PublishedonWeb03/03/2004 Design and Use of Fluorogenic Aldehydes for Monitoring the Progress of Aldehyde Transformations Fujie Tanaka,* Nobuyuki Mase, and Carlos F. Barbas, III* TheSkaggsInstituteforChemicalBiologyandtheDepartmentsofChemistryandMolecularBiology, TheScrippsResearchInstitute,10550NorthTorreyPinesRoad,LaJolla,California92037 ReceivedJanuary20,2004; E-mail:[email protected];[email protected] Simple and rapid methods for monitoring the progress of Scheme2 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 increaseinfluorescence.2Wehavepreviouslydevelopedfluorescent detectionstrategiestomonitorMichaelandDiels-Alderreactions usingfluorogenicR,(cid:226)-unsaturatedcarbonylcompounds3andhave demonstrated that the system is useful for evaluation of catalysts andreactionconditions.4Aldehydesareversatileandareusedfor Table1. FluorescenceofAldehydesandAldolsa many types of reactions. To develop systems for monitoring the wavelength(nm) fluorescenceintensity progressofaldehydetransformations,anentirelynewapproachwas required. Here we report the first design, synthesis, and use of solvent (cid:236)ex (cid:236)em cb aldehyde aldol foldc fluorogenicaldehydesfordirectmonitoringofaldehydetransforma- 1,2 DMSO 282 360 50 1.7(cid:2)103 1.3(cid:2)104 8 tionsbyfluorescencegrowth. DMF 282 360 50 1.3(cid:2)103 1.2(cid:2)104 9 pH7 250 352 50 74d 1.9(cid:2)103d 26 Our design is based on resonance energy transfer5 between a 5,6 DMSO 300 360 50 1.1(cid:2)102 5.7(cid:2)102 5 fluorophoreandanaldehydeinasinglemolecule.Thefluorogenic 7,8 DMSO 265 385 5 4.9(cid:2)102 8.7(cid:2)103 19 aldehydesarecomposedofafluorophoreandanaldehydemoiety DMF 265 385 5 4.6(cid:2)102d 4.2(cid:2)103d 9 coupled by a linker. When intact, the aldehyde moiety acts as a pH7 250 380 5 57d 4.4(cid:2)103d 78 9,10 DMSO 315 360 5 2.5(cid:2)103 4.5(cid:2)104 18 quencherofthefluorophore’sfluorescence;however,thereaction DMF 315 360 5 2.4(cid:2)103 4.5(cid:2)104 18 productofthealdehydemoietydoesnotquenchfluorescenceand pH7 315 360 5 6.5(cid:2)102 4.2(cid:2)103 6 fluorescence is “turned-on” in the product. We reasoned that an 11,12 DMSO 260 380 25 2.5(cid:2)102d 8.6(cid:2)102d 3 arylaldehydewouldquenchthefluorescenceofaproximalfluoro- DMSO 260 450 25 1.5(cid:2)104d 8.2(cid:2)103d 0.5 phore,andthatasimplearylgroupwithoutacarbonylwouldnot.6 aThefluorescencewasrecordedonamicroplatespectrophotometerusing To test this hypothesis, we prepared the aldehyde 1 and aldol 2 100(cid:237)Lofsolutioncomposedof0.5%CH3CN,0.5%2-PrOH,and99%of shown in Scheme 1. As expected, aldol 2 showed a higher fluo- theindicatedsolventina96-wellpolypropyleneplateat26 (cid:176) C.Solvent pH7refersto50mMsodiumphosphate,pH7.0.Thedataareshownafter rescence than aldehyde 1 (Table 1). On the other hand, neither backgroundcorrectionexceptwherenoted.bc)concentrationofaldehyde aldehyde 3 nor aldol 4 was fluorescent. Note that in 4, the aryl oraldol((cid:237)M).cfold)fluorescenceintensityofaldol/fluorescenceintensity groupconjugatedtothefluorophoreviaanamidebondquenched ofaldehyde.dThedatawithoutbackgroundcorrection. thefluorophore’sfluorescence. Scheme1 