Anal. Chem. 1994,66, 1473-1484
Macrocyclic Antibiotics as a New Class of Chiral Selectors for Liquid Chromatography Danlel W. Armstrong,' Yublng Tang, Shush1 Chen, rlwen Zhou, Christina Bagwlll, and Jlng-Ran Chen Department of Chemistw, University of MissourkRolla, Rolla, Missouri 6540 1
There are a large number of antibiotic macrocycles which include several different structural types. Three representative macrocyclic compounds were covalently linked to silica gel and evaluated by HPLC for their ability to resolve racemic mixtures as well as for their stability and loadability. Over 70 compounds were resolved. In some cases separations were achieved that have not been reported on any other chiral stationary phase (CSP). These stationary phases appear to be multimodal in that they can be used in both the normalphase and reversed-phase modes. Different compounds are resolved in each mode. There does not appear to be any deleterious effects to the stationary phases or any irreversible changes in the enantioselectivity when changing from one mode to another. The diversity of functionality of some of these chiral selectors is only approached by that of glycoproteins. Consequently, enantioseparation may be possible via several different mechanisms including ?r-r complexation, hydrogen bonding, inclusion in a hydrophobic pocket, dipole stacking, steric interactions, or combinations tl~ereof. While all other CSPs avail themselves of the same type of interactions, they are not all necessarily available in a single chiral selector and in relatively close proximity to one another. Macrocyclic antibiotics seem to have many of the useful enantioselectivity properties of proteins and other polymeric chiral selectors without their inherent problems of instability and low capacities. Although a tremendous number of LC chiral stationary phases (CSPs) are now available, they tend to be derived from relatively few classes of compounds. Most chiral selectors are based on amino acids (native or derivatized),I-s proteins,"1° cyclodextrins(native or derivatized),11-13andderivatized linear or branched carbohydrates (e.g., cellulose or amylose).lk16 A few other CSPs have been reported that do not directly fit (1) Davankov, V. A. In Advances in Chromatography; Giddings, J. C., Grushka, E., C a m , J., Brown, P. R., Eds.; Marcel Dekker: New York, 1980; Vol. 18, p 139. (2) Davankov, D.; Bochkov, A.; Kurganov, A.; Roumeliotis, P.; Unger, K. Chromatographia 1980, 13, 677. (3) Pirkle, W. H.; House, D. W.; Fin, J. M. J . Chromarogr. 1980, 103, 143. (4) Oi, N.; Kitahara, H. J. J . Chromatogr. 1986, 9, 511. (5) Pirkle, W. H.; Burke, J. A., 111. J . Chromatogr. 1991, 557, 173. (6) Allenmark, S.; Bomgren, B.; Boren, H. J. Chromatogr. 1983, 269, 71. (7) Hermansson, J. J. Chromatogr. 1983, 269, 71. (8) Miwa, T.; Kuroda, H.; Sakashita,S.;Asakawa, N.; Miyake, Y. J . Chromatogr. 1990.51I , 89. (9) Jadaud, P.; Wainer, I. W. J . Chromatogr. 1989, 476, 165. (10) Domenici, E.; Bertucci, C.; Salvadori,P.; Felix, G.; Cahagne, I.; Motellier, S.; Wainer, I. W. Chromatographia 1990, 29, 170. (1 1) Armstrong, D. W.; DeMond, W. J . Chromatogr. Sci. 1984,22,411. (12) Amstrong, D. W.; Stalcup, A. M.; Hilton. M. L.; Duncan, J. D.; Faulkner, J. R., Jr.; Chang, S.-C. Anal. Chem. 1990, 62, 1612. (13) Pawlowska, M.; Chen, S.; Armstrong, D. W. J . Chromatogr. 1993,641,257. (14) Linder, K. R.; Mannschrek, A. J. Chromatogr. 1980, 193, 308. (15) Okamoto, Y.; Hatada, K.; Aburazani, R. J . Chromatgr. 1988, 448, 454. (16) Okamoto, Y.; Hatado, K. D., Patent JW1,165,569, June 29, 1989. 0003-2700/94/0368-1473$04.50/0 0 1994 American Chemlcai Socletv
