High-performance liquid chromatographic separation of paclitaxel intermediate phenylisoserine derivatives on macrocyclic glycopeptide and cyclofructan-based chiral stationary phases

Journal of Pharmaceutical and Biomedical Analysis 114 (2015) 312–320

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High-performance liquid chromatographic separation of paclitaxel intermediate phenylisoserine derivatives on macrocyclic glycopeptide and cyclofructan-based chiral stationary phases István Ilisz a , Nóra Grecsó a,b , Eniko˝ Forró b , Ferenc Fülöp b , Daniel W. Armstrong c , Antal Péter a,∗ a

Department of Inorganic and Analytical Chemistry, University of Szeged, Dóm tér 7, H-6720 Szeged, Hungary Institute of Pharmaceutical Chemistry, University of Szeged, Eötvös utca 6, H-6720 Szeged, Hungary c Department of Chemistry and Biochemistry, University of Texas at Arlington, Arlington, TX 76019-0065, USA b

a r t i c l e

i n f o

Article history: Received 23 April 2015 Received in revised form 3 June 2015 Accepted 4 June 2015 Available online 10 June 2015 Keywords: Column liquid chromatography Phenylisoserine analogs Macrocyclic glycopeptide-based chiral stationary phases Cyclofructan-based chiral stationary phases

a b s t r a c t High-performance liquid chromatographic methods were developed for the separation of enantiomers of four unnatural paclitaxel precursor phenylisoserine analogs on chiral stationary phases containing macrocyclic glycopeptides and cyclofructans as chiral selectors. The effects of the mobile phase composition, the nature and concentration of different mobile phase additives (alcohols, amines and acids) in different chromatographic modes, temperature and the structures of the analytes on the separations were investigated. Separations were carried out at constant mobile phase compositions in the temperature range 10–50 ◦ C on macrocyclic antibiotic-based and 5–35 ◦ C on cyclofructan-based columns and the changes in enthalpy, (H◦ ), entropy, (S◦ ), and free energy, (G◦ ), were calculated. The elution sequence was determined in most cases; no general rule could be observed. © 2015 Elsevier B.V. All rights reserved.

1. Introduction Chiral ␤-amino acids and their derivatives are of great interest from both pharmaceutical and chemical perspectives [1–7]. Some of them exhibit antibacterial activity (e.g., cispentacin and icofungipen) [1,8,9], but they can also have wide-ranging uses in peptide [10,11], heterocyclic [12,13] and combinatorial chemistry [14,15] and drug research [16,17]. In recent years, acyclic ␤-amino acids have been recognized as an important class of compounds in the synthesis of potential pharmaceuticals, such as paclitaxel (Taxol® ) and its analogue docetaxel (Taxotère® ), currently considered to be among the most efficient drugs in cancer chemotherapy [18]. To support the paclitaxel needs, researchers have been working on semisynthetic methods involving synthetic side-chain coupling to the C13 O of the more readily available baccatin III derivatives. Since the stereochemistry determines the biological and physicochemical properties of molecules, enantioselective highperformance liquid chromatography (HPLC) is routinely used as an

∗ Corresponding author at: Department of Inorganic and Analytical Chemistry, University of Szeged, H-6720 Szeged, Dóm tér 7, Hungary. Fax: +36 62 544340. http://dx.doi.org/10.1016/j.jpba.2015.06.007 0731-7085/© 2015 Elsevier B.V. All rights reserved.

analytical method for the selective analysis of enantiomers. HPLC enantioseparations of ␤-amino acids have been performed by both indirect and direct methods and the results have been surveyed in several review articles [19–23]. Cinchona alkaloid [24–26], macrocyclic glycopeptide [27] and polysaccharide-based [28,29] CSPs have been used for the enantioseparation of ␤-amino acids. For the HPLC separation of ␤-lactam stereoisomers, different types of selectors have been applied: (R)-N-(3,5-dinitrobenzoyl) phenylglycine [30], tris-carbamates of cellulose or amylose polysaccharides [31–37], ␤-cyclodextrin analogues in HPLC [38,39] and capillary electrophoresis [40,41] and macrocyclic glycopeptide [42,43]. The effects of temperature in enantioselective separations have been studied extensively and a number of papers have been published on this issue [44–50] recently. The thermodynamic characteristics of the chromatographic process and the mechanistic aspects of chiral recognition can be expressed by means of the modified van’t Hoff equation [51]: ln˛ = −

(H ◦ ) (S ◦ ) + RT R

where, R is the gas constant, T is temperature in Kelvin, ˛ is the selectivity factor (H◦ ) and (S◦ ) the differences in the changes in standard enthalpy and standard entropy, respectively.