Figure1. Fluorescenceemissionspectra((cid:236)ex250nm)ofaldehyde7(0), We prepared candidate fluorogenic aldehydes and their aldols (5-12,Scheme2)byusingaseriesoffluorophoresandcompared a2l-dPorlOH8-(499),%an5d0mfluMorsoopdhiourme 1p3ho(sOph)ataet,5pH(cid:237)M7.0.in 0.5% CH3CN-0.5% theirfluorescence(Table1).Aldehyde7,preparedastheamideof 9-aminophenanthrene(13),wasthemostpromisingofthealdehydes bufferandinorganicsolvents,andthefluorescenceintensityof8 prepared. The reaction product, aldol 8, showed (cid:24)80-fold higher didnotvarywithinthepHrangeof5.3-8.0inaqueousbuffer.In fluorescence((cid:236)ex250nm,(cid:236)em380nm)thanaldehyde7inaqueous addition,thefluorescenceofaldol8differedfromthatoffluoro- buffer (pH 7.0) and (cid:24)20-fold higher ((cid:236)ex 265 nm, (cid:236)em 385 nm) phore13asshowninFigure1.Aldol10showed(cid:24)20-foldhigher inDMSO.Althoughthefluorescenceintensityvariedwithsolvent, fluorescence than aldehyde 9 in DMSO. In contrast, aldehyde11 aldol8/aldehyde7hadanexcellentfluorogenicrangeinaqueous showedhigherfluorescencethanaldol12at(cid:236)ex260nmand(cid:236)em 3692 9 J.AM.CHEM.SOC. 2004,126,3692-3693 10.1021/ja049641aCCC:$27.50 ©2004AmericanChemicalSociety COMMUNICATIONS Scheme3 Figure3. Fluorescence assay of reduction of aldehyde 7 with alcohol Table2. FluorescenceofCompounds14-16a dehydrogenase(ADH)fromThermoanaerobiumbrockii.‡(A)Timecourse, (B)emissionspectra((cid:236)ex250nm)at50min.Conditions: (a)[ADH]0.235 solvent (cid:236)ex (cid:236)em cb fluorescence foldc unit/mL, [NADPH] 40 (cid:237)M, [aldehyde 7] 12.5 (cid:237)M, 0.5% CH3CN-0.5% 14 DMSO 265 385 5 6.5(cid:2)103 13 2-PrOH-99% 50 mM sodium phosphate, pH 7.0; (b) reaction without DMF 265 385 5 2.6(cid:2)103d 6 additionofNADPH;(c)reactionusing3insteadof7;(d)reactionwithout pH7 250 380 5 4.4(cid:2)103 77 ADH; (e) reaction without ADH and NADPH. ‡The UV (340 nm) and 15 DMSO 265 385 5 1.3(cid:2)104 26 fluorescence((cid:236)em450nm)studiessuggestedthatthisenzymecontained DMF 265 385 5 5.6(cid:2)103d 12 somereducingcofactor. pH7 250 380 5 3.2(cid:2)103d 57 fashiontodirectlyfollowthereductionofthealdehyde.Formation 16 DMSO 265 385 5 5.8(cid:2)103 12 of less than 0.2 (cid:237)M of product 16 was readily detected in a 100 DMF 265 385 5 2.7(cid:2)103d 6 pH7 250 380 5 3.0(cid:2)103d 53 (cid:237)L-scalereactionina96-wellplate. Wehavedevelopedfluorogenicaldehydesthatcanbeusedfor a,bSeeTable1legend.cfold)fluorescenceintensityof14,15,or16/ monitoring reactions through increased fluorescence. These fluo- fluorescenceintensityofaldehyde7.dSeeTable1legend. rogenic aldehydes should be useful for screening of catalysts in approachesusinglibraries.3,9,10Ourstrategyforaccessingfluoro- genic aldehydes should also be applicable to the preparation of fluorogenic substrates that allow the transformations of other functionalgroupstobedirectlymonitored. Acknowledgment. ThisstudywassupportedinpartbytheNIH (CA27489)andTheSkaggsInstituteforChemicalBiology. SupportingInformationAvailable: Fluorescencespectra,graphs ofstandardsof8and16,synthesisandcharacterizationofcompounds (PDF).ThismaterialisavailablefreeofchargeviatheInternetathttp:// Figure2. Fluorescenceassayofantibody38C2-catalyzedaldolreaction