractions more significantly increased for slightly hexane/acetone from the in initial the ratio level of 99.7: of 49.0% for hexane0.3 (to/ acetone51.0% and= 54.0%,99:1 and respectively). more significantly The values for of hexanethe SDE/ acetonemagnitude in ( the∆ee) ratio parameter of 99.7: (defined 0.3 (to as 51.0% and 54.0%,the difference respectively). between The the valueshighest ofand the the SDE lowest magnitude value of (the∆ee enantiomeric) parameter excess (defined of the as thecollected difference betweenfractions) the highest equaled and 1.8% the lowestand 7.0% value for of hexane/acetone the enantiomeric = excess99:1 and of thehexane/acetone collected fractions) = 99.7:0.3, equaled 1.8%respectively. and 7.0% for These hexane results/acetone indicate= 99:1 that andthe eluent hexane used/acetone in the= first99.7:0.3, seriesrespectively. of the experiment These was results characterized by a polarity too high to allow effectively the occurrence of SDE. In contrast to the first indicate that the eluent used in the first series of the experiment was characterized by a polarity too series, it is noticeable that the utilization of a less polar eluent leads to obtaining a compound with a high to allow effectively the occurrence of SDE. In contrast to the first series, it is noticeable that the slightly higher ee value in the latter fractions. These results immediately allowed us to state that the utilizationpurification of a less process polar of eluent the cr leadsude reaction to obtaining mixture a compoundon achiral silica with agel slightly may affect higher theee eevalue value. in the latterSecondly, fractions. the These SDE resultsof compound immediately 1 depends allowed on theus eluent to state composition that the used purification in the purification process of the crudeprocess; reaction the mixture less polar on eluent achiral facilitates silica gel the may SDE aphenomenon.ffect the ee value. Secondly, the SDE of compound 1 depends on the eluent composition used in the purification process; the less polar eluent facilitates the SDE phenomenon.3.2. Study on the SDE of Atropisomeric Biaryl: 2,2′-Dimethoxy-1,1′-Binaphthyl (2) Inspired by the initial results, we decided to investigate whether the SDE phenomenon may be 3.2. Studyapplicable on the to SDE the enantiopurification of Atropisomeric Biaryl: of 2,2′-dimethoxy-1,1 2,20-Dimethoxy-1,1′-binaphthyl0-Binaphthyl (2) enriched (2) in the (R)-isomer Inspired(Scheme 2). by Compound the initial results,2 was obtained we decided in a benchmark to investigate Suzuki-Miyaura whether the reaction SDE phenomenon which is usually may be used to assess the efficiency of a palladium catalyst in cross-coupling reactions. The reaction that was applicable to the enantiopurification of 2,20-dimethoxy-1,10-binaphthyl (2) enriched in the (R)-isomer (Scheme 2). Compound 2 was obtained in a benchmark Suzuki-Miyaura reaction which is usually used to assess the efficiency of a palladium catalyst in cross-coupling reactions. The reaction that was run in the presence of 0.3% SDS and Na2CO3 in an aqueous medium at ambient temperature utilized Symmetry 2018, 10, x FOR PEER REVIEW 6 of 20 Symmetry 2019, 11, 680 6 of 20 run in the presence of 0.3% SDS and Na2CO3 in an aqueous medium at ambient temperature utilized the (S)-SymmetryBisNap 2018-Phos, 10, x ligand FOR PEER (1 REVIEW mol%) and Pd(C6H5CN)2Cl2 pre-catalyst (0.5 mol%). The enantiomeric6 of 20 the (S)-BisNap-Phos ligand (1 mol%) and Pd(C6H5CN)2Cl2 pre-catalyst (0.5 mol%). The enantiomeric excess of 22 was determined by an HPLC technique using a Chiralpak®® AS-RH column, and the mobile excessrun of in wasthe presence determined of 0.3% by SDS an HPLC and Na technique2CO3 in an usingaqueous a Chiralpak medium at ambientAS-RH temperature column, and utilized the mobile phase H2O/CH3CN = 50/50 (Figure 2(ESI)). The initial enantiomeric excess was assigned as 41.0% ee. phasethe H 2(OS)-/CHBisNap3CN-Phos= 50 ligand/50 (Figure (1 mol%) S2). and The Pd(C initial6H5CN) enantiomeric2Cl2 pre-catalyst excess (0.5 was mol%). assigned The enantiomeric as 41.0% ee . excess of 2 was determined by an HPLC technique using a Chiralpak® AS-RH column, and the mobile phase H2O/CH3CN = 50/50 (Figure 2(ESI)). The initial enantiomeric excess was assigned as 41.0% ee.

SchemeScheme 2. 2. SynthesisSynthesis of of ( (RR)-2,2)-2,2′-dimethoxy-1,10-dimethoxy-1,1′-binaphthalene0-binaphthalene (2 (2).).

Further, 100.0 mg ofScheme compound 2. Synthesis2 was of (R fractionated)-2,2′-dimethoxy-1,1 on a′-binaphthalene 12 150 mm (2 column). filled with 10.0 g Further, 100.0 mg of compound 2 was fractionated on a 12 ×× 150 mm column filled with 10.0 g silica gel, using a mixture of hexane/ethyl acetate = 97:3 as an eluent. Considering the low solubility silica gel, using a mixture of hexane/ethyl acetate = 97:3 as an eluent. Considering the low solubility of compoundFurther, 2 in 100.0 the eluent,mg of compound the compound 2 was fractionated was applied on ona 12 the × 150 gel mm and column loaded filled onto with the column10.0 g in of compound 2 in the eluent, the compound was applied on the gel and loaded onto the column in this form.silica gel, The using separations a mixture were of hexane/ethyl run at the eluentacetate flow= 97:3 rate as an of eluent. 18 mL Considering/min and the the collected low solubility fractions this form.of compound The separations 2 in the wereeluent, run the at compound the eluent was flow applied rate of on 18 the mL/min gel and and loaded the collectedonto the column fractions in had had the volume of 18 mL. The SDE profile of compound 2 is shown in Figure3 (the numeric data the volumethis form. of 18 The mL. separations The SDE were profile run of at compoundthe eluent flow 2 is rate shown of 18 mL/minin Figure and 3 (thethe collected numeric fractions data are had given are given in Table S2). As one can see, the enantiomeric excess of the examined compound increased in Tablethe 2(ESI)).volume ofAs 18 one mL. canThe see,SDE theprofile enantiomeric of compound excess 2 is shown of the in examinedFigure 3 (the compound numeric data increased are given with with the elution volume from 21.0% ee in fraction 32 (in which compound 3 was first detected by the elutionin Table volume 2(ESI)). from As one 21.0% can see,ee in the fraction enantiomeric 32 (in excesswhich of compound the examined 3 was compound first detected increased by withTLC) to TLC) to 63.0% ee in the last fraction (fraction 52). The SDE magnitude (∆ee) in the studied cases 63.0%the ee in elution the last volume fraction from (fraction 21.0% ee 52). in fraction The SDE 32 (inmagnitude which compound (Δee) in the3 was studied first detected cases equaled by TLC) 42.0%.to equaled63.0% 42.0%. ee in the The last chiral fraction HPLC (fraction chromatograms 52). The SDE magnitude of the first (Δ andee) in last the fractions studied cases are depictedequaled 42.0%. in Figure The chiral HPLC chromatograms of the first and last fractions are depicted in Figure 2(ESI). The final S2. TheThe finalchiral fractionHPLC chromatograms (fraction 52) of is the 22.0% first and more last enantiomerically fractions are depicted enriched in Figure in 2(ESI). comparison The final to the fraction (fraction 52) is 22.0% more enantiomerically enriched in comparison to the initial sample of initialfraction sample (fraction of 41.0% 52) eeis. 22.0% Such more highly enantiomerically significant value enriched of ∆ eein couldcomparison be caused to the byinitial the sampleπ-π stacking of 41.0% ee. Such highly significant value of Δee could be caused by the π-π stacking interaction between interaction41.0% ee between. Such highly the significant naphthyl value moieties of Δee [ 62could] as be it caused has been by the previously π-π stacking observed interaction for between other types the naphthylthe naphthyl moieties moieties [62] [62] as asit ithas has been been prev previouslyiously observed forfor other other types types of ofsubstituted substituted of substituted naphthalenes [63]. On the other hand, 2,20-dimethoxy-1,10-binaphthyl could easily naphthalenesnaphthalenes [63]. [63].On Onthe theother other hand, hand, 2,22,2′-dimethoxy-1,1′-dimethoxy-1,1′-binaphthyl′-binaphthyl could could easily easily undergo undergo undergo crystallization, and it is also theoretically possible that a multiple micro recrystallization crystallization,crystallization, and andit is italso is also theoretically theoretically possible possible that that aa multiplemultiple micro micro recrystallization recrystallization process process may may process may have taken place on the column and eventually influenced the enantiomeric distribution have takenhave taken place place on theon thecolumn column and and eventually eventually ininfluencedfluenced thethe enantiomericenantiomeric distribution distribution in thein the in the collected fractions. In any case, the obtained results indicate a possibility of the enantiomeric collectedcollected fractions. fractions. In anyIn any case, case, the the obtained obtained resultsresults indicate aa possibilitypossibility of ofthe the enantiomeric enantiomeric enrichment of potentially crystalline compounds (or even a separation of the enantiomeric fraction enrichmentenrichment of potentially of potentially crystall crystallineine compounds compounds (or (or eveneven a separationseparation of of the the enantiomeric enantiomeric fraction fraction fromfrom the racemate) the racemate) using using the the methodology methodology based based onon the achiral achiral chromatographic chromatographic technique. technique. from the racemate) using the methodology based on the achiral chromatographic technique.

