Preparation and evaluation of novel stationary phases for improved chromatographic purification of pneumocandin B0

Journal of Chromatography A, 1101 (2006) 204–213

Preparation and evaluation of novel stationary phases for improved chromatographic purification of pneumocandin B0 Christopher J. Welch ∗ , Jimmy O. DaSilva, Joseph Nti-Gyabaah, Firoz Antia, Kent Goklen, Russell Boyd Merck Research Laboratories, Merck & Co. Inc., Rahway, NJ 07065, USA Received 25 August 2005; received in revised form 4 October 2005; accepted 6 October 2005 Available online 27 October 2005

Abstract Preparation and evaluation of a number of stationary phases for improved chromatographic purification of pneumocandin B0 , a key intermediate in the synthesis of the antifungal agent, Cancidas, has led to the identification of several materials with potential for improved performance. © 2005 Elsevier B.V. All rights reserved. Keywords: Pneumocandin B0 ; Cancidas; Preparative chromatography; Stationary phase

1. Introduction The role of the stationary phase in preparative chromatographic purifications can scarcely be overemphasized [1–3]. Nevertheless, the choice of affordable commercially available preparative stationary phases that are available in bulk quantities is far from ideal. The situation is particularly unsatisfactory with regard to stationary phases for achiral normal phase purifications. In addition to silica itself, which dominates this stationary phase category, only a few functionalized silicas (diol, cyano, amino, etc.) are available, and of those, a paucity of highly polar stationary phases is found. Consequently, normal phase achiral chromatography is often poorly suited for carrying out purifications where polar solvents must be used to keep the compound of interest in solution. Chiral stationary phases (CSPs) often possess sufficient polarity and retention to be useful for such purifications, and are often used for this purpose during early chemical development. For industrial-scale achiral purifications, the use of an expensive CSP may be cost-prohibitive. In recent years, several polar achiral stationary phases have been introduced (e.g. www.esind.com, www.pci-hplc.com), but it is our belief that additional products of this class are needed (Fig. 1). Pneumocandin B0 is an intermediate in the production of the antifungal drug, Cancidas [4,5]. We have previously reported

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Corresponding author.

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on the chromatographic purification of pneumocandin B0 using a silica stationary phase, with proline as an important additive modulating retention and selectivity [6]. While the proline additive plays a valuable role in the chromatographic purification, it also introduces significant complexities into the process. The need to pre-treat the column by proline addition, the need to remove proline from the isolated product stream, and the need to periodically wash off and ‘reprolinate’ the column means that the column is used for the actual chromatographic purification only a fraction of the time, reducing the overall productivity of the method. We were intrigued by the idea that the proline additive might somehow be immobilized on the stationary phase so as to afford the desired chromatographic properties, without the need for the cumbersome prolination/proline recovery/reprolination steps. Preliminary evaluation of a number of commercial stationary phases had led to the finding, illustrated in Fig. 2, that commercial Tosoh Amide 80 stationary phase, described somewhat vaguely as a ‘polycarbamate’ (www.tosohbiosep.com) provided retention comparable with the adsorbent used in the existing purification process (prolinated silica). Although not always the case, greater retention in this purification was believed to generally result in improved chromatographic productivity, owing to improved solubility of the target compound in highly polar chromatographic eluents. We reasoned that further improvements in the pneumocandin B0 purification might be possible with relatively simple stationary phases prepared from inexpensive

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250 mm × 4.6 mm I.D.) used in normal phase analysis of collected fractions were obtained from YMC. HPLC-grade solvents and water used for the experiments were obtained from EM Industries or Fisher Scientific. Volumetric compositions of solutions are designated as volumes before mixing, and were not adjusted for non-ideal mixing effects. For example, an ethyl acetate–methanol–water solution designated as 87/9/7 (EtOAc/MeOH/water) was prepared by mixing 87 volumes of EtOAc, 9 volumes of MeOH and 7 volumes of water. These solutions were prepared at ambient temperature (∼24 ◦ C). (Note that whole number proportions were used for convenience and the sum of the volumes does not necessarily add up to 100.) 2.2. Equipment Fig. 1. Pneumocanding B0 .