I. Ilisz et al. / Journal of Pharmaceutical and Biomedical Analysis 114 (2015) 312–320

If (H◦ ) is invariant with temperature, a plot of R ln ˛ vs 1/T has slope −(H◦ ) and intercept (S◦ ). For the purpose of this study the above mentioned c¨ lassical” van’t Hoff approach assuming only one site was utilized, however we are aware of the fact, that for a more realistic approach the contribution of the enantioselective and non-selective sites should be distinguished. In the present paper, direct HPLC methods are described for the enantioseparation of new racemic phenylisoserine analogues, with the application of different macrocyclic antibiotic- and cyclofructan (CF)-based chiral stationary phases (CSPs). For comparison purposes, most of the separations were carried out at constant mobile phase compositions. The effects of the mobile phase composition, the nature and concentration of different additives (alcohols, amines and acids) in different chromatographic modes, the specific structural features of the analytes and temperature on the retention are discussed on the basis of the experimental data. The separation of the stereoisomers was optimized by variation of the chromatographic parameters. The elution sequence was in all cases determined by spiking the racemate with an enantiomer with known absolute configuration. 2. Experimental 2.1. Synthesis of phenylisoserine analogues Since a 3-phenylisoserine-derived side-chain is essential for the antitumour activity of paclitaxel, there is a need for the development of efficient processes for the preparation of (2R,3S)3-amino-3-phenyl-2-hydroxypropionic acid (4A) or its direct enantiopure sources [52], which supposes an adequate analytical method for its analytical enantioseparation. Some very efficient enzyme-catalysed direct and indirect strategies have been published for the preparation of (2R,3S)-3-phenylisoserine (4A), either through the Burkholderia cepacia lipase (PS-IM)-catalysed hydrolysis of the ester function of racemic ethyl 3-amino-3phenyl-2-hydroxypropionate (3) [53] or through the Candida antarctica lipase B (CAL-B)-catalysed ring cleavage of racemic cis-3-hydroxy-4-phenylazetidin-2-one (2) [53] or through the CAL-B-catalysed two-step cascade hydrolysis of racemic cis-3acetoxy-4-phenylazetidin-2-one (1) [54]. Racemic cis-3-acetoxy-1-(4-methoxyphenyl) azetidin-2-one was prepared according to the literature [55]. The hydrolysis of 1 in methanol (MeOH) with saturated aqueous NaHCO3 and Na2 CO3 furnished racemic cis-3-hydroxy-4-phenylazetidin-2-one (2), while the acidic hydrolysis of (2) with 22% ethanolic HCl or 18% HCl resulted in 3 and 4, respectively. The enantiomeric 1A, 2A, 3A and 4A (ee > 98%, determined by using GC [56]) have been prepared through the above-mentioned direct and indirect enzymatic strategies [53,54]. 2.2. Chemicals and reagents Acetonitrile (MeCN), MeOH, n-hexane, ethanol (EtOH), 1propanol (PrOH), 2-propanol (2-PrOH,) of HPLC grade, and 1-butanol (BuOH), 2-methyl-2-propanol (t-BuOH), triethylamine (TEA), formic acid (FA), acetic acid (AcOH), trifluoroacetic acid (TFA) and perchloric acid (HClO4 ) of analytical reagent grade were purchased from VWR International (Radnor, PA, USA). The Milli-Q water was further purified by filtration on a 0.45-␮m filter, type HV, Millipore (Molsheim, France). 2.3. Apparatus and chromatography Two Waters HPLC systems were applied: (a) consisted of an M-600 low-pressure gradient pump, an M-2996 photodiode-array detector and an Empower 2 Chromatography Manager data system

313

and (b) Waters Breeze system consisted of a 1525 binary pump, a 487 dual-channel absorbance detector, a 717 plus autosampler and Empower 2 data manager software (both systems from Waters Chromatography, Milford, MA, USA). Both chromatographic systems were equipped with Rheodyne Model 7125 injectors (Cotati, CA, USA) with 20-␮l loops. The macrocyclic antibiotic-based CSPs used were teicoplanincontaining Chirobiotic T, teicoplanin aglycone-containing Chirobiotic TAG, vancomycin-containing Chirobiotc V and vancomycinaglycone-containing Chirobiotic VAG columns, 250 × 4.6 mm I.D., 5 ␮m particle size (for each column) (Astec, Whippany, NJ, USA). Recently developed isopropyl carbamate-CF6-based (Larihc CF6P), (R)-naphthylethyl carbamate-CF6-based (Larihc CF6-RN) and dimethylphenylcarbamate-CF7-based (Larihc CF7-DMP) CSPs, all 250 × 4.6 mm I.D., with a particle size of 5 ␮m, were from Larihc (AZYP, Arlington, TX, USA). The dead-times (t0 ) of the Chirobiotic and Larihc columns were determined by injecting aceton dissolved in methanol or tri-t-butylbenzene, respectively. The columns were thermostated in a Spark Mistral column thermostat (Spark Holland, Emmen, The Netherlands). The precision of the temperature adjustment was ±0.1 ◦ C. For the Chirobiotic columns mobile phases in the reversed phase mode (RPM) were mixed from MeOH and 0.1% aqueous solution of TEA adjusted to a certain pH with AcOH, leading to the formation of triethylammonium acetate (TEAA). The ratio of TEAA solution and MeOH in the range 90/10–10/90 (v/v) were varied. In the polar-ionic mode (PIM) AcOH and TEA were dissolved in MeOH in different concentrations. For the Larihc columns mobile phases based on (i) n-hexane containing alcoholic modifiers (EtOH, 1-PrOH, 2-PrOH, BuOH or t-BuOH) and acidic modifiers (AcOH, TFA, HCOOH, HClO4 ) or (ii) MeOH/MeCN in different ratio as bulk solvent and AcOH or TFA and TEA as acid and base modifiers were applied.