Figure 3. SDE of compound 2 in achiral MPLC conditions: the elution profile. The fitting equation is y = 2.089x -46.242 (R2 = 0.979).

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Figure 3. SDE of compound 2 in achiral MPLC conditions: the elution profile. The fitting equation is Symmetry 2019, 11, 680 7 of 20 y = 2.089x -46.242 (R2 = 0.979).

3.3. Study on the SDE of β-Hydroxy Ketone: (4R)-(4-nitrophenyl)-4-Hydroxy-2-Butanone (4R)-(4-nitrophenyl)-4-Hydroxy-2-Butanone (3) (3) Taking into account account a a possible possible strong strong effect effect of ofthe the π-π- πforcesforces in inthe the crystal crystal net, net, we turned we turned our attentionour attention to another to another class classof organic of organic compounds. compounds. The next The chiral next model chiral compound model compound (4R)-(4- (4nitrophenyl)-4-hydroxy-2-butanoneR)-(4-nitrophenyl)-4-hydroxy-2-butanone (3) was (3) wassynthesized synthesized under under aldol aldol condensation condensation conditions (Scheme3 )[3) 64[64]] in in a reactiona reaction between between 4-nitrobenzaldehyde 4-nitrobenzaldehyde and technical and technical grade acetonegrade acetone in the presence in the presenceof 20 mol% of of20L -proline.mol% of InL- thisproline. benchmark In this organocatalyticbenchmark organocatalytic reaction the chiral reaction organic the moleculechiral organic plays moleculethe role of plays a catalyst, the role which of a is catalyst, more inherent which is to more biomimetic inherent processes. to biomimetic As a result, processes. the reaction As a result, products the reactionare not contaminated products are with not toxiccontaminated metal impurities, with toxic the metal catalyst impurities, does not undergothe catalyst decomposition, does not undergo and in decomposition,general, the process and itselfin general, is compatible the process with the itself requirements is compatible of green with chemistry, the requirements as well as chemicalof green processeschemistry, occurring as well onas thechemical early Earth. processes The optical occurri purityng on of synthesizedthe early Earth. compound The optical3 was determined purity of synthesized compound 3 was determined by® an HPLC technique using a Chiralpak® AD-RH column, by an HPLC technique using a Chiralpak AD-RH column, and the CH3CN/H2O = 30/70 mobile phaseand the [65 CH] (Figure3CN/H2 S3).O = 30/70 mobile phase [65] (Figure 3(ESI)).

Scheme 3. Synthesis of (R)-4-hydroxy-4(nitrophenyl)butan-2-one (3). Scheme 3. Synthesis of (R)-4-hydroxy-4(nitrophenyl)butan-2-one (3). The ratio of the amount of the studied compound to the amount of the stationary phase used The ratio of the amount of the studied compound to the amount of the stationary phase used may affect the efficiency of SDE. To explore this dependence three different quantities of 3 (50.0 mg, may affect the efficiency of SDE. To explore this dependence three different quantities of 3 (50.0 mg, 100.0 mg and 150.0 mg supported on 1.0 g of silica) were eluted with hexane/MTBE = 85:15 in flash 100.0 mg and 150.0 mg supported on 1.0 g of silica) were eluted with hexane/MTBE = 85:15 in flash chromatography conditions on a 12 150 mm column filled with 10.0 g silica gel. On the basis of the chromatography conditions on a 12 ×× 150 mm column filled with 10.0 g silica gel. On the basis of the obtained results (Table S3) the enantiomeric distribution profiles are presented in Figure4. In all cases a obtained results (Table 3(ESI)) the enantiomeric distribution profiles are presented in Figure 4. In all clear decreasing dependence of the enantiomeric excess was observed. First fractions exhibited a higher cases a clear decreasing dependence of the enantiomeric excess was observed. First fractions enantiomeric enrichment in the (R)-isomer than the latter ones. The values of ∆ee were 23.6%, 17.2% exhibited a higher enantiomeric enrichment in the (R)-isomer than the latter ones. The values of Δee and 16.8% for 50.0 mg, 100.0 mg and 150.0 mg of the examined compound sample size, respectively. were 23.6%, 17.2% and 16.8% for 50.0 mg, 100.0 mg and 150.0 mg of the examined compound sample In the case of the sample with the smallest quantity of the compound the highest ee value of 55.6% size, respectively. In the case of the sample with the smallest quantity of the compound the highest occurred in fraction 37 (first fraction in which compound 3 was detected by TLC), while the lowest in ee value of 55.6% occurred in fraction 37 (first fraction in which compound 3 was detected by TLC), fraction 70 and equaled 32.0% ee. In comparison with the initial value of 47.0% ee, the first fractions while the lowest in fraction 70 and equaled 32.0% ee. In comparison with the initial value of 47.0% ee, were enantiomerically enriched in (R)-isomer by 8.6% (for a 50.0 mg sample), 5.5% (for a 100.0 mg the first fractions were enantiomerically enriched in (R)-isomer by 8.6% (for a 50.0 mg sample), 5.5% sample), 1.8% (for a 150.0 mg sample), while the last fractions were enantiomerically depleted by 15% (for a 100.0 mg sample), 1.8% (for a 150.0 mg sample), while the last fractions were enantiomerically (for the 50.0 mg and 150.0 mg sample), 10.6% (for a 50.0 mg sample). As it can be seen in Figure4, for depleted by 15% (for the 50.0 mg and 150.0 mg sample), 10.6% (for a 50.0 mg sample). As it can be 100.0 mg and 150.0 mg of the compound in the sample the SDE phenomenon occurred, yet to a lesser seen in Figure 4, for 100.0 mg and 150.0 mg of the compound in the sample the SDE phenomenon degree. In consequence, these outcomes indicate that the magnitude of the SDE phenomenon depends occurred, yet to a lesser degree. In consequence, these outcomes indicate that the magnitude of the on the ratio of the compound amount to the silica gel amount in the chromatographic purification SDE phenomenon depends on the ratio of the compound amount to the silica gel amount in the process. The observed relationship between the SDE and the amount of the separated compound chromatographic purification process. The observed relationship between the SDE and the amount in comparison to the achiral stationary phase may be due to the fact that a smaller quantity has a of the separated compound in comparison to the achiral stationary phase may be due to the fact that larger number of unsaturated adsorption centers on a gel, which leads to the intensification of the SDE a smaller quantity has a larger number of unsaturated adsorption centers on a gel, which leads to the process. However, when the separation is performed using a larger compound to stationary phase intensification of the SDE process. However, when the separation is performed using a larger ratio, an increase in gel loading and therefore saturation of the adsorption centers could be observed, compound to stationary phase ratio, an increase in gel loading and therefore saturation of the which may result in the slowdown of the SDE process. adsorption centers could be observed, which may result in the slowdown of the SDE process.