reagents containing appropriate polar groups, and therefore initiated a general investigation into this area. 2. Experimental 2.1. Materials Reagents were purchased from Aldrich (Milwaukee, WI, USA) and used without further purification. l-Proline was supplied by Kyowa Hakko Kogyo. Chromatography solvents (HPLC-grade) were purchased from EM Industries or Fisher Scientific. Aminopropyl silica columns used in the on-column functionalization experiments were obtained from ES industries, West Berlin, NJ, USA (Chromegabond amine). Loose silica and aminopropyl silica were Kromasil (Eka Chemicals, Molndal, Sweden). The Amide-80 column (250 mm × 4.6 mm ˚ spherical) was obtained from TosoBioI.D. and 10 ␮m × 80 A, science. The silica grade-631 column (250 mm × 4.6 mm I.D. ˚ irregular) was obtained from Princeton and 16–20 ␮m × 60 A, Chromatography (Cranbury, NJ, USA). The J’Sphere ODS˚ pore diameter × 4 ␮m particle diameter; M80 column (80 A 250 mm × 4.6 mm I.D.) used in reversed phase analysis and the ˚ pore diameter × 5 ␮m particle diameter; silica column (120 A

HPLC studies were carried out with Agilent 1100 HPLC systems, equipped with autosampler and diode array detectors. LC–MS studies were carried out using an Agilent 1100 MSD system fitted with a MacMod Extend C18 50 mm × 4.6 mm I.D. column operating at a flow rate of 1.5 ml/min with gradient eluent with ACN/water containing 2 mM ammonium formate at pH 3.5 or 6.5. Preparative evaluation of stationary phases was carried out using a Waters FractionLynx preparative HPLC system. NMR analysis was carried out using a Bruker Avance 400 spectrometer operating at 400 MHz using CDCl3 referenced to the chloroform signal at 7.27 1 H. In situ column functionalization was carried out using a freestanding retired Shimadzu HPLC pump for reagent flow. Column packing was carried out using an Alltech (Deerfield, IL, USA) column packer. 2.3. On-column preparation of amide, carbamate and urea phases (SP 1–8) On-column acylation of commercial amino silica columns ˚ pore size; (Chromegabond Amine; 5 ␮m particle size; 60 A 250 mm × 4.6 mm I.D.) via the corresponding anhydrides was used in the preparation of the acetamide (SP 1), trifluoroacetamide (SP 2) and succinamide (SP 5) phases. In the typical procedure, the column is flushed with dichloromethane at a flow rate of 2 ml/min for a period of 30 min. A 1 M solution of the

Fig. 2. Tosoh Amide 80 stationary phase affords comparable retention and selectivity in the chromatographic purification of pneumocandin B0 . For each run, 1.2 mL of the feed solution was injected (representing ∼15 g pneumocandin B0 /L bed). The Amide 80 run employed an 88/9/7 (v/v/v) (ethyl acetate–methanol–water; e–m–w) mobile phase, and for the prolinated silica run, the mobile phase was modified by adding l-proline to the 88/9/7 e–m–w mobile phase then used for the run. The column was presaturated with proline before the run. For both cases, the mobile phase flow rate was ∼7.5 column volumes per hour.

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anhydride in dichloromethane containing one equivalent of triethylamine is then passed though the column at a flow rate of 2 ml/min. The column is then flushed with dichloromethane for 10 min at 2 ml/min, followed by flushing with 20% methanol in dichloromethane for 20 min at 2 ml/min. A comparable approach was used for preparation of the 3,5-dinitrobenzoyl (SP 3) and palmitoyl (SP 4) stationary phases, except that the corresponding acid chlorides were used. In an analogous fashion, carbamate phases SP 6 and SP 7 were prepared from the corresponding chloroformates, and the urea phase SP 8 was prepared from the corresponding isocyanate. 2.4. On-column preparation of N-acetyl-d-Asn stationary phase (SP 9) Preparation of the N-acetyl-d-Asn stationary phase (SP 9) followed the general approach described previously [7] and outlined in Fig. 4. Coupling of the N-acetyl-d-Asn to amino silica was accomplished by passing a solution of the N-acetyl-d-Asn (1.75 g in 210 ml of 95% acetonitrile/water) along with one equivalent of the peptide coupling agent, EDC (1.92 g) through the column at a flow rate of 2 ml/min. The column was then flushed with acetonitrile for 10 min, dichloromethane for 10 min, then 20% methanol in dichloromethane for 20 min, all at a flow rate of 2 ml/min. 2.5. On-column preparation of Boc-amino acid stationary phases (SP 10, 12, 14) Preparation of the amino acid stationary phases followed the general approach described previously [7] and outlined in Fig. 4. Coupling of protected amino acids to amino silica was accomplished by passing a solution of the 10 mmol of amino acid (4.06 mmol for the Boc-iso-Gln-OH stationary phase – SP 14), in acetonitrile, along with an equivalent of the peptide coupling agent, EDC, through the column at a flow rate of 2 ml/min. The column was then flushed with acetonitrile for 10 min, then dichloromethane for 10 min, followed with 20% methanol in dichloromethane for 20 min, all at a flow rate of 2 ml/min. 2.6. On-column preparation of deprotected amino acid stationary phases (SP 11, 13 and 15) Removal of the Boc protecting group from SP 10, 12, 14 was accomplished by pumping a solution of 3% TFA in dichloromethane through the column at a flow rate of 2 ml/min for 30 min, followed by 20% methanol in dichloromethane for 15 min, followed by 10% N,N-diisopropylethylamine in dichloromethane for 15 min, followed by 20% methanol in dichloromethane for 15 min, thereby affording deprotected amino acid stationary phases SP 11, 13 and 15. 2.7. On-column preparation of 2,4-DNP phase SP 16 Preparation of the 2,4-DNP stationary phase followed the general approach described previously [8].

