Published on Web 03/03/2004
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, 10550 North Torrey Pines Road, La Jolla, California 92037
Received January 20, 2004; E-mail: carlos@scripps.edu; ftanaka@scripps.edu
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 compounds3 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 transfer5 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 Aldolsa
wavelength (nm)
solvent
1,2
5,6
7,8
9,10
11,12
DMSO
DMF
pH 7
DMSO
DMSO
DMF
pH 7
DMSO
DMF
pH 7
DMSO
DMSO
λex
282
282
250
300
265
265
250
315
315
315
260
260
λem
360
360
352
360
385
385
380
360
360
360
380
450
fluorescence intensity
cb
aldehyde
aldol
foldc
50
50
50
50
5
5
5
5
5
5
25
25
1.7 ×
1.3 × 103
74d
1.1 × 102
4.9 × 102
4.6 × 102 d
57d
2.5 × 103
2.4 × 103
6.5 × 102
2.5 × 102 d
1.5 × 104 d
1.3 ×
1.2 × 104
1.9 × 103 d
5.7 × 102
8.7 × 103
4.2 × 103 d
4.4 × 103 d
4.5 × 104
4.5 × 104
4.2 × 103
8.6 × 102 d
8.2 × 103 d
8
9
26
5
19
9
78
18
18
6
3
0.5
103
104
a The fluorescence was recorded on a microplate spectrophotometer using
100 µL of solution composed of 0.5% CH3CN, 0.5% 2-PrOH, and 99% of
the indicated solvent in a 96-well polypropylene plate at 26 °C. Solvent
pH 7 refers to 50 mM sodium phosphate, pH 7.0. 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 ∼80-fold higher
fluorescence (λex 250 nm, λem 380 nm) than aldehyde 7 in aqueous
buffer (pH 7.0) and ∼20-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
3692
9
J. AM. CHEM. SOC. 2004, 126, 3692-3693
Figure 1. Fluorescence emission spectra (λex 250 nm) of aldehyde 7 (0),
aldol 8 (4), and fluorophore 13 (O) at 5 µM in 0.5% CH3CN-0.5%
2-PrOH-99% 50 mM sodium phosphate, pH 7.0.
buffer and in organic solvents, and the fluorescence intensity of 8
did not vary within the pH range of 5.3-8.0 in aqueous buffer. In
addition, the fluorescence of aldol 8 differed from that of fluorophore 13 as shown in Figure 1. Aldol 10 showed ∼20-fold higher
fluorescence than aldehyde 9 in DMSO. In contrast, aldehyde 11
showed higher fluorescence than aldol 12 at λex 260 nm and λem
10.1021/ja049641a CCC: $27.50 © 2004 American Chemical Society
COMMUNICATIONS
Scheme 3
Table 2. Fluorescence of Compounds 14-16a
solvent
14
15
16
DMSO
DMF
pH 7
DMSO
DMF
pH 7
DMSO
DMF
pH 7
λex
265
265
250
265
265
250
265
265
250
λem
385
385
380
385
385
380
385
385
380
cb
fluorescence
foldc
5
5
5
5
5
5
5
5
5
6.5 ×
2.6 × 103 d
4.4 × 103
1.3 × 104
5.6 × 103 d
3.2 × 103 d
5.8 × 103
2.7 × 103 d
3.0 × 103 d
13
6
77
26
12
57
12
6
53
103
a,b See Table 1 legend. c fold ) fluorescence intensity of 14, 15, or 16/
fluorescence intensity of aldehyde 7. d See Table 1 legend.
Figure 3. Fluorescence assay of reduction of aldehyde 7 with alcohol
dehydrogenase (ADH) from Thermoanaerobium brockii.‡ (A) Time course,
(B) emission spectra (λex 250 nm) at 50 min. Conditions: (a) [ADH] 0.235
unit/mL, [NADPH] 40 µM, [aldehyde 7] 12.5 µM, 0.5% CH3CN-0.5%
2-PrOH-99% 50 mM sodium phosphate, pH 7.0; (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 (340 nm) and
fluorescence (λem 450 nm) studies suggested that this enzyme contained
some reducing cofactor.
fashion to directly follow the reduction of the aldehyde. Formation
of less than 0.2 µM of product 16 was readily detected in a 100
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.3,9,10 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.
