Anal. Chem. 1998, 70, 4967-4973
Combinatorial Synthesis of Highly Selective Cyclohexapeptides for Separation of Amino Acid Enantiomers by Capillary Electrophoresis Marcella Chiari,* Viviana Desperati, Ernesto Manera, and Renato Longhi
Institute of Biocatalysis and Molecular Recognition, CNR, Milano, Italy
Cyclic peptide libraries dissolved in the electrolyte solution can be used as chiral selectors in capillary electrophoresis. In the present investigation, the resolution obtained in capillary electrophoresis for a set of dinitrophenyl-D,L-amino acids was the parameter used to screen for the most effective selectors contained in a mixture of thousands of components of a cyclic hexapeptide sublibrary with three randomized positions. The deconvolution procedure was simplified by fractionating the sublibrary components according to the hydrophobicity of the amino acids in the randomized positions through reversedphase HPLC. By comparing the resolution obtained with the separated fractions, a set of hydrophobic amino acids was recognized as essential to achieve adequate enantioselectivity. The whole deconvolution process, which made it possible to select two highly selective cyclopeptides, required the synthesis and the evaluation of 15 sublibraries instead of the 54 syntheses required by a classical procedure of serial deconvolution. Determination of the optical purity and separation of enantiomers has long been a challenge to analytical chemists. Chiral capillary electrophoresis has achieved remarkably rapid development since its introduction in 1985, and further developments can be expected in future years due to the numerous advantages provided by CE over other techniques presently available to separate enantiomers. CE is a conceptually simple technique that offers highly efficient chiral separations with the requirement of only small amounts of chiral selectors. In addition, CE is recognized as an effective tool for the study of selector-selectand interactions and for the determination of thermodynamic parameters such as enantiomer-selector binding constants.1 Direct separation of optical isomers requires a chiral enviroment. The differences in electrophoretic mobility of enantiomers in a chiral background electrolyte (BGE) arise from the different stability of transient diastereomeric complexes which the analytes form with the chiral selector contained in the BGE. A wide variety of complexion mechanisms and chiral selectors have been exploited for chiral separations, but host-guest interactions have proved to be the most effective to date. Although some chiral * Corresponding author: (tel) ++39 2 2800034; (fax) ++39 2 2800036; (e-mail)
[email protected]. (1) Chankvetadze, B. Capillary Electrophoresis in Chiral Analysis; Wiley and Sons: Chichester, England, 1997; Chapter 2. 10.1021/ac9806557 CCC: $15.00 Published on Web 10/24/1998
© 1998 American Chemical Society
separations have been obtained in CE with chiral selectors incorporated into a gel matrix or covalently linked to the wall of an open column, the vast majority of authors have made use of chiral compounds added to the BGE. Beside cyclodextrins, which are the most popular chiral selectors, crown ethers, proteins, and linear saccharides have been successfully adopted in chiral CE separations.2 More recently macrocyclic antibiotics were introduced as CE chiral selectors by Armstrong and co-workers.3 In particular, glycopeptide antibiotics with their unique structural features and functionalities are among the more useful chiral selectors presently available for use in capillary electrophoresis (ref 4 and references therein). A recent article reported on enantiomeric resolutions achieved through a cyclopetide library produced by combinatorial chemistry.5 The idea of using a muticomponent chiral selector is not new, and there are some examples in inclusion gas chromatography and complexation gas chromatography; however, it had never been applied before to capillary electrophoresis. We too have been intrigued by the possibility offered by combinatorial chemistry in the synthesis of peptides and, more specifically, cyclopeptides to be used as chiral selectors. The technique involves the preparation of large numbers of peptides synthesized as mixtures in the same reaction vessel.6-7 For instance, by using all the natural amino acids with the exception of cysteine and trypthophan, in the synthesis of cyclohexapetides, 186 chemical species are simultaneously produced. Each of the peptides is represented only to a very limited extent, but the probability of finding in the mixture a series of cyclopeptides whose conformation is suitable for chiral recognition toward a specific class of analytes is extremely high. In the present work, using a deconvolution strategy, preceded by a proline scan step and accompanied by HPLC fractionation, we have selected the most active chiral discriminats among 5832 individual cyclohexapeptides present in a sublibrary with three defined positions. Although the application of cyclic peptides for (2) Vespalec, R.; Bocek, P. Electrophoresis 1997, 18, 843-852. (3) Armstrong, D. W.; Rundlett, K.; Reid, G. L., III Anal. Chem. 1994, 66, 16901695. (4) Armstrong, D. W.; Nair, U. B. Electrophoresis 1997, 18, 2331-2342. (5) Jung, G.; Hofstetter, H.; Feiertag, S.; Stoll, D.; Hofstetter, O.; Wiesmuller, K.-H.; Schurig V. Angew. Chem. 1996, 108, 2261-2263. (6) Combinatorial Peptide and Nonpeptide Libraries; Jung, G., Ed.; VCH Weinheim, 1996. (7) Eichler, J.; Houghten, R. A. Biochemistry 1993, 32, 11035-11041.