2.8. On-column preparation of acrylamide-derived phase (SP 17) A 10 M solution of acrylamide in acetonitrile was circulated through an ES Chromegabond amino column (250 mm × 4.6 mm I.D.) at a flow rate of 2 ml/min, the effluent from the column being directed to the inlet reservoir so as to allow reagent recirculation. Flow of the reagent solution was continued for a period of 4 h, whereupon an increase in column backpressure was noted. Flow was stopped, the column removed and flow direction reversed, and the column was washed with a solution of first dichloromethane at 2 ml/min for 20 min, then 20% methanol in dichloromethane for 20 min, whereupon the column backpressure returned to normal levels. 2.9. Preparation of SP 6 using direct bonding approach To a stirring, inerted (N2 ) mixture of 4.51 mmol of 3aminopropyltriethoxysilane and 1.1 equivalents of triethylamine in 20 ml dichloromethane on an ice bath was added dropwise 1.5 equivalents of methyl chloroformate in 5 ml of dichloromethane. Reaction progress was monitored by LC–MS and 1 H NMR, showing completion at about 1 h. The reaction mixture was washed with 15 ml of water to remove triethylammonium hydrochloride, followed by 15 ml of brine. The organic layer was dried over anhydrous magnesium sulfate and concentrated to dryness under vacuum. The resulting material was resuspended in toluene and filtered to remove residual triethylammonium hydrochloride. Following evaporation, the resulting organosilane 18 (Fig. 8) showed NMR and MS data consistent with the proposed structure. 1 H NMR (400 MHz, CDCl3) δ: 4.97 ppm (bs, 1H), 3.80 (q, 6H), 3.63 (s, 3H), 3.16 (q, 2H), 1.60 (m, 2H), 1.22 (t, 3H), 0.61 (m, 2H). ESI-MS 302 amu (M + Na). Bonding of organosilane 18 to silica to afford SP 6 was car´˚ in ried out by refluxing 5 g of silica (Kromasil 16 ␮m, 60 A) toluene using a Dean Stark trap for 1 h, then adding a toluene solution of the organosilane 18 followed by gentle refluxing for 2 h. The mixture was filtered on a sintered glass funnel, then washed three times with 20% methanol in dichloromethane. Following drying, the resulting material was slurried in 2propanol and packed into a 250 mm × 4.6 mm I.D. HPLC column for evaluation. Residual stationary phase taken from the column packer reservoir was dried overnight under high vacuum, then submitted for combustion analysis (C 2.88%; N 0.36%). 2.10. Preparation of SP 13 using direct bonding approach To a stirring, inerted (N2 ) mixture of 4.51 mmol of 3aminopropyltriethoxysilane in dichloromethane with 1 equivalent of the peptide coupling agent EDC was added 1 equivalent of N-(tert-butoxy carbonyl)-l-proline. Reaction progress was monitored by LC–MS and FT-NMR, showing completion after about 5 h. Upon completion, the sample was extracted with water, washed with brine, dried over anhydrous magnesium sul-