3. Results and discussion 3.1. Separation of enantiomers on macrocyclic glycopeptide-based columns Analytes 1 and 2 in this study (Fig. 1) possess a ␤-lactam skeleton with phenyl and hydroxy groups, while analytes 3 and 4 are phenylisoserine analogues. This distinction results in different steric effects and hydrogen-bonding, and influences the hydrophobicity, bulkiness and rigidity of the molecules. The experimental conditions investigated included the pH of the mobile phase and the concentration of the organic modifier in RPM, and the nature and concentration of acid and base additives in PIM. Investigations of the effects of temperature on the separation were carried out in both RPM and PIM. The effects of pH were investigated for analyte 4 on the Chirobiotic TAG. In the RPM, a decrease of pH in the aqueous phase of the 0.1% aqueous TEAA/MeOH (70/30 v/v) eluent system from 5.0 to 3.0 slightly increased the retention factors, while the selectivity and resolution decreased, as illustrated in Fig. 2. Similar results were obtained by Armstrong et al. [57] on a teicoplanin CSP for analytes with free carboxylic acid groups. The pH that produced the highest ˛ also yielded the best resolution. At low pH k , ␣ and RS changed in different ways. Protonation of teicoplanin aglycone at low pH either directly affects the ionic or dipolar interactions between the analyte and the CSP, or indirectly influences the separation by changing the conformation of the selector. The effects of the organic modifier content on the separation were investigated by variation of the ratio of 0.1% aqueous TEAA (pH 4.1) and MeOH in the range 90/10–10/90 (v/v); as the MeOH content of the mobile phase was increased, the retention factor

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Fig. 1. Structure of analytes.

Fig. 2. Effect of pH on chromatographic parameters, k1 , ˛ and RS of analyte 4. Chromatographic conditions: column, Chirobiotic TAG; compound 4; mobile phase, 0.1% aqueous TEAA (pH 3.0–5.0)/MeOH (70/30 v/v); flow rate, 0.8 ml min−1 ; detection at 215 and 230 nm. symbols, k1 : ; ˛: ; RS :

for analyte 3 on Chirobiotic V and VAG and for 4 on Chirobiotic TAG increased (analyte 3 was not separable on Chirobiotic T and TAG, and analyte 4 was not on Chirobiotic T) (Fig. 3); this suggests that the separation may be controlled by a hydrophilic interaction liquid chromatography (HILIC) than by the RPM mechanism

at high MeOH contents. For analytes 1 and 2 on both Chirobiotic T and TAG columns, k increased with increasing water content in the mobile phase, indicating the importance of hydrophobic interactions in the chromatographic process. The ˛ values generally increased with increasing MeOH content (the only exception was analyte 3 on Chirobiotic V), while the RS values progressively increased with increasing MeOH content, except for analyte 1 on Chirobiotic T and TAG and for analyte 3 on Chirobiotic VAG, when decreases in RS were observed above 50% MeOH content. PIM was applied on Chirobiotic T and TAG CSPs with the use of MeOH/AcOH/TEA (a, 100/0.05/0.05, b, 100/0.1/0.1 and c, 100/0.2/0.2 v/v/v) (Table 1). Increase of the TEA/AcOH content in the mobile phase resulted in a slight decrease in retention, indicating the importance of ionic interactions in the mobile phase. However, partial or baseline separation was observed on Chirobiotic T and TAG columns in the PIM as compared with the RPM for analytes 3 and 4, indicating that chiral discrimination was better in the PIM (Table 1). In summary, the macrocyclic glycopeptide-based CSPs display a complementary character. Elution sequences were determined in most cases. For analytes 1 and 2 on the Chirobiotic columns, the elution sequence was RS < SR, while for analyte 4 it was SR < RS. The elution sequence did not differ when the mobile phase was changed from RPM to PIM.

Fig. 3. Dependence of the retention factor of the first-eluting enantiomer (k1 ), the separation factor (˛) and the resolution (RS ) of analytes 1–4 on the MeOH content in the hydro-organic mobile phase. Chromatographic conditions: column, Chirobiotic T, TAG, V and VAG; compounds, 1–4; mobile phase, 0.1% aqueous TEAA (pH 4.1)/MeOH (90/10–10/90 v/v); flow rate, 0.8 ml min−1 ; detection at 215 and 230 nm.