pubs.acs.org. ofacetoneandaldehyde7.Conditions: [antibody]2(cid:237)M(activesite),[7] 50(cid:237)M,[acetone]5%(v/v)(680mM),2.5%CH3CN-2.5%2-PrOH/PBS References (pH7.4).0: 38C2;O: nonaldolaseantibodyIgG(control);]: reaction with38C2intheabsenceofacetone;4: reactionwithoutantibody(blank). (1) (a)Matayoshi,E.;Wang,G.T.;Krafft,G.;Erickson,J.Science1990, 247, 954. (b) Taylor, S. J.; Morken, J. P. Science 1998, 280, 267. (c) RFU)relativefluorescenceintensity. 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. 450nm,although12showedaslightlyhigherfluorescenceat(cid:236)ex Chem.Soc.2001,123,6496.(e)Das,G.;Talukdar,P.;Matile,S.Science 260 nm and (cid:236)em 380 nm. These results indicate that the proper 2002,298,1600.(f)Stauffer,S.R.;Hartwig,J.F.J.Am.Chem.Soc. 2003,125,6977.(g)Konarzycka-Bessler,M.;Bornscheuer,U.Angew. selectionoffluorophoresisimportantforthepreparationofuseful Chem.,Int.Ed.2003,42,1418. fluorogenicaldehydes. (2) Nishino,N.;Powers,J.J.Biol.Chem.1980,255,3482.List,B.;Barbas, Toexaminetheapplicabilityofthefluorogenicaldehydestoother 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. reactions,aldehyde7wastransformedtoaldol14byaldolreaction Svensson,R.;Greno,C.;Johansson,A.;Mannervik,B.;Morgenstern,R. withhydroxyacetone,toallylalcohol15byIn-mediatedallylation,7 Anal. Biochem. 2002, 311, 171. Onoda, M.; Uchiyama, S.; Endo, A.; Tokuyama,H.;Santa,T.;Imai,K.Org.Lett.2003,5,1459. and to alcohol 16 by reduction (Scheme 3). These products were (3) Tanaka,F.;Thayumanavan,R.;Barbas,C.F.,III.J.Am.Chem.Soc.2003, allfluorescent(Table2),indicatingthatthelossof(cid:240)-conjugation 125,8523. (4) Mase,N.;Tanaka,F.;Barbas,C.F.,III.Org.Lett.2003,5,4369.Tanaka, between the aldehyde carbonyl and the aryl group is key to F.;Thayumanavan,R.;Mase,N.;Barbas,C.F.,III.TetrahedronLett. fluorescence and that aldehyde 7 can be used as a fluorogenic 2004,45,325. (5) Lakowicz,J.R.PrinciplesofFluorescenceSpectroscopy,2nded.;Kluwer substrateformanyreactions. Academic: NewYork,1999;p80.Seealsoref1fandreferencestherein. Tomonitorthetime-courseofanaldolreaction,westudiedthe (6) BenzaldehydehasanRband((cid:236)max328nminalcohol).Silverstein,R. M.;Bassler,G.C.;Morrill,T.C.SpectrometricIdentificationofOrganic reactionofacetoneandaldehyde7catalyzedbyaldolaseantibody Compounds,5thed.;JohnWiley&Sons: NewYork,1991;p308. 38C28 (Figure 2). The reaction with antibody 38C2 showed a (7) Chan,T.H.;Yang,Y.J.Am.Chem.Soc.1999,121,3228. (8) Wagner,J.;Lerner,R.A.;Barbas,C.F.,III.Science1995,270,1797. significantincreaseinfluorescence,whilereactionwithacontrol Tanaka,F.;Barbas,C.F.,III.J.Immunol.Methods2002,269,67. antibody,reactionwithoutacetone,andreactionwithoutantibody (9) Nakadai, M.; Saito, S.; Yamamoto, H. Tetrahedron 2002, 58, 8167. Kofoed,J.;Nielsen,J.;Reymond,J.-L.Bioorg.Med.Chem.Lett.2003, allshowedlittleornoincreaseinfluorescence.Catalyticreduction 13,2445.Tanaka,F.;Barbas,C.F.,III.J.Am.Chem.Soc.2002,124, of7withalcoholdehydrogenaseinthepresenceofNADPHwas 3510.Gildersleeve,J.;Varvak,A.;Atwell,S.;Evans,D.;Schultz,P.G. successfully monitored by observing an increase in fluorescence 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.; (Figure3).Althoughreactionswiththisenzymecanbemonitored Machajewski,T.D.;Mak,C.C.;Wong,C.