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Figure 4. SDE of compound 3 inin achiral achiral MPLC conditions: the the elution elution profile. profile. The The fitting fitting equation for Figure50.0 mg 4. of ofSDE sample sample of compound is is y y == −0.676x0.676x 3 in +achiral 77.190+ 77.190 MPLC (R2 (R= 0.909)2 conditions:= 0.909) and andfor the 100.0 for elution 100.0 mg ofprofile. mg sample ofsample The is yfitting = is−0.352x y equation= 0.352x+ 57.612 for+ − 2 − 50.057.612(R2 = mg 0.966) (R of2 =sample and0.966) for is and150.0 y = for− 0.676xmg 150.0 of sample+ mg 77.190 of sampleis (R y == −0.909)0.283x is y = and + 0.283x58.408 for 100.0 +(R58.4082 =mg 0.972). of (R sample2 = 0.972). is y = −0.352x + 57.612 (R2 = 0.966) and for 150.0 mg of sample is y = −0.283x −+ 58.408 (R2 = 0.972). 3.4. Study on the SDE of P-Chiral Compounds: tert-Butylphenylphosphinothioic acid (4) (4) 3.4. Study on the SDE of P-Chiral Compounds: tert-Butylphenylphosphinothioic acid (4) The influenceinfluence of the starting material’s enantiomericenantiomeric composition on the magnitude of SDE was examinedThe influence with the oftert the-butylphenylphosphinothioic starting material’s enantiomeric acidacid ( (4composition4)) (Figure(Figure5 5).). TheonThe the chosenchosen magnitude modelmodel of compoundcompound SDE was examinedwas earlier with synthesized the tert-butylphenylphosphinothioic in accordance with the literatureliterature acid procedure(4) (Figure [[41].5).41 ].The This chosen compound model hascompound already wasfound earlier itsits applicationapplication synthesized asas a ina chiral chiralaccordance solvating solvating with agent agent the (CSA)lit (CSA)erature for for procedure the the enantiomeric enantiomeric [41]. This excess excess compound determination determination has already by by an foundNMRan NMR techniqueits techniqueapplication [66 ].[66]. as It is aIt worthchiralis worth solvating noting noting that agentthat the the enantiomeric(CSA) enantiomeric for the purity enantiomeric purity of 4ofcould 4 could excess also also determination be determinedbe determined by anby NMR an NMR technique technique [66]. since since It4 ismay worth4 may simultaneously noting simultaneously that the play enantiomeric play a role a role of the of purity analyzedthe analyzed of 4 could compound compound also be and determined and CSA CSA [67] by(Figure[67] an (Figure NMR S4). 4(ESI)).technique The reason The since forreason the4 may observedfor simultaneouslythe observ chiraled recognition chiral play recognition a role lays of inthe lays the analyzed abilityin the ability ofcompound4 to of act 4 asto and aact donor CSA as a [67]anddonor (Figure an and acceptor an 4(ESI)). acceptor of hydrogen The of reason hydrogen bonds for the andbonds observ hence anded the hence chiral formation the recognition formation of homochiral lays of homochiralin the and ability heterochiral and of heterochiral4 to dimericact as a donorspeciesdimeric and (Figurespecies an acceptor5 (Figure). Taking of5). intohydrogen Taking account into bonds account the propertiesand thehence properties ofthe the formation selected of the selectedof compound, homochiral compound, we and thought heterochiral we thought that it dimericwouldthat it would prove species anprove (Figure ideal an candidate ideal5). Taking candidate for into investigating accountfor invest the theigating properties dependence the dependence of ofthe the selected SDE of phenomenonthe compound, SDE phenomenon onwe the thought initial on thatenantiomericthe initial it would enantiomeric prove excess an of theideal excess tested candidate of samples.the tested for invest samples.igating the dependence of the SDE phenomenon on the initial enantiomeric excess of the tested samples.

Figure 5.5. (R)-tert-butylphenylphosphinothioic acid acid ( (4):): chemical chemical structures structures on on the homochiral and Figureheterochiral 5. (R )- aggregates.tert-butylphenylphosphinothioic acid (4): chemical structures on the homochiral and heterochiral aggregates.

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For the purpose of this study, three samples of (R)-4 inin thethe amountamount of 100.0 mg and with the optical purity of 25.0% ee, 60.0% ee and 75.0% ee were prepared, supported on 1.0 g of silica, and then subjected to the purification process driven by flash chromatography on a 12 150 mm column filled subjected to the purification process driven by flash chromatography on a 12 ×× 150 mm column filled with 10.0 g achiral silica gel.gel. The samplessamples werewere then then eluted eluted in in gradient gradient conditions conditions using using a mixture a mixture of cyclohexane of cyclohexane and tertand-butyl tert- methylbutyl methyl ether ether in the in ratio the ratio from from 100/0 100/0 to 0/ 100.to 0/100. The The separations separations were were run run at theat the eluent eluent flow flow rate rate of 18of mL18 /mL/minmin and and the collectedthe collected fractions fractions had the had volume the volume of 18 mL. of 18 To ourmL. disappointment,To our disappointment, the resulting the ∆resultingee was not Δee significant was not significant and reached and only reached about only 3.0% about (Figure 3.0%6). The(Figure first 6). fractions The first in fractions which compound in which 4compound was detected 4 was by detected TLC were by fractions TLC were 7, 6,fractions 9 of 25.0, 7,60.0, 6, 9 andof 25.0, 75.0% 60.0, ee, and respectively. 75.0% ee, Compoundrespectively. 4 obtainedCompound in 4 the obtained course in of the the course purification of the purificati exhibitedon a slightlyexhibited lower a slightly enantiomeric lower enantiomeric purity in the purity last fractionin the last (Figures fraction6 and (Figures7, Table 6 and S4). 7, Table 4(ESI)).

Figure 6. SDE of compound 4 of 25.0% and 60.0% ee inin achiral MPLC MPLC conditions: conditions: the the elution elution profiles. profiles. The model of the fittingfitting isis polynomial.polynomial.