Figure 2. Fluorescence assay of antibody 38C2-catalyzed aldol reaction
of acetone and aldehyde 7. Conditions: [antibody] 2 µM (active site), [7]
50 µM, [acetone] 5%(v/v) (680 mM), 2.5% CH3CN-2.5% 2-PrOH/PBS
(pH 7.4). 0: 38C2; O: nonaldolase antibody IgG (control); ]: reaction
with 38C2 in the absence of acetone; 4: reaction without antibody (blank).
RFU ) relative fluorescence intensity.
450 nm, although 12 showed a slightly higher fluorescence at λex
260 nm and λem 380 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
38C28 (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 (340 nm) and fluorescence (λem 450 nm) of
NADPH, fluorogenic aldehyde 7 can be used in a complementary
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 http://
pubs.acs.org.
References
(1) (a) Matayoshi, E.; Wang, G. T.; Krafft, G.; Erickson, J. Science 1990,
247, 954. (b) Taylor, S. J.; Morken, J. P. Science 1998, 280, 267. (c)
Reetz, M. T.; Kuhling, K. M.; Deege, A.; Hinrichs, H.; Belder, D. Angew.
Chem., Int. Ed. 2000, 39, 3891. (d) Copeland, G. T.; Miller, S. J. J. Am.
Chem. Soc. 2001, 123, 6496. (e) Das, G.; Talukdar, P.; Matile, S. Science
2002, 298, 1600. (f) Stauffer, S. R.; Hartwig, J. F. J. Am. Chem. Soc.
2003, 125, 6977. (g) Konarzycka-Bessler, M.; Bornscheuer, U. Angew.
Chem., Int. Ed. 2003, 42, 1418.
(2) Nishino, N.; Powers, J. J. Biol. Chem. 1980, 255, 3482. List, B.; Barbas,
C. F., III; Lerner, R. A. Proc. Natl. Acad. Sci. U.S.A. 1998, 95, 15351.
Carlson, R. P.; Jourdain, N.; Reymond, J.-L. Chem. Eur. J. 2000, 6, 4154.
Svensson, R.; Greno, C.; Johansson, A.; Mannervik, B.; Morgenstern, R.
Anal. Biochem. 2002, 311, 171. Onoda, M.; Uchiyama, S.; Endo, A.;
Tokuyama, H.; Santa, T.; Imai, K. Org. Lett. 2003, 5, 1459.
(3) Tanaka, F.; Thayumanavan, R.; Barbas, C. F., III. J. Am. Chem. Soc. 2003,
125, 8523.
(4) Mase, N.; Tanaka, F.; Barbas, C. F., III. Org. Lett. 2003, 5, 4369. Tanaka,
F.; Thayumanavan, R.; Mase, N.; Barbas, C. F., III. Tetrahedron Lett.
2004, 45, 325.
(5) Lakowicz, J. R. Principles of Fluorescence Spectroscopy, 2nd ed.; Kluwer
Academic: New York, 1999; p 80. 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; p308.
(7) Chan, T. H.; Yang, Y. J. Am. Chem. Soc. 1999, 121, 3228.
(8) Wagner, J.; Lerner, R. A.; Barbas, C. F., III. Science 1995, 270, 1797.
Tanaka, F.; Barbas, C. F., III. J. Immunol. Methods 2002, 269, 67.
(9) Nakadai, M.; Saito, S.; Yamamoto, H. Tetrahedron 2002, 58, 8167.