Analytical Chemistry, Vol. 70, No. 23, December 1, 1998 4967
chiral resolution is not novel per se, the novelty in this approach lies in the synthesis of “artificial” cyclic peptide libraries and the step-by-step selection of the best enantiselective cyclic peptides tailor-made for a given class of analytes. EXPERIMENTAL SECTION Reagents. Water and all chemicals were analytical grade. 2-(1H-Benzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate (HBTU), 1-hydroxybenxotriazole (HOBt), o-7-azabenzotriazol-1-yl-1,1,3,3-tetramethyluronium hexafluorophosphate (HATU), N-(Fmoc-amino acid) derivatives, and reagents and resin for peptide synthesis were from Novabiochem (Laufelfingen, CH). Apparatus. Matrix-assisted laser desorption mass spectra (MALDI-TOF MS) were acquired on a Voyager-RP Biospectrometry workstation (PerSeptive Biosystem, Framingham, MA). NMR spectra were acquired on a Bruker DMX-500 spectrometer equipped with a Silicon Graphics Indy workstation and processed with a Bruker XWINMR software program. All chemical shifts were referred to methyl resonance of trimethylsilyl-3propionic acid. Two-dimensional NMR spectra were acquired in the phase-sensitive mode with quadrature detection in both dimensions by using the time proportional phase increment method8 and with pulsed-field gradients for water elimination (WATERGATE).9,10 Typically, 2048 t2 points were acquired using 16 scans and 512 increments over 6000 Hz of spectral width in F1. Squared µ/3 shifted sine bell was applied in both dimensions and zero filling to 1K was performed in the F1 dimension before the Fourier transformation. Gradient-enhanced TOCSY9,10 and NOESY9,10 spectra were acquired with 0.08 and 1 s, respectively. Amino acid analysis were carried out according to refs 11 and 12 using a homemade system equipped with an ion-exchange MCI gel CK10U column and a fluorescence detector FP-110 (Jasco); the data were acquired on a Wyer 740 data module. CE separations were done in a HP 3D capillary electrophoresis system model G 1600 A (Hewlett-Packard, Wilmington, DE). Data were collected on a PC computer using HP 3D ChemStation. In all the experiments, 50-µm fused-silica capillaries (Polymicro Technologies, Phoenix, AZ) were used, some of them coated as described in ref 13. The samples were detected at 340 nm. Before each run, the capillary, 65 cm long (57 cm to the window), was filled with a solution of cyclohexapeptides in sodium phosphate buffer by applying a pressure of 15 psi for 5 min. Synthesis of Cyclohexapeptide Sublibraries. Linear haxapeptide sublibraries were manually synthesized by the solidphase14 Fmoc/tBu method.15 The peptide chain was assembled stepwise on a chlorotrityl chloride-polystyrene-1% divinylbenzene resin16 derivatized with NRFmoc-β-alanine (0.8 mequiv/g). (8) Marion, D.; Wuthroch, K. Biochem. Biophys. Res. Commun. 1983, 113, 967971. (9) Piotto, M.; Saudek, V.; Sklenar, V. J. Biomol. NMR 1992, 2, 661-666. (10) Sklenar, V.; Piotto, M.; Lappik, R.; Saudek, V. J. Magn. Reson. Ser. A 1993, 102, 241-245. (11) Spackman, D. H.; Moore S.; Stein W. H. Anal. Chem. 1958, 30, 11901206. (12) Fujiwara, M.; Ishida, Y.; Nimura N.; Toyama, A.; Kinoshiota, T. Anal. Biochem. 1987, 166, 72-78. (13) Chiari, M.; Nesi, M.; Sandoval, J. J. E.; Pesek, J. J. J. Chromatogr., A 1995, 717, 1-13. (14) Bruce, R. B. J. Am. Chem. Soc. 1963, 85, 2149-2154. (15) Atherton, E.; Sheppard, R. C. In The Peptides; Underfriens, S., Meienhofer, J., Eds.; Academic Press: New York, 1987; Vol. 9, pp 1-39.
