Determination of Enantioselectivities by Means of Chiral Stationary Phase HPLC in Order to Identify Effective Proline-Derived Organocatalysts

The pyrrolidine fragment is a privileged scaffold within chiral ligands employed in coordination complexes exhibiting catalytic activity in asymmetric reactions and, more recently, as enantioselective organocatalysts per se. Likewise, the employment of (S)-proline as starting material constitutes the most direct form to synthesize those chiral derivatives. Afterwards, a preliminary evaluation of the catalytic performance of proline-derived compounds consists of screening many prochiral substrates in well standardized model reactions such as Michael additions and Mannich reactions, with the aim of identifying “broad spectrum” catalysts for more complex synthetic applications. Therefore, a central part of this process involves the fast and direct measurement of enantioselectivities of optically active adducts. The growing development of chiral stationary phases and thus, the wide commercial availability of chiral columns have consolidated high performance liquid chromatography (HPLC) as the preferred technique to identify the most effective catalysts.


Introduction
In recent times, a growing demand for enantiopure, value-added chiral compounds such as pharmaceuticals, food additives and agrochemicals has been registered. 1 Likewise, one of the major aims of organic synthesis is the creation of molecular diversity and complexity from simple and readily available substrates. 2 Therefore, the development of stereoselective synthetic strategies focused on those classes of organic molecules has increased in an extraordinary way.
Asymmetric synthesis makes use of different analytical techniques such as X-ray diffraction, 3 chiral nuclear magnetic resonance (NMR) shift reagents, 4 chiral chromatography, 5 among others, 6 with the aim of evaluating the efficiency of a given strategy, either via chiral auxiliaries, 7 asymmetric catalysis mediated by metal complexes, 8 enzymatic catalysis 9 and, a more recently developed methodology, organocatalysis. 10

Brief Overview of Chiral Stationary Phases
In the beginnings of asymmetric synthesis, enantiomeric purities of chiral compounds were usually determined by comparison of experimental optical rotations or via the preparation of diastereomeric derivatives followed by analysis of their 1 H NMR spectra.This situation gradually changed since Gil-Av et al. (1966)  11 achieved the analytical separation of single enantiomers from racemic α-amino acids by means of a chiral stationary phase for gas chromatography (GC).Thus, chiral chromatography currently allows a direct comparison of chromatograms obtained from enantioenriched samples with those recorded from the corresponding racemates.High performance liquid chromatography (HPLC) is the preferred technique over GC for most of the chiral analytes since it not only allows the analysis of enantiopurity, but also the easy recovery of the sample or even the enantio-enrichment of optically active compounds at the semipreparative or preparative scale. 12GC is ideal for the analytical resolution of volatile substances, especially chiral hydrocarbons, which pose a special challenge due to the lack of functional groups that could reversibly interact with a chiral selector and thus lead to usual chiral recognition strategies. 13n general, the separation of enantiomeric compounds via chiral stationary phases is based on the formation of transient diastereomeric complexes (of different bonding energies) in a thermodynamic equilibrium, which in turn results from the different fitting onto the structures of chiral selectors, depending on the configurational complementarity with the functional groups belonging to the analyte. 14Therefore, one of the two transient diastereomeric complexes formed by each of the enantiomers comprising a racemate will be more stabilized by means of potential intermolecular interactions such as hydrogen bonding, π-π complexation, dipole stacking, ionic and/or steric interactions, and others. 14In this regard, in-depth studies have allowed a sophisticated understanding of the chiral recognition mechanisms performed by enantioselective stationary phases, though forefront research continues emerging. 15Hence, it is plausible to achieve the analytical resolution of almost any existent chiral compound given the presently available chiral stationary phases.
Two main groups of chiral stationary phases (CSPs) for HPLC can be recognized: 5 (i) Brush-type chiral stationary phases (or selectorbased chiral sorbents) that usually consist of relatively small chiral molecules immobilized onto an achiral support (e.g., organic polymers or silica gel particles).Chiral metal complexes, 16 crown ethers, 17 cyclodextrins, 18 cyclofructans, 19 antibiotics, 20 Pirkle-type receptors, 21 zwitterionic quinine-based selectors, 22 among others enter in this category. 23ii) Sorbents based on optically active polymers, which can be synthetic such as the molecularly imprinted polymers 24 or obtained from natural sources, e.g.8][29][30][31][32][33]    the recognition ability of these polymers depends on the employed synthetic methods since the chiral recognition sites within the CSPs must be formed during the polymerization process.
the polymers exhibited a remarkably higher chiral recognition when prepared by the radical polymerization of optically active monomers in comparison to those prepared by the reaction of poly(acryloyl chloride) with the corresponding chiral amines.][36][37][38][39][40][41][42][43][44][45][46][47][48][49][50][51][52] From stationary phases based on naturally occurring chiral compounds such as α-amino acid derivatives to the design of synthetic chiral receptors, chiral chromatography constitutes a medullar part in the assessment of new asymmetric synthetic strategies through the fast determination of enantiomeric excesses (ee) in routine reaction tests encompassing multiple samples.Currently, the most widely employed chiral columns are those presenting polysaccharide-derived stationary phases due to their broad-spectrum applicability for optically active analytes of almost any nature.

