Communications Masavuki matsushita.kazuhiro yoshida (C) y with regard to ast na-basec PTC rea ps Re oll.CA 92037 (USA) 月85-784259 4ppda 5984 Wiley-VCH Verlag CmbH&Co.KCaA,Weinhein DOl:10.1002/anie.200352793 Cm.E2o35934-598
Sensors High-Throughput Screening by Using a BlueFluorescent Antibody Sensor** Masayuki Matsushita,* Kazuhiro Yoshida, Noboru Yamamoto, Peter Wirsching, Richard A. Lerner, and Kim D. Janda* Phase-transfer catalysis (PTC) has received increasing attention in recent years, particularly with regard to asymmetric synthesis, because of its simplicity, mild reaction conditions, and moderate-to-high product yields.[1, 2] Cinchona alkaloidderived quaternary ammonium salts have been the most frequently employed chiral catalysts given their efficiency, low cost, ease of preparation, and suitability for introducing structural diversity.[2] Notably, a number of important natural and nonnatural amino-acid derivatives have been synthesized by a-CC bond formation by quaternary Cinchona-based catalysts.[2, 3] Yet, despite their successful application to catalytic asymmetric synthesis, dramatic effects of the catalyst structure, solvent, temperature, and metal-ion base on the enantioselectivity have often been problematic with regard to PTC reaction optimization.[2–5] Combinatorial synthesis can afford large libraries of molecules as a source of new and improved catalysts.[6] However, considerable effort is often entailed in the screen- [*] Dr. M. Matsushita, Prof. Dr. K. D. Janda, Dr. K. Yoshida, Dr. N. Yamamoto, Dr. P. Wirsching, Prof. Dr. R. A. Lerner Department of Chemistry and The Skaggs Institute for Chemical Biology The Scripps Research Institute 10550 N. Torrey Pines Road, La Jolla, CA 92037 (USA) Fax: (+1) 858-784-2595 E-mail: masayuki@scripps.edu kdjanda@scripps.edu [**] This work was supported by funding from the Skaggs Institute for Chemical Biology. We thank Prof. Hyung-geun Park (Seoul National University, Korea) and Prof. Nobuyuki Mase (Shizuoka University, Japan) for helpful discussions. Supporting information for this article is available on the WWW under http://www.angewandte.org or from the author. Communications 5984 2003 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim DOI: 10.1002/anie.200352793 Angew. Chem. Int. Ed. 2003, 42, 5984 –5987
Angewandte ctermir the has ea ee values by using UV/Vis oth ar dic 12 scence mas pectrom etry. Scheme.Bluefuorescent mAb 19G2 hapen and chiral ligands for app nodslisl have emer ed for par icular applicatio to screen the of N-(diph knowledge.HTS of the ee values of pro rming reacton has no eer v.we d scril of monoclo al ant dies R)-2 was p I the se ().in hich the xture m47% 03% 410a nd a (S)-2 and (R) end.(S)-and (RH d (R)-2(Se bind to 2.but only the 19G2 inc dust.followed by Pd-catalvzed cross-coupling to 19G oul lysts u the synthes s of (S)-2 Here lkaloids in the PTC tion by usins a HTS u plate-r the rapi catalyst derive dby individual nin ninea as the parent ther alkyl substituents.and finally O ated.or the A9 Out of a possible Cinchon 8F96 were obtained in high yield and ong thes reported and 24 were nev A B G H akaod-derived 5934 angewandte.org a003 Wiley-VCH bH& 598
ing of these libraries, particularly with regard to enantioselective reactions. In high-throughput screening (HTS) for the evaluation of catalyst libraries, the determination of the enantiomeric excess (ee) of the products is generally the ratelimiting procedure.[6c] Hence, the application of HTS to enantioselective transformations has not been straightforward. In 1997, Reetz and coworkers developed the first HTS of reaction ee values by using UV/Vis spectroscopy.