Design and Use of Fluorogenic Aldehydes for Monitoring the Progress of Aldehyde Transformations

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1 Published on Web 3/3/24 Design and Use of Fluorogenic Aldehydes for Monitoring the Progress of Aldehyde Transformations Fujie Tanaka,* Nobuyuki Mase, and Carlos F. Barbas, III* The Skaggs Institute for Chemical Biology and the Departments of Chemistry and Molecular Biology, The Scripps Research Institute, 155 North Torrey Pines Road, La Jolla, California 9237 Received January 2, 24; Simple and rapid methods for monitoring the progress of chemical reactions are critical for high-throughput screening of catalysts as well as for characterization of catalysts on a small scale. 1,2 Fluorogenic substrates that increase in fluorescence as reactions progress provide a straightforward method of reaction monitoring because reaction progress is directly observed as an increase in fluorescence. 2 We have previously developed fluorescent detection strategies to monitor Michael and Diels-Alder reactions using fluorogenic R,β-unsaturated carbonyl compounds 3 and have demonstrated that the system is useful for evaluation of catalysts and reaction conditions. 4 Aldehydes are versatile and are used for many types of reactions. To develop systems for monitoring the progress of aldehyde transformations, an entirely new approach was required. Here we report the first design, synthesis, and use of fluorogenic aldehydes for direct monitoring of aldehyde transformations by fluorescence growth. Our design is based on resonance energy transfer 5 between a fluorophore and an aldehyde in a single molecule. The fluorogenic aldehydes are composed of a fluorophore and an aldehyde moiety coupled by a linker. When intact, the aldehyde moiety acts as a quencher of the fluorophore s fluorescence; however, the reaction product of the aldehyde moiety does not quench fluorescence and fluorescence is turned-on in the product. We reasoned that an arylaldehyde would quench the fluorescence of a proximal fluorophore, and that a simple aryl group without a carbonyl would not. 6 To test this hypothesis, we prepared the aldehyde 1 and aldol 2 shown in Scheme 1. As expected, aldol 2 showed a higher fluorescence than aldehyde 1 (Table 1). On the other hand, neither aldehyde 3 nor aldol 4 was fluorescent. Note that in 4, the aryl group conjugated to the fluorophore via an amide bond quenched the fluorophore s fluorescence. Scheme 2 Table 1. Fluorescence of Aldehydes and Aldols a wavelength (nm) fluorescence intensity solvent λex λem c b aldehyde aldol fold c 1,2 DMSO DMF ph d d 26 5,6 DMSO ,8 DMSO DMF d d 9 ph d d 78 9,1 DMSO DMF ph ,12 DMSO d d 3 DMSO d d.5 a The fluorescence was recorded on a microplate spectrophotometer using 1 µl of solution composed of.5% CH 3CN,.5% 2-PrOH, and 99% of the indicated solvent in a 96-well polypropylene plate at 26 C. Solvent ph 7 refers to 5 mm sodium phosphate, ph 7.. The data are shown after background correction except where noted. b c ) concentration of aldehyde or aldol (µm). c fold ) fluorescence intensity of aldol/fluorescence intensity of aldehyde. d The data without background correction. Scheme 1 We prepared candidate fluorogenic aldehydes and their aldols (5-12, Scheme 2) by using a series of fluorophores and compared their fluorescence (Table 1). Aldehyde 7, prepared as the amide of 9-aminophenanthrene (13), was the most promising of the aldehydes prepared. The reaction product, aldol 8, showed 8-fold higher fluorescence (λex 25 nm, λem 38 nm) than aldehyde 7 in aqueous buffer (ph 7.) and 2-fold higher (λex 265 nm, λem 385 nm) in DMSO. Although the fluorescence intensity varied with solvent, aldol 8/aldehyde 7 had an excellent fluorogenic range in aqueous Figure 1. Fluorescence emission spectra (λex 25 nm) of aldehyde 7 (), aldol 8 (4), and fluorophore 13 (O) at5µm in.5% CH 3CN-.5% 2-PrOH-99% 5 mm sodium phosphate, ph 7.. buffer and in organic solvents, and the fluorescence intensity of 8 did not vary within the ph range of in aqueous buffer. In addition, the fluorescence of aldol 8 differed from that of fluorophore 13 as shown in Figure 1. Aldol 1 showed 2-fold higher fluorescence than aldehyde 9 in DMSO. In contrast, aldehyde 11 showed higher fluorescence than aldol 12 at λex 26 nm and λem J. AM. CHEM. SOC. 24, 126, /ja49641a CCC: $ American Chemical Society