Kofoed, J.; Nielsen, J.; Reymond, J.-L. Bioorg. Med. Chem. Lett. 2003,
13, 2445. Tanaka, F.; Barbas, C. F., III. J. Am. Chem. Soc. 2002, 124,
3510. Gildersleeve, J.; Varvak, A.; Atwell, S.; Evans, D.; Schultz, P. G.
Angew. Chem., Int. Ed. 2003, 42, 5971. Tanaka, F.; Fuller, R.; Shim, H.;
Lerner, R. A.; Barbas, C. F., III. J. Mol. Biol. 2004, 335, 1007. Fong, S.;
Machajewski, T. D.; Mak, C. C.; Wong, C.-H. Chem. Biol. 2000, 7, 873.
(10) Tsukiji, S.; Pattnaik, S. B.; Suga, H. Nat. Struct. Biol. 2003, 10, 713.
JA049641A
J. AM. CHEM. SOC.
9
VOL. 126, NO. 12, 2004 3693
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, 10550 North Torrey Pines Road, La Jolla, California
92037
Corresponding author e-mail: carlos@scripps.edu, ftanaka@scripps.edu
Supporting Information
Fluorescence Spectra ----------------------------------------- S2
Graphs of Standards of 8 and 16 --------------------------- S6
Synthesis and Characterization of Compounds ----------- S7
Hard copy of NMR ------------------------------------------- S11
S1
Fluorescence Spectra. Fuorescence was recorded on Spectra Max Gemini (Molecular Devices)
using 100 µL of a solution in a 96-well polypropyrene plate (Thomson Instrument Company
923175) at 26 °C. The data are shown after background correction.
Fluorescence intensity
60000
40000
20000
0
350
400
450
Emission wavelength (nm) (Excitation 282 nm)
Figure S1. Fluorescence emission spectra (λex 282 nm) of 1, 2, and 2-aminonaphthalene in 0.5%
CH3CN-0.5% 2-PrOH-99% DMSO. Square, 1 (50 µM); triangle, 2 (50 µM); circle, 2aminonaphthalene (50 µM).
Fluorescence intensity
6000
5000
4000
3000
2000
1000
0
320
340
360
380
400
420
440
460
Emission wavelength (nm) (Excitation 250 nm)
Figure S2. Fluorescence emission spectra (λex 250 nm) of 1, 2, and 2-aminonaphthalene in 0.5%
CH3CN-0.5% 2-PrOH-99% (50 mM Na phosphate, pH 7.0). Square, 1 (50 µM); triangle, 2 (50
µM); circle, 2-aminonaphthalene (50 µM).
S2
Fluorescence intensity
10000
8000
6000
4000
2000
0
320
340
360
380
400
420
440
460
480
Emission wavelength (nm) (Excitation 265 nm)
Figure S3. Fluorescence emission spectra (λex 265 nm) of 7, 8, and 13 in 0.5% CH3CN-0.5% 2PrOH-99% DMSO. Square, 7 (5 µM); triangle, 8 (5 µM); circle, 13 (5 µM).
Fluorescence intensity
4000
3000
2000
1000
0
320
340
360
380
400
420
440
460
480
Emission wavelength (nm) (Excitation 265 nm)
Figure S4. Fluorescence emission spectra (λex 265 nm) of 7, 8, and 13 in 0.5% CH3CN-0.5% 2PrOH-99% DMF. Square, 7 (5 µM); triangle, 8 (5 µM); circle, 13 (5 µM).
Fluorescence intensity
10000
8000
6000
4000
2000
0
250
260
270
280
290
300
310
320
330
Excitation wavelength (nm) (Emission 385 nm)
Figure S5. Fluorescence excitation spectra (λem 385 nm) of 7, 8, and 13 in 0.5% CH3CN-0.5% 2PrOH-99% DMSO. Square, 7 (5 µM); triangle, 8 (5 µM); circle, 13 (5 µM).
S3
Fluorescence intensity
5000
4000
3000
2000
1000
0
250
260
270
280
290
300
310
320
330
Excitation wavelength (nm) (Emission 385 nm)
Figure S6. Fluorescence excitation spectra (λem 385 nm) of 7, 8, and 13 in 0.5% CH3CN-0.5% 2PrOH-99% (50 mM Na phosphate, pH 7.0). Square, 7 (5 µM); triangle, 8 (5 µM); circle, 13 (5
µM).