Since the concentration of the compounds in late laterr fractions was low, those fractions (14–23 for 25.0% ee sample, 12–22 for 60.0% ee sample, and 12–24 for 75.0% ee sample) were combined before the ee determination. The distribution of the enantiomer in consequent fractions was determined in the experiment wherewhere samplesample ofof4 4with with 75.0% 75.0%ee eewas was used. used. As As it it could could be be seen seen from from Figure Figure7 and 7 and Table Table S4 the4(ESI) majority the majority of the sample of the of sample slightly of higher slightlyee was higher eluted ee in was fractions eluted 9–10, in fractions while a less 9–10, enantioenriched while a less compoundenantioenriched was eluted compound with thewas next eluted 12 fractions.with the next Such 12 behavior fractions. of theSuch compounds behavior of may the indicatecompounds that partmay ofindicate the sample that part was adsorbedof the sample on stationary was adsorbed phase on and stationary slowly liberated phase and upon slowly elution. liberated upon elution.

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FigureFigure 7. SDE 7. SDE of compoundof compound4 of4 of 75.0% 75.0%ee eein in achiral achiral MPLC MPLC conditions:conditions: the the elution elution profile. profile. The The model model of the fittingof the fitting is polynomial. is polynomial.

An analysisAn analysis of theof the collected collected data data showedshowed that in in the the case case of ofcompound compound 4, even4, even at a low at a polarity low polarity of theof eluent,the eluent, the the magnitude magnitude of of SDE SDE is is insignificantlyinsignificantly influenced influenced byby the the enantiomeric enantiomeric purity purity of the of the startingstarting material. material. Additionally, Additionally, it was was observed observed that that the theamount amount of the of compound the compound collected collected after the after elution constitutes only around 64.0% of the weight loaded onto the column. This is caused by strong the elution constitutes only around 64.0% of the weight loaded onto the column. This is caused interactions between the examined compound and the stationary phase and therefore its irreversible by strong interactions between the examined compound and the stationary phase and therefore its adsorption. irreversibleIn the adsorption. cases of compounds 1–3, where the separation process was carried out with the isocratic elution,In the casesthe phenomenon of compounds of SDE1– 3occurs, where in a the linea separationr manner in process time. This was may carried mean out that with the process the isocratic elution,takes the place phenomenon at the same speed of SDE during occurs the inseparation a linear procedure. manner in The time. gradient This of may a straight mean line that informs the process takesus place about at whether the same the speed enrichment during or thedepletion separation ee of the procedure. fraction is The observed, gradient and of its a value straight represents line informs us aboutthe speed whether of these the changes. enrichment The linear or depletion effect of theee ofSDE the overtime fraction changes is observed, can be explained and its value by the represents fact the speedthat during of these the changes. isocratic elution, The linear the econstantsffect of the of adsorption SDE overtime and changesdesorption can do be not explained change. In by the the fact thatcase during of gradient the isocratic elution elution, conditions, the as constants it had been of adsorptionpresented for and the desorptioncompounds do4, the not constants change. of In the adsorption are change during the elution process and therefore no linear correlation was observed. case of gradient elution conditions, as it had been presented for the compounds 4, the constants of adsorption3.5. Study are on change the SDE during of Amino the Acid elution Derivatives: process N-Acetyl-Phenylalanine and therefore no linear (5) correlation was observed. 3.5. StudyBiologically on the SDE important of Amino small Acid Derivatives:molecules are N-Acetyl-Phenylalanine of especial interest in (terms5) of the SDE studies. Therefore, the possibility of enantioseparation of the amino acid derivatives has been studied. N- acetyl-phenylalanineBiologically important (5) was small selected molecules as a model are of since especial it is readily interest available in terms from of thenatural SDE and studies. Therefore,synthetic the amino possibility acid [68], of 5 is enantioseparation also a product of widely of the used amino benchmark acid derivativesasymmetric hydrogenation has been studied. N-acetyl-phenylalaninereaction [69]. In theory, ( 5strong) was intermolecular selected as a hy modeldrogen since bonding it is could readily be re availablesponsible for from the enantio natural and syntheticrecognition amino in acid the [case68], of5 isamino also aacids. product The simplest of widely possible used benchmarkhomochiral ( asymmetricA) and heterochiral hydrogenation (B) reactiondimeric [69 ].aggregates In theory, formed strong for intermolecular N-acetyl-phenylalanine hydrogen are bonding depicted couldin Figure be responsible8. At the same for time, the the enantio recognitioncrystallographic in the case analysis of amino indicates acids. that The in simplestthe solid possiblestate, N-acetyl-amino homochiral (acidsA) and form heterochiral a more (B) dimericcomplicated aggregates net formedof hydrogen for N bonds-acetyl-phenylalanine in which the moieties are depicted of amino in acids Figure are8. usually At the samegroups time, in the tetrameric cycles (C) [70] [CSD Refcodes: COQHAR, DADGUK01, ACNVAL]. Interestingly, in the crystallographic analysis indicates that in the solid state, N-acetyl-amino acids form a more complicated presence of protic solvents, the latter could replace one (or more) of the amino acid derivative net of hydrogen bonds in which the moieties of amino acids are usually groups in tetrameric cycles (C)[70 ] [CSD Refcodes: COQHAR, DADGUK01, ACNVAL]. Interestingly, in the presence of protic solvents, the latter could replace one (or more) of the amino acid derivative molecules to form simpler aggregates such as e.g., trimers (D) [CSD Refcode: ACDAHO]. For comparison, similar studies on the Symmetry 2019, 11, 680 11 of 20 Symmetry 2018, 10, x FOR PEER REVIEW 11 of 20

SDEmolecules of N-acetylated to form simpler 1-phenylethylamine aggregates such [71], andas e.g., some trimers other amides (D) [CSD [72– 74Refcode:], which ACDAHO]. show a simpler For modecomparison, of coordination, similar studies have already on the beenSDE reported.of N-acetylated 1-phenylethylamine [71], and some other amides [72–74], which show a simpler mode of coordination, have already been reported.