-H.Chem.Biol.2000,7,873. by changes in UV (340 nm) and fluorescence ((cid:236)em 450 nm) of (10) Tsukiji,S.;Pattnaik,S.B.;Suga,H.Nat.Struct.Biol.2003,10,713. NADPH,fluorogenicaldehyde7canbeusedinacomplementary JA049641A J.AM.CHEM.SOC.9VOL.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: [email protected], [email protected] 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. 60000 y sit n e nt 40000 e i c n e c es 20000 or u Fl 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% CH CN-0.5% 2-PrOH-99% DMSO. Square, 1 (50 µM); triangle, 2 (50 µM); circle, 2- 3 aminonaphthalene (50 µM). 6000 sity 5000 n e nt 4000 e i nc 3000 e c s e 2000 or u Fl 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% CH CN-0.5% 2-PrOH-99% (50 mM Na phosphate, pH 7.0). Square, 1 (50 µM); triangle, 2 (50 3 µM); circle, 2-aminonaphthalene (50 µM). S2 10000 y 8000 sit n e nt 6000 e i c n e 4000 c s e or u 2000 Fl 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% CH CN-0.5% 2- 3 PrOH-99% DMSO. Square, 7 (5 µM); triangle, 8 (5 µM); circle, 13 (5 µM). 4000 y sit 3000 n e nt e i 2000 c n e c es 1000 or u Fl 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% CH CN-0.5% 2- 3 PrOH-99% DMF. Square, 7 (5 µM); triangle, 8 (5 µM); circle, 13 (5 µM). 10000 sity 8000 n e nt 6000 e i c n ce 4000 s e or u 2000 Fl 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% CH CN-0.5% 2- 3 PrOH-99% DMSO. Square, 7 (5 µM); triangle, 8 (5 µM); circle, 13 (5 µM). S3 5000 sity 4000 n e nt 3000 e i c n e 2000 c s e or u 1000 Fl 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% CH CN-0.5% 2- 3 PrOH-99% (50 mM Na phosphate, pH 7.0). Square, 7 (5 µM); triangle, 8 (5 µM); circle, 13 (5 µM). 50000 sity 40000 n e nt 30000 e i c n ce 20000 s e or u 10000 Fl 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-2- yl)aniline in 0.5% CH CN-0.5% 2-PrOH-99% DMSO. Square, 9 (5 µM); triangle, 10 (5 µM); 3 circle, 4-(1H-benzimidazol-2-yl)aniline (5 µM). S4 6000 y sit n e e int 4000 c n e c es 2000 or u Fl 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% CH CN-0.5% 2- 3 PrOH-99% DMSO. 15000 y sit n e 10000 nt e i c n e c s 5000 e or u Fl 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% CH CN-0.5% 2- 3 PrOH-99% DMSO. 6000 y sit 5000 n e nt 4000 e i c 3000 n e c s 2000 e or u 1000 Fl 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% CH CN-0.5% 2- 3 PrOH-99% DMSO. S5 nm) 10000 m) 2500 y = 2270.231x + 90.592 385 8000 85 n r2 = 0.995 em m 3 2000 λnm, 6000 λm, e 1500 65 4000 5 n 1000 2 6 ex x 2 λFU ( 2000 λU (e 500 R 0 RF 0 0 1 2 3 4 5 0 0.2 0.4 0.6 0.8 1 8 (µM) 8 (µM) Figure S11. Standard of aldol 8 in 0.5% CH CN-0.5% 2-PrOH-99% DMSO. 3 m) n y = 2105.942x + 48.084 5 2000 2 8 r = 0.994 3 m e 1500 λ m, n 5 1000 6 2 x e 500 λ U ( F R 0 0 0.2 0.4 0.6 0.8 1 16 (µM) Figure S12. Standard of alcohol 16 in 0.5% CH CN-0.5% 2-PrOH-99% DMSO. 3 S6 3-(4-Formylphenyl)-N-naphthalen-2-yl-propionamide (1). A mixture of 3-(4- formylphenyl)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 CH Cl (8.0 mL) was stirred at room temperature for 2.5 h. The 2 2 reaction mixture was added to H O and extracted with CH Cl . The organic layers were washed 2 2 2 with brine, dried over MgSO , filtered, concentrated in vacuo, and flash chromatographed 4 (EtOAc/hexane = 2:3) to afford 1 (83.5 mg, 70%). 