Fluorescence intensity
50000
40000
30000
20000
10000
0
340
360
380
400
420
440
460
480
Emission wavelength (nm) (Excitation 315 nm)
Figure S7. Fluorescence emission spectra (λex 315 nm) of 9, 10, and 4-(1H-benzimidazol-2yl)aniline in 0.5% CH3CN-0.5% 2-PrOH-99% DMSO. Square, 9 (5 µM); triangle, 10 (5 µM);
circle, 4-(1H-benzimidazol-2-yl)aniline (5 µM).
S4
Fluorescence intensity
6000
4000
2000
0
320
340
360
380
400
420
440
460
480
Emission wavelength (nm) (Excitation 265 nm)
Figure S8. Fluorescence emission spectra (λex 265 nm) of 14 (5 µM) in 0.5% CH3CN-0.5% 2PrOH-99% DMSO.
Fluorescence intensity
15000
10000
5000
0
320
340
360
380
400
420
440
460
480
Emission wavelength (nm) (Excitation 265 nm)
Figure S9. Fluorescence emission spectra (λex 265 nm) of 15 (5 µM) in 0.5% CH3CN-0.5% 2PrOH-99% DMSO.
Fluorescence intensity
6000
5000
4000
3000
2000
1000
0
320
340
360
380
400
420
440
460
480
Emission wavelength (nm) (Excitation 265 nm)
Figure S10. Fluorescence emission spectra (λex 265 nm) of 16 (5 µM) in 0.5% CH3CN-0.5% 2PrOH-99% DMSO.
S5
RFU (λex 265 nm, λem 385 nm)
RFU (λex 265 nm, λem 385 nm)
10000
8000
6000
4000
2000
0
0
1
2
3
4
2500
y = 2270.231x + 90.592
r2 = 0.995
2000
1500
1000
500
0
0
5
0.2
8 (µM)
0.4
0.6
0.8
8 (µM)
RFU (λex 265 nm, λem 385 nm)
Figure S11. Standard of aldol 8 in 0.5% CH3CN-0.5% 2-PrOH-99% DMSO.
y = 2105.942x + 48.084
r2 = 0.994
2000
1500
1000
500
0
0
0.2
0.4
0.6
0.8
1
16 (µM)
Figure S12. Standard of alcohol 16 in 0.5% CH3CN-0.5% 2-PrOH-99% DMSO.
S6
1
3-(4-Formylphenyl)-N-naphthalen-2-yl-propionamide (1). A mixture of 3-(4formylphenyl)propionic acid (70.0 mg, 0.393 mmol), 2-aminonaphthalene (57.1 mg, 0.399 mmol),
1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (109.5 mg, 0.571 mmol), and
DMAP (1.0 mg, 0.008 mmol) in CH2Cl2 (8.0 mL) was stirred at room temperature for 2.5 h. The
reaction mixture was added to H 2O and extracted with CH2Cl2. The organic layers were washed
with brine, dried over MgSO4, filtered, concentrated in vacuo, and flash chromatographed
(EtOAc/hexane = 2:3) to afford 1 (83.5 mg, 70%). 1H NMR (400 MHz, CDCl3): δ 9.98 (s, 1H),
8.17 (s, 1H), 7.84-7.77 (m, 5H), 7.48-7.36 (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 C20H18NO2 (MH+) 304.1332, found 304.1333.
3-[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 1H NMR (500 MHz, CDCl3): δ 8.15 (s, 1H), 7.78-7.75 (m, 3H), 7.47-7.35 (m, 3H),
7.30-7.22 (m, 5H), 5.12 (ddd, J = 2.3 Hz, 2.6 Hz, 7.3 Hz, 1H), 3.27 (d, J = 2.3 Hz, 1H), 3.08 (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), 2.70 (t, J = 6.2
Hz, 2H), 2.18 (s, 3H).
13
C NMR (100 MHz, CDCl3): δ 209.2, 170.4, 140.8, 140.1, 135.1, 133.8,
130.6, 128.7, 128.6, 127.6, 127.5, 126.5, 126.0, 125.0, 119.7, 116.6, 69.6, 51.8, 39.4, 31.1, 30.7.