4968 Analytical Chemistry, Vol. 70, No. 23, December 1, 1998
Removal of NRFmoc protecting groups was achieved by a 15-min treatment with piperidine-N,N′-dimethylformamide (DMF) (1: 1). Coupling reactions of defined positions (40 min) were performed with the HBTU-HOBt activation procedure17 according to the FastMoc protocol18 and with a 6-fold molar excess of NRFmoc-protected amino acids. Equimolarly premixed NRFmoc-amino acids (0.4 mmol of each R N -Fmoc-amino acid, in a 1:1 solution of DMA-DCM) were activated with HBTU-HOBt17 and added to the resin for coupling of mixed positions.6 Double couplings (2 h each) of random positions were performed using a 1:1 molar ratio of amino groups on the resin and NRFmoc amino acids. After peptide library assembly was completed, the side-chain-protected peptides were deblocked from the resin (250 mg) with 40 mL of hexafluoro-2propanol in CH2Cl2 (1:4).19 Peptides (1 mM) were cyclized in DMF using a 3-fold excess of HATU-HOAt in the presence of a 6-fold excess of diisopropylethylamine (DIEA).20 After 12 h, the solvent was removed in a vacuum concentrator and the residual reaction products were dissolved in dichloromethane (DCM). The organic phase was washed three times with KHSO4 (5% in water) and water up to neutrality. DCM was then removed in vacuo using the rotating vacuum concentrator. Side-chain-protected cyclopeptides (100 µmol) were cleaved according to ref 21 using an optimized mixture of reagents called “reagent K”. Cyclopeptide sublibraries were precipitated by adding 100 mL of ice-cooled tertbutyl methyl ether (TBE). The crude precipitates were collected by centrifugation, washed three times with TBE, dissolved in water, and freeze-dried. The crude material (100 µmol, 50% yield) was quantified by amino acid analysis and characterized by MALDI-TOF spectrometry. Mass spectra showed an identical characteristic peak pattern for linear and cyclic peptide mixtures with the peaks corresponding to the cyclic peptides shifted by 18 amu to lower m/z values. Cycolpeptide sublibraries cyclo(Arg-Lys-X-Pro-X-β-Ala) and cyclo(Arg-Lys-Tyr-Pro-X-β-Ala) were fractionated by semipreparative reverse-phase liquid chromatography on a Whatman Partisil 10 ODS-2 (1 × 50 cm). The material corresponding to the major chromatographic peak was eluted in the interval from 3 to 18% acetonitrile by applying, within 40 min, a linear gradient of acetonitrile in water (0-25%) at a flow rate of 7 mL/min. Fractions, collected every 2 min, were pooled to give three peptide mixtures differing in the hydrophilicity of the amino acids in the randomized positions. The cyclo(Arg-Lys-Tyr-Pro-Tyr-β-Ala) peptide was synthesized (50% yield) and purified by reversed-phase HPLC under the conditions used to fractionate the sublibrary and characterized by MALDI-TOF spectrometry: m/z 779 (M + H)+ (calculated 778, average isotopic composition). The amino acid sequence was confirmed by means of proton 2D NMR experiments (TOCSY and NOESY) and the complete peak assignment is given in Table 1. (16) Barlo, K.; Chatzi, O.; Gatos, D.; Stavropoulos, G. Int. J. Pept. Protein Res. 1991, 37, 513-520. (17) Knorr, K.; Trzeciak, A.; Bannwarth W.; Gillessen, D. Tetrahedron Lett. 1989, 30, 1927-1930. (18) Applied Biosystem User Bulletin No. 30, 1990. (19) Grell, E.; Barlos, K. J. Chem. Soc., Chem. Commun. 1994, 2559-260. (20) Ehrlich, A.; Rothemund, S.; Brudel, M.; Beyermann, M.; Carpino, L. A.; Bienert, M. Tetrahedron Lett. 1993, 34, 4781-4784. (21) King, D. S.; Fields, C. G.; Fields, G. B. Int. J. Peptide Protein Res. 1990, 36, 255-266.