these ~ategories.l~-~l Of these, the most successful have been a chiral crown ether phase for the resolution of primary amine containing comp~undsl~-*~ and second or third generation r-complex/hydrogen-bonding stationary phases that are conceptuallyrelated to earlier derivatized amino acid CSPs.22~23 Regardless of their particular class, chiral selectors that are the most useful in HPLC have some common characteristics. Generally they are able to affect a reasonable number of enantiomeric separations that are of interest to the scientific community. They must be sufficiently stable to withstand immobilization and packing procedures as well as normal HPLC operating conditions (e.g., appropriate solvents, temperatures, pressures, pHs, ionic strengths, etc.). Also they must be available in adequate quantities and at reasonable cost. Every class of chiral selectors has strengths and weaknesses in regard to selectivity, stability, loadability, and so forth. Hence some CSPs are complementary to one another while others tend to duplicate existing separations and/or stationary phases. In this work we describe another class of chiral selectors for HPLC. The main relationship between them is that they are all macrocyclic antibiotics. There are literally hundreds of these compounds, many of which meet the general criteria for useful chiral selectors. Unlike other classes of chiral selectors, macrocyclic antibiotics comprise a large variety of structural types including macrocyclic polyenepolyols, ansa compounds (e.g., aliphatic bridged aromatic ring systems), macrocyclic glycopeptides, peptides, peptide-heterocycle conjugates, and so forth. In general, these compounds have molecular weights greater than 600 but less than 2200. There are acidic, basic, and neutral types. Some macrocyclic antibiotics absorb strongly in the UV and visible spectral regions while others are fairly transparent. In this report we describe the use of three representative but structurally different macrocycles as covalently bonded chiral selectors in HPLC. In addition to showing a high degree of enantioselectivity toward a variety of different compounds, these CSPs can be used in both reversed-phase and normaiphase condition^.^^^^^ Also, we have shown that macrocyclic antibiotics are extremely useful as chiral additives in the (17) Okamoto, Y.; Honda, S.; Yuki, H.; Nakamura, H.; Iitaka, Y.; N o m , T. Chem. Lett. 1W, 1149. (18) Dobashi, Y.; Hara, S. J. Am. Chem. Soc. 1985, 197, 3406.
(19) Shinbo, T.; Yamaguchi, T.;Nishimura, K.; Suguira, M. J . Chromatogr. 1987, 405, 145. (20) Hilton, M.; Armstrong, D. W. J . Uq. Chromatogr. 1991, 14, 9. (21) Hilton, M.; Armstrong, D. W. J . Lip. Chromatogr. 1991, 14, 3673. (22) Pirkle, W. H.; Welch, C. J.; Lamm, B. J . Org. Chem. 1992, 57, 3854. (23) Gasprrini, F.; Misiti, D.; Willani, C. Chirality 1992, I , 447. (24) Armstrong, D. W.; Hilton, M.; Coffin, L. LC-GC 1992, 9, 646. (25) Armstrong, D. W.; Chen, S.;Chang, C.; Chang, S. J . Uq. Chromatogr. 1992, 15, 545.
Analytical Chemistry, Vol. 66, No. 9, May 1, 1994
1475
Bo COOH
I
H I
O-CH3 OH
0 ‘C-CH,
8
RIFAMYCIN B
VANCOMYCIN OH
THI OSTREPTON Flgurr 1. Structures of the macrocycles used as bonded stationary-phase chlral selectors In thls study. Vancomycln consists of three fused macrocyclic rings, wiostrepton has a large macrocyclic ring, while rlfamycln B is a smaller macrocycllc compound.
solutions used for CE, TLC, and LC as well as effective stationary phases for GC, SFC, and CE. These results are to be reported in a series of subsequent papers. To our knowledge, the use of macrocyclic antibiotics as silica gel bonded CSPs for the resolution of enantiomeric molecules has not been reported. However, vancomycin coupled to polyacrylamide resin was previously found to be useful (as compared to titration assays) in determining binding to some derivatized peptides.26 EXPERIMENTAL SECTION Materials. All racemic analytes resolved in this study were obtained from Aldrich (Milwaukee, WI) or Sigma (St. Louis, MO). All HPLC grade solventsand N,N-dimethylformamide (DMF)were obtained from Fisher (Pittsburgh, PA). All organosilanecompoundswere obtained from Petrarch (Bristol, (26) Smith, P. W.;Chang, G.;Still, C. J. Org. Chem. 1988, 53, 1590.