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315

Table 1 Chromatographic data, retention factor (k), selectivity factor (˛), resolution (RS ) and elution sequence on Chirobiotic T and TAG column in polar-ionic mode. Compound

Column

Eluent

k1

k2

˛

RS

Elution sequence

1

T

a b c a b c a b c a b c b b a b c a b c

0.10 0.08 0.09 0.21 0.21 0.20 0.16 0.15 0.14 0.35 0.34 0.33 1.61 2.00 1.59 1.52 1.39 1.15 1.14 0.99

0.35 0.32 0.29 0.53 0.51 0.47 0.64 0.55 0.55 1.29 1.2 1.17 1.75 2.01 1.70 1.6 1.49 1.34 1.42 1.17

3.52 4.00 3.42 2.46 2.44 2.37 4.05 3.67 3.85 3.66 3.52 3.57 1.09 1.01 1.07 1.05 1.07 1.17 1.25 1.18

2.48 2.91 2.06 4.15 3.23 2.56 5.38 3.41 4.93 10.00 5.81 8.91 <0.2 <0.2 0.36 <0.2 0.28 1.23 0.76 0.72

RS < SR RS < SR RS < SR RS < SR RS < SR RS < SR RS < SR RS < SR RS < SR RS < SR RS < SR RS < SR – – – – – SR < RS SR < RS SR < RS

TAG

2

T

TAG

3 4

T TAG T

TAG

Chromatographic conditions: column,; Chirobiotic T and TAG; mobile phase, MeOH/TEA/AcOH, (a, 100/0.05/0.05 v/v/v), b, (100/0.1/0.1 v/v/v) c, (100/0.2/0.2 v/v/v); flow rate, 0.8 ml min−1 ; detection 215 and 230 nm.

Fig. 4. Effects of the amount of the alcoholic modifier on the retention factor of the first-eluting enantiomer (k1 ), the separation factor (˛) and the resolution (RS ) for analytes 3 and 4 on the CF6-P column. Chromatographic conditions: column CF6-P; mobile phase, n-hexane/2-PrOH/TFA (50/50/0.1, 30/70/0.1, 20/80/0.1 and 10/90/0.1 v/v/v); flow rate, 0.5 ml min−1 ; detection, 206 nm; symbols, k1 : : for 3 and :4; ˛: : for 3 and :4;.

3.2. Separation of enantiomers on cyclofructan-based columns Derivatized CF-based CSP permit effective enantiomeric separations, especially of chiral molecules containing a primary amine group [58]. Chiral separations of analytes 1–4 were performed on three CF-based CSPs (CF6-RN, CF6-P and CF7-DMP) with mobile phases of n-hexane containing alcoholic modifiers (EtOH, PrOH, IPA, BuOH or t-BuOH). For comparison of the performances of the three CF columns, separations were carried out with mobile phases containing n-hexane/IPA/TFA (30/70/0.1 v/v/v) (data not shown). The CF6-P column provided the largest retention factors, and DMPCF7 the smallest. IP-CF6 appeared to be the most effective in the separation of the enantiomers of 3 and 4, whereas CF7-DMP exhibited low separation efficiency in the separation of 3 and 4. Analytes 1 and 2 were not separable on CF-based CSPs, probably because of the lack of a primary amino group. The alcohol content of the mobile phase strongly influenced the chromatographic behaviour. As can be seen in Fig. 4 for analytes 3 and 4, k decreased strongly with increasing alcohol content on

Fig. 5. Effects of the nature of the alcohol modifier on the retention factor of the firsteluting enantiomer (k1 ), the separation factor (˛) and the resolution (RS ) for analytes 3 and 4 on the CF6-P column. Chromatographic conditions: column, CF6-P; mobile phase, n-hexane/EtOH/TFA (36/64/0.1 (v/v/v), n-hexane/1-PrOH/TFA (30/70/0.1 v/v/v), n-hexane/2-PrOH/TFA (30/70/0.1 v/v/v), n-hexane/BuOH/TFA (26/74/0.1 v/v/v), n-hexane/t-BuOH/TFA (26/74/0.1 v/v/v); alcoholic modifiers, EtOH, PrOH, 2PrOH, BuOH and t-BuOH; flow rate, 0.5 ml min−1 ; detection, 206 nm; symbols, k1 : : for 3 and : 4; ˛: : for 3 and : 4.

CF6-P in n-hexane/IPA/TFA as mobile phase system (typical normal phase behaviour), while the changes in ˛ and RS differed. For the ˛ values, slight increases were registered with increasing IPA content, while RS exhibited a maximum (analyte 3) or a minimum curve (analyte 4) with increasing alcohol content. In summary, the alcohol concentration exerted a slight effect on the enantioselectivity, i.e., the ratio of the non-chiral and chiral interactions between the CSP and the analytes depended only slightly on the alcohol concentration. The nature of the alcoholic modifier exerted a considerable effect on the retention. Fig. 5 reveals that, for analytes 3 and 4 on CF6-P at constant alcohol concentration (0.91 mol L−1 ), the application of EtOH (64 vol.%), PrOH (70 vol.%), 2-PrOH (70 vol.%), BuOH (74 vol.%) or t-BuOH (74 vol.%) in the n-hexane/alcohol/TFA mobile phase system influenced the retention, selectivity and resolution in different ways. The retention factors for the first-eluting enantiomers for analytes 3 and 4 were smaller in the EtOH and PrOH-containing mobile phases and higher in the eluents containing 2-PrOH, BuOH or t-BuOH. In general, the more apolar

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Fig. 6. Effects of the nature (A) and concentration (B) of the acid modifier on the retention factor of the first-eluting enantiomer (k1 ), the separation factor (˛) and the resolution (RS ) for analytes 3 (A) and 4 (B) on the CF6-P column. Chromatographic conditions: column, CF6-P; mobile phase, n-hexane/2-PrOH/acid (10/90/0.1 v/v/v), acid modifiers, AcOH, HCOOH, TFA and HClO4 ; flow rate, 0.5 ml min−1 ; detection, 206 nm; symbols, k1 : : for 3 and 4; ˛: : for 3 and 4; RS : : for 3 and 4.