[7] Following their pioneering work, other methods that use, for example, IR-thermography,[8] circular dichroism,[9] capillary electrophoresis,[10] fluorescence,[11] mass spectrometry,[12] chemosensing,[13] competitive immunoassay,[14] and enzymatic methods[15] have emerged for particular applications. Most of these techniques have been used to screen the ee values of asymmetric reduction or hydrolysis reactions, but, to our knowledge, HTS of the ee values of products from an asymmetric CC bond forming reaction has not been reported. Recently, we described a series of monoclonal antibodies (mAbs), for example, mAb 19G2, prepared against the transstilbene hapten 1 (Scheme 1), in which the 19G2-1 complex produced a blue fluorescence in high quantum yield (lex = 327 nm, lem = 410 nm, Ff = 0.78).[16] During subsequent studies to find alternative ligands for these mAbs, we discovered that each of the chiral trans-stilbene amino acid esters (S)-2 and (R)-2 (Scheme 1) could bind to 19G2, but only the 19G2- (S)-2 complex afforded a blue fluorescence. Hence, it occurred to us that 19G2could act as a sensor in the HTS of chiral catalysts used for the synthesis of (S)-2 and (R)-2. Herein, the aim was to evaluate a panel of derivatized Cinchona alkaloids in the PTC of an asymmetric a-alkylation reaction by using a HTS fluorescence plate-reader format for the rapid estimation of ee values of products. A catalyst library derived from Cinchona alkaloids was constructed by individual compound synthesis. Four natural Cinchona alkaloids (cinchonidine, cinchonine, quinidine, quinine) and one nonnatural Cinchona-type alkaloid[17] were hydrogenated, or left as the parent compound, then derivatized with four different Nalkyl substituents, and finally Oalkylated, or the hydroxyl was kept unmodified (Scheme 2). Out of a possible 40 Cinchona alkaloid ammonium salts, 35 were obtained in high yield and purity. Among these 35 catalysts, 11 had been previously reported[3, 4, 18] and 24 were new compounds. Each catalyst was then employed in PTC for the alkylation of N-(diphenylmethylene)glycine methyl ester 3 with 4-bromomethyl-trans-stilbene 4 (Scheme 3). After the alkylations were complete, the benzophenone Schiff base group was hydrolyzed and the product mixture of (S)-2 and (R)-2 was purified. The simplicity of the sequence allowed us to carry out 35 parallel reactions in two days. Yields of the isolated product mixture ranged from 47% to 83%. To determine the ee values of the 35 product mixtures, we constructed a calibration curve by using independently synthesized (S)-2 and (R)-2. To this end, (S)- and (R)-NBoc-iodoalanine methyl ester (Fluka) were each reacted with zinc dust, followed by Pd-catalyzed cross-coupling to 4- Scheme 1. Blue-fluorescent mAb 19G2 hapten and chiral ligands for sensor applications. Scheme 2. Preparation of a Cinchona alkaloid-derived catalyst library. Angewandte Chemie Angew. Chem. Int. Ed. 2003, 42, 5984 –5987 www.angewandte.org 2003 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim 5985
Communications Ko oH 4 (S2+-2 3 m地1c2me apeeqmedoreah a day.and 3)accurate,and with a wide ated by the ther of a reference reac tion. Then the wa ey in 150 mM NaCl,pH7.4)with 5 DME HTS ha 416m %-well p acid synthe xan resc on Finally are als ow th preparation.plate reading.and catalyst evaluation ymmetric c alysis for other CC bond-formnre ction the method.10samples were (S)-2and HPLC The flue sensor and HPLC =0.99 ote that the ter high electivities in mmetric PTO the mAb high-per d h alyst give excellent enantioselectivity,as well as on catalyst the 210% 02 meml :78%e.HPLC)wa O'Donnell on 85%ee HPLC) also observed by QN4 (63%ee.fluc cence:68 ee HPLC)is Figure The evalues from Cinchon-ased PTC measured with the etdescribed a catalyst of this type that Received:September 4.2003 [752793] 5986 ndte.or