1474 AnaljltcaiChemlstry. Voi. $6, No. 9,M y 1, 1994
PA). Cyclic antibiotics containing amine, hydroxyl, or carboxylic acid functionalities can be linked to silica gel in a variety of different ways. The three cyclic antibiotics discussed in this work (vancomycin, thiostrepton, and rifamycin B from Sigma) all contain one or more of these functional groups. Structures for these compounds are shown in Figure 1. Carboxylic acid terminated organosilanes (e.g., [ 1-(carbomethoxy)ethyl] methyldichlorosilane, [2-(carbomethoxy)ethyl]trichlorosilane, etc.) can be used to immobilize vancomycin and thiostrepton, while amine-terminated organosilanes (e.g., (3-aminopropyl)dimethylethoxysilane,(3-aminopropy1)triethoxysilane, etc.) can be used for rifamycin B. In a typical reaction, 4 g of dry silica gel is slurried on 50 mL of dry toluene in a 250-mL three-neck flask. Two grams of the desired organosilane is dissolved in 15 mL of dry toluene contained in a dropping flask. The organosilane solution is added dropwise over -30 min to the refluxing toluenesilica
-
gel slurry. The mixture is allowed to reflux (- 110 "C) for 2 h and is then cooled, filtered, and washed with methanol, 50% aqueous methanol, and methanol again and then dried. The silanized silica gel can be slurried in anhydrous DMF. One gram of the desired cyclic antibiotic is added along with an appropriate carbodiimide dehydrating agent. After 6 h the CSP material is filtered and washed with methanol and then aqueous methanol. The cyclic antibiotics also can be attached to silica gel via epoxy-terminated organosilanes as has been described previously for cyclodextrins. Another approach involves reacting the macrocycle with a 2-3 molar excess of an isocyanateterminated organosilane (e.g., (3-isocyanatopropy1)triethoxysilane) in anhydrous DMF. This product is then added to a dry DMF slurry of silica gel (-2 g of modified cyclic antibiotic to 4 g of silica gel). The solution is stirred and allowed to react for 20 h at 107 "C. Subsequently the CSP was filtered and washed as indicated previously. Although other attachment chemistries are possible, these are the ones used in the initial studies. Methods. The CSPs (5-pm particles) were slurry packed into 25 cm X 0.44cm (id.) stainless steelcolumns. Separations were achievedusing a Shimadzu LC 6A liquid chromatograph with UV detection (254 nm) and a C-R3A chromatopac data station or with a Waters Model 590 HPLC with a 745B data module. Separations were carried out at a flow rate of 1.0 mL/min and at room temperatures (-22 "C) unless noted otherwise. Mobile-phase compositions are listed in the appropriate tables and figures.