Synopsis of the Development of Organocatalysts and their Applications
Organocatalysis, conventionally defined as the use of small organic molecules as catalysts to promote asymmetric organic transformations, is now considered a stablished strategy for asymmetric synthesis. 53Though the term organocatalysis was introduced by Ostwald in 1900, 54 it remained relatively forgotten until the 1970's when Hajos and Parrish (Hoffmann-La Roche), 55 and Eder et al. (Schering) 55 independently reported the intramolecular aldol reaction catalyzed by (S)-proline, whose product was obtained in 99% yield and 93% enantiomeric excess.This asymmetric approach experienced a rebirth since 2000, when List et al. 56 published their seminal work describing the employment of proline as organocatalyst in the enantioselective intermolecular aldol reaction between acetone and different aldehydes.
Proline is an abundant α-amino acid available in both enantiomeric forms.Its functional amino and carboxylic groups situated at a convenient distance confer the proline a great versatility as organocatalyst since on one side of the molecular structure, the carboxylic acid fragment allows the formation of hydrogen bonds with one heteroatom from a non-enolizable electrophile and on the other side, the secondary amine functionality participates in the formation of a nucleophilic enamine with an enolizable aldehyde or ketone. 57The modification of the carboxylic acid functionality can modulate the capability of forming hydrogen bonds, in turn improving the solubility of resulting proline-derived catalysts.With respect to the second point, bifunctional pyrrolidine catalysts have been also obtained from trans-hydroxyproline, whose hydroxy group enables the support of the organocatalyst onto silica gel (heterogeneous catalysis) or ionic tags (ionic liquid catalytic systems), thus facilitating their recovery.][60][61][62][63][64][65][66][67][68][69][70][71] Given the great diversity of reactions in which it is possible to employ (R)-or (S)-proline and its derivatives as organocatalysts, it is worthy to mention that relatively few methods of activation were initially identified. 10,71,72For example, the stereoinduction observed in reactions listed in Figure 3 is ruled by a simplified rationalization of the mechanistic principle of catalysis via enamine.
On the other hand, the applications of chiral organocatalysts have not been limited to the development of asymmetric methodologies, but have also fruitfully extended to the asymmetric synthesis of various chiral natural and synthetic bioactive compounds. 73or instance, Hong et al. 74 developed the enantioselective total synthesis via cascade threecomponent organocatalysis of (+)-conicol [(+)-26, Scheme 1], an interesting chiral compound isolated from the ascidian Aplidium conicum, that has exhibited antiproliferative activity against human acute lymphoblastic  activity, in which the evaluation of enantioselectivities via chiral HPLC plays a central role.

Diazabicycloheptanes as Organocatalysts
(1S,4S)-2,5-Diazabicyclo[2.2.1]heptane, (1S,4S)-29, was deemed a promising chiral scaffold for diverse applications in asymmetric catalysis.This compound can be easily prepared from trans-4-(S)-hydroxyproline (Scheme 2), and several derivatives were tested as chiral ligands coordinating metal reagents or as organocatalysts themselves in different asymmetric reactions, inducing enantioselectivities with varying levels of success.In particular, diethylzinc addition to carbonyls of aldehydes was the most successful. 75rthermore, in one of the first examples of organocatalyzed asymmetric Biginelli reaction, the hydrobromide of diazabicycloheptane (1S,4S)-(R)-30 afforded moderate results (Scheme 3). 76Outstandingly good Scheme 2. Synthesis of chiral diazabicycloheptanes ligands.resolutions were achieved for the series of chiral cyclic ureas (35) by means of using Chirobiotic TM T column.Figure 4 shows a typical example of chromatograms for a product of the tested Biginelli reaction.
More recently, it was found that diastereomeric salts of diazabicycloheptane (1S,4S)-31 combined with (R)-mandelic acid [(R)-38] successfully organocatalyzed the Michael addition reaction under solvent-free conditions. 77 general overview of performance of (1S,4S)-31 in the aforementioned reaction is depicted in Scheme 4. These results were interesting since it has been known that the structural nature of an acidic proton source had no influence on stereoselectivity given that acid additives tend to carry out general acid catalysis type. 72