bromo-trans-stilbene in analogy with reported procedures.[19] Both (S)-2 and (R)-2 were obtained in > 99% ee as analyzed by chiral HPLC. Precise mixtures of these isomers were then prepared (0, 25, 50, 75, 100% (S)-2, corresponding to 100% ee (R)-2, 50% ee (R)-2, 0% ee, 50% ee (S)-2, 100% ee (S)-2) as solutions in DMF. Each calibration standard (50 mm) was mixed with 19G2(25 mm; 50 mm binding sites) in PBS (10 mm sodium phosphate, 150 mm NaCl, pH 7.4) with 5% DMF cosolvent, and the fluorescence intensities (lex = 327 nm, lem = 416 nm) were measured by using a 96-well plate reader. The data fitted well to a hyperbolic, as required by the specific binding of (S)-2 and (R)-2 by the mAb 19G2. The fluorescence values for each of the 35 product mixtures were then obtained in the same way as above, and the corresponding ee values were then calculated (Figure 1). The process of sample preparation, plate reading, and catalyst evaluation took less than one hour. To confirm the validity of the method, 10 samples were randomly chosen across the range of ee values of (S)-2 and (R)-2 and reanalyzed by chiral HPLC. The fluorescence sensor and HPLC measurements varied, on the average, 10% and afforded an excellent linear correlation (slope = 0.92, r 2 = 0.99). We note that the tertbutyl ester analogue of 3 generally gave higher enantioselectivities in asymmetric PTC alkylations.[3] However, it was necessary to use 3 as the alkylation substrate, as 19G2did not show selective fluorescence with the tert-butyl ester derivatives corresponding to (S)-2 and (R)-2. Significantly, the blue-fluorescent mAb sensor identified high-performance catalysts that were the same as those found by others to give excellent enantioselectivity, as well as one previously unknown catalyst. If we choose 70% ee as a preliminary cut-off point for catalyst efficiency, then, based on our test panel, such a value would give a reasonable 5–10% member selection from large libraries for further analysis. The catalyst CD2 (68% ee, fluorescence; 78% ee, HPLC) was reported by O'Donnell et al. in their seminal work on Cinchona-based PTC.[18b] CD8 (75% ee, fluorescence; 85% ee, HPLC) was also observed by Park et al. to exhibit the highest enantioselectivity among this class of catalysts.[18a] Notably, QN4 (63% ee, fluorescence; 68% ee, HPLC) is a new quinine-based structure for PTC. Lygo et al.[4b] described a catalyst of this type that afforded a > 80% ee in reactions by using the tert-butyl ester analogue of 3. We have presented the foundation for a novel HTS method that uses a blue-fluorescent mAb to assay the enantionselectivity of products from, for example, a catalytic asymmetric alkylation. The method is: 1) sensitive, as expected for fluorescence detection, with only 10 nmol of sample required for each measurement, 2) rapid, hundreds of catalysts could potentially be screened and ranked in less than a day, and 3) accurate, and with a wide dynamic range, as demonstrated by the comparison to HPLC determinations from 100% ee (S)-2 to 100% ee (R)-2. However, since the method is based on the catalysis of a reference reaction, the intent is to provide a HTS to obtain a subset of catalysts that produce the highest ee values. Then, these candidates can be tested and rigorously analyzed, in this case, for efficiency in preparing other amino acids of interest. Importantly, bluefluorescent HTS has broad applicability. By using the preparation of the same reference compounds, catalysts of other types could be evaluated in both alkylation and nonalkylation strategies for amino acid syntheses. For example, the latter could include Jacobsen-type catalysts for the Strecker reaction.[20] Finally, we are also currently extending the diversity of the trans-stilbene moiety, which will allow the application of the blue-fluorescent mAb sensor in screening asymmetric catalysis for other CC bond-forming reactions, as well as other classes of reactions, such as oxidations, reductions, and hydrolyses. Received: September 4, 2003 [Z52793] Scheme 3. Synthesis of chiral ligands for mAb 19G2 by using Cinchona-based PTC. Figure 1. The ee values from Cinchona-based PTC measured with the mAb 19G2 sensor. Communications 5986 2003 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim www.angewandte.org Angew. Chem. Int. Ed. 2003, 42, 5984 –5987
Angewandte CH.New York 4.3-519m.19 R.Chinchilla.C Najera.G.Guillena.R.Kreiter .2003.7 BI 001 9.2961-296 [10 M.T Ree A homp Z Hoff A.E.B 别 Yak Hatake a.I.Am .J-H.Lee.M.k.Park.Y. Kim, 6 58:b)R.F W.lackson.K. JChem.Soe.Chem.Com 199.644 .12 Am.Chem.5oc198.2.4901-49d www.angewandte.org 03 Verlag CmbH&Co.KGaA Weinheim 597