RESULTS AND DISCUSSION Vancomycin (vancosin) is an amphoteric glycopeptide produced by Streptomyces orientalis (Figure l).27 It has a molecular weight of 1449. There are three macrocyclic portions to the molecule, which also contains five aromatic rings. Also, there are two side chains, one of which is a carbohydrate dimer and the other an N-methylamino acid. Upon heating in neutral or basic conditions, aspartic acid is thought to be lost, thereby opening at least one of the macrocyclic rings. This may occur during the silica gel immobilization reaction step (see Experimental Section). Native vancomycin contains 18 stereogeniccenters, 9 hydroxyl groups, 2 amine groups (one primary and one secondary), 7 amido groups, and 2 chlorine moieties (substituents on two different aromatic rings). The type of groups mentioned here are known to beuseful for stereoselectivemolecularinteractions with chiral analytes. Vancomycin is soluble in water, somewhatsolublein methanol, and insoluble in higher alcohols and other less polar organic solvents. Rifamycin B is one of the more common members of a family of Rifamycin antibiotics (Figure 1 ) . They are macrocyclic compounds produced by Nocardia mediterranei.2a*29Generally, they are characterized by a naphthohydroquinone chromophore with a long aliphatic "bridge". Rifamycin B has a molecular weight of 755.8 and is slightly soluble in water and somewhat more so in ethanol and (27) Higgins, H. M.; Harrison, W. H.; Wild, G. M.; Bungay, H. R.; McCormick, M. H. Anribior. Ann. 1 9 S 5 9 . 906. (28) Semi. P.; et al. Anribior. Ann. 195960, 262. (29) Rinehart. K. L., Jr.; Shield, L. S. Forrschr. Chem. Org. Nurursr. 1976,33,23 1.
13
0
I/
N H C C H ~ N H C C HBr ~
G
Ill1
B
26
0
15
0 T I M E ,
30
M I N
Figure 2. (A) Reversed-phase enantiomeric separation of (from left to right) bromocii, devrinol, and coumachlor. The column was a 25 cm X 0.44 cm (1.d.) vancomycin CSP (5-pm silica gel). The mobile phase consisted of 10:90 acetonitrile-1 % pH 7 trlethylammonium acetate buffer (by volume). The flow rate was 1.O mL/min at amblent temperature (22 "C). UV absorbance detection (254 nm) was used. (6)NormaCphaseenantiomeric separation of (from left to right) N43,5dinitfobenroyi)-a-methyibenzylamine and 3-[2-(2-bromoacetamMo)acetamidol-WIOXYLon the same column as In (A). The mobile phase consisted of 5050 2-propanol-hexane (by volume). The flow rate was 1.0 mL/min at ambient temperature (22 "C). UV absorbance
detection (254 nm) was used.
methanol. Although it is a yellow solid, it forms yellow to orange solutions (depending on the conditions). Rifamycin B has nine stereogenic centers, four hydroxyl groups, one carboxylic acid moiety, and one amide bond. This particular macrocycle may be particularly useful as a chiral solution additive for the separation of chiral amino alcohols.30 Thiostrepton is produced by Streptomyces azureu~.3~,~2 It is a macrocyclic polypeptide antibiotic of 1665 molecular weight which contains five thiazole rings and one quinoline ring. Although it is a white solid, it absorbs significantly in the UV region. Thiostrepton is poorly soluble in water, alcohols, and nonpolar organic solvents. It contains 17 stereogenic centers, 5 hydroxyl groups, 10 amide linkages, and 1 secondary amine (Figure 1). Figure 2A shows the resolution of bromacil, devrinol and coumachlor enantiomers in the reversed-phase mode on the vancomycin bonded phase column. Figure 2B shows the resolution of 3- [2-(2-bromoacetamido)acetamido]-PROXYL (an ESR probe) and N-(3,5-dinitrobenzoy1)-ar-methylben(30) Armstrong, D. W.; Rundlctt, K.; Reid, G. L., 111 AMI. Chem., in press. ( 3 1 ) Pagano, J. F.; Weinstein, M. J.; Stout, H. A.; Donovick, R. Anribior. Ann. 1955-56, 554.
( 32) Vandcputte, J.; Dutcher, J. D. Anribior. Ann. 1955-56, 560.