alcohols with longer or branched alkyl side-chains resulted in larger retention factors for polar analytes. However, the enantioselectivity decreased slightly when more apolar alcohols were present in the same molar concentration, i.e., the nature of the alcohol slightly affected the ratio of non-enantioselective and enantioselective interactions. The nature of the alcohol had an effect on the resolution also: it generally decreased with increasing alcohol carbon number, but the application of 2-PrOH sometimes resulted in higher RS values. The effects of acidic modifiers in the mobile phase on the separation for analytes 3 and 4 were tested with AcOH, HCOOH, TFA and HClO4 using a n-hexane/2-PrOH/acidic modifier (10/90/0.1 v/v/v) system. Fig. 6A presents a comparison of the chromatographic data for analyte 3 by using the acidic modifiers at a constant 0.1 vol%. It was demonstrated that larger k values were in most cases obtained with HClO4 than with AcOH, TFA or HCOOH. However, the ˛ values did not change significantly with variation of the acid, while the highest RS values were obtained when HClO4 was used. Similar results were obtained for analyte 4 (data not

shown). An increase in the HClO4 content from 0.05% to 0.3% in the n-hexane/2-PrOH/HClO4 (10/90/0.05-0.3 v/v/v) mobile phase system resulted in a slight change in the retention factor, but for both 3 and 4 the largest k1 was obtained on the application of 0.1 vol.% HClO4 (Fig. 6B). Application of PIM on CF6-P with MeOH/MeCN 40/60 (v/v) as bulk solvent and AcOH/TEA 0.075/0.05 or 0.15/0.1 (v/v) as acid and base modifiers, or MeOH/MeCN 10/90 (v/v) as bulk solvent and TFA/TEA 0.075/0.05, 0.15/0.1 and 0.3/0.2 (v/v) as acid and base modifiers, revealed that increase of the AcOH/TEA or TFA/TEA content in the mobile phase resulted in a slight decrease in retention, indicating the importance of ionic interactions in the mobile phase (data not shown). However, on change of the acid and base concentrations, the ˛ values varied in only a small range: for analyte 3 it ranged between 1.05 and 1.12, and for analyte 4 between 1.14 and 1.16. In all cases when separations occurred on CF-based columns, the elution sequence was determined, but no general regularities could be established. The elution sequences for analytes

Table 2 Temperature dependence of retention factor of first eluting enantiomer (k1 ), second eluting enantiomer (k2 ), separation factor (˛), resolution (RS ) and elution sequence of analytes 1–4 on Chirobiotic columns. Compound

1

Column

T

TAG

2

T

TAG

3

V

VAG

4

TAG

k, ˛, RS

k1 ˛ RS k1 ˛ RS k1 ˛ RS k1 ˛ RS k1 ˛ RS k1 ˛ RS k1 ˛ RS

Temperature (◦ C)

Elution sequence

10

20

30

40

50

0.46 3.79 7.05 1.00 3.32 6.52 0.25 2.89 4.44 0.68 3.81 7.05 0.84 1.27 1.71 4.15 1.09 1.43 0.42 1.45 0.74

0.34 3.36 5.68 0.76 2.94 6.63 0.19 2.69 3.44 0.52 3.41 7.44 0.81 1.21 1.64 3.90 1.08 1.14 0.36 1.29 0.62

0.24 3.06 4.44 0.58 2.60 6.38 0.14 2.52 2.61 0.40 3.08 7.07 0.79 1.17 0.93 3.74 1.07 0.86 0.31 1.18 0.30

0.18 2.77 2.82 0.45 2.33 5.28 0.10 2.41 1.79 0.31 2.78 6.46 0.77 1.11 0.77 3.59 1.06 0.60 0.28 1.00 0.00

0.13 2.58 2.00 0.35 2.09 3.67 0.08 2.32 1.50 0.25 2.50 4.75 0.76 1.08 0.57 3.46 1.05 0.29 0.25 1.00 0.00

RS < SR

RS < SR

RS < SR

RS < SR

–

–

SR < RS

Chromatographic conditions: column, Chirobiotic T, TAG, V and VAG; mobile phase, d, 0.1% aqueous TEAA (pH 4.1)/MeOH (50/50 v/v); flow rate 0.8 ml min−1 ; detection, 215 and 230 nm.

