Enantioresolution of a Series of Chiral Benzyl Alcohols by HPLC on a Dinitrobenzoylphenylglycine Stationary Phase after Achiral Pre-Column Derivatization*

High performance liquid chromatography method for the separation of a series of chiral benzyl alcohols on N-(3,5-dinitrobenzoyl)-D-phenylglycine stationary phase (Macherey Nagel, Chiral-2) after pre-column achiral derivatization was developed. Cheap and easy available aromatic acid chlorides were used as derivatization agents. Good to excellent separations of the enantiomers were achieved in all cases in relatively short analytical runs. It was shown that the enantiorecognition depends on the substituents both in the starting alcohol and in the acid chloride. The method presents an efficient alternative to the direct analyses on polysaccharide and cyclodextrine-derived stationary phases.


Introduction
Chiral benzyl alcohols are an important class of organic compounds, which exist as structural subunits in many natural and biologically active compounds.They are also widespread products from variety of test transformations used for determination of asymmetric induction caused by different asymmetric catalysts [1][2][3][4][5][6][7].Therefore; the determination of the enantiomeric purity of the products is a crucial step in the preparation of single-enantiomer drugs and chiral catalysts and the development of new and more versatile methods continues to attract attention.
High performance liquid chromatography (HPLC) on chiral stationary phases (CSPs) is among the most general and powerful techniques for separation of optical isomers [8][9][10][11][12][13].The efficiency of the separation is strongly dependant on the structure of the stationary phase (SP) used.Of the numerous CSPs available on the market, Brush-type or Pirkle-type columns, containing a low-molecular-weight chiral molecule (chiral selector) covalently bound to the silica gel surface, are the most widely investigated [14][15][16].Among them, the phases based on derivatives of α-amino acids, inexpensive and readily available enantiomerically pure materials, are the most broadly exploited."Pirkle I-phases" based on dinitrobenzoylphenylglycine (DNBPG) selector, covalently bonded to aminopropyl silica via a spacer, are one of the first and intensively used.The main advantages of these phases are the relatively low cost of the columns and their availability in both enantiomeric forms, which is of great importance for trace analysis where the small peak should be eluted first.However, their application is limited in respect to analyte character.DNBPG has two amide groups, which can undergo dipole-dipole interactions and/or hydrogen bonding with suitable molecules.As these interactions are responsible for the separation, the phases are generally inefficient for direct analysis of some important classes of compounds, such as amines, amino acids, alcohols, amino alcohols, etc.
In this paper, we present an effective liquid chromatography method for enantioseparation of benzyl alcohols on one of the cheapest dinitrobenzoylphenylglycine chiral stationary phase (Macherey Nagel, Chiral-2) after achiral pre-column derivatizations as benzoates, chlorobenzoates and naphthoates.The protocol, we believe, is of practical significance as an alternative to the highly efficient direct enantiorecognitions on polysaccharide and cyclodextrinederived phases.