MALDI-FTMS: calcd for C23H23NO3Na (MNa+) 384.1570, found 384.1579.
4-Formyl-N-naphthalen-2-yl-benzamide (3). 1H NMR (400 MHz, CDCl3): δ 10.1 (s,
1H), 8.36 (brs, 1H), 8.10-8.00 (m, 4H), 7.88-7.82 (m, 3H), 7.60 (dd, J = 2.0 Hz, 8.8 Hz, 1H), 7.537.43 (m, 2H). MALDI-FTMS: calcd for C18H14O2N (MH+) 276.1019, found 276.1022.
4-(1-Hydroxy-3-oxobutyl)-N-naphthalen-2-yl-benzamide (4). 1H NMR (400 MHz,
CDCl3-CD3OD): δ 9.21 (s, 1H x 0.7), 8.32 (s, 1H), 7.90 (d, J = 8.2 Hz, 2H), 7.83-7.78 (m, 3H),
7.66 (dd, J = 2.0 Hz, 8.8 Hz, 1H), 7.49-7.40 (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 C21H20NO3 (MH+) 334.1438, found 334.1440.
3-(4-Formylphenyl)-N-naphthalen-1-yl-propionamide (5).
1
H NMR (400 MHz,
CDCl3-CD3OD): δ 9.98 (s, 1H), 7.86-7.83 (m, 3H), 7.71 (d, J = 8.2 Hz, 1H), 7.67 (d, J = 7.3 Hz,
S7
1H), 7.57 (d, J = 8.2 Hz, 1H), 7.50-7.38 (m, 5H), 3.20 (t, J = 7.6 Hz, 2H), 2.87 (t, J = 7.6 Hz, 2H).
MALDI-FTMS: calcd for C20H18NO2 (MH+) 304.1332, found 304.1331.
3-[4-(1-Hydroxy-3-oxobutyl)-phenyl]-N-naphthalen-1-yl-propionamide
(6).
1
H
NMR (400 MHz, CDCl3-CD3OD): δ 7.85 (m, 1H), 7.75-7.68 (m, 2H), 7.57 (m, 1H), 7.49-7.43 (m,
3H), 7.33-7.27 (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), 2.80 (t, J = 7.6 Hz, 2H), 2.77 (dd, J = 3.2 Hz, 17 Hz, 1H), 2.19 (s, 3H).
MALDI-FTMS: calcd for C23H23NO3Na (MNa+) 384.1570, found 384.1578.
3-(4-Formylphenyl)-N-phenanthren-9-yl-propionamide (7). 1H NMR (400 MHz,
CDCl3-CD3OD): δ 9.99 (s, 1H), 8.71 (d, J = 8.8 Hz, 1H), 8.63 (d, J = 7.9 Hz, 1H), 8.03 (s, 1H),
7.88-7.84 (m, 3H), 7.67-7.49 (m, 7H), 3.24 (t, J = 7.6 Hz, 2H), 2.91 (t, J = 7.6 Hz, 2H). 13C NMR
(100 MHz, CDCl3-CD3OD): δ 192.5, 171.8, 148.3, 134.5, 131.2, 130.8, 130.2, 130.0, 129.1, 128.8,
128.3, 127.5, 126.7, 126.5, 126.3, 122.8, 122.5, 122.2, 121.9, 37.6, 31.6. MALDI-FTMS: calcd for
C24H20NO2 (MH+) 354.1488, found 354.1488.
3-[4-(1-Hydroxy-3-oxobutyl)phenyl]-N-phenanthren-9-yl-propionamide
(8).