2 COMMUNICATIONS Scheme 3 Table 2. Fluorescence of Compounds a solvent λex λem c b fluorescence fold c 14 DMSO DMF d 6 ph DMSO DMF d 12 ph d DMSO DMF d 6 ph d 53 a,b See Table 1 legend. c fold ) fluorescence intensity of 14, 15, or16/ fluorescence intensity of aldehyde 7. d See Table 1 legend. Figure 2. Fluorescence assay of antibody 38C2-catalyzed aldol reaction of acetone and aldehyde 7. Conditions: [antibody] 2 µm (active site), [7] 5 µm, [acetone] 5%(v/v) (68 mm), 2.5% CH 3CN-2.5% 2-PrOH/PBS (ph 7.4). : 38C2; O: nonaldolase antibody IgG (control); ]: reaction with 38C2 in the absence of acetone; 4: reaction without antibody (blank). RFU ) relative fluorescence intensity. 45 nm, although 12 showed a slightly higher fluorescence at λex 26 nm and λem 38 nm. These results indicate that the proper selection of fluorophores is important for the preparation of useful fluorogenic aldehydes. To examine the applicability of the fluorogenic aldehydes to other reactions, aldehyde 7 was transformed to aldol 14 by aldol reaction with hydroxyacetone, to allyl alcohol 15 by In-mediated allylation, 7 and to alcohol 16 by reduction (Scheme 3). These products were all fluorescent (Table 2), indicating that the loss of π-conjugation between the aldehyde carbonyl and the aryl group is key to fluorescence and that aldehyde 7 can be used as a fluorogenic substrate for many reactions. To monitor the time-course of an aldol reaction, we studied the reaction of acetone and aldehyde 7 catalyzed by aldolase antibody 38C2 8 (Figure 2). The reaction with antibody 38C2 showed a significant increase in fluorescence, while reaction with a control antibody, reaction without acetone, and reaction without antibody all showed little or no increase in fluorescence. Catalytic reduction of 7 with alcohol dehydrogenase in the presence of NADPH was successfully monitored by observing an increase in fluorescence (Figure 3). Although reactions with this enzyme can be monitored by changes in UV (34 nm) and fluorescence (λem 45 nm) of NADPH, fluorogenic aldehyde 7 can be used in a complementary Figure 3. Fluorescence assay of reduction of aldehyde 7 with alcohol dehydrogenase (ADH) from Thermoanaerobium brockii. (A) Time course, (B) emission spectra (λex 25 nm) at 5 min. Conditions: (a) [ADH].235 unit/ml, [NADPH] 4 µm, [aldehyde 7] 12.5 µm,.5% CH 3CN-.5% 2-PrOH-99% 5 mm sodium phosphate, ph 7.; (b) reaction without addition of NADPH; (c) reaction using 3 instead of 7; (d) reaction without ADH; (e) reaction without ADH and NADPH. The UV (34 nm) and fluorescence (λem 45 nm) studies suggested that this enzyme contained some reducing cofactor. fashion to directly follow the reduction of the aldehyde. Formation of less than.2 µm of product 16 was readily detected in a 1 µl-scale reaction in a 96-well plate. We have developed fluorogenic aldehydes that can be used for monitoring reactions through increased fluorescence. These fluorogenic aldehydes should be useful for screening of catalysts in approaches using libraries. 