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317

Table 3 Thermodynamic parameters, (H◦ ), (S◦ ), T × (S◦ ), (G◦ ), correlation coefficients (R2 ) and Q values of analytes 1–4 on Chirobiotic and CF6-P columns. Analyte

Column

Mobile phase

Correlation coefficients (R2 )

−(H◦ ) (kJ mol−1 )

−(S◦ ) (J mol−1 K−1 )

−Tx(S◦ )298K (kJ mol−1 )

−(G◦ )298K (kJ mol−1 )

Q (H◦ ) /T x (S◦ )298K

1

d

4

T TAG T TAG V VAG TAG

0.9978 0.9997 0.9943 0.9988 0.9907 0.9907 0.9975*

7.3 8.8 4.2 7.9 3.0 0.7 7.2

15.0 21.0 6.0 16.9 8.7 1.7 22.3

4.4 6.3 1.8 5.0 2.6 0.5 6.7

2.9 2.5 2.4 2.9 0.4 0.2 0.5

1.7 1.4 2.3 1.6 1.2 1.4 1.1

3

CF6-P

e

0.9989** 0.9974*** 0.9941 0.9961 0.9990** 0.9980*** 0.9981** 0.9918*** 0.9979** 0.9976***

−1.2 2.0 2.4 1.3 −1.6 3.6 1.3 4.9 0.5 2.5

−7.0 4.3 6.5 3.5 −9.4 8.7 1.4 14.1 0.3 7.4

−2.1 1.3 1.9 1.0 −2.8 2.6 0.3 4.2 0.1 2.2

0.9 0.8 0.5 0.3 1.2 1.0 1.0 0.7 0.4 0.3

0.6 1.5 1.3 1.3 0.6 1.4 3.3 1.2 5.0 1.1

2 3

4

f g e f g

Chromatographic conditions: column, Chirobiotic T, TAG, V, VAG and CF6-P; mobile phase, d, 0.1% TEAA (pH = 4.1)/MeOH (50/50 v/v), e, hexane/2-PrOH/TFA (10/90/0.1 v/v/v), f, hexane/2-PrOH/HClO4 (10/90/0.1 v/v/v), g, MeOH/MeCN/TFA/TEA (10/90/0.3/0.2 v/v/v/v), flow rate, 0.5 ml min−1 ; detection 206 nm; R2 , correlation coefficient of van’t Hoff plot, ln ˛ – 1/T curves; * Temperature range 10–40 ◦ C. ** Temperature range 5–15 ◦ C. *** Temperature range 15–35 ◦ C; Q = (H◦ )/T × (S◦ )298K .

1, 2 and 4 were the same as observed on Chirobiotic CSPs. 3.3. Effects of temperature and thermodynamic parameters In order to investigate the effects of temperature on the chromatographic parameters on macrocyclic glycopeptide-based Chirobiotic T, TAG, V and VAG columns, a variable-temperature study was carried out over the temperature range 10–50 ◦ C (in 10 ◦ C increments). Experimental data for mobile phase d, 0.1% aqueous TEAA (pH 4.1)/MeOH (50/50 v/v), are listed in Table 2. A comparison of the retention factors in Table 2 reveals that all of the recorded values decreased with increasing temperature. Differences in the changes in enthalpy and entropy, (H◦ ) and (S◦ ), are presented in Table 3. The (H◦ ), provides information on the relative ease of transfer of analytes from the mobile to

the stationary phase. The (H◦ ) values on the Chirobiotic CSPs ranged from −0.7 to −8.8 kJ mol−1 . The interactions of 1, 2 and 4 with Chirobiotic TAG were characterized by the highest negative (H◦ ) values, while 3 on Chirobiotic VAG exhibited the smallest negative (H◦ ). Negative (H◦ ) values for a pair of enantiomers are accompanied by a negative (S◦ ). A negative (S◦ ) reflects an increase in order and/or a loss in the degrees of freedom in the course of enantioselective selector–selectand interactions. The trends in the change in (S◦ ) showed that 1, 2 and 4 on Chirobiotic TAG displayed the largest negative entropies. The thermodynamic parameter (G◦ ) suggests that for analytes 1 and 2 the Chirobiotic T and TAG provided the strongest binding to the selector, as reflected by the large negative values, whereas the complexation of analyte 4 with the selector to the Chirobiotic TAG was less efficient. Analyte 3 was separable on Chi-

Table 4 Temperature dependence of retention factor of first eluting enantiomer (k1 ), separation factor (␣), resolution (RS ) and elution sequence of analytes 3 and 4 on CF6-P column. Compound

3

Eluent

e

f

g

4

e

f

g

k, ˛, RS

k1 ˛ RS k1 ˛ RS k1 ˛ RS k1 ˛ RS k1 ˛ RS k1 ˛ RS

Temperature (◦ C)

Elution sequence

5

10

15

25

35

0.85 1.37 0.41 2.94 1.30 1.26 2.24 1.16 1.09 0.63 1.57 0.55 2.79 1.45 1.25 3.81 1.194 1.48

0.76 1.38 0.44 2.91 1.29 1.27 2.11 1.14 1.07 0.62 1.59 0.59 2.78 1.44 1.32 3.40 1.190 1.47

0.68 1.39 0.42 2.73 1.26 1.03 1.94 1.13 1.05 0.59 1.61 0.57 2.55 1.43 1.38 3.06 1.186 1.47

0.57 1.36 0.28 1.99 1.22 1.25 1.60 1.11 0.94 0.53 1.54 0.52 1.77 1.35 1.48 2.55 1.14 1.15

0.50 1.32 <0.2 1.35 1.18 1.00 1.25 1.09 0.85 0.41 1.46 <0.2 1.30 1.25 1.26 2.16 1.11 0.95

–

–

–

SR < RS

SR < RS

SR < RS

Chromatographic conditions: column, CF6-P; mobile phase, e, hexane/2-PrOH/TFA (10/90/0.1 v/v/v), f, hexane/2-PrOH/HClO4 (10/90/0.1 v/v/v), g, MeOH/MeCN/TFA/TEA (10/90/0.3/0.2 v/v/v/v); flow rate, 0.5 ml min−1 ; detection 206 nm.