1
H
NMR (500 MHz, CDCl3): δ 8.69 (d, J = 8.1 Hz, 1H), 8.60 (d, J = 7.7 Hz, 1H), 8.13 (s, 1H), 7.83
(d, J = 7.3 Hz, 1H), 7.66-7.53 (m, 5H), 7.38 (s, 1H), 7.34-7.27 (m, 4H), 5.14 (m 1H), 3.30 (1H),
3.15 (t, J = 7.6 Hz, 2H), 2.88-2.75 (m, 2H), 2.84 (t, J = 7.6 Hz, 2H), 2.17 (s, 3H). 13C NMR (100
MHz, CDCl3): δ 209.2, 171.0, 140.9, 140.1, 131.6, 131.0, 130.0, 128.7, 128.6, 127.0, 126.9, 126.7,
126.3, 126.1, 123.3, 122.3, 121.2, 121.1, 69.6, 51.8, 39.3, 31.4, 30.7. MALDI-FTMS: calcd for
C27H25NO3Na (MNa+) 434.1727, found 434.1732.
N-[4-(1H-Benzoimidazol-2-yl)phenyl]-3-(4-formylphenyl)-propionamide (9).
1
H
NMR (400 MHz, CDCl3-CD3OD): δ 9.95 (s, 1H), 8.01 (d, J = 8.5 Hz, 2H), 7.83 (d, J = 7.8 Hz,
2H), 7.69 (d, J = 8.5 Hz, 2H), 7.62-7.60 (m, 2H), 7.46 (d, J = 7.8 Hz, 2H), 7.28-7.25 (m, 2H), 3.14
(t, J = 7.6 Hz, 2H), 2.76 (t, J = 7.6 Hz, 2H). MALDI-FTMS: calcd for C 23H 20N 3O 2 (MH+)
370.1550, found 370.1548.
N-[4-(1H-Benzoimidazol-2-yl)phenyl]-3-(4-formylphenyl)-propionamide (10). 1H
NMR (400 MHz, CDCl3-CD3OD): δ 8.01 (d, J = 8.1 Hz, 2H), 7.68 (d, J = 8.1 Hz, 2H), 7.65-7.56
(m, 2H), 7.30-7.23 (m, 6H), 5.10 (m, 1H), 3.04 (t, J = 8.0 Hz, 2H), 2.93 (m, 1H), 2.76 (m, 1H), 2,70
S8
(t, J = 8.0 Hz, 2H), 2.19 (s, 3H). MALDI-FTMS: calcd for C 26H 26N 3O 3 (MH+) 428.1969, found
428.1978.
N-Fluoranthen-3-yl-3-(4-formylphenyl)-propionamide (11). 1H NMR (400 MHz,
CDCl3-CD3OD): δ 9.96 (s, 1H), 7.99-7.35 (m, 13H), 3.21 (t, J = 7.6 Hz, 2H), 2.89 (t, J = 7.6 Hz,
2H). MALDI-FTMS: calcd for C26H20NO2 (MH+) 378.1488, found 378.1485.
N-Fluoranthen-3-yl-3-[4-(1-hydroxy-3-oxobutyl)phenyl]-propionamide
(12).
1
H
NMR (400 MHz, CDCl3-CD3OD): δ 8.01-7.28 (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 29H 25NO 3 (M+) 435.1834, found
435.1830.
3-[4-(1,2-Dihydroxy-3-oxobutyl)phenyl]-N-phenanthren-9-yl-propionamide
(14).
H NMR (300 MHz, CDCl3) δ 8.69 (d, J = 8.0 Hz, 1H), 8.60 (d, J = 7.4 Hz, 1H), 8.10 (brs, 1H),
1
7.90-7.80 (m, 1H), 7.78-7.45 (m, 5H), 7.45-7.25 (m, 4H), 5.10-4.85 (m, 1H), 4.50-4.25 (m, 1H),
3.80-2.72 (m, 6H), 2.22 (s, 3H x 1/4), 1.96 (s, 3H x 3/4). MALDI-FTMS calcd for C 27H 25NO 4
(MNa+): 450.1676, found: 450.1677.
3-[4-(1-Hydroxybut-3-enyl)phenyl]-N-phenanthren-9-yl-propionamide
(15).