3,9,1 Our strategy for accessing fluorogenic aldehydes should also be applicable to the preparation of fluorogenic substrates that allow the transformations of other functional groups to be directly monitored. Acknowledgment. This study was supported in part by the NIH (CA27489) and The Skaggs Institute for Chemical Biology. Supporting Information Available: Fluorescence spectra, graphs of standards of 8 and 16, synthesis and characterization of compounds (PDF). This material is available free of charge via the Internet at pubs.acs.org. References (1) (a) Matayoshi, E.; Wang, G. T.; Krafft, G.; Erickson, J. Science 199, 247, 954. (b) Taylor, S. J.; Morken, J. P. Science 1998, 28, 267. (c) Reetz, M. T.; Kuhling, K. M.; Deege, A.; Hinrichs, H.; Belder, D. Angew. Chem., Int. Ed. 2, 39, (d) Copeland, G. T.; Miller, S. J. J. Am. Chem. Soc. 21, 123, (e) Das, G.; Talukdar, P.; Matile, S. Science 22, 298, 16. (f) Stauffer, S. R.; Hartwig, J. F. J. Am. Chem. Soc. 23, 125, (g) Konarzycka-Bessler, M.; Bornscheuer, U. Angew. Chem., Int. Ed. 23, 42, (2) Nishino, N.; Powers, J. J. Biol. Chem. 198, 255, List, B.; Barbas, C. F., III; Lerner, R. A. Proc. Natl. Acad. Sci. U.S.A. 1998, 95, Carlson, R. P.; Jourdain, N.; Reymond, J.-L. Chem. Eur. J. 2, 6, Svensson, R.; Greno, C.; Johansson, A.; Mannervik, B.; Morgenstern, R. Anal. Biochem. 22, 311, 171. Onoda, M.; Uchiyama, S.; Endo, A.; Tokuyama, H.; Santa, T.; Imai, K. Org. Lett. 23, 5, (3) Tanaka, F.; Thayumanavan, R.; Barbas, C. F., III. J. Am. Chem. Soc. 23, 125, (4) Mase, N.; Tanaka, F.; Barbas, C. F., III. Org. Lett. 23, 5, Tanaka, F.; Thayumanavan, R.; Mase, N.; Barbas, C. F., III. Tetrahedron Lett. 24, 45, 325. (5) Lakowicz, J. R. Principles of Fluorescence Spectroscopy, 2nd ed.; Kluwer Academic: New York, 1999; p 8. See also ref 1f and references therein. (6) Benzaldehyde has an R band (λmax 328 nm in alcohol). Silverstein, R. M.; Bassler, G. C.; Morrill, T. C. Spectrometric Identification of Organic Compounds, 5th ed.; John Wiley & Sons: New York, 1991; p38. (7) Chan, T. H.; Yang, Y. J. Am. Chem. Soc. 1999, 121, (8) Wagner, J.; Lerner, R. A.; Barbas, C. F., III. Science 1995, 27, Tanaka, F.; Barbas, C. F., III. J. Immunol. Methods 22, 269, 67. (9) Nakadai, M.; Saito, S.; Yamamoto, H. Tetrahedron 22, 58, Kofoed, J.; Nielsen, J.; Reymond, J.-L. Bioorg. Med. Chem. Lett. 23, 13, Tanaka, F.; Barbas, C. F., III. J. Am. Chem. Soc. 22, 124, 351. Gildersleeve, J.; Varvak, A.; Atwell, S.; Evans, D.; Schultz, P. G. Angew. Chem., Int. Ed. 23, 42, Tanaka, F.; Fuller, R.; Shim, H.; Lerner, R. A.; Barbas, C. F., III. J. Mol. Biol. 24, 335, 17. Fong, S.; Machajewski, T. D.; Mak, C. C.; Wong, C.-H. Chem. Biol. 2, 7, 873. (1) Tsukiji, S.; Pattnaik, S. B.; Suga, H. Nat. Struct. Biol. 23, 1, 713. JA49641A J. AM. CHEM. SOC. 9 VOL. 126, NO. 12,