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fate, then evaporated to dryness. The resulting organosilane 19 (Fig. 8) showed NMR data consistent with the proposed structure. 1 H NMR (400 MHz, CDCl3) δ: 6.0–9.5 ppm (m, 1H), 3.78 (q, 6H), 3.1–3.6 (m, 5H), 2.20 (m, 2H), 1.82 (M, 2H), 1.58 (m, 2H), 1.42 (t, 9H), 1.15 (m, 9H), 0.57 (m, 2H). Bonding of the organosilane 19 to silica to afford SP 12 was carried out by refluxing 5 g of silica (Kromasil 16 ␮m, ´˚ in toluene using a Dean Stark trap for 1 h, then adding 60 A) a toluene solution of the organosilane, evaporating to dryness, and heating the resulting mixture in a Kugehlrohr distillation apparatus for 2 days (100 ◦ C, 10 mmHg). The resulting mixture was slurried in 20% methanol in dichloromethane, filtered on a sintered glass funnel, washed three times with 20% methanol in dichloromethane, then dried under high vacuum to afford SP 12. Removal of the Boc group to afford SP 13 was carried out by slurrying SP 12 with 45 ml of a 35% solution of trifluoroacetic acid (TFA) in dichloromethane, then shaking at room temperature for 45 min. The slurry was then filtered, and the recovered stationary phase (SP 13) was washed three times with 20% methanol in dichloromethane, then with a solution of 10% Hunig’s base in dichloromethane, then taken up in a slurry with 2-propanol, and packed into a 250 mm × 4.6 mm I.D. HPLC column for evaluation. 2.11. Preparation of SP 17 using direct bonding approach To a stirring, inerted (N2 ) mixture of 4.51 mmol of 3aminopropyltriethoxysilane in dichloromethane was added 10 equivalents of acrylamide 97%, stabilized w/25–30 ppm cupric ion. Reaction progress was monitored by LC–MS and FTNMR, showing initial formation of the mono-Michael adduct 20 (Fig. 8) to be complete in about 5 h, with only trace amounts of the bis-Michael adduct being formed, even under more forcing conditions. 1 H NMR (400 MHz, CDCl3) δ: 7.76 (bs, 1H), 5.37 (bs, 1H). 3.83 (q, 6H), 2.88 (t, 2H), 2.62 (t, 2H), 2.35 (t, 2H), 1.58 (m, 2H), 1.23 (t, 9H), (m, 9H), 0.67 (m, 2H). ESI-MS 293 amu (M + H). Bonding of organosilane 20 to silica to afford SP 17 was car´˚ in ried out by refluxing 5 g of silica (Kromasil 16 ␮m, 60 A) toluene using a Dean Stark trap for 3 h, then adding a toluene solution of the organosilane 20 followed by gentle refluxing for 2 h. The mixture was filtered on a sintered glass funnel, then washed three times with 20% methanol in dichloromethane. Following drying, the resulting material was slurried in 2-propanol and packed into a 250 mm × 4.6 mm I.D. HPLC column for evaluation. Residual stationary phase taken from the column packer reservoir was dried overnight under high vacuum, then submitted for combustion analysis (C 3.3%; N 0.79%). 2.12. Preparation of SP 6 using ‘on-column’ bonding approach ˚ was placed in Kromasil amino silica (5 g, 10 ␮m, 100 A) a 100 ml round bottom flask, to which was added 25 ml of dichloromethane. Following complete wetting of the stationary phase, aided by gentle swirling, a solution of methyl chloro-

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formate (10 mmol) and triethylamine (10 mmol, 1.0 equivalent) in 25 ml of dichloromethane was added, and the mixture was rotated overnight on a rotary evaporator apparatus at room temperature without applied vacuum. The following morning, the mixture was filtered on a sintered glass funnel, washed three times with 20% methanol in dichloromethane, taken up in a slurry with 2-propanol, and packed into a 250 mm × 4.6 mm I.D. HPLC column for evaluation. Residual stationary phase taken from the column packer reservoir was dried overnight under high vacuum, then submitted for combustion analysis (C 6.0%; N 1.4%). 2.13. Preparation of SP 13 using ‘on-column’ bonding approach ˚ was placed in Kromasil amino silica (5 g, 10 ␮m, 100 A) a 100 ml round bottom flask, to which was added 25 ml of dichloromethane. Following complete wetting of stationary phase, aided by gentle swirling, a solution of Boc-lPro (4.7 mmol) and 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC, 4.7 mmol, 1.0 equivalent) in 25 ml of dichloromethane was added, and the mixture was rotated overnight on a rotary evaporator apparatus at room temperature and without applied vacuum. The following morning, the mixture was filtered on a sintered glass funnel, washed three times with 20% methanol in dichloromethane, and dried under high vacuum to afford SP 12. Following drying, 45 ml of a 35% solution of trifluoroacetic acid (TFA) in dichloromethane was added, and the resulting slurry was shaken at room temperature for 45 min. The slurry was then filtered, and the recovered stationary phase (SP 13) was washed three times with 20% methanol in dichloromethane, then with a solution of 10% Hunig’s base in dichloromethane, then taken up in a slurry with 2-propanol, and packed into a 250 mm × 4.6 mm I.D. HPLC column for evaluation. Residual stationary phase taken from the column packer reservoir was dried overnight under high vacuum, then submitted for combustion analysis (C 7.5%; N 2.0%). 2.14. Preparation of SP 17 using ‘on-column’ bonding approach on bulk amino silica ˚ was placed in Kromasil amino silica (5 g, 10 ␮m, 100 A) a 100 ml round bottom flask, to which was added 25 ml of dichloromethane. Following complete wetting of stationary phase, aided by gentle swirling, a solution of acrylamide containing 25–30 ppm cupric ion as a free radical inhibitor (14 mmol) in 25 ml of dichloromethane was then added, and the mixture was rotated overnight on a rotary evaporator apparatus at room temperature without applied vacuum. The following morning, the mixture was filtered on a sintered glass funnel, washed three times with 20% methanol in dichloromethane, slurried with 2-propanol, and packed into a 250 mm × 4.6 mm I.D. length HPLC column for evaluation. Residual stationary phase taken from the column packer reservoir was dried overnight under high vacuum, then submitted for combustion analysis (C 6.3%; N 1.8%).