Table 1. Proton Resonance Assignment of Cyclo(Arg1-Lys2-Tyr3-Pro4-Tyr5-β-Ala6) at 285 K and pH 7.0 3J
Arg1 Lys2 Tyr3 Pro4 Tyr5 β-Ala6
NHR
8.98 7.33
NH
R
β-β′
8.60 7.75 8.88
4.02 4.58 4.8 3.77 4.34 3.62/3.37
1.84 1.91 3.16/2.81 1.75/1.41 3.30/2.99 2.64/2.49
8.72 7.87
γ-γ′
δ-δ′
1.70 1.45
3.24 1.75
0.56/0.50
3.01/2.51
-′
2.6/3.5
3.07 7.11/6.84 7.06/6.83
Table 2. List of Sublibraries and Cyclopeptides Used in the Deconvolution Processa
Figure 1. Structure of the DNP-D,L-amino acids contained in the analyte test mixture.
All the sublibraries and the cyclopeptides used in the present investigation showed good solubility in water; their solutions, up to 40 mM, did not show any significant turbidity. Synthesis of DNP-amino Acids. To a suspension of 0.5 mmol of amino acid in 10 mL of NaHCO3, 11 mL of a solution obtained by dissolving 0.25 mL of 2,4 dinitrofuorobenzene (DNFB) in 49 mL of acetone was added. The reaction mixture was stirred for 2 h at 30-40 °C and the progress of the reaction evaluated by RP-HPLC. After the reaction was completed, 0.5 mL of 2 N HCl was added and the solvent evaporated under vacuum. The reaction product was purified by semipreparative RP-HPLC and quantified by UV spectroscopy ( )18 180 L mol-1 cm-1, λ ) 362.4 nm).22 RESULTS It was recently reported that three, randomly chosen, synthetic cyclohexapeptide sublibraries were used as chiral selectors in capillary electrophoresis.5 In practice, the addition of 10 mM sublibraries with the general formula cyclo(O-O-X-X-X-O) to the mobile phase induced a complete enantiomeric resolution of a series of amino acid derivatives. In particular, the sublibrary cyclo(Arg-Lys-X-X-X-D-Ala) provided a baseline separation of N-dinitrophenyl (DNP)-D,L-glutamic acid enantiomers. The present work proposes a simplified procedure of serial deconvolution to select, among the 5832 (183 combinations) constituents of a sublibrary with three randomized positions, the individual components able to resolve in CE a set of DNP-amino acid enantiomers (Figure 1). The entire procedure required two steps: (i) the synthesis of sublibraries with a progressively increased number of defined positions; (ii) the evaluation of their ability to act as chiral selectors in CE. After identification and isolation of the most effective selectors, a systematic investigation on the CE separation parameters such as pH, ionic strength, temperature, and selector concentration was carried out. Table 2 lists all the sublibraries and the cyclopetides synthesized and evaluated in the present work; the amino acid residues are numbered from the amino terminus of the corresponding linear peptide. (22) Berman, J.; Green, M.; Sugg, E.; Andregg, R.; Millington, D. S.; Norwood, D. L.; McGreehan, J.; Wiseman, J. J. Biol. Chem. 1992, 267, 1434-1437.