Figure 8.8. Chemical structures of the possible aggregatesaggregates ofof NN--acetyl-phenylalanineacetyl-phenylalanine ((55).). For the purposes of this study, the samples of 5 with the initial value of ee 50.0% were evaluated in For the purposes of this study, the samples of 5 with the initial value of ee 50.0% were evaluated different eluent systems. First elution was carried out using hexane/i-PrOH = 92/8 and the second series in different eluent systems. First elution was carried out using hexane/i-PrOH = 92/8 and the second employed dichloroethene as the mobile phase. Since compound 5 is not soluble in the mobile phase, series employed dichloroethene as the mobile phase. Since compound 5 is not soluble in the mobile 100.0 mg of it was initially dissolved in methanol, and the solvent was evaporated in the presence of phase, 100.0 mg of it was initially dissolved in methanol, and the solvent was evaporated in the about 1.0 g of silica, then the compounds supported on the stationary phase were loaded at the top of a presence of about 1.0 g of silica, then the compounds supported on the stationary phase were loaded 12 150 mm column filled with additional 10.0 g silica gel. The separations were run at the eluent at the× top of a 12 × 150 mm column filled with additional 10.0 g silica gel. The separations were run flow rate of 18 mL/min and the collected fractions had the volume of 18 mL. In both cases 26 fractions at the eluent flow rate of 18 mL/min and the collected fractions had the volume of 18 mL. In both were collected, compound 5 first appeared in fraction 9 (hexane/i-PrOH) and in fraction 12 (ClC H Cl). cases 26 fractions were collected, compound 5 first appeared in fraction 9 (hexane/i-PrOH) 2and4 in The enantiomeric composition of the consecutive fractions was determined be means of a chiral HPLC 2 4 fraction 12® (ClC H Cl). The enantiomeric composition of the consecutive fractions was determined be Chiralpak OJ-RH column, with the mobile phase using H2O/CH3CN = 85/15 at the flow rate equal to means of a chiral HPLC Chiralpak® OJ-RH column, with the mobile phase using H2O/CH3CN = 85/15 0.3 mL/min (Figure S5). at the flow rate equal to 0.3 mL/min (Figure 5(ESI)). The amount of compound 5 and its enantiomeric composition in the consecutive fractions were The amount of compound 5 and its enantiomeric composition in the consecutive fractions were evaluated and are presented in Table S5. The SDE profiles are presented in Figure9. Whenever the evaluated and are presented in Table 5(ESI). The SDE profiles are presented in Figure 9. Whenever mixture hexane/i-PrOH = 92/8 was used as the eluent, the value ∆ee was 5.9% and the enantiomeric the mixture hexane/i-PrOH = 92/8 was used as the eluent, the value Δee was 5.9% and the enantiomeric excess of the most enriched fraction 9 exceeded the initial mixture by 3.5%. Using dichloroethane excess of the most enriched fraction 9 exceeded the initial mixture by 3.5%. Using dichloroethane allowed us to reach a greater magnitude of the SDE phenomenon with the 7.5% value of the ∆ee allowed us to reach a greater magnitude of the SDE phenomenon with the 7.5% value of the Δee parameter. The enantiomeric excess of fraction 12 deviated the initial by 7.2%. The use of an aprotic parameter. The enantiomeric excess of fraction 12 deviated the initial by 7.2%. The use of an aprotic solvent strongly affected the SDE, and (in contrast to where hexane/i-PrOH was applied) furnished an solvent strongly affected the SDE, and (in contrast to where hexane/i-PrOH was applied) furnished elution with the opposite profile, where the initial fractions were enantiomerically depleted and the an elution with the opposite profile, where the initial fractions were enantiomerically depleted and last ones were enantiomerically enriched. the last ones were enantiomerically enriched. The total amounts of compound 5 recovered after the purification were 65.7% (in the case of The total amounts of compound 5 recovered after the purification were 65.7% (in the case of hexane/i-PrOH), and only 47.0% (in the case of dichloroethane) of the weight loaded onto the column. hexane/i-PrOH), and only 47.0% (in the case of dichloroethane) of the weight loaded onto the column. The results presented in Table S5 clearly indicate that an irreversible sorption of 5 on the stationary phase The results presented in Table 5(ESI) clearly indicate that an irreversible sorption of 5 on the was much bigger when the aprotic solvent was used. It also facilitated the formation of heterochiral stationary phase was much bigger when the aprotic solvent was used. It also facilitated the formation aggregates (R,S)- in the solution which (being symmetric and less polar—see Figure 10) are eluting of heterochiral aggregates (R,S)- in the solution which (being symmetric and less polar—see Figure faster to form first fractions of a lower ee. 10) are eluting faster to form first fractions of a lower ee.

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Figure 9. SDE of compound 5 in achiral MPLC conditions: the elution profiles. Fitting equation for FigureFigure 9. SDE 9. SDE of compound of compound5 in 5 achiralin achiral MPLC MPLC conditions: conditions: the elution elution profiles. profiles. Fitting Fitting equation equation for for dichloroethane is y = 0.730x + 32.462 (R2 = 0.261), and for hexane/i-PrOH is y = −0.557x + 56.961 (R2 = dichloroethanedichloroethane is y is= y0.730x = 0.730x+ +32.462 32.462 (R2 == 0.261),0.261), and and for forhexane/i-PrOH hexane/i-PrOH is y = is−0.557x y = +0.557x 56.961 +(R256.961 = 0.428). − (R2 = 0.428).

The experimental The experimental observations observations correlate correlate well well with with the the thermodynamic analysis analysis of theof thepotential potential equilibriumequilibrium between between possible possible stereo stereoisomeric stereoisomericisomeric aggregates aggregates of 5 5 (Figure (Figure(Figure 10). 1010). ).It Itwas It was was found found found that that the the bimolecularbimolecular ((RR,,SS)-aggregate )-aggregate(R,S)-aggregate isis more more is more stable stable stable than than than the the the isomeric isomeric isomeric (S ((,SS,)-,SS)-)- or or or ( R( R(,R,R)-aggregates.,)-aggregates.R)-aggregates. In In particular,In particular, particular, an an advanced M062x/6-311++G(d,p)(PCM) computational study showed that the differences between anadvanced advanced M062x M062x/6-311++G(d,p)(PCM)/6-311++G(d,p)(PCM) computational computationa studyl study showed showed that that the the diff differenceserences between between the the values of Gibbs free energies of the formation of both molecular systems equals 4.0 and 2.5 thevalues values of Gibbs of Gibbs free energiesfree energies of the of formation the formatio of bothn of molecular both molecular systems systems equals 4.0equals and 2.54.0 kcaland/mol 2.5 kcal/molkcal/mol respectively respectively (Table (Table 1). 1).Additionally, Additionally, we we analyzed analyzed keykey distancesdistances between between the the substructures substructures respectivelywithin the (Table considered1). Additionally, aggregates. weIt was analyzed found that key these distances distances between are practically the substructures identical in both within within the considered aggregates. It was found that these distances are practically identical in both the consideredcases. So, the aggregates. difference between It was found thermodynamic that these stabilities distances is area not practically a consequence identical of the inpower both of cases. cases. So, the difference between thermodynamic stabilities is a not a consequence of the power of So, thelocal diff intermolecularerence between interactions, thermodynamic but rather stabilitiesa more favorable is a not arrangement a consequence of the structural of the power elements of local localintermolecular intermolecularin space, which interactions, provides interactions, more but ratherfavorablebut rather a more electrostatic a more favorable favorable interactions arrangement arrangement and/or lower of theof steric the structural structural repulsions. elements elements Two in inspace, space,aggregates which which provides providesare also morecharacterized more favorable favorable with electrostatic electrostatic different po interactionslarities interactions where and heterochiraland/or/orlower lower aggregate stericsteric repulsions.repulsions. has a lower Two aggregatesdipole are moment also characterized (Figure 10). with different different po polaritieslarities where heterochiral aggregate has a lower dipole moment (Figure 1010).).

R,R-aggregate R,S-aggregate S,S-aggregate

Figure 10. Views of the homo- and heterochiral aggregates of compound 5 in the DCM Rsolution,R-aggregate optimized at the M062x/6-311++G(d,p)(PCM)R,S-aggregate level of theory. S,S-aggregate Figure 10. Figure 10. ViewsViews ofof thethe homo- homo and- and heterochiral heterochiral aggregates aggregates of compound of compound5 in the 5 DCMin the solution DCM solutionoptimized optimized at the M062x at/ 6-311the M062x/6-311++G(d,p)(PCM)++G(d,p)(PCM) level of theory. level of theory.

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Table 1.1. DiDifferencesfferences between between the the heterochiral heterochiral and and homochiral homochiral aggregates aggregates of compound of compound5 in the 5 in DCM the DCMsolution solution optimized optimized at the M062x at the/6-311 M062x/6-311++G(d,p)(PCM)++G(d,p)(PCM) level of theory. level of theory.

TransitionTransition ∆∆GG [kcal/mol] [kcal/mol] (R(R,,SS)) → (S(S,S,S)) 4.0 4.0 → (R(R,S,S)) → (R(R,R,R)) 2.5 2.5 → The limited solubility of compound 5, and its tendency to form diverse and intricate crystalline The limited solubility of compound 5, and its tendency to form diverse and intricate crystalline forms, make the SDE process less efficient and make its study very complicated. Taking that into forms, make the SDE process less efficient and make its study very complicated. Taking that into consideration, a simpler liquid derivative of α-amino acids should be selected to eliminate the consideration, a simpler liquid derivative of α-amino acids should be selected to eliminate the possible possible separation via micro crystallization on the stationary phase, and facilitate enantioseparation separation via micro crystallization on the stationary phase, and facilitate enantioseparation by limiting by limiting the number of possible aggregation models which may be in an equilibrium and have an the number of possible aggregation models which may be in an equilibrium and have an opposite opposite effect on the SDE efficiency. effect on the SDE efficiency.