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2.15. Chromatographic evaluation of the bonded phases Mixtures of ethyl acetate, methanol and water were prepared mixed at various compositions, and employed as mobile phase for the runs. For the prolinated silica run, proline was added to the ternary component mobile phase. All solvents were HPLC-grade (from Fisher Scientific). Proline was obtained from Ajinomoto (Japan). Feed solution for the experiments was derived from crude solids containing the compound of interest, pneumocandin B0 , at 60% purity in addition to more than 10 other analogue impurities. A method for the production and isolation of the pneumocandin B0 crude solids from the fermentation broth is described elsewhere [6]. A Waters FractionLynx preparative HPLC system was used for evaluation of each stationary phase. Before use, all columns were flushed with 10 column volumes of methanol and equilibrated with 10 column volumes of the mobile phase. For each injection, 1.2 ml of the feed solution was injected (representing a loading of ∼15 g/l bed). The mobile phase flow rate was ∼7.5 column volumes per hour. For each run, fractions were collected and analyzed to assess the quality of the purification. Each injection was repeated to ensure a consistent elution profile. For the prolinated run, the prolinated mobile phase was pumped through a bare silica column until proline saturation as achieved. The run was carried out by injecting the feed and then eluting it with the prolinated eluent. 2.16. Analytical methods To assess retention and selectivity, fractions were collected from each run, then analyzed by reversed and normal phase HPLC methods. The protocols for the assays are described elsewhere [5]. 3. Results and discussion We have previously utilized a strategy in which preparation and evaluation of a group of rationally designed chromatographic media can be used for the identification of materials with improved performance for a given purification task [8–11]. We have more recently developed approaches in which microscale solid phase synthesis and high throughput screening may be used for the preparation and evaluation of larger libraries of candidate chromatographic adsorbents [12–15]. The purification of pneumocandin B0 is considerably more complex than the two component separations that we have heretofore studied, and proved not readily amenable to high throughput screening. We therefore adopted a more direct strategy in which a smaller number of candidate adsorbents were directly evaluated using chromatographic separation under actual preparative loading conditions. In addition to evaluation of commercial adsorbents in our collection, we prepared a number of additional stationary phases for evaluation, focusing on inexpensive and simple to produce materials containing strong hydrogen-bonding interaction sites. We employed an on-column functionalization approach in which

a variety of inexpensive and readily available reagents were pumped through amino silica HPLC columns using an HPLC pump. Despite a number of known limitation and drawbacks of this experimental approach [16], it does offer the advantage of speed and convenience. Fig. 3 illustrates 17 different stationary phases prepared for evaluation by on-column functionalization. In most cases, simple pumping of a solution of the appropriate reagent through an amino column using an HPLC pump afforded the requisite stationary phase. 3.1. Drawbacks of the on-column functionalization approach Multistep solid phase synthesis, also known as the on-column bonding approach, is often plagued by the problem of incomplete reaction (Fig. 4). The resulting heterogeneously functionalized surfaces may afford inferior chromatographic performance. We have previously shown that that elimination of nonproductive adsorption sites can afford improves chromatographic performance [17]. In the present case, complete acylation of commercial amino silica is known to be problematic, the surface density of amino groups being too high to allow complete reaction. 3.2. On-column synthesis of 17 investigational stationary phases Preparation of the amide phases (SP 1–5) proceeded in a fairly straightforward fashion via acylation of commercial aminopropyl silica columns using the corresponding anhydrides or acid chlorides. In an analogous fashion, carbamate phases SP 6 and SP 7 were prepared from the corresponding chloroformates, and the urea phase SP 8 was prepared from the corresponding isocyanate. Preparation of the amino acid stationary phases followed the general approach described previously [7] and outlined in Fig. 5. We selected a group of stationary phases with high potential for hydrogen-bonding, choosing a diverse family of structures accessible from commercially available starting materials. Proline was included in the study group, owing to its demonstrated importance as a moderator of retention and selectivity in the existing chromatographic purification of pneumocandin B0 . Coupling of protected amino acids to amino silica utilizes chemistry that is well known, and closely analogous to that used in Merrifield solid phase peptide synthesis. In each case, an Nprotected amino acid is coupled with the amino silica, followed in a subsequent step by removal of the protecting group. We opted to evaluate each stationary phase both before and after removal of the protecting group, so as to better understand the influence of structure on separation. Removal of the Boc protecting group was accomplished by pumping a solution of TFA in dichloromethane through the column. We previously have found that the 2,4-DNP stationary phase, SP 16, is often a useful material [8] and included it in this study. Finally, we prepared an additional stationary phase derived from acrylamide, SP 17. In this example, both a mono- and a bis-

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Fig. 3. Functionalization of amino silica HPLC columns affords 17 different stationary phases for evaluation.