a
S.1.1:
cyclo(Arg-Lys-X-X-X-β-Ala)
S.2.1: S.2.2: S.2.3:
cyclo(Arg-Lys-Pro-X-X-β-Ala) cyclo(Arg-Lys-X-Pro-X-β-Ala) cyclo(Arg-Lys-X-X-Pro-β-Ala)
S.3.1: S.3.2: S.3.3: S.3.4: S.3.5: S.3.6:
cyclo(Arg-Lys-Val-Pro-X-β-Ala) cyclo(Arg-Lys-Met-Pro-X-β-Ala) cyclo(Arg-Lys-Ile-Pro-X-β-Ala) cyclo(Arg-Lys-Leu-Pro-X-β-Ala) cyclo(Arg-Lys-Tyr-Pro-X-β-Ala) cyclo(Arg-Lys-Phe-Pro-X-β-Ala)
S.4.1: S.4.2: S.4.3: S.4.4: S.4.5: S.4.6:
cyclo(Arg-Lys-Tyr-Pro-Val-β-Ala) cyclo(Arg-Lys-Tyr-Pro-Met-β-Ala) cyclo(Arg-Lys-Tyr-Pro-Ile-β-Ala) cyclo(Arg-Lys-Tyr-Pro-Leu-β-Ala) cyclo(Arg-Lys-Tyr-Pro-Tyr-β-Ala) cyclo(Arg-Lys-Tyr-Pro-Phe-β-Ala)
X stands for all the natural amino acids except Trp and Cys.
Three of the six positions of the initial sublibrary, 1, 2, and 6, were defined and contained the amino acids Arg, Lys, and β-Ala. The use of β-Ala in position 6 was preferred over that of D-Ala as it improved resolution of DNP-glutamic acid enatiomers. The combinatorial synthesis of cyclic hexapeptides was carried out on a solid phase using, for the randomized positions, a premix method with equimolar amino acid mixtures. The linear peptides with protected lateral chains were cyclized in solution. The presence of a turn structure-inducing amino acid, such as proline, facilitates cyclization.23 For this reason, we investigated the role of proline in the three randomized positions on the separation of the test analytes. The sublibrary with proline in position 5 did not separate DNP-glutamic acid enantiomers whereas those with proline in 3 and especially 4 provided resolution; therefore, proline was assigned in position 4. Table 3 shows the values of enantiomeric resolution of the DNP-glutamic acid calculated as follows:
Rs ) 2(t2 - t1)/(w1 + w2)
(1)
where t1 and t2 are migration times and w1 and w2 are baseline peak widths for the two enantiomers. It has to be noted that the sublibrary with two randomized positions contains 182 different hexapeptides and that it is very likely that only some of the species contribute to the chiral recognition of DNP-amino acid derivatives. As a result of this, the cyclopetide sublibrary had to be used at a high concentration, 30 mM, to achieve acceptable resolution. (23) Rose, G. D.; Gierasch, L. M.; Smith, J. A. Adv. Protein Chem. 1985, 37, 1-109.
Analytical Chemistry, Vol. 70, No. 23, December 1, 1998
4969
Table 3. Values of Enantiomeric Resolution of DNP-glutamic Acid in Running Electrolytes Containing Different Sublibraries sublibrary cyclo(Arg-Lys-Pro-X-X-β-Ala) cyclo(Arg-Lys-X-Pro-X-β-Ala) cyclo(Arg-Lys-X-X-Pro-β-Ala)
Table 4. Amino Acid Composition (µmol) of the Semipreparative HPLC Fractions of the Sublibrary Cyclo(Arg-Lys-X-Pro-X-β-Ala)
resolutiona
fraction
1.05 2.06 0
a Resolution values were measured under the following experimental conditions: fused-silica capillary, 70 cm long, 50 µm i.d.; applied voltage, -20 kV; running buffer, 20 mM sodium phosphate buffer pH 7.0 containing 30 mM sublibraries.
a
AA
1
2
3
Asp/Asn Thr Ser Glu/Gln Pro Gly Ala Val Met Ile Leu Tyr Phe His Lys Arg β-Alaa
19.3 7.1 10.8 19.8 n.d. 12.3 10.8 0.3 0.4 0.0 0.0 0.0 0.0 5.0 60.6 63.6 54.7
8.5 3.9 5.0 9.6 n.d. 5.0 6.4 7.3 5.3 6.4 9.8 9.6 5.3 6.3 52.2 56.4 51.7
3.1 2.1 2.5 3.7 n.d. 2.6 2.8 2.5 3.4 3.2 6.8 3.8 8.4 2.5 18.6 22.5 23.8
The amount of β-Ala gives the total peptide content of each fraction.