Organocatalysis via Proline Dipeptide Derivatives Assisted by Mechanochemistry
Mechanochemical synthesis involves mechanical grinding of the corresponding reagents under solvent free conditions or in the presence of molar equivalents of a suitable solvent (e.g.water), either generated during the reaction or added (minimal solvent).The reaction usually proceeds with no heating other than that produced from the conversion of the mechanical energy of milling into heat, being the dispersion and an incremented surface area the determining factors in reactions subjected to the mechanical action. 78A wide range of applications of mechanochemistry have been found, not only in areas typically related to mechanical grinding such as in the preparation of oxides, 79 metal complexes, 80 energy-related or environmental heterogeneous catalysts 81 and metal-organic frameworks or hosts for molecular inclusions, 82 but also in non-traditional fields such as molecular co-crystal formation 83 and production of pharmaceutical materials. 84In recent years, mechanochemistry has also constituted a developing field of interest in organic synthesis, 85,86 thus ball-milling has been successfully employed in the synthesis of peptides and aromatic amides, 87 in the preparation of substituted hydantoins from dipeptides 88 in carbon-heteroatom bond forming reactions, in the synthesis of heterocycles, 89 in the synthesis of Ugi 4-CR and Passerini 3-CR adducts, 90 in cross-coupling reactions as well as in other metal-catalyzed organic processes. 91ikewise, asymmetric organocatalysis can also take advantage of mechanochemical tools to carry out solvent free (or minimal solvent versions) of existing reactions which proceed via enamine formation among other activation mechanisms. 92In this regard, it should be noted the pioneering work implemented by Bolm and co-workers, 93 and Nájera and co-workers 94 in aldol and Michael reactions under ball-milling activation.
Our research group evaluated the organocatalytic activity of the methyl ester of (S)-proline-(S)-phenylalanine dipeptide (S,S)-39 in the asymmetric aldol reactions between cyclohexanone or acetone together with various aromatic aldehydes under ball-milling, solvent-free conditions. 95Using a milling frequency of 2760 rpm (46 Hz) at -20 °C, dipeptide (S)-39 stereoselectively catalyzed the formation of aldol products in yields as high as 94%, with up to 91:9 anti:syn d.r.(diastereomeric ratio) and up to 95% ee.Furthermore, (S)-proline-containing thiodipeptides could also be employed for the mechanochemical asymmetric aldol reaction, which in some cases proved to be better organocatalysts relative to their amide analogues [(S,S)-39 vs. (S,S)-43]. 96Equally, the methyl ester of (S)-proline-(S)-tryptophan (S,S)-44 combined with benzoic acid as additive and a small amount of water, afforded higher diastereo-and enantioselectivities (up to 98:2 anti:syn d.r. and up to 98% ee). 97More recently, O-methyl esters of proline-derived α,β-dipeptides, e.g.(S)-45, have been evaluated, 98 as well as amides supported on MBHA (4-methylbenzhydrylamine) resin, (S)-46. 99    Table 2. Organocatalyzed direct aldol reaction between cyclohexanone and aryl-aldehydes aryl-substituted with electron-withdrawing groups (respecting to carbonyl electrophilicity) (cont.) Figure 5 collects representative chromatograms of enantioenriched diastereomeric mixtures generated with chiral dipeptides as organocatalysts.It is worthy to note that a slight difference in the substitution pattern may affect the elution order of the aldol products.For example, p-nitro substituted (2S,1'R)-42a (Table 2, entries 1-5) is last eluted under the chromatographic conditions employed with a Chiralpak AD-H chiral column while on the contrary, orthoand meta-nitro substituted [(2S,1'R)-42b and (2S,1'R)-42c, respectively] elute first.Taking u-42c as example of analyte, Table 3 collects diverse chromatographic conditions employed for analysis of aldol reactions catalyzed by selected organocatalysts as recently described in the literature.Best chromatographic conditions for the analytical resolution of (±)-42c were reported by Pedotti and Patti, 105 who employed a Lux Cellulose-2 column (chiral selector: cellulose tris-3-chloro-4-methylphenylcarbamate, 250 × 4.6 mm, 5 μm particle size), achieving resolution factors as high as 4.53 with hexane/i-propanol (9:1) as eluent.Resolution factors (R S ) calculated from chromatograms available in their corresponding supporting information files by using the formula: R S = 2(t 2 -t 1 )/w 1 + w 2 , wherein, t 1 and t 2 are the retention times of the enantiomers and w 1 and w 2 are the peak widths at their baselines.d.r.: diastereomeric ratio.
When evaluating new ligands as organocatalysts, unambiguous assignment of the absolute configuration of products obtained from organocatalytic reactions is crucial to make an appropriate analysis of chromatograms corresponding to racemic and enantioenriched samples.For example, Gandhi and Singh 106 developed an enantioselective synthetic route to prepare the bicyclic azetidine (R,S,S)-55e from aldol product (S,R)-42e, that had been obtained in a reaction catalyzed by diamino-sulfonamide (S,R,R)-52 (Scheme 5).Gandhi and Singh 106 assigned the configuration of the new chiral centers by means of nuclear Overhauser effect (nOe) experiments; in particular, they observed an enhancement in the peak intensity of H 2 by irradiating H 1 , and vice versa.
In the case of organocatalyst (S,S)-44, chromatographic examination of the experimental stereochemical results (see Table 2, conditions described in entry 12) led to propose a reasonable transition state to explain the observed stereocontrol (Figure 6).Thus, the creation of a hydrophobic pocket enhances non-covalent π-π interactions between aromatic rings present both in the catalyst and in the aldehyde, so that the interaction between these fragments leads to a more rigid transition state, which is translated into a higher stereoselectivity. 97t is worth mentioning that high-speed ball milling has been recognized as an environment-friendly mechanochemical technique given that it enhances atom economy by diminishing or eliminating solvent usage when carrying out organocatalytic reactions. 107At this point, it is appropriate to mention that recent advances on separation techniques such as supercritical fluid extraction, 108 solid phase extraction, 109 among other practices 110 might permit a greater level of sustainability in chemical reactions in general.