Michael adduct are possible, as illustrated in Fig. 6. This situation highlights one of the key drawbacks of the on-column bonding approach, namely the difficulty in determining exactly what is taking place at the surface. We were able to resolve some of these issues relating to stationary phase structure using standard solution phase laboratory approaches to show that the predominant species present in SP 17 is likely the mono-Michael adduct.

Fig. 4. Direct bonding vs. multistep ‘on-column’ bonding approach to functionalized silica surfaces.

3.3. Evaluation of on-column generated stationary phases Evaluations of the 17 investigational stationary phases were carried out using various EtOAc/MeOH/water mobile phase combinations for the separation of crude pneumocandin B0 under preparative loading conditions. The results, summarized in Table 1, showed that three of the stationary phases (SP 6, 13 and 17) significantly surpassed the performance of both the prolinated silica and the Amide 80 stationary phase that had earlier been found to be of interest. The superior performance of the immobilized proline stationary phase, SP 13, was to some degree predicted, although some of the other results were somewhat surprising. For example, the methyl carbamate phase (SP 6) was shown to have outstanding performance, whereas closely related phases such as SP 1 or SP 7 did not perform nearly as well. Similarly the nitroaromatic phases SP 3 and SP 16, which were expected to perform reasonably well, were both, in fact, quite poor. Among the amino acid-derived stationary phases, the stationary phases ‘loaded’ with strong hydrogen-bonding groups performed relatively poorly. These difference of expectations from experimental outcome only serve to emphasize the importance of screening a diverse sampling of potential solutions to any given separation problem. Chromatograms comparing the performance of two of the best stationary phases (methyl carbamate, SP 6 and l-Pro, SP

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Fig. 5. Preparation of amino acid-derived stationary phases.

13) with that of the commercial Tosoh Amide 80 stationary phase are depicted in Fig. 7. Data for acrylamide phase (SP 17) are not shown owing to overly long retention. The retention afforded by the investigational stationary phases greatly exceeds that of the commercial material, and of the prolinated silica used in the previously described purification process. The increased retention afforded by these phases allows an increase in the methanol content of the eluent, enabling greater analyte solubility and affording a better match between eluent and feed compositions. Comparative chromatograms of the three best investigational stationary phases operating at higher methanol eluents are depicted in Fig. 8. Eluent compositions were adjusted so that all chromatograms occur in approximately 14 column volumes, thus the less retentive l-Pro stationary phase (SP 13) is

operated with 15.5% methanol, while the carbamate (SP 6) and acrylamide adduct (SP 17) stationary phases are operated at 17% methanol (versus 8.6% methanol in the original eluent). Analysis of collected fractions revealed that for all three stationary phases, most of the main peak can be included in the rich cut, although some shaving of the tail portion may be necessary to reduce impurity levels. 3.4. Preparation and evaluation of direct bonding analogs of top three stationary phases These results suggest that any of these three stationary phases may be of interest for improved preparative purification of pneumocandin B0 . As noted previously, there are a number

Fig. 6. Preparation of acrylamide derived stationary phase, SP 17.

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Table 1 Qualitative assessment of retention and selectivity of 17 experimental stationary phases for the preparative purification of pneumocandin B0 SP#

Name

Retention

Selectivity

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17

Acetamide Trifluoroacetamide 3,5-DNB Palmitamide Succinamide Me carbamate i-Bu carbamate Et urea N-acetyl-d-Asn Boc-D-Gln D-Gln Boc-l-Pro l-Pro Boc-iso-Gln iso-Gln 2,4-DNP Acrylamide

Good Good Poor Poor Good Excellent Poor Poor Good Poor Good Poor Excellent Poor Good Poor Excellent

Poor Poor Poor Poor Poor Excellent Poor Poor Poor Poor Poor Poor Excellent Poor Poor Poor Excellent

The designation of ‘good’, ‘poor’ and ‘excellent’ denotes retention or selectivity comparable to, inferior to, or superior to that obtained using prolinated silica.