Table 5. Values of Enantiomeric Resolution of DNP-amino Acids in a Running Electrolyte Containing the Three Fractions 1°, 2°, and 3° of the Cyclo(Arg-Lys-X-Pro-X-β-Ala) Sublibrary Separated by Preparative RP-HPLC resolutiona Figure 2. Semipreparative RP-HPLC profile of cyclo(Arg-Lys-XPro-X-β-Ala). The crude sublibrary (160 µmol) was dissolved in 0.1% (v/v) TFA and applied to a Whatman Partisil 10 ODS-2 (1 × 50 cm) column. The material corresponding to the major chromatographic peaks was eluted in the interval between 3 and 18% acetonitrile by applying, within 40 min, a linear gradient of acetonitrile in water (025%) at a flow rate of 7 mL/min. Fractions, collected every 2 min, were pooled in three fractions as indicated by arrows, 130 µmol of peptides was recovered (yield 81%), and the amino acid composition of each fraction is given in Table 4.
The next step was the definition of the amino acid residue in position 3. A complete evaluation of the influence of the amino acid in this position would require the synthesis of 18 sublibraries and the evaluation of their performance as chiral selectors toward the analyte test mixture. However, we have shortened this procedure by separating the sublibrary in three fractions, by semipreparative reversed-phase HPLC. The sublibrary cyclo(ArgLys-X-Pro-X-β-Ala), was separated according to the hydrophobicity of the amino acids in the randomized positions. Figure 2 shows the chromatographic pattern of the sublibrary with positions 3 and 5 randomized eluted with a linear gradient of acetonitrile. The three fractions, pooled as indicated by arrows (Figure 2), were subjected to amino acid analysis and the results are summarized in Table 4. Some hydrophobic amino acids, Val, Met, Ile Leu, Tyr, and Phe, were not represented in the HPLC fraction with the lower retention time (fraction 1), and this fraction did not show any ability to separate the analyte text mixture. On the contrary, the two fractions with all the amino acids represented (fractions 2 and 3) provided good enantioselectivity. Table 5 shows the resolution values for different enantiomer pairs as a function of the sublibrary fraction obtained by HPLC used as a chiral selector. 4970 Analytical Chemistry, Vol. 70, No. 23, December 1, 1998
analytes
cyclo(R-K-X-P-X-β-A)
1°
2°
3°
Dnp-D,L-Glu Dnp-D,L-Ala Dnp-D,L-Leu
2.05 0.80 0
1.09 0 0
4.05 5.54 3.53
1.69 2.45 0.89
a
For experimental conditions, see Table 3.
Figure 3 exemplifies the separations of several D and L DNP-amino acids obtained with the sublibrary collected as the intermediate fraction in preparative RP-HPLC (fraction 2). The HPLC fractionation demonstrated in a simple way that some or all of the six amino acids, Val, Met, Ile, Leu Tyr, and Phe, were deeply involved in the chiral recognition of the analytes. Therefore, six sublibraries of general structure cyclo(Arg-Lys-O-Pro-X-β-Ala), with O equal to one of the six above-mentioned amino acids and X equal to a randomized position containing 18 natural amino acids, were synthesized and checked in CE at 30 mM concentration. The resolution values of a set of racemates obtained in a 20 mM phosphate buffer pH 7.0 containing up to 30 mM of each sublibrary (Figure 4) indicated that all the six selected amino acids possess similar separation capacities. However, the experiments indicated that the exclusion of hydrophilic amino acids improved enantioselectivity and binding capacity. Since the insertion of an aromatic amino acid facilitates quantitation of the peptides through UV spectroscopy,24 Tyr was assigned in position 3. A similar procedure was adopted to assess what amino acid in position 5 conferred the highest enantioselectivity. The sublibrary cyclo(Arg-Lys-Tyr-Pro-X-β-Ala) was fractionated by reversed-phase (24) Mach, H.; Middaugh, C. R.; Lewis, R. V. Anal. Biochem. 1992, 200, 7480.