Thiohydantoin (S)-Proline Derivatives as Organocatalyst
Kokotos et al. 111 have synthesized diverse (S)-proline derivatives containing a thiohydantoin fragment and tested them as organocatalyst in the asymmetric Michael addition reaction.Similarly, in our research group, a different series of thiohydantoins derived from proline was prepared by means of the synthetic route presented in Scheme 6. 112 Various techniques including X-ray diffraction structural analysis, 13 C NMR and MS-TOF (time-of-flight mass spectrometry) helped confirm the formation of the thiohydantoin scaffold rather than isothioureas, a result that was explained in terms of the hard and soft acid and base theory (HSAB theory) proposed by Pearson,113 considering that nitrogen (a hard nucleophile) preferably attacks the carbonyl group (a hard electrophile). 114These thiohydantoin derivatives were tested as organocatalysts in the asymmetric Michael reaction, and variables such as solvents, acidic additives and temperature were modified in order to find the most optimal conditions.The importance of solvent-free reaction conditions to maximize the suitable intermolecular interactions affording the desired stereocontrol constitute salient features of these catalytic systems (Scheme 7).

Diaza-Analogues of gem-Diphenyl Prolinols and their Application as Organocatalysts
α,α-Diarylprolinol derivatives are well-established families of catalysts, which are widely used to promote diverse asymmetric reactions. 63The enantioinduction generated from these catalysts is mainly due to the gem-diphenyl carbinol fragment, that may be considered as a chiral amplifier. 115Hence, the synthesis of chiral diaza analogues of the classical and privileged amino alcohols seemed an evident goal to address in the development of alternative chiral ligands.In this regard, in our group there has been a continuous interest in the synthesis of α-phenyl and α,α-diphenyl prolinamines and its derivatives. 116n particular, in 2008 we accomplished the substitution of the tertiary hydroxyl group within N-benzyl α,α-diphenylprolinol (S)-64-I by an azide ion [(S)-65-I] in the presence of high concentrations of sulfuric acid (to provoke S N 1 type reaction, see Scheme 8).The resulting aminoazide was reduced and deprotected to obtain diamine (S)-68a, that was used as precursor of a diazaborolidine, in turn tested as catalyst in the asymmetric reduction of prochiral ketones. 117An alternative route was developed to carry out the OH→N 3 substitution directly from (S)-diphenyl(pyrrolidin-2-yl)methanol (S)-64-II by using trifluoroacetic acid, this in order to afford the pyrrolidinederived azide (S)-65-II, which could be N-Boc protected and then reduced to the diamine derivative (S)-66.The N-Boc protecting group on the pyrrolidine nitrogen allowed the functionalization of the primary amino group into various amide, alkylated amine, sulfonamide and triazole derivatives [(S)-68a-f] (Scheme 8).In each case, carefully controlled conditions were required to generate the desired derivatives from the sterically hindered benzhydrylamine moiety. 118 is worth mentioning that by-product (S)-68d' formed as a consequence of the Thorpe-Ingold effect. 119The enantiomeric purity of amidine (S)-68d' was evaluated by HPLC (Figure 7) in order to correlate the ee with the enantiopurity of amino azide (S)-65-II and its derivatives.