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Fig. 7. van’t Hoff plots for the separation factor (˛) of 3 and 4 on CF6-P column. Chromatographic conditions: column, CF6-P; mobile phase, for analyte 3 (A) and for analyte 4 (B), e, n-hexane/IPA/TFA (10/90/0.1 v/v/v), for analyte 4 (C), f, n-hexane/IPA/HClO4 (10/90/0.1 v/v/v), and for analyte 4 (D), g, MeOH/MeCN/TFA/TEA (10/90/0.3/0.2 v/v/v/v); flow rate 0.5 ml min−1 ; detection 206 nm.

robiotic V and VAG, but the selector-selectand complex formation was manifested by small −(G◦ ) values. The relative contributions of the enthalpic and entropic terms to the free energy of adsorption can be visualized by the enthalpy/entropy ratios Q [Q = (H◦ )/[298 × (S◦ )] (Table 3). Comparison of the Q values for the individual analytes revealed that the enantioselective discrimination was in all cases enthalpically driven (Q > 1.0), but for analytes 3 and 4 on Chirobiotic V and TAG the relative contribution of the entropy terms were higher. On the CF6-P column, the effects of temperature on the chromatographic parameters for analytes 3 and 4 were studied by the application of NPM and PIM over the temperature range 5–35 ◦ C (Table 4). The mobile phases applied in NPM were e, hexane/2PrOH/TFA (10/90/0.1 v/v/v) and f, hexane/2-PrOH/HClO4 (10/90/0.1 v/v/v), while in PIM it was g, MeOH/MeCN/TFA/TEA (10/90/0.3/0.2 v/v/v/v). A comparison of the retention factors in Table 4 reveals that most of the recorded values decreased with increasing temperature. To shed light on the effects of temperature on the separation, accurate chromatographic data were collected, from which van’t Hoff plots were constructed. The ln ˛ vs 1/T plots for analyte 3 in mobile phase systems f and g exhibited linear fits in all temperature ranges, while for analyte 3 in system e and for analyte 4 in all three mobile phases (e, f and g) the ln ˛ vs 1/T plots could be divided into two linear regions, which means that the linear van’t Hoff plots reflect different overall binding situations in the examined temperature ranges (Fig. 7). (In these cases, Table 3 presents values calculated for the two temperature ranges independently.) The (H◦ ) values ranged from −4.9 to 1.6 kJ mol−1 and were slightly more negative in the temperature range 15–35 ◦ C. In eluent system e, analytes 3 and 4 in the temperature range 5–15 ◦ C exhibited positive (H◦ ) values. The trends in the (S◦ ) and (H◦ )

were similar. Under the conditions where (H◦ ) was negative, (S◦ ) was also negative, and the largest positive (H◦ ) was accompanied by the largest positive (S◦ ). The value of (S◦ ) is governed by the difference in the number of degrees of freedom between the stereoisomers on the CSP, and mainly by the numbers of solvent molecules released from the chiral selector and the analyte when the molecule is associated with the CSP. The relative contributions of the enthalpic and entropic terms to the free energy of adsorption can be visualized through the enthalpy/entropy ratio calculated at 298 K (Table 3). With a few exceptions it can be concluded from the −(H◦ ) and −T(S◦ ) data for all the analytes that the enantioresolution is predominantly enthalpically driven, and the selectivity decreases with increasing temperature. For analytes 3 and 4 in eluent system e in the temperature range 5–15 ◦ C the separations exhibited positive (H◦ ) and positive T × (S◦ ) values, i.e. a larger contribution of entropy to the enantioseparation was observed in this temperature region. In this temperature range, the enantioresolution was entropically driven, and the selectivity increased with increasing temperature (Table 3 and Fig. 7). The thermodynamic parameter −(G◦ )298 suggests that, for 3 and 4 in the NPM, the binding to the selector was stronger as reflected by the larger −(G◦ ) values than in PIM. 4. Conclusion The enantiomers of four ␤-lactam and phenylisoserine analogues were separated by using columns containing CSPs consisting of macrocyclic glycopeptides and cyclofructans. On Chirobiotic CSPs, ␤-lactams and phenylisoserine were separable in the RPM and PIM, and the separation was influenced by the pH of the eluent and the MeOH content of the aqueous 0.1% TEAA (pH 4.1) mobile