3 Design and Use of Fluorogenic Aldehydes for Monitoring the Progress of Aldehyde Transformations Fujie Tanaka,* Nobuyuki Mase, Carlos F. Barbas, III* The Skaggs Institute for Chemical Biology and the Departments of Chemistry and Molecular Biology, The Scripps Research Institute, 155 North Torrey Pines Road, La Jolla, California 9237 Corresponding author Supporting Information Fluorescence Spectra S2 Graphs of Standards of 8 and S6 Synthesis and Characterization of Compounds S7 Hard copy of NMR S11 S1

4 Fluorescence Spectra. Fuorescence was recorded on Spectra Max Gemini (Molecular Devices) using 1 µl of a solution in a 96-well polypropyrene plate (Thomson Instrument Company ) at 26 C. The data are shown after background correction. 6 Fluorescence intensity Emission wavelength (nm) (Excitation 282 nm) Figure S1. Fluorescence emission spectra (λex 282 nm) of 1, 2, and 2-aminonaphthalene in.5% CH 3 CN-.5% 2-PrOH-99% DMSO. Square, 1 (5 µm); triangle, 2 (5 µm); circle, 2- aminonaphthalene (5 µm). 6 Fluorescence intensity Emission wavelength (nm) (Excitation 25 nm) Figure S2. Fluorescence emission spectra (λex 25 nm) of 1, 2, and 2-aminonaphthalene in.5% CH 3 CN-.5% 2-PrOH-99% (5 mm Na phosphate, ph 7.). Square, 1 (5 µm); triangle, 2 (5 µm); circle, 2-aminonaphthalene (5 µm). S2

5 1 Fluorescence intensity Emission wavelength (nm) (Excitation 265 nm) Figure S3. Fluorescence emission spectra (λex 265 nm) of 7, 8, and 13 in.5% CH 3 CN-.5% 2- PrOH-99% DMSO. Square, 7 (5 µm); triangle, 8 (5 µm); circle, 13 (5 µm). 4 Fluorescence intensity Emission wavelength (nm) (Excitation 265 nm) Figure S4. Fluorescence emission spectra (λex 265 nm) of 7, 8, and 13 in.5% CH 3 CN-.5% 2- PrOH-99% DMF. Square, 7 (5 µm); triangle, 8 (5 µm); circle, 13 (5 µm). 1 Fluorescence intensity Excitation wavelength (nm) (Emission 385 nm) Figure S5. Fluorescence excitation spectra (λem 385 nm) of 7, 8, and 13 in.5% CH 3 CN-.5% 2- PrOH-99% DMSO. Square, 7 (5 µm); triangle, 8 (5 µm); circle, 13 (5 µm). S3

6 5 Fluorescence intensity Excitation wavelength (nm) (Emission 385 nm) Figure S6. Fluorescence excitation spectra (λem 385 nm) of 7, 8, and 13 in.5% CH 3 CN-.5% 2- PrOH-99% (5 mm Na phosphate, ph 7.). Square, 7 (5 µm); triangle, 8 (5 µm); circle, 13 (5 µm). 5 Fluorescence intensity Emission wavelength (nm) (Excitation 315 nm) Figure S7. Fluorescence emission spectra (λex 315 nm) of 9, 1, and 4-(1H-benzimidazol-2- yl)aniline in.5% CH 3 CN-.5% 2-PrOH-99% DMSO. Square, 9 (5 µm); triangle, 1 (5 µm); circle, 4-(1H-benzimidazol-2-yl)aniline (5 µm). S4

7 Fluorescence intensity Emission wavelength (nm) (Excitation 265 nm) Figure S8. Fluorescence emission spectra (λex 265 nm) of 14 (5 µm) in.5% CH 3 CN-.5% 2- PrOH-99% DMSO. 15 Fluorescence intensity Emission wavelength (nm) (Excitation 265 nm) Figure S9. Fluorescence emission spectra (λex 265 nm) of 15 (5 µm) in.5% CH 3 CN-.5% 2- PrOH-99% DMSO. 6 Fluorescence intensity Emission wavelength (nm) (Excitation 265 nm) Figure S1. Fluorescence emission spectra (λex 265 nm) of 16 (5 µm) in.5% CH 3 CN-.5% 2- PrOH-99% DMSO. S5