3.6. Study on the SDE of Aamino Acid Derivatives: Methyl Methyl Phenylalaninate Phenylalaninate (6) (6) Thus, methyl phenylalaninate phenylalaninate (6 (6) )was was chosen chosen to to be be examined. examined. The The compound compound was was obtained obtained by esterificationby esterification [75] [75 of] ofee ee= =36.0%36.0% (S) (S-phenylalanine)-phenylalanine in in standard standard condit conditionsions for for this this sequence sequence of of the reactions (Scheme4 4).).

Scheme 4. Synthesis of methyl phenylalaninate (6). Scheme 4. Synthesis of methyl phenylalaninate (6). Since 6 is a liquid well soluble in the majority of organic solvents there was no need to support Since 6 is a liquid well soluble in the majority of organic solvents there was no need to support the compound on the stationary phase before the separation. The solution of 6 in 0.5 mL of the mobile the compound on the stationary phase before the separation. The solution of 6 in 0.5 mL of the mobile phase with the (S)- enantiomer of ee = 36.0% in the amount of 100.0 mg was subjected to the separation phase with the (S)- enantiomer of ee = 36.0% in the amount of 100.0 mg was subjected to the separation process on an achiral 12 150 mm column filled with additional 11.0 g silica gel and prewashed with process on an achiral 12 × 150 mm column filled with additional 11.0 g silica gel and prewashed with the mobile phase. The separations were carried out at the eluent flow rate of 18 mL/min and the the mobile phase. The separations were carried out at the eluent flow rate of 18 mL/min and the collected fractions had the volume of 18 mL. In this step, diethyl ether (Rf = 0,43) was used as the collected fractions had the volume of 18 mL. In this step, diethyl ether (Rf = 0,43) was used as the mobile phase. The presence of the compound in the obtained fraction was verified on the basis of the mobile phase. The presence of the compound in the obtained fraction was verified on the basis of the TLC technique. At first compound 6 appeared in fraction 12. The optical purity of the fractions was TLC technique. At first compound 6 appeared in fraction 12. The optical purity of the fractions was determined based on the 1H and 13C NMR techniques using, as CSA, 100 mol% of enantiomericaly determined based on the 1H and 13C NMR techniques using, as CSA, 100 mol% of enantiomericaly pure 4 (Figure S6). pure 4 (Figure 6(ESI)).13 In the case of C NMR, two sets of signals corresponding to the OCH3 (51.41/51.45 ppm) and In the case of 13C NMR, two sets of signals corresponding to the OCH3 (51.41/51.45 ppm) and NCH (54.29/54.34 ppm) carbon atoms were analyzed, while in the case of 1H NMR only signals NCH (54.29/54.34 ppm) carbon atoms were analyzed, while in the case of 1H NMR only signals corresponding to NCH proton at 3.95/4.01 (t, J = 6.5 Hz) ppm were used for the ee calculation (Figure corresponding to NCH proton at 3.95/4.01 (t, J = 6.5 Hz) ppm were used for the ee calculation (Figure S6). The individual optical purities, measured by NMR, and their average values are shown in Table S6. 6(ESI)). The individual optical purities, measured by NMR, and their average values are shown in As a result of this separation (Figure 11, and Table S6), 11 fractions were collected, in which the Table 6(ESI). total amount of the compound was 68.1 mg (where: the mass of the (R)-enantiomer was 21.8 mg, and As a result of this separation (Figure 11, and Table 6(ESI)), 11 fractions were collected, in which the mass of the S enantiomer was 46.3 mg). This means that during the separation 31.0 mg of compound the total amount of the compound was 68.1 mg (where: the mass of the (R)- enantiomer was 21.8 mg, 6 was permanently adsorbed on the stationary phase, including 10.2 mg of the (R)-enantiomer and and the mass of the S enantiomer was 46.3 mg). This means that during the separation 31.0 mg of 21.7 mg of the (S)-enantiomer. The ∆ee value for this separation was 13.8% ee (S). The differentially compound 6 was permanently adsorbed on the stationary phase, including 10.2 mg of the (R)- calculated enantiomeric composition of compound 6 irreversibly adsorbed on the stationary phase is enantiomer and 21.7 mg of the (S)-enantiomer. The Δee value for this separation was 13.8% ee (S).The the same as for the initial sample and constitutes 36.0% ee (S), which means that the adsorption process differentially calculated enantiomeric composition of compound 6 irreversibly adsorbed on the is not selective and the amount of the adsorbed enantiomer depends only on the initial composition of stationary phase is the same as for the initial sample and constitutes 36.0% ee (S), which means that the starting mixture. the adsorption process is not selective and the amount of the adsorbed enantiomer depends only on the initial composition of the starting mixture.

Symmetry 2019, 11, 680 14 of 20 Symmetry 2018, 10, x FOR PEER REVIEW 14 of 20

FigureFigure 11. 11.SDE SDE of of compound compound6 6using using diethyldiethyl ether:ether: the the elution elution pr profile.ofile. The The fitting fitting equation equation is isy = y = 1.426x−1.426x+ +59.275 59.275 (R (R22 == 0.836). − SinceSince the the initial initial compound compound is is not not racemic, racemic, itsits irreversibleirreversible nonstereoselective adsorption adsorption leads leads to to a changea change in the in the column’s column’s character character from from achiral achiral to to chiral. chiral. The enantiomeric enantiomeric purity purity of of the the chiral chiral selector selector onon a newly-formeda newly-formed chiral chiral surface surface dependsdepends on the enantiomeric composition composition of of the the mixture mixture during during loadingloading and and on on the the adsorptive adsorptive capacity capacity ofof thethe stationarystationary phase. phase. Such Such a anewly-created newly-created chiral chiral column column diffdifferentiateserentiates the the enantiomers enantiomers of theof chromatographedthe chromatographed compound compound and causes and theircauses natural their separation.natural Thisseparation. could be aThis good could alternative be a explanation good alternative of the SDE explanation phenomenon of underthe SDE chromatographic phenomenon condition.under chromatographicIn order to confirm condition. this hypothesis and explain the mechanism of the SDE process, the following experimentsIn order were to confirm carried this out. hypothesis Compound and6 explainof the same the mechanism initial enantiocomposition of the SDE process, (36.0% the followingee,(S) was elutedexperiments by tert-butyl were carried methyl out. ether. Compound In the first6 of the separation same initial experiment, enantiocomposition 100.0 mg of(36.0%6 with ee, ( anS) was initial eluted by tert-butyl methyl ether. In the first separation experiment, 100.0 mg of 6 with an initial purity of 36.0% ee (S) in 0.5 mL of hexane was injected into a 12 150 mm column packed with 11.0 g purity of 36.0% ee (S) in 0.5 mL of hexane was injected into a 12 ×× 150 mm column packed with 11.0 g of spherical achiral silica gel, washed with the mobile phase. The separation was performed using of spherical achiral silica gel, washed with the mobile phase. The separation was performed using tert-butyl methyl ether (MTBE) as the mobile phase (Rf = 0,4) and the eluent flow rate of 18 mL/min, tert-butyl methyl ether (MTBE) as the mobile phase (Rf = 0,4) and the eluent flow rate of 18 mL/min, 18 mL of the fractions were collected. As a result of this separation, 8 fractions containing 6 were 18 mL of the fractions were collected. As a result of this separation, 8 fractions containing 6 were obtainedobtained (fractions (fractions from from 7 to7 to 14). 14). The The presence presence of of the the compound compound in in the the fractions fractionswas was verifiedverified basedbased on theon TLC the technique.TLC technique. Then, Then, when when no more no compoundmore compound was found was infound the subsequentin the subsequent fractions fractions collected, thecollected, column wasthe column used for was the secondused for separation the second experiment, separation whereexperiment, the same where portion the same of compound portion of6 (as incompound the case of 6 the (as firstin the separation) case of the was first injected. separation) The wa seconds injected. separation The second experiment separation was experiment carried out usingwas thecarried same out mobile using phasethe same MTBE, mobile as phase in the MTBE, first separation as in the first process. separation As a resultprocess. of As this a separation,result of 10this fractions separation, were 10 collected fractions (fractions were collected from (fractions 36 to 45). from The 36 optical to 45). purityThe optical of the purity fractions of thecollected fractions in thecollected first and in second the first separation and second experiments separation wasexperiments determined wasbased determined on the based1H 13 Con NMR the 1H spectroscopic 13C NMR techniquespectroscopic with CSAtechnique4. The with data CSA collected 4. The indata the collected course of in both the experimentscourse of both was experiments analyzed, was and is presentedanalyzed, in and Table is presented S7, while in the Table elution 7(ESI), profiles while are the shown elution in profiles Figure are12. shown in Figure 12.