I. Ilisz et al. / Journal of Pharmaceutical and Biomedical Analysis 114 (2015) 312–320

phase. For the cyclofructan-based columns, CF6-P provided the best separation efficiency for phenylisoserine analogues in the NPM and PIM. The separation was affected by the nature and concentration of the alcohol and acid modifiers in n-hexane as bulk solvent. Separations were carried out at constant mobile phase compositions in the temperature range 10-50 ◦ C on macrocyclic antibiotic-based columns, and 5–35 ◦ C on cyclofructane-based columns, and the changes in standard enthalpy, (H◦ ), standard entropy, (S◦ ), and free energy, (G◦ ), were calculated. In most cases, the separations proved to be enthalpically driven, but on CF6-P in NPM for analytes 3 and 4, entropically driven separation was also observed in the temperature range 5–15 ◦ C. The elution sequence was determined in most cases, but no general trends were observed. Acknowledgment This work was supported by Hungarian national Grants OTKA K 108847. References [1] F. Fülöp, The chemistry of 2-aminocycloalkanecarboxylic acids, Chem. Rev. 101 (2001) 2181–2204. [2] E. Forró, F. Fülöp, Direct and indirect enzymatic methods for the preparation of enantiopure cyclic ␤-amino acids and derivatives from ␤-lactams, Mini Rev. Org. Chem. 1 (2004) 93–102. [3] A. Liljeblad, L.T. Kanerva, Biocatalysis as a profound tool in the preparation of highly enantiopure ␤-amino acids, Tetrahedron 62 (2006) 5831–5854. [4] L. Kiss, E. Forró, F. Fülöp, Synthesis of carbocyclic ␤-amino acids, in: A.B. Hughes (Ed.), Amino Acids, Peptides and Proteins in Organic Chemistry, Wiley, Weinheim, 2009, pp. 367–409. [5] D. Fernandez, E. Torres, F.X. Aviles, R.M. Ortuno, J. Vendrell, Cyclobutane-containing peptides: evaluation as novel metallocarboxypeptidase inhibitors and modelling of their mode of action, Bioorg. Med. Chem. 17 (2009) 3824–3828. [6] E. Forró, F. Fülöp, Recent lipase-catalyzed hydrolytic approaches to pharmacologically important beta-and gamma-amino acids, Curr. Med. Chem. 19 (2012) 6178–6187. [7] L. Kiss, F. Fülöp, Synthesis of carbocyclic and heterocyclic beta-aminocarboxylic acids, Chem. Rev. 111 (2014) 1116–1169. [8] M. Zegarac, E., Mestrovic, N.K., Hulita, D., Filic, M., Dumic, A., Grunenberg, B., Keil, H. Ceric, PCT WO 2,005,100,302 A1 20,051,027. ˇ ´ V. Sunji ´ Quinine-mediated parallel kinetic [9] Z. Hamerˇsak, M. Roje, A. Avdagic, c, resolution of racemic cyclic anhydride: stereoselective synthesis, relative and absolute configuration of novel alicyclic beta-amino acids, Tetrahedron: Asymmetry 18 (2007) 635–644. [10] M. Mándity, E. Wéber, T.A. Martinek, G. Olajos, G.K. Tóth, E. Vass, F. Fülöp, Design of peptidic foldamer helices: a stereochemical patterning approach, Angew. Chem. Int. Ed. 48 (2009) 2171–2175. [11] T. Martinek, F. Fülöp, Peptidic foldamers: ramping up diversity, Chem. Soc. Rev. 41 (2012) 687–702. [12] P.H. Carter, U.S. Patent, 277 (2005) 666; Chem. Abstr. 144 (2005) 51,452. [13] L. Kiss, M. Nonn, E. Forró, R. Sillanpää, F. Fülöp, Synthesis of novel isoxazoline-fused cispentacin stereoisomers, Tetrahedron Lett. 50 (2009) 2605–2608. [14] I. Kanizsai, S. Gyónfalvi, Z. Szakonyi, R. Sillanpää, F. Fülöp, Synthesis of bi- and tricyclic beta-lactam libraries in aqueous medium, Green Chem. 9 (2007) 357–360. [15] L. Kiss, E. Forró, R. Sillanpää, F. Fülöp, Diastereo- and enantioselective synthesis of orthogonally protected 2,4-diaminocyclopentanecarboxylates: a flip from beta-amino- to beta,gamma-diaminocarboxylates, J. Org. Chem. 72 (2007) 8786–8790. [16] E. Juaristi, V.A. Soloshonok, Enantioselective Synthesis of ␤-amino Acids, Wiley-Interscience, New York, 2005. [17] D. Fernandez, E. Torres, F.X. Aviles, R.M. Ortuno, J. Vendrell, Cyclobutane-containing peptides: evaluation as novel metallocarboxypeptidase inhibitors and modelling of their mode of action, Bioorg. Med. Chem. 17 (2009) 3824–3828. [18] D.G.I. Kingston, D.J. Newman, Taxoids: cancer-fighting compound from nature, Curr. Opin. Drug Disc. Dev. 10 (2007) 130–144. [19] M.H. Hyun, Development and application of crown ether-based HPLC chiral stationary phases, Bull. Korean Chem. Soc. 26 (2005) 1153–1163. [20] I. Ilisz, R. Berkecz, A. Péter, Application of chiral derivatizing agents in the high-performance liquid chromatographic separation of amino acid enantiomers: a review, J. Pharm. Biomed. Anal. 47 (2008) 1–15. [21] I. Ilisz, R. Berkecz, A. Péter, Retention mechanism of high-performance liquid chromatographic enantioseparation on macrocyclic glycopeptide-based chiral stationary phases, J. Chromatogr. A 1216 (2009) 1854–1860.

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