8 RFU (λex 265 nm, λem 385 nm) RFU (λex 265 nm, λem 385 nm) y = x r 2 = (µm) 8 (µm) Figure S11. Standard of aldol 8 in.5% CH 3 CN-.5% 2-PrOH-99% DMSO. RFU (λex 265 nm, λem 385 nm) y = x r 2 = (µm) Figure S12. Standard of alcohol 16 in.5% CH 3 CN-.5% 2-PrOH-99% DMSO. S6

9 3-(4-Formylphenyl)-N-naphthalen-2-yl-propionamide (1). A mixture of 3-(4- formylphenyl)propionic acid (7. mg,.393 mmol), 2-aminonaphthalene (57.1 mg,.399 mmol), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (19.5 mg,.571 mmol), and DMAP (1. mg,.8 mmol) in CH 2 Cl 2 (8. ml) was stirred at room temperature for 2.5 h. The reaction mixture was added to H 2 O and extracted with CH 2 Cl 2. The organic layers were washed with brine, dried over MgSO 4, filtered, concentrated in vacuo, and flash chromatographed (EtOAc/hexane = 2:3) to afford 1 (83.5 mg, 7%). 1 H NMR (4 MHz, CDCl 3 ): δ 9.98 (s, 1H), 8.17 (s, 1H), (m, 5H), (m, 5H), 7.22 (s, 1H), 3.19 (t, J = 7.6 Hz, 2H), 2.76 (t, J = 7.6 Hz, 2H). MALDI-FTMS: calcd for C 2 H 18 NO 2 (MH + ) , found [4-(1-Hydroxy-3-oxobutyl)phenyl]-N-naphthalen-2-yl-propionamide (2). Aldol 2 was prepared by the proline-catalyzed aldol reaction of acetone and aldehyde 1 as described previously. S1 1 H NMR (5 MHz, CDCl 3 ): δ 8.15 (s, 1H), (m, 3H), (m, 3H), (m, 5H), 5.12 (ddd, J = 2.3 Hz, 2.6 Hz, 7.3 Hz, 1H), 3.27 (d, J = 2.3 Hz, 1H), 3.8 (t, J = 6.2 Hz, 2H), 2.87 (dd, J = 7.3 Hz, 14.1 Hz, 1H), 2.79 (dd, J = 2.6 Hz, 14.1 Hz, 1H), 2.7 (t, J = 6.2 Hz, 2H), 2.18 (s, 3H). 13 C NMR (1 MHz, CDCl 3 ): δ 29.2, 17.4, 14.8, 14.1, 135.1, 133.8, 13.6, 128.7, 128.6, 127.6, 127.5, 126.5, 126., 125., 119.7, 116.6, 69.6, 51.8, 39.4, 31.1, 3.7. MALDI-FTMS: calcd for C 23 H 23 NO 3 Na (MNa + ) , found Formyl-N-naphthalen-2-yl-benzamide (3). 1 H NMR (4 MHz, CDCl 3 ): δ 1.1 (s, 1H), 8.36 (brs, 1H), (m, 4H), (m, 3H), 7.6 (dd, J = 2. Hz, 8.8 Hz, 1H), (m, 2H). MALDI-FTMS: calcd for C 18 H 14 O 2 N (MH + ) , found (1-Hydroxy-3-oxobutyl)-N-naphthalen-2-yl-benzamide (4). 1 H NMR (4 MHz, CDCl 3 -CD 3 OD): δ 9.21 (s, 1H x.7), 8.32 (s, 1H), 7.9 (d, J = 8.2 Hz, 2H), (m, 3H), 7.66 (dd, J = 2. Hz, 8.8 Hz, 1H), (m, 2H), 7.46 (d, J = 8.2 Hz, 2H), 5.19 (dd, J = 3.8 Hz, 9.1 Hz, 1H), 2.99 (s, 1H), 2.91 (dd, J = 8.9 Hz, 16.7 Hz, 1H), 2.79 (dd, J = 3.7 Hz, 16.7 Hz, 1H), 2.21 (s, 3H). MALDI-FTMS: calcd for C 21 H 2 NO 3 (MH + ) , found (4-Formylphenyl)-N-naphthalen-1-yl-propionamide (5). 1 H NMR (4 MHz, CDCl 3 -CD 3 OD): δ 9.98 (s, 1H), (m, 3H), 7.71 (d, J = 8.2 Hz, 1H), 7.67 (d, J = 7.3 Hz, S7