Figure 3. Enantiomeric resolution of DNP-D,L-amino acids with cyclic hexapeptide sublibraries with two randomized positions. Conditions: 30 mM cyclopeptide sublibrary cyclo(Arg-Lys-X-Pro-X-β-Ala) eluted as the second fraction in RP-HPLC, in 20 mM sodium phosphate buffer, pH 7.0; capillary, 50 µm i.d., 65 cm total length, 57 cm to the window; V ) -20 kV, I ) 40; electrokinetic injection, -10 kV, 3 s; detection at 340 nm.
Figure 4. Values of enantiomeric resolution of different DNP-D,Lamino acids in running electrolytes containing cyclic hexapeptide sublibraries with one randomized position. Resolution values with 30 mM cyclo(Arg-Lys-X-Pro-X-β-Ala) in the first line are compared to those obtained with 30 mM sublibraries of general formula cyclo(ArgLys-O-Pro-X-β-Ala), where O is Val, Met, Ile, Leu, Tyr, or Phe. CE separation conditions as in Figure 3.
HPLC, and the three fractions obtained were assessed for enantioselectivity and then subjected to amino acid analysis. Analogous to our observations for position 3, it appears that one or more of the six amino acids are essential to chiral recognition. The last step was the synthesis of six cyclohexapeptides, cyclo(Arg-Lys-Tyr-Pro-O-β-Ala), with O equal to Val, Met, Ile, Leu, Tyr, or Phe. In this case, the presence of an aromatic ring on the amino acid lateral chain is essential for the cyclohaxapeptide to be an efficient chiral selector (Figure 5). Only the cyclopeptides with Tyr or Phe in position 5 showed a marked enantioselectivity and provided excellent resolution of the test sample at a concentration ranging from 1 to 30 mM. The graph in Figure 6 summarizes the resolution values obtained for different DNPamino acids at different stages of the deconvolution process.
Figure 5. Values of enantiomeric resolution of different DNP-D,Lamino acids obtained with cyclic hexapeptides with all the positions defined. Resolution values with a 30 mM cyclo(Arg-Lys-Tyr-Pro-Xβ-Ala) sublibrary in the first line are compared to those obtained with cyclopeptides of general formula cyclo(Arg-Lys-Tyr-Pro-O-β-Ala), where O is Val, Met, Ile, Leu, Tyr, or Phe. The sublibraries with Tyr or Phe in position 5 were 10 mM in the BGE; all the others were 30 mM. CE separation conditions as in Figure 3.
Figure 6. Comparison of the values of enantiomeric resolution of different DNP-D,L-amino acids at different deconvolution stages of a cyclic hexapeptide sublibrary. Resolution values in a cyclo(Arg-LysX-X-X-β-Ala) sublibrary, in the first line, are compared to those obtained in sublibraries with a progressively increasing number of defined positions. All the sublibraries were 30 mM in the running buffer while the completely defined cyclo(Arg-Lys-Tyr-P-Tyr-β-Ala) peptide is used at 10 mM concentration. CE separation conditions as in Figure 3.
When the initial sublibrary with 5832 constituents was added to the BGE, only one of the racemates, DNP-glutamic acid, could be partially resolved. The insertion of proline in position 4 increased the resolution of DNP-glutamic and provided resolution of DNP-alanine and DNP-aspartic racemates. A significant increase in the enantiselectivity was obtained with the sublibrary with a hydrophobic amino acid (e.g., Tyr) in position 3. However, the most dramatic improvement in the properties of the selector was achieved when a residue with an aromatic ring was present in position 5. For example, with tyrosine all the racemates could be very efficiently separated with resolution values up to 30 Analytical Chemistry, Vol. 70, No. 23, December 1, 1998
4971
Figure 7. Separation of DNP-D,L-amino acids in a running electrolyte containing cyclo(Arg-Lys-Tyr-Pro-Tyr-β-Ala). Conditions: 10 mM cyclopeptide in 20 mM sodium phosphate buffer, pH 7.0; capillary, 50 µm i.d., 65 cm total length, 57 cm to the window; -20 kV; electrokinetic injection, -10 kV 3 s; detection at 340 nm, 15 °C.