1H NMR (400 MHz, CDCl ): δ 9.98 (s, 1H), 3 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 C H NO (MH+) 304.1332, found 304.1333. 20 18 2 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, CDCl ): δ 8.15 (s, 1H), 7.78-7.75 (m, 3H), 7.47-7.35 (m, 3H), 3 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). 13C NMR (100 MHz, CDCl ): δ 209.2, 170.4, 140.8, 140.1, 135.1, 133.8, 3 130.6, 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 C H NO Na (MNa+) 384.1570, found 384.1579. 23 23 3 4-Formyl-N-naphthalen-2-yl-benzamide (3). 1H NMR (400 MHz, CDCl ): δ 10.1 (s, 3 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), 7.53- 7.43 (m, 2H). MALDI-FTMS: calcd for C H O N (MH+) 276.1019, found 276.1022. 18 14 2 4-(1-Hydroxy-3-oxobutyl)-N-naphthalen-2-yl-benzamide (4). 1H NMR (400 MHz, CDCl -CD OD): 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), 3 3 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 C H NO (MH+) 334.1438, found 334.1440. 21 20 3 3-(4-Formylphenyl)-N-naphthalen-1-yl-propionamide (5). 1H NMR (400 MHz, CDCl -CD OD): 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, 3 3 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 C H NO (MH+) 304.1332, found 304.1331. 20 18 2 3-[4-(1-Hydroxy-3-oxobutyl)-phenyl]-N-naphthalen-1-yl-propionamide (6). 1H NMR (400 MHz, CDCl -CD OD): δ 7.85 (m, 1H), 7.75-7.68 (m, 2H), 7.57 (m, 1H), 7.49-7.43 (m, 3 3 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 C H NO Na (MNa+) 384.1570, found 384.1578. 23 23 3 3-(4-Formylphenyl)-N-phenanthren-9-yl-propionamide (7). 1H NMR (400 MHz, CDCl -CD OD): 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), 3 3 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, CDCl -CD OD): δ 192.5, 171.8, 148.3, 134.5, 131.2, 130.8, 130.2, 130.0, 129.1, 128.8, 3 3 128.3, 127.5, 126.7, 126.5, 126.3, 122.8, 122.5, 122.2, 121.9, 37.6, 31.6. MALDI-FTMS: calcd for C H NO (MH+) 354.1488, found 354.1488. 24 20 2 3-[4-(1-Hydroxy-3-oxobutyl)phenyl]-N-phenanthren-9-yl-propionamide (8). 1H NMR (500 MHz, CDCl ): δ 8.69 (d, J = 8.1 Hz, 1H), 8.60 (d, J = 7.7 Hz, 1H), 8.13 (s, 1H), 7.83 3 (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, CDCl ): 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, 3 126.3, 126.1, 123.3, 122.3, 121.2, 121.1, 69.6, 51.8, 39.3, 31.4, 30.7. MALDI-FTMS: calcd for C H NO Na (MNa+) 434.1727, found 434.1732. 27 25 3 N-[4-(1H-Benzoimidazol-2-yl)phenyl]-3-(4-formylphenyl)-propionamide (9). 1H NMR (400 MHz, CDCl -CD OD): δ 9.95 (s, 1H), 8.01 (d, J = 8.5 Hz, 2H), 7.83 (d, J = 7.8 Hz, 3 3 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 H N O (MH+) 23 20 3 2 370.1550, found 370.1548. N-[4-(1H-Benzoimidazol-2-yl)phenyl]-3-(4-formylphenyl)-propionamide (10). 1H NMR (400 MHz, CDCl -CD OD): δ 8.01 (d, J = 8.1 Hz, 2H), 7.68 (d, J = 8.1 Hz, 2H), 7.65-7.56 3 3 (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