10 1H), 7.57 (d, J = 8.2 Hz, 1H), (m, 5H), 3.2 (t, J = 7.6 Hz, 2H), 2.87 (t, J = 7.6 Hz, 2H). MALDI-FTMS: calcd for C 2 H 18 NO 2 (MH + ) , found [4-(1-Hydroxy-3-oxobutyl)-phenyl]-N-naphthalen-1-yl-propionamide (6). 1 H NMR (4 MHz, CDCl 3 -CD 3 OD): δ 7.85 (m, 1H), (m, 2H), 7.57 (m, 1H), (m, 3H), (m, 4H), 5.13 (dd, J = 3.2 Hz, 9.1 Hz, 1H), 3.11 (t, J = 7.6 Hz, 2H), 2.89 (dd, J = 9.1 Hz, 17 Hz, 1H), 2.8 (t, J = 7.6 Hz, 2H), 2.77 (dd, J = 3.2 Hz, 17 Hz, 1H), 2.19 (s, 3H). MALDI-FTMS: calcd for C 23 H 23 NO 3 Na (MNa + ) , found (4-Formylphenyl)-N-phenanthren-9-yl-propionamide (7). 1 H NMR (4 MHz, CDCl 3 -CD 3 OD): δ 9.99 (s, 1H), 8.71 (d, J = 8.8 Hz, 1H), 8.63 (d, J = 7.9 Hz, 1H), 8.3 (s, 1H), (m, 3H), (m, 7H), 3.24 (t, J = 7.6 Hz, 2H), 2.91 (t, J = 7.6 Hz, 2H). 13 C NMR (1 MHz, CDCl 3 -CD 3 OD): δ 192.5, 171.8, 148.3, 134.5, 131.2, 13.8, 13.2, 13., 129.1, 128.8, 128.3, 127.5, 126.7, 126.5, 126.3, 122.8, 122.5, 122.2, 121.9, 37.6, MALDI-FTMS: calcd for C 24 H 2 NO 2 (MH + ) , found [4-(1-Hydroxy-3-oxobutyl)phenyl]-N-phenanthren-9-yl-propionamide (8). 1 H NMR (5 MHz, CDCl 3 ): δ 8.69 (d, J = 8.1 Hz, 1H), 8.6 (d, J = 7.7 Hz, 1H), 8.13 (s, 1H), 7.83 (d, J = 7.3 Hz, 1H), (m, 5H), 7.38 (s, 1H), (m, 4H), 5.14 (m 1H), 3.3 (1H), 3.15 (t, J = 7.6 Hz, 2H), (m, 2H), 2.84 (t, J = 7.6 Hz, 2H), 2.17 (s, 3H). 13 C NMR (1 MHz, CDCl 3 ): δ 29.2, 171., 14.9, 14.1, 131.6, 131., 13., 128.7, 128.6, 127., 126.9, 126.7, 126.3, 126.1, 123.3, 122.3, 121.2, 121.1, 69.6, 51.8, 39.3, 31.4, 3.7. MALDI-FTMS: calcd for C 27 H 25 NO 3 Na (MNa + ) , found N-[4-(1H-Benzoimidazol-2-yl)phenyl]-3-(4-formylphenyl)-propionamide (9). 1 H NMR (4 MHz, CDCl 3 -CD 3 OD): δ 9.95 (s, 1H), 8.1 (d, J = 8.5 Hz, 2H), 7.83 (d, J = 7.8 Hz, 2H), 7.69 (d, J = 8.5 Hz, 2H), (m, 2H), 7.46 (d, J = 7.8 Hz, 2H), (m, 2H), 3.14 (t, J = 7.6 Hz, 2H), 2.76 (t, J = 7.6 Hz, 2H). MALDI-FTMS: calcd for C 23 H 2 N 3 O 2 (MH + ) , found N-[4-(1H-Benzoimidazol-2-yl)phenyl]-3-(4-formylphenyl)-propionamide (1). 1 H NMR (4 MHz, CDCl 3 -CD 3 OD): δ 8.1 (d, J = 8.1 Hz, 2H), 7.68 (d, J = 8.1 Hz, 2H), (m, 2H), (m, 6H), 5.1 (m, 1H), 3.4 (t, J = 8. Hz, 2H), 2.93 (m, 1H), 2.76 (m, 1H), 2,7 S8