Chiral diamines (S)-65-II and (S)-68a,b,e,f were evaluated as bifunctional organocatalysts in the asymmetric Michael addition (Table 4).(S)-2-(Azidodiphenylmethyl) pyrrolidine (S)-65-II was identified as the most efficient organocatalyst.As it could be anticipated, stereocontrol is mainly directed by steric hindrance.Diamine (S)-68a was the only derivative where hydrogen bonds apparently play a significant role according to the stereoselectivity observed (Figure 8).Table 5 presents selected results regarding the enantioselectivities observed with diverse substrates by employing organocatalyst (S)-65-II.In addition, Figure 9 includes chromatograms pertinent to Table 5.

Conclusions
The development of chiral stationary phases since the second half of the twentieth century constitutes an indispensable tool that is frequently taken by granted.Nevertheless, without chiral chromatography, the enormous advance registered in several areas of asymmetric synthesis such as organocatalysis would not have been possible.Diverse standard compounds such as α-amino acids, Tröger's base, benzoin, Pirkle's alcohol, among others, have been conventionally employed to evaluate newly designed chiral selectors.Continuing studies with available techniques have also allowed a better understanding of specific mechanisms of chiral recognition.
Organocatalysis has been a buoyant area in asymmetric synthesis during the last 15 years.An immense quantity of data used to evaluate new ligands and reactions is available thanks to the employment of chiral chromatography.It would be interesting to develop "tailor-made" chiral selectors for e.g.chiral Michael adducts, which should be feasible considering the principle of reciprocity (Pirkle concept) employed in the design of selectors.Combined techniques such as HPLC-circular dichroism (CD) together with quantum chemical CD calculations 123 will help evaluate stereoinduction from a newly developed ligand as potential organocatalyst.

Figure 1 .
Figure 1.Some relevant examples of chiral selectors developed for their use in liquid chromatography (all structures are adapted from the corresponding references).

a
Determined by1 H NMR spectroscopy of the crude product; b determined by chiral column HPLC of the predominant anti product.d.r.: diastereomeric ratio; e.r.: enantiomeric ratio; U: volumetric flow rate of the mobile phase.

Figure 7 .
Figure 7. Confirmation of enantiopurity of a derivative obtained from azide (S)-65-II.The absolute configuration of (S)-68d' was also corroborated by X-ray diffraction analysis.

Table 4 .a
Asymmetric Michael reaction catalyzed by pyrrolidine derivatives Determined by HPLC with chiral column OD-H; b IPA:H 2 O (3:1) was employed as solvent; c configuration confirmed based on previous reports in literature. 120e.r.: enantiomeric ratio.

Table 5 .
Salient examples of Michael adducts generated by azide (S)-65comparing optical rotation from literature, which is in accordance with the observed stereoinduction promoted by steric hindrance.e.r.: enantiomeric ratio; U: volumetric flow rate of the mobile phase; t R : retention time.

Table 1 .
22lient developments regarding chiral selectors for HPLC (based on the summary table compiled by Lämmerhofer)22

Table 1 .
22lient developments regarding chiral selectors for HPLC (based on the summary table compiled by Lämmerhofer)22(cont.)

Table 2
summarizes the selected results regarding the obtained enantioselectivities induced by diverse organocatalysts in aldol reactions.Table2also includes the available details of the chromatographic separations of the resulting stereoisomeric products.