Symmetry 2019, 11, 680 15 of 20 Symmetry 2018, 10, x FOR PEER REVIEW 15 of 20

Figure 12. SDE of compound 6 by tert-butyl methyl ether: the elution profile. The fitting equation for Figure 12. SDE of compound 6 by tert-butyl methyl ether: the elution profile. The fitting equation for 1st separation is y = 2.284x + 56.615 (R2 = 0.988) and for 2nd separation is y = 3.863x + 184.578 1st separation is y = −−2.284x + 56.615 (R2 = 0.988) and for 2nd separation is y = −3.863x− + 184.578 (R2 = (R2 = 0.936). 0.936). As a result of the first separation experiment, 8 fractions were obtained in which the total mass of As a result of the first separation experiment, 8 fractions were obtained in which the total mass the compound was 68.0 mg (68.0% of initial), where the calculations were as follows: the amount of of the compound was 68.0 mg (68.0% of initial), where the calculations were as follows: the amount the (R)-enantiomer was 22.3 mg and the amount of the (S)-enantiomer was 46.7 mg (of 35.1% ee (S), of the (R)-enantiomer was 22.3 mg and the amount of the (S)-enantiomer was 46.7 mg (of 35.1% ee calculated on the basis of each fraction). The value of the ∆ee for the first separation experiment was (S), calculated on the basis of each fraction). The value of the Δee for the first separation experiment 16.0%, and the most enriched fraction 7 ee equaled 40.5%, while the less enriched fraction 14 equaled was 16.0%, and the most enriched fraction 7 ee equaled 40.5%, while the less enriched fraction 14 24.2%. This means that during the first separation 31 mg (31.0%) of compound 6 (of 37.0% ee (S)) was equaled 24.2%. This means that during the first separation 31 mg (31.0%) of compound 6 (of 37.0% ee permanently adsorbed on the stationary phase, including 9.7 mg of the (R)-enantiomer and 21.3 mg of (S)) was permanently adsorbed on the stationary phase, including 9.7 mg of the (R)-enantiomer and the (S)-enantiomer. In the first separation experiment, similarly as it had been observed in the case 21.3 mg of the (S)-enantiomer. In the first separation experiment, similarly as it had been observed in of the elution with diethyl ether, during the chromatographic process part of the eluted compounds the case of the elution with diethyl ether, during the chromatographic process part of the eluted (31.0 mg) was adsorbed on the stationary phase not enantio-selectively with the ratio R/S depending compounds (31.0 mg) was adsorbed on the stationary phase not enantio-selectively with the ratio R/S only on the initial composition of the mixture. depending only on the initial composition of the mixture. Notably, in the case of the second separation experiment, the same column was used, but with Notably, in the case of the second separation experiment, the same column was used, but with already adsorbed compound 6. The total amount of 6 collected after the separation was much higher already adsorbed compound 6. The total amount of 6 collected after the separation was much higher and equal to 91.0 mg (91.0%). The weighted arithmetic mean ratio of the (R)-enantiomer to the and equal to 91.0 mg (91.0%). The weighted arithmetic mean ratio of the (R)-enantiomer to the (S)- (S)-enantiomer was 30.7 to 60.3 mg (33.0% ee (S)). The value of the ∆ee for this separation calculated enantiomer was 30.7 to 60.3 mg (33.0% ee (S)). The value of the Δee for this separation calculated based based on the ee of fraction 36 (42.7%) and fraction 45 (9.0%) equaled 33.7%. Thus, only 9.0 mg (9.0%) of on the ee of fraction 36 (42.7%) and fraction 45 (9.0%) equaled 33.7%. Thus, only 9.0 mg (9.0%) of compound 6 of 71.0% ee (S) was lost on the stationary phase. compound 6 of 71.0% ee (S) was lost on the stationary phase. The efficiency of the second separation experiment was therefore notably higher than of the first The efficiency of the second separation experiment was therefore notably higher than of the first one. The simple rationalization of this phenomenon could be as follows: the effect can be explained one. The simple rationalization of this phenomenon could be as follows: the effect can be explained by the formation of a new interaction between the enantiomers and the modified stationary phase by the formation of a new interaction between the enantiomers and the modified stationary phase which formed after part of the chiral compound was adsorbed on the stationary phase surface. The which formed after part of the chiral compound was adsorbed on the stationary phase surface. The homochiral and heterochiral aggregates are formed during the separation between the free compound homochiral and heterochiral aggregates are formed during the separation between the free in the mobile phase and the chiral selectors adsorbed on the stationary phase. Such process follows compound in the mobile phase and the chiral selectors adsorbed on the stationary phase. Such the rule of three points (Pirkle rule) [76,77]. According to this principle, for the chiral separation of process follows the rule of three points (Pirkle rule) [76,77]. According to this principle, for the chiral enantiomers, it is necessary to have a minimum of three interactions between the enantiomers and separation of enantiomers, it is necessary to have a minimum of three interactions between the enantiomers and the chiral selector, as shown in Figure 13, while at least one of these interactions

Symmetry 20192018,, 1110,, 680x FOR PEER REVIEW 1616 of of 20 dependsSymmetry 2018 on ,the 10, xconfiguration FOR PEER REVIEW of the chiral centers of the selector and the selectand. The separation16 of of20 the enantiomers chiral selector, by as HPLC shown with in Figurethe chiral 13 ,stationa while atry least phases one depends of these on interactions the formation depends of transient on the depends on the configuration of the chiral centers of the selector and the selectand. The separation of configurationdiastereoisomeric of the linkages chiral centers (in our of case the selectorthey are andhomochiral the selectand. and heterochiral The separation aggregates). of the enantiomers Different the enantiomers by HPLC with the chiral stationary phases depends on the formation of transient bystabilities HPLC withof these the complexes chiral stationary result in phases the possib dependsility onof the chiral formation separation of transient of enantiomers. diastereoisomeric linkagesdiastereoisomeric (in our case linkages they are (in homochiral our case they and are heterochiral homochiral aggregates). and heterochiral Different aggregates). stabilities Different of these complexesstabilities of result these in complexes the possibility result of in the the chiral possib separationility of the of chiral enantiomers. separation of enantiomers.