11 (t, J = 8. Hz, 2H), 2.19 (s, 3H). MALDI-FTMS: calcd for C 26 H 26 N 3 O 3 (MH + ) , found N-Fluoranthen-3-yl-3-(4-formylphenyl)-propionamide (11). 1 H NMR (4 MHz, CDCl 3 -CD 3 OD): δ 9.96 (s, 1H), (m, 13H), 3.21 (t, J = 7.6 Hz, 2H), 2.89 (t, J = 7.6 Hz, 2H). MALDI-FTMS: calcd for C 26 H 2 NO 2 (MH + ) , found N-Fluoranthen-3-yl-3-[4-(1-hydroxy-3-oxobutyl)phenyl]-propionamide (12). 1 H NMR (4 MHz, CDCl 3 -CD 3 OD): δ (m, 13H), 5.12 (dd, J = 3.5 Hz, 9.1 Hz, 1H), 3.12 (t, J = 7.3 Hz, 2H), 2.91 (dd, J = 9.1 Hz, 16.6 Hz, 1H), 2.86 (t, J = 7.3 Hz, 2H), 2.76 (dd, J = 3.5 Hz, 16.6 Hz, 1H), 2.19 (s, 3H). MALDI-FTMS: calcd for C 29 H 25 NO 3 (M + ) , found [4-(1,2-Dihydroxy-3-oxobutyl)phenyl]-N-phenanthren-9-yl-propionamide (14). 1 H NMR (3 MHz, CDCl 3 ) δ 8.69 (d, J = 8. Hz, 1H), 8.6 (d, J = 7.4 Hz, 1H), 8.1 (brs, 1H), (m, 1H), (m, 5H), (m, 4H), (m, 1H), (m, 1H), (m, 6H), 2.22 (s, 3H x 1/4), 1.96 (s, 3H x 3/4). MALDI-FTMS calcd for C 27 H 25 NO 4 (MNa + ): , found: [4-(1-Hydroxybut-3-enyl)phenyl]-N-phenanthren-9-yl-propionamide (15). A mixture of aldehyde 7 (9.3 mg,.26 mmol), allylbromide (5 µl,.58 mmol), In (9.1 mg,.79 mmol) in DMF (.4 ml)-h 2 O (.5 ml) was stirred at room temperature for 1.5 h. S2 The reaction mixture was added to sat-nh 4 Cl and extracted with EtOAc. The organic layers were washed with brine, dried over MgSO 4, filtered, concentrated in vacuo, and flash chromatographed (EtOAc/hexane = 2:3) to afford 15 (1. mg, 96%). 1 H NMR (5 MHz, CDCl 3 ): δ 8.72 (d, J = 8.1 Hz, 1H), 8.63 (d, J = 8.1 Hz, 1H), 8.19 (s, 1H), 7.87 (d, J = 7.4 Hz, 1H), (m, 6H), (m, 4H), 5.8 (m, 1H), (m, 2H), 4.75 (m, 1H), 3.18 (t, J = 7.4 Hz, 2H), 2.88 (t, J = 7.4 Hz, 2H), (m, 2H), 2. (brs, 1H). 13 C NMR (1 MHz, CDCl 3 ): δ 171.1, 142.1, 139.8, 134.4, 131.6, 131., 13., 128.6, 128.5, 126.9, 126.7, 126.3, 126.2, 123.3, 122.3, 121.3, 121.2, 118.5, 73., 43.8, 39.3, MALDI-FTMS calcd for C 27 H 25 NO 2 Na (MNa + ): , found: S9