Figure 8. Dependency of electroosmotic flow on the concentration of cyclo(Arg-Lys-Tyr-Pro-Tyr-β-Ala) added to a 20 mM sodium phosphate buffer, pH 7.0.
(Figure 6). Due to the high enantiselettivity of the cyclopeptides with Tyr or Phe in position 5, these selectors could be used at a concentration as low as 1 mM. A systematic study of the effects of temperature, pH, ionic strength, and selector concentration on separation was then carried out, and optimum conditions were used in the separations depicted in Figure 7. A series of D,L-DNP-derivatives were successfully separated at 15 °C in an uncoated capillary using 20 mM phosphate buffer at pH 7 containing 10 mM cyclo(Arg-LysTyr-Pro-Tyr-β-Ala). The cyclopeptides used in this investigation are positively charged at the selected operative pH. The influence of pH on the separation suggests that electrostatic interactions between selector and analyte are implicated in the chiral recognition process. The resolution capacity was lost at pH 3 and pH 9 for all the enantiomers (data not shown) where the carboxyl groups of the analytes and the amino groups of Lys are respectively protonated and deprotonated. An important issue when positively charged cyclopetides are used is their tendency to adsorb onto the silica wall, resulting in a suppression of the electroosmotic flow (EOF). EOF values measured in an uncoated capillary at different concentrations of S.4.6 cyclopeptide and corrected for solution viscosity are reported in Figure 8. A significant and concentration-dependent EOF suppression is already observed at 2.5 mM cyclopeptide concentration. Panels a and b of Figure 9 show, as an example, the dependency of the resolution of a series of D and L DNP-amino acids from the S.4.6 concentration in coated and uncoated capillaries, respectively. Although a higher selectivity was found in the uncoated capillary, the use of a coated capillary provided better efficiency due to the absence of analyte-wall interactions so that a similar resolution was obtained in the two different capillaries.
Figure 9. Resolution values of different DNP-amino acid enantiomers (key in panel b) as a function of cyclo(Arg-Lys-Tyr-Pro-Pheβ-Ala) concentration in (a) uncoated and (b) coated capillaries. CE separation conditions as in Figure 3.
CONCLUSIONS The present investigation demonstrated the usefulness of combinatorial chemistry for the synthesis of CE chiral selectors. By applying a normal procedure of serial deconvolution, 18 × 3 syntheses would be required for the evaluation of the 5832 individual components that are present as a mixture in a cyclo-
peptide sublibrary with three randomized positions. However, we have simplified the process first by applying a proline scan followed by a fractionation of the sublibrary with two randomized positions in three fractions by means of preparative RP-HPLC. This approach made it possible separate the sublibrary constituents according to the hydrophobicity of the amino acids present in the
4972 Analytical Chemistry, Vol. 70, No. 23, December 1, 1998
randomized positions. The independent evaluation of the three fractions allowed the assessment of the contribution of a group of residues with homogeneous characteristics to the enantioselctive properties of the selector. In this way, the number of sublibrary syntheses required for deconvolution was dramatically reduced (from 64 to 15), rendering the whole procedure applicable in practice. It is likely that the present approach can be applied to different classes of analytes and the unlimited potentiality of combinatorial chemistry exploited to produce different types of complexing agents.
ACKNOWLEDGMENT This research was funded in part by the CNR Target Project on “Biotechnology”. We thank Dr. G. Carrea and Prof. B. Danieli for their helpful suggestions concerning this work.
Received for review June 15, 1998. Accepted September 10, 1998. AC9806557
Analytical Chemistry, Vol. 70, No. 23, December 1, 1998
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