Results and Discussion
A series of known racemic chiral benzyl alcohols 1-5 was obtained by reduction of the parent ketones with NaBH 4 according to a standard procedure.The alcohols were easily converted into the ester derivatives 7-11 by refluxing with an acid chloride in pyridine, as shown on Scheme 1.
These derivatization agents were chosen in an attempt to increase the interactions between the analyte and the π-acceptor DNBPG stationary phase.Two alternative work-up protocols were applied for the isolation of the Scheme 1. Preparation of benzyl alcohols 1-5 and deriva- tives 7-12.
products.When ethyl acetate was used for extraction, the esters 7-11 were isolated in high to excellent yields (80-95%) after purification by HPFC on silica gel.In the second scheme, hexane was used instead of ethyl acetate and the target derivatives were isolated in lower yields (50-65%) due to their limited solubility in hexane, but pure enough to be analyzed without chromatography purification, which shortened significantly the general analyzing process.Despite reducing the yield of the esters, the results are explicit as indicated by the same chromatograms obtained after both purification ways.The latter shows that the hexane extraction is the preferable work-up, except for alcohols available in a highly limited scale.
The ester derivatives 7-11 were analyzed by HPLC on Chiral-2 MN column, consisting DNBPG chiral selector, at 25℃ with mobile phases composed of hexane, i-propanol, and trifluoroacetic acid (TFA) in varied proportions.Excellent to very good separations were achieved in all cases (Table 1).The retention factors k 1 and k 2 were recalculated towards T o , which was determined by using benzene as a standard.All resolution parameters were calculated by the software, adjacent to the apparatus, ChemStation for LC 3D Rev. B.01.01.
As a first series, the esters with phenyl alcohols 1-3, derivatives possessing substituents only at the acid component, were analyzed.The derivatives 7-9 were eluted with hexane/iPr-OH/TFA 100:0.03:0.05 and effective separations were achieved in fast analytical runs, retention times of 5-16 min.
As a second series, the esters of alcohols containing nitro-substituent at the aromatic ring, 4 and 5, were obtained and analyzed.To achieve effective combination separation/retention time, more polar mobile phase was used, hexane/iPr-OH/TFA 100:0.1:0.05.As seen on Table 1, the two groups of derivatives, 10a-10f and 11a-11f, follow different separation patterns.Commensurable resolution factors were obtained for the esters with 2-nitrophenyl alcohol 10a-10f.The retention times of 25- 27 min were observed for 10b-10e, while slower elution was detected for 10a and 10f, 36-40 and 53-57 min, respectively.These results show that the monochlorobenzoates 10b-10d are the derivatives of choice within this series, 10b being the preferable example.In the case of the esters with 4-nitrophenyl alcohol 5, the simple benzoate 11a and naphthoate 11f showed the best resolution factors, 1.34 and 1.32, respectively (Table 1), while 2chlorobenzoate 11b and 2,4-dichlorobenzoate 11e were the less effective derivatives, 0.78.Thus, 11a and 11f are the preferred derivatives of 5 despite the slower elution process in respect to 11b-11e, 40-46 vs 21-26 min.The separations of the enantiomers of 10b and 11a are illustrated on Figure 2.
The method was further extended towards the nonracemic 1-(2-methoxyphenyl)propanol 6, obtained by addition of diethylzink to the corresponding aldehyde in the presence of а chiral catalyst by our colleagues [69], who supplied us with a sample.The unknown ester de-rivatives 12a-12f were obtained and purified via the same protocols (Scheme 1) and were afterward analyzed.Relatively polar mobile phase was used, hexane/iPr-OH/TFA 100:0.5:0.05, and efficient separations were achieved in very fast elution, 4-8 min.As seen on Table 1, benzoate 12a was the less effective, 1.23, while the best separation was achieved for naphthoate 12f, R S 3.22, which is consistent with the initial expectations.Inside chlorinated derivatives, 2-chlorobenzoate 12b showed the best resolution factor, while dichlorosubstituted compound 12e was the less efficient.The chromatograms of the frontier examples 12a and 12f are given on Figure 3.
As seen, the separation is good enough even in the case of 12a to be used for an explicit determination of the enantiomeric excess.The latter is confirmed by the fact that the obtained ee values of the derivatives 12a-12f are in full congruence with the enantiomeric excess of the starting alcohol 6, determined by chiral GC analysis [69].The  ppearance of the minor (R)-isomer as a first signal preseries of chiral racemic benzyl alcohols and a non-

Experimental
ts were purchased from Aldrich, erck and Fluka and were used without any further putions were performed on an Agilent 1100 Sy a sents an additional advantage of the particular analysis protocol especially when high degree of enantioselectivity is achieved.The same pattern is valid for the non-racemic derivatives 7, where the minor (R)-enantiomer elutes first.
General: All reagen M rification.Fluka silica gel/TLC-cards 60778 with fluorescent indicator 254 nm were used for TLC chromatography and Rf-values determination.The high performance flash chromatography (HPFC) purifications were carried out on a Biotage HorizonTM system (Charlottesville, Virginia, USA) on silica gel.The melting points were determined in capillary tubes on SRS MPA100 OptiMelt (Sunnyvale, CA, USA) automated melting point system.The NMR spectra were recorded on a Bruker Avance DRX 250 and Bruker Avance II+ 600 (where indicated) spectrometers (Rheinstetten, Germany) in deuterochloroform; the chemical shifts were quoted in ppm in δ-values against tetramethylsilane (TMS) as an internal standard and the coupling constants were calculated in Hz.
The high performance liquid chromatography (HPLC) enantiosepara

Table 1 . Resolution of the enantiomers of the benzyl alcohol derivatives 7-12.
5-dinitrobenzoyl)-D-phenylglycine.The analyses were performed at 25 ℃ with a flow rate of 1.0 mL/min.