Figure 13. The model of the interactions between selectand and chiral selector. Figure 13. The model of the interactions between selectand and chiral selector. The efficiencyFigure of 13. the The enentiomer model of the separation interactions can betw beeen explained selectand basedand chiral on selector.the quantumchemical calculations,The effi ciencyas in the of case the enentiomerof 6. In particular, separation the M062x/6-311++G(d,p) can be explained based computational on the quantumchemical study showed The efficiency of the enentiomer separation can be explained based on the quantumchemical calculations,that the homochiral as in the aggregate case of 6. is In more particular, stable thethan M062x the isomeric/6-311++ heterochiralG(d,p) computational one (Table study2). The showed model calculations, as in the case of 6. In particular, the M062x/6-311++G(d,p) computational study showed thatof coordination the homochiral was aggregate based on iscr moreystallographic stable than data the isomeric[70] [CSD heterochiral Refcodes: IWOMIS] one (Table 2where). The hydrogen model of that the homochiral aggregate is more stable than the isomeric heterochiral one (Table 2). The model coordinationbonds NH---O=C was basedwith the on crystallographicbond length of 2.28 data Å [ 70we]re [CSD responsible Refcodes: for IWOMIS] the crystal where net formation. hydrogen bondsNext, of coordination was based on crystallographic data [70] [CSD Refcodes: IWOMIS] where hydrogen NH—Othe quantumchemical=C with the bond study length confirmed of 2.28 Åthat were the responsible less polar homochiral for the crystal aggregates net formation. (Figure Next, 14) theare bonds NH---O=C with the bond length of 2.28 Å were responsible for the crystal net formation. Next, quantumchemicaleluting faster than studythe heterochiral confirmed ones. that theThe less DFT polar calculation homochiral also indicated aggregates that (Figure the thermodynamic 14) are eluting the quantumchemical study confirmed that the less polar homochiral aggregates (Figure 14) are fasterstabilities than of the the heterochiral formed aggregates ones. The are DFT dependent calculation on also the indicatedarrangement that of the their thermodynamic structural elements stabilities in eluting faster than the heterochiral ones. The DFT calculation also indicated that the thermodynamic ofspace. the formed aggregates are dependent on the arrangement of their structural elements in space. stabilities of the formed aggregates are dependent on the arrangement of their structural elements in space.

R,R-aggregate R,S-aggregate S,S-aggregate

Figure 14.R ,RViewsViews-aggregate ofof thethe homo- homo and- and heterochiral heterochiralR,S-aggregate aggregates aggregates of compound of compound6 inS,S the- aggregate6 DCMin the solution DCM solution optimized at the M062x/6-311++G(d,p)(PCM) level of theory. optimizedFigure 14. atViews the M062x of the/6-311 homo++G(d,p)(PCM)- and heterochiral level oftheory. aggregates of compound 6 in the DCM Tablesolution 2.2. Di Differencesoptimizedfferences between atbetween the M062x/6-311++G(d,p)(PCM) heterochiral heterochiral and and homochiral homochiral level aggregates ofaggregates theory. of compoundof compound6 in the6 in DCM the solutionDCM solution optimized optimized at the M062x at the/6-311 M062x/6-311++G(d,p)(PCM)++G(d,p)(PCM) level of theory. level of theory. Table 2. Differences between heterochiral and homochiral aggregates of compound 6 in the DCM solution optimized at theTransition TransitionM062x/6-311++G(d,p)(PCM) ∆∆GG [kcal/mol] [kcal/mol] level of theory. (R(R,,RR)) → (R(R,S,)S) 2.2 2.2 Transition→ ∆G [kcal/mol] (S(S,,SS)) → (R(R,S,)S) 3.3 3.3 (R,R) →→ (R,S) 2.2 (S,S) → (R,S) 3.3 4. Conclusions 4. ConclusionsThe data presented above indicate that the optical purificationpurification approach based on SDE has a potential and in some cases could be applied as an alternative to the conventional procedures of chiral potentialThe anddata in presented some cases above could indicate be applied that as the an optical alternative purification to the conventional approach based procedures on SDE of chiralhas a separations. The SDE phenomenon of six non-racemic compounds on achiral silica gel was explored, separations.potential and The in some SDE phenomenoncases could be of applied six non-racemic as an alternative compounds to the onconventional achiral silica procedures gel was explored, of chiral and some correlation between the eluent used, ratio of the compounds to the stationary phases and andseparations. some correlation The SDE betweenphenomenon the eluent of six used,non-racemic ratio of compounds the compounds on achiral to the silica stationary gel was phases explored, and compound structures was found to influence the magnitude of the enantiomer self- compoundand some correlation structures wasbetween found the to eluent influence used, the rati magnitudeo of the ofcompounds the enantiomer to the self-disproportionation stationary phases and disproportionation process. An explanation of a possible mechanism of this phenomenon, which process.compound An explanationstructures was of a possiblefound mechanismto influenc ofe thisthe phenomenon,magnitude of which the involvesenantiomer the in self- situ involves the in situ formation of the chiral surface on the achiral stationary phase, was also provided. formationdisproportionation of the chiral process. surface An on explanation the achiral of stationary a possible phase, mechanism was also of provided.this phenomenon, The presented which The presented results may mean that the SDE phenomenon should not only be explained by the involves the in situ formation of the chiral surface on the achiral stationary phase, was also provided. The presented results may mean that the SDE phenomenon should not only be explained by the

Symmetry 2019, 11, 680 17 of 20 results may mean that the SDE phenomenon should not only be explained by the formation of the aggregates between the enantiomers in a mobile phase, but also by the change of the column character from achiral to chiral. Since crystallization and sublimation are purification methods applicable only for enantiomerically enriched crystalline samples, SDE-based chromatographic methodology could be applied as a general tool for practical enantiopurification of organic compounds enriched in one of the enantiomers. Such a self enantio-enriching process may take place in nature, and could be responsible for the formation of the first chiral non-racemic compounds on the early Earth. Additionally, it worth mentioning that, taking into consideration the fact that in many publications on asymmetric synthesis, the optical purity of the furnished products exceeds 99% ee, it is important to measure the enantiomeric purity before any purification of the product obtained in an asymmetric reaction to achieve reliable results of the measurement of the level of an asymmetric induction. From a practical point of view, to facilitate a SDS effect, is may be recommend to use an aprotic solvents of low polarity and higher ratio of the stationary phase to the separated compound. The mobile phases should have a Rf value in the range of 0.2–0.4 and the resulting TLC spot should be wider rather than narrow. Probably, better separations may be obtained in cases of substances forming weak intermolecular hydrogen bonds.

Supplementary Materials: Supplementary materials can be accessed at: http://www.mdpi.com/2073-8994/11/5/680/s1. Author Contributions: Conceptualization, O.M.D.; methodology, O.M.D. and M.B.; investigation, O.M.D., M.B., K.K., R.J., W.K., S.K.; writing—original draft preparation, O.M.D., M.B., D.S.; writing—review and editing, O.M.D.; visualization, M.B., O.M.D.; supervision, O.M.D.; inspiration and discussions: K.M.P. Funding: This research received no external funding. The research was carried out in part using the PLGrid (‘Prometheus’ cluster) infrastructure (ACK ‘Cyfronet’ in Cracov), and the equipment purchased thanks to the financial support of the European Regional Development Fund under the framework of the Operational Program Development of Eastern Poland 2007–2013 (Contract No. POPW.01.03.00-06-009/11-00, equipping the laboratories of the Faculties of Biology and Biotechnology, Mathematics, Physics and Informatics, and Chemistry for studies of biologically active substances and environmental samples) as well as the Polish National Science Centre research grant (2012/05/B/ST5/00362). Conflicts of Interest: The authors declare no conflict of interest.

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