12 3-(4-Hydroxymethylphenyl)-N-phenanthren-9-yl-propionamide (16). A mixture of aldehyde 7 (1. mg,.28 mmol) and NaBH 3 CN (6. mg,.95 mmol) in THF (.5 ml)-2- PrOH (.1 ml)-5 mm Na phosphate, ph 7. (.2 ml) was stirred at room temperature for 1 day. The reaction mixture was added to sat-nh 4 Cl and extracted with EtOAc. The organic layers were washed with brine, dried over MgSO 4, filtered, concentrated in vacuo, and flash chromatographed (EtOAc/hexane = 1:1.2) to afford 16 (4.3 mg, 43%). 1 H NMR (5 MHz, CDCl 3 -CD 3 OD): δ 8.72 (d, J =8.1 Hz, 1H), 8.65 (d, J = 7.7 Hz, 1H), 7.9 (s, 1H), 7.86 (d, J = 7. Hz, 1H), (m, 5H), 7.35 (d, J = 8.5 Hz, 2H), 7.33 (d, J = 8.5 Hz, 2H), 4.65 (s, 2H), 3.14 (t, J = 7.5 Hz, 2H), 2.88 (t, J = 7.5 Hz, 2H). MALDI-FTMS: calcd for C 24 H 21 NO 2 Na (MNa + ) , found References (S1) Sakthivel, K.; Notz, W.; Bui, T.; Barbas, C. F., III J. Am. Chem. Soc. 21, 123, 526. (S2) Chan, T. H.; Yang, Y. J. Am. Chem. Soc. 1999, 121, S1

13 Compound 1, 1 H NMR (4MHz, CDCl 3 ) Compound 2, 1 H NMR (5MHz, CDCl 3 ) S11

14 Compound 2, 13 C NMR (1MHz, CDCl 3 ) Compound 3, 1 H NMR (4MHz, CDCl 3 ) S12

15 Compound 4, 1 H NMR (4MHz, CDCl 3 -CD 3 OD) Compound 5, 1 H NMR (4MHz, CDCl 3 -CD 3 OD) S13

16 Compound 6, 1 H NMR (4MHz, CDCl 3 -CD 3 OD) Compound 7, 1 H NMR (4MHz, CDCl 3 -CD 3 OD) S14

17 Compound 7, 13 C NMR (1MHz, CDCl 3 -CD 3 OD) Compound 8, 1 H NMR (5MHz, CDCl 3 ) S15

18 Compound 8, 13 C NMR (1MHz, CDCl 3 ) Compound 9, 1 H NMR (4MHz, CDCl 3 -CD 3 OD) S16

19 Compound 1, 1 H NMR (4MHz, CDCl 3 -CD 3 OD) Compound 11, 1 H NMR (4MHz, CDCl 3 -CD 3 OD) S17

20 Compound 12, 1 H NMR (4MHz, CDCl 3 -CD 3 OD) Compound 14, 1 H NMR (3MHz, CDCl 3 ) S18

21 Compound 15, 1 H NMR (5MHz, CDCl 3 ) Compound 15, 13 C NMR (1MHz, CDCl 3 ) S19

22 Compound 16, 1 H NMR (5MHz, CDCl 3 -CD 3 OD) S2

Fluorescent Detection of Carbon-Carbon Bond Formation

Published on Web 06/21/2003 Fluorescent Detection of Carbon-Carbon Bond Formation Fujie Tanaka,* Rajeswari Thayumanavan, and Carlos F. Barbas III* Contribution from The Skaggs Institute for Chemical Biology