.Keywords: alkaloids · antibodies · enantioselectivity · high-throughput screening · phase-transfer catalysis [1] For recent reviews of PTC, see: a) K. Maruoka, T. Ooi, Chem. Rev. 2003, 103, 3013 – 3028; b) P. I. Dalko, L. Moisan, Angew. Chem. 2001, 113, 3840 – 3864; Angew. Chem. Int. Ed. 2001, 40, 3726 – 3748; c) M. J. O'Donnell, J. Martin in Catalytic Asymmetric Synthesis, 2nd ed. (Ed.: I. Ojima), Wiley-VCH, New York, 2000, pp. 727 – 755; d) T. Shioiri, S. Arai in Stimulating Concepts in Chemistry (Eds.: F. Vogtle, J. F. Stoddart, M. Shibasaki), Wiley-VCH, Weinheim, 2000, pp. 123 – 143. [2] M. J. O'Donnell, Aldrichimica Acta 2001, 34, 3 – 15. [3] E. J. Corey, F. Xu, M. C. Noe, J. Am. Chem. Soc. 1997, 119, 12414 – 12415. [4] a) P. Mazon, R. Chinchilla, C. Najera, G. Guillena, R. Kreiter, R. J. M. K. Gebbink, G. van Koten, Tetrahedron: Asymmetry 2002, 13, 2181 – 2185; b) B. Lygo, P. G. Wainwright, Tetrahedron Lett. 1997, 38, 8595 – 8598. [5] T. Okino, Y. Takemoto, Org. Lett. 2001, 3, 1515 – 1517. [6] a) J. P. Stambuli, J. F. Hartwig, Curr. Opin. Chem. Biol. 2003, 7, 420 – 426; b) A. Berkessel, Curr. Opin. Chem. Biol. 2003, 7, 409 – 419; c) M. T. Reetz, Angew. Chem. 2001, 113, 292 – 320; Angew. Chem. Int. Ed. 2001, 40, 284 – 310; d) K. W. Kuntz, M. L. Snapper, A. H. Hoveyda, Curr. Opin. Chem. Biol. 1999, 3, 313 – 319; e) A. H. Hoveyda, Chem. Biol. 1999, 6, R305 – R308. [7] M. T. Reetz, A. Zonta, K. Schimossek, K. Liebeton, K.-E. Jaeger, Angew. Chem. 1997, 109, 2961 – 2963; Angew. Chem. Int. Ed. Engl. 1997, 36, 2830 – 2832. [8] M. T. Reetz, M. H. Becker, K. M. Kuhling, A. Holzwarth, Angew. Chem. 1998, 110, 2792 – 2795; Angew. Chem. Int. Ed. 1998, 37, 2647 – 2650. [9] K. Ding, A. Ishii, K. Mikami, Angew. Chem. 1999, 111, 519 – 523; Angew. Chem. Int. Ed. 1999, 38, 497 – 501. [10] M. T. Reetz, K. M. Kuhling, A. Deege, H. Hinrichs, D. Belder, Angew. Chem. 2000, 112, 4049 – 4052; Angew. Chem. Int. Ed. 2000, 39, 3891 – 3893. [11] G. Klein, J.-L. Reymond, Helv. Chim. Acta 1999, 82, 400 – 407. [12] a) J. Guo, J. Wu, G. Siuzdak, M. G. Finn, Angew. Chem. 1999, 111, 1868 – 1871; Angew. Chem. Int. Ed. 1999, 38, 1755 – 1758; b) M. T. Reetz, M. H. Becker, H.-W. Klein, D. Stockigt, Angew. Chem. 1999, 111, 1872– 1875; Angew. Chem. Int. Ed. 1999, 38, 1758 – 1761. [13] G. T. Copeland, S. J. Miller, J . Am. Chem. Soc. 1999, 121, 4306 – 4307. [14] F. Taran, C. Gauchet, B. Mohar, S. Meunier, A. Valleix, P. Y. Renard, C. Creminon, J. Grassi, A. Wagner, C. Mioskowski, Angew. Chem. 2002, 114, 132– 135; Angew. Chem. Int. Ed. 2002, 41, 124 – 127. [15] P. Abato, C. T. Seto, J. Am. Chem. Soc. 2001, 123, 9206 – 9207. [16] A. Simeonov, M. Matsushita, E. A. Juban, E. H. Thompson, T. Z. Hoffman, A. E. Beuscher, M. J. Taylor, P. Wirsching, W. Rettig, J. K. McCusker, R. C. Stevens, D. P. Millar, P. G. Schultz, R. A. Lerner, K. D. Janda, Science 2000, 290, 307 – 313. [17] Y. Iwabuchi, M. Nakatani, N. Yokoyama, S. Hatakeyama, J. Am. Chem. Soc. 1999, 121, 10 219 – 10 220. [18] a) H.-g. Park, B.-S. Jeong, M.-S. Yoo, J.-H. Lee, M.-k. Park, Y.-J. Lee, M.-J. Kim, S.-s. Jew, Angew. Chem. 2002, 114, 3162– 3164; Angew. Chem. Int. Ed. 2002, 41, 3036 – 3038; b) M. J. O'Donnell, W. D. Bennett, S. Wu, J. Am. Chem. Soc. 1989, 111, 2353 – 2355. [19] a) Y. Tamaru, H. Ochiai, T. Nakamura, Z. Yoshida, Tetrahedron Lett. 1986, 27, 955 – 958; b) R. F. W. Jackson, K. James, M. J. Wythes, A. Wood, J. Chem. Soc. Chem. Commun. 1989, 644 – 645. [20] a) P. Vachal, E. N. Jacobsen, J. Am. Chem. Soc. 2002, 124, 10 012– 10 014; b) M. S. Sigman, P. Vachal, E. N. Jacobsen, Angew. Chem. 2000, 112, 1336 – 1338; Angew. Chem. Int. Ed. 2000, 39, 1279 – 1281; c) M. S. Sigman, S. Mattew, E. N. Jacobsen, J. Am. Chem. Soc. 1998, 120, 4901 – 4902. Angewandte Chemie Angew. Chem. Int. Ed. 2003, 42, 5984 –5987 www.angewandte.org 2003 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim 5987