A
mixture of aldehyde 7 (9.3 mg, 0.026 mmol), allylbromide (50 µL, 0.58 mmol), In (9.1 mg, 0.079
mmol) in DMF (0.4 mL)-H2O (0.05 mL) was stirred at room temperature for 1.5 h.S2 The reaction
mixture was added to sat-NH4Cl and extracted with EtOAc. The organic layers were washed with
brine, dried over MgSO4, filtered, concentrated in vacuo, and flash chromatographed
(EtOAc/hexane = 2:3) to afford 15 (10.0 mg, 96%). 1H NMR (500 MHz, CDCl3): 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), 7.70-7.55 (m, 6H),
7.37-7.30 (m, 4H), 5.80 (m, 1H), 5.20-5.12 (m, 2H), 4.75 (m, 1H), 3.18 (t, J = 7.4 Hz, 2H), 2.88 (t,
J = 7.4 Hz, 2H), 2.55-2.42 (m, 2H), 2.00 (brs, 1H). 13C NMR (100 MHz, CDCl3): δ 171.1, 142.1,
139.8, 134.4, 131.6, 131.0, 130.0, 128.6, 128.5, 126.9, 126.7, 126.3, 126.2, 123.3, 122.3, 121.3,
121.2, 118.5, 73.0, 43.8, 39.3, 31.4. MALDI-FTMS calcd for C 27H 25NO 2Na (MNa+): 418.1777,
found: 418.1777.
S9
3-(4-Hydroxymethylphenyl)-N-phenanthren-9-yl-propionamide (16). A mixture of
aldehyde 7 (10.0 mg, 0.028 mmol) and NaBH3CN (6.0 mg, 0.095 mmol) in THF (0.5 mL)-2PrOH (0.1 mL)-50 mM Na phosphate, pH 7.0 (0.2 mL) was stirred at room temperature for 1 day.
The reaction mixture was added to sat-NH4Cl and extracted with EtOAc. The organic layers were
washed with brine, dried over MgSO4, filtered, concentrated in vacuo, and flash chromatographed
(EtOAc/hexane = 1:1.2) to afford 16 (4.3 mg, 43%).
1
H NMR (500 MHz, CDCl3-CD3OD): δ
8.72 (d, J =8.1 Hz, 1H), 8.65 (d, J = 7.7 Hz, 1H), 7.90 (s, 1H), 7.86 (d, J = 7.0 Hz, 1H), 7.71-7.55
(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 24H 21NO 2Na (MNa+) 378.1464, found
378.1468.
References
(S1) Sakthivel, K.; Notz, W.; Bui, T.; Barbas, C. F., III J. Am. Chem. Soc. 2001, 123, 5260.
(S2) Chan, T. H.; Yang, Y. J. Am. Chem. Soc. 1999, 121, 3228.
S10
Compound 1, 1H NMR (400MHz, CDCl3)
Compound 2, 1H NMR (500MHz, CDCl3)
S11
Compound 2, 13C NMR (100MHz, CDCl3)
Compound 3, 1H NMR (400MHz, CDCl3)
S12
Compound 4, 1H NMR (400MHz, CDCl3-CD3OD)
Compound 5, 1H NMR (400MHz, CDCl3-CD3OD)
S13
Compound 6, 1H NMR (400MHz, CDCl3-CD3OD)
Compound 7, 1H NMR (400MHz, CDCl3-CD3OD)
S14
Compound 7, 13C NMR (100MHz, CDCl3-CD3OD)
Compound 8, 1H NMR (500MHz, CDCl3)
S15
Compound 8, 13C NMR (100MHz, CDCl3)
Compound 9, 1H NMR (400MHz, CDCl3-CD3OD)
S16
Compound 10, 1H NMR (400MHz, CDCl3-CD3OD)
Compound 11, 1H NMR (400MHz, CDCl3-CD3OD)
S17
Compound 12, 1H NMR (400MHz, CDCl3-CD3OD)
Compound 14, 1H NMR (300MHz, CDCl3)
S18
Compound 15, 1H NMR (500MHz, CDCl3)
Compound 15, 13C NMR (100MHz, CDCl3)
S19
Compound 16, 1H NMR (500MHz, CDCl3-CD3OD)
S20