Analyticel Chemistry, Vol. 66,No. 9, M y 1, 1994
1475
Table 1. Chromatographic Data for the Reversed-Phase Resolution of Racemic Compounds on Macrocycllc Antlblotlc Bonded Slatlonary Phawr
compoundsate
yy qy
kl’*
a
mobile phasec
pH
columnd
3.27
2.0
1090
4.1
Van
1.98 2.27
1.70 1.44
1090 1090
7.0 4.1
van van
&--
1.40
1.62
1090
4.1
Van
(4) 5-methyl-5-phenylhydantoin
0.38 0.24
1.41 1.36
1090 10:90
7.0 4.1
Van Van
(5) proglumide
1.17 1.18
1.40 1.75
1090 1090
7.0 4.1
Van Van
(6) a-(l-aminoethyl)-ahydroxybenzyl alcohol (7) bendroflumethiazide
0.39
1.30
1090
7.0
Van
1.58
1.25
10:90
7.0
Van
(8) bromacil
0.67
1.21
1090
7.0
Van
(9) idazoxan
0.38
1.21
1090
7.0
Rif
0.32
1.21
1090
7.0
Van
0.84
1.20
1090
7.0
Van
(12) N-carbamyl-D,L-phenylalanine
0.31
1.20
1090
4.1
Van
(13) aminoglutethimide
0.79
1.15
1090
7.0
Van
(14) N-benzoylalanine methyl ester
0.47
1.15
1090
4.1
Van
(15) coumafuryl
0.68
1.15
1090
7.0
van
(1) coumachlor
0
U
(2) warfarin
0
(3) devrinol
8
H
(10) 3-methyl-5-cyano-6-
methoxy-3,4-dihydro-2-pyridone
(11) pyridoglutethimide
CP
0
H
1476 Ana~IcaIChemIstty,Vol. 66, No. 9, M y 1, 1994
Tablr 1 (Conllnued)
compounds0.e kl'b
(16) dansyl-a-amino-n-butyric acid
~ - ~ - c o I H
(Y
mobile phasee p H columnd
3.29 1.15 1090 4.1
Van
3.00 1.15 1090 4.1
Van
1.55 1.15 1090 4.1
Van
H(%
(17) dansylasparticacid
HO&--cH-CY-W
I
HI%
(18) N-(3,5-dinitrobenzoyl)phenylglycine
22.0
(19) thioridazine
1.15 1090 7.0
Thio
6.17 1.14 1090 4.1
Van
0.78 1.14 1090 7.0
Van
2.09 1.12 1090 4.1
Van
1.35 1.12 1090 7.0
Van
0.80 1.11 1090 7.0 1.90 1.10 1090 4.1
Van Van
(26) N-(3,5-dinitrobenzoyl)leucine
1.44 1.10 1090 4.1
Van
(27) methsuximide
0.39 1.10 1090 7.0
Van
(28) dansylvaline
3.97 1.09 1090 4.1
Van
(29) indoprofen
1.55 1.09 1090 4.1 2.85 1.06 1090 7.0
Van Van
(30) N-benzoylphenylalanine
2.10 1.08 1090 4.1
Van
0.68 1.08 1090 4.1
Van
3.75 1.07 1090 4.1 1.63 1.05 1090 7.0
Van Van
(20) dansylnorleucine (21) 5-(4-hydroxyphenyl)-5-phenylhydandoin
(22) dansyleerine
OH I
HO-CH2-CH-C0,H
I
NHRJ
(23) indapamide
(24) benzoin methyl ester (25) N-benzoylleucine
CeH&H(OCHs)COCaHs
Gwm-%-y-m& NHRz
(31) N-benzoylvaline
(cH3ta(--cH--co&
I
g&::6.5. A final, but not insignificant, aspect of cyclic antibiotics is that their primary structures are usually In some cases the X-ray crystal structures are available as well.38 This greatly facilitates molecular modeling and the study of molecular interactions that affect chiral recognition. Molecular modeling and interaction studies currently are underway for vancomycin and rifamycin B.
CONCLUSIONS Macrocyclic antibiotics are viable chiral selectors for HPLC. They can be bonded to silica gel via linkage chains using a variety of chemistries. They can be used in either the reversed-phase mode or the normal-phase mode and have different enantioselectivities in each. Also, these macrocycles can be derivatized in order to change their enantioselectivity. Macrocyclic antibiotic bonded stationary phases have many of the characteristics of protein-based stationary phases, but with greater stability and much higher capacities. As a result of their relatively small size and the fact that their structures are known, basic studies on chiral recognition should be feasible. Received for revlew December 14, 1993. Accepted February 4, 1994." ~~~~
~
*Abstract published in Advance ACS Abstracts. March 15, 1994.