Fig. 7. Investigational stationary phases SP 13, SP 6 and SP 17 offer superior chromatographic separation of pneumocandin B0 than commercial Tosoh Amide 80 stationary phase (data for SP 17 not shown). For each run, 1.2 mL of the feed solution was injected. The eluent flow rate was ∼7.5 cv/h.

Fig. 8. Comparative assessment of retention and selectivity of top three experimental stationary phases for the preparative purification of pneumocandin B0 using high methanol eluents. Separation with SP 13 has been adjusted to lower methanol concentration to afford comparable run time. For each run, 1.2 ml of the feed solution was injected (representing ∼15 g pneumocandin B0 /l bed). The eluent flow rate was ∼7.5 cv/h.

of problems associated with the on-column multistep surface functionalization of silica, including limited ability to nondestructively characterize the materials inside the column, and a tendency to form heterogeneous materials containing residual reactive groups, which often leads to decreased chromatographic performance. In previous studies, we have found that these limitations are acceptable in a screening program, but once the lead stationary phase(s) has been identified, preparation using the direct bonding approach to silica surface functionalization should be carried out. In general, this results in stationary phases with modestly to substantially improved performance. In the present study, we adopted a direct bonding surface functionalization approach to the preparation of the lead stationary phases, SP6, SP 13 and SP 17 which is illustrated in Fig. 9. Solution acylation of commercial aminopropyl triethoxy silane (APTES) with methyl chloroformate gave carbamate, ˚ Kromasil) under 18, which was bound to silica (16 ␮m, 60 A Dean-Stark conditions to afford SP 6, which was slurry packed into an HPLC column for evaluation. Similarly, EDC mediated coupling of APTES with Boc-l-Pro in toluene afforded amide 19, which was bonded to silica using by vacuum heating in a Kugehlrohr apparatus for 2 days (100 ◦ C, 10 mmHg). The resulting Boc-l-Pro stationary phase (SP 12) was deprotected with TFA in dichloromethane to afford l-Pro stationary phase SP 13, which was slurry packed into an HPLC column for evaluation. Developing a direct bonding approach for SP 17 required a bit more experimentation, as we were unsure of the exact structure of the stationary phase produced by on-column modification of amino silica with acrylamide, with mono-Michael adduct, bisMichael adduct, or possibly even a supported polyacrylamide all being possibilities. Investigations into the solution reaction of APTES with acrylamide under a variety of conditions showed that while formation of both the mono- and bis-Michael adducts

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Fig. 9. Direct bonding surface functionalizations approach to the preparation of the lead stationary phases, SP6, SP, 13 and SP 17.

is possible, it is predominantly the mono adduct 20 that is formed under the non-forcing conditions used in the preparation of SP 19. Reaction of APTES with 10 equivalents of acrylamide in dichloromethane at room temperature results in essentially complete conversion to mono-Michael adduct 20 after 1 h, with only a trace amount of the bis adduct visible by LC–MS, and a 1 H NMR spectrum confirming the mono-Michael adduct structure. ˚ KroThe resulting product was bound to silica (16 ␮m, 60 A masil) under Dean-Stark conditions with refluxing toluene to afford SP 17, which was slurry packed into a 250 mm × 4.6 mm I.D. HPLC column for evaluation. As each of the stationary phases produced by direct bonding surface functionalization was prepared first as loose material and then packed into HPLC columns for evaluation, we had the opportunity to carry out combustion analysis to evaluate the efficacy of surface immobilization. The results indicate a level of surface modification between 0.2 and 0.3 mmol/g, which lies within the typical range of what can be expected by this technique. It, therefore, came as a great surprise to us that each of these newly prepared stationary phases showed very little retention upon chromatographic evaluation, whereas the analogous phases prepared by the on-column bonding approach displayed high retention and selectivity. We initially suspected problems with the chemistry or bonding, or issues relating to surface density, but after careful analysis and follow up experiments, have concluded that the heterogeneous stationary phases prepared by on-column bonding may actually be advantageous for chromatographic purification of pneumocandin B0 . In other words, the presence of both unreacted amine groups (which work only modestly alone) and functionalized groups (proline, carbamate or acrylamide adduct) may be required for effective retention and separation of pneumocandin B0 . This result is in sharp contrast to the typical cases known for the separation of small molecule analytes on brush type chiral stationary phases [16], but may per-

haps be rationalized if one considers the very large ‘footprint’ and wide distribution of potential interaction sites on the analyte, and the relatively small size of the stationary phase selector. It would not be unreasonable in such a situation to imagine analyte adsorption to be dominated by interactions in which two or more surface functionalized groups simultaneously interact with the analyte molecule. 3.5. A scaleable bonding approach to SP6, SP13 and SP17 Realizing that bulk preparation of a stationary phase by oncolumn bonding within a column is not an attractive option for preparation of bulk media at larger scale, we next investigated the possibility of carrying out chemical derivatization of loose amino silica in a flask. In this instance, Kromasil amino silica ˚ was placed in a flask, and reactions analogous (10 ␮m, 100 A) to the flow treatments utilized in the preparation of SP 6, SP 13 and SP 17 were performed. The resulting materials were comparable in performance to the original on-column functionalized materials. This result suggests that bulk stationary phase can be prepared simply by scale up of these reactions. Table 2 Raw materials costs relating to preparation of the three top performing stationary phases Stationary phase

Steps

Reagents

Approximate $/kg

SP 6

1

Methyl chloroformate Triethylamine

$60 $60

SP13

2

Boc-l-Pro EDC TFA

$1000 $2200 $400

Acrylamide

$60

SP 17

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While the performance of the three lead stationary phases (SP 6, SP 13 and SP 17) is similar, there are considerable differences in the projected costs for larger scale preparation. A preliminary evaluation of materials pricing shows that in addition to requiring two separate steps (coupling, deprotection) and an expensive coupling agent (EDC, although this could probably be replaced with a less expensive reagent), the L-Boc-Pro starting material used in the preparation of SP 13 is also quite expensive (Table 2). By comparison, raw materials for preparation of SP 6 and SP 17 are quite inexpensive, and fewer steps are required in their preparation. 4. Conclusions The somewhat surprising apparent role of residual amines in benefiting retention and separation of pneumocandin B0 is an interesting phenomenon that may merit further study. One could imagine preparation of libraries of mixed mode phases to further explore this issue, perhaps allowing the identification of optimal ratios of the two selectors. In this study, initial hypotheses concerning retention interactions for pneumocandin B0 were only partially borne out. Nevertheless, simple and potentially inexpensive stationary phases that do afford improved preparative chromatographic separation of pneumocandin B0 were successfully identified. Further study of the utility of these and other stationary phases for carrying out preparative purifications will be the subject of future investigations.

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References [1] E. Bayer, W. Jennings, R.E. Majors, J. Kirkland, K.K. Unger, H. Engelhardt, G. Schomburg, W.H. Pirkle, C.J. Welch, D.W. Armstrong, J.O. Porath, J.B. Sjovall, C.W. Gehrke in, C.W. Gehrke, R.L. Wixom, E. Bayer (Eds.), Chromatography: A Century of Discovery 1900–2000 (The Bridge to Sciences/Technology), Elsevier, New York, 2002. [2] E. Francotte, in: G. Cox (Ed.), Preparative Enantioselective Chromatography, Blackwell, London, 2005. [3] C.J. Welch, J. Chromatogr. 666 (1994) 3. [4] R.E. Schwartz, D.F. Sesin, H. Joshua, K.E. Wilson, A.J. Kempf, K.A. Goklen, D. Kuehner, P. Gailliot, C. Gleason, J. Antibiot. 45 (1992) 1853. [5] D.J. Roush, F.D. Antia, K.E. Goklen, J. Chromatogr. 827 (1998) 373. [6] J. Nti-Gyabaah, F.D. Antia, M.E. Dahlgren, K.E. G¨oklen, Biotechnol. Progr. (in press). [7] C.J. Welch, G.A. Bhat, M.N. Protopopova, Enantiomer 3 (1998) 463. [8] C.J. Welch, W.H. Pirkle, J. Chromatogr. 609 (1992) 89. [9] W.H. Pirkle, C.J. Welch, J.A. Burke, B. Lamm, Anal. Proc. 29 (1992) 225. [10] W.H. Pirkle, C.J. Welch, B. Lamm, J. Org. Chem. 57 (1992) 3854. [11] W.H. Pirkle, C.J. Welch, J. Liq. Chromatogr. 15 (1992) 1947. [12] C.J. Welch, M.N. Protopopova, G.A. Bhat, Enantiomer 3 (1998) 471. [13] C.J. Welch, M.N. Protopopova, G.A. Bhat, J. Comb. Chem. 1 (1999) 364. [14] C.J. Welch, M.N. Protopopova, G.A. Bhat, in: J.P. Blitz, C.B. Little (Eds.), Fundamental and Applied Aspects of Chemically Modified Surfaces, Royal Society of Chemistry, London, 1999, p. 129. [15] C.J. Welch, S.D. Pollard, D.J. Mathre, P.J. Reider, Org. Lett. 3 (2001) 95. [16] C.J. Welch, S.R. Perrin, J. Chromatogr. 690 (1994) 218. [17] W.H. Pirkle, C.J. Welch, J. Chromatogr. 589 (1992) 45.