MAGNETIC RESONANCE IN CHEMISTRYMagn. Reson. Chem.2000;38: 51–54

Note

Structural elucidation of two novel products from thesoybean lipoxygenase-catalysed dioxygenation of curcumin

Gabor Toth,1∗ Monika Roth,2 Bernhard Weckerle2 and Peter Schreier2

1 Technical Analytical Research Group of the Hungarian Academy of Sciences, Institute for General and Analytical Chemistry, TechnicalUniversity of Budapest, Szent Gellert ter 4, H-1111 Budapest, Hungary2 Lehrstuhl fur Lebensmittelchemie, Universitat Wurzburg, Am Hubland, D-97074 Wurzburg, Germany

Received 2 June 1999; revised 27 August 1999; accepted 30 August 1999

ABSTRACT: The conversion of curcumin by soybean lipoxygenase leads to three stereoisomers of 2-[(40-hydroxy-30-methoxy)phenoxy]-4-(400-hydroxy-300-methoxy-(phenyl)-8-hydroxy-6-oxo-3-oxabicyclo[3.3.0]-7-octene. Their struc-tures and conformational behaviour were elucidated by various1H and13C NMR methods and high-performance liquidchromatography–electrospray ionization tandem mass spectrometry. Copyright 2000 John Wiley & Sons, Ltd.

KEYWORDS: NMR; 1H NMR; 13C NMR; stereochemistry; conformation, 2-[(40-hydroxy-30-methoxy)phenoxy]-4-(400-hydroxy-300-methoxyphenyl)-8-hydroxy-6-oxo-3-oxabicyclo[3.3.0]-7-octene

INTRODUCTION

Natural curcumin, a mixture of at least three relatedcompounds [curcumin (1), demethoxycurcumin and bis-demethoxycurcumin] is widely used as a spice andfood colouring agent. Its antioxidant and antitumori-genetic properties have recently been summarized.1 Inaddition, 1 exhibits anti-inflammatory effects2 and is apotent inhibitor of cyclooxygenase and lipoxygenase inrat colon mucosa.3 In the course of studies on the antioxi-dant mechanism of curcumin, its radical reactions wereinvestigated.4 Our recent investigations on unconven-tional lipoxygenase (LOX) substrates revealed1 to be anexcellent substrate for the enzyme from soybean, a non-haem, iron-containing dioxygenase. The major productof the LOX-catalysed conversion of1 has been char-acterized as 2-[(40-hydroxy-30-methoxy)phenoxy]-4-(400-hydroxy-300-methoxyphenyl)-8-hydroxy-6-oxo-3-oxabi-cyclo[3.3.0]-7-octene (2).5 In this paper, we describe theisolation and characterization of two novel products,3and 4 (Scheme 1). Their structures were elucidated byextensive one- and two-dimensional1H and 13C NMRspectroscopy and on-line high-performance liquid chro-matography–electrospray ionization tandem mass spec-trometry (HPLC–ESI-MS/MS).

* Correspondence to: G. Toth, Technical and Analytical ResearchGroup of the Hungarian Academy of Sciences, Institute for General andAnalytical Chemistry, Technical University of Budapest, Szent Gellertter 4, H-1111 Budapest, Hungary.E-mail: g-toth@chem.bme.huContract/grant sponsor: Deutsche Forschungsgemeinschaft;Con-tract/grant number: SFB 347.Contract/grant sponsor: Hungarian Scientific Research Fund;Con-tract/grant number: OTKA T026264.

EXPERIMENTAL

Conversion of curcumin

An ethanolic solution of 100 mg.272µmol/ of 1 and50 mg .0.52µmol/ of LOX was added to 1 l of 0.1Mborate buffer (pH 8.3). The mixture was stirred underoxygen for 3 h at ambient temperature. The reaction wasstopped by adjusting the pH to 4 (hydrochloric acid),then sodium chloride was added up to saturation andthe mixture was extracted three times with diethyl etherand three times with ethyl acetate. The combined organicextracts were dried over anhydrous sodium sulphate, thesolvent was evaporated under vacuum and the residue wastaken up in ethanol. Large-scale isolation of a mixtureof 3 and 4 was performed by preparative HPLC on aLiChrospher 100 NH2 column (250ð 16 mm i.d., 5µm)(Knauer, Berlin, Germany) using isocratic elution withethanol–water (90 : 10) at a flow-rate of 5 ml min�1 (detec-tion at 230 nm).

(HPLC–ESI-MS/MS)

Mass spectrometry was performed on a Finnigan triple-stage quadrupole TSQ 7000 HPLC–MS/MS system(Finnigan MAT, Bremen, Germany) with electrospray ion-ization (ESI) as interface in positive mode. For HPLC, anApplied Biosystems (Foster City, CA, USA) Model 140Bpump was used. For ESI in the positive-ion mode thespray capillary voltage was set to 4 kV and the temperatureof the heated inlet capillary was 200°C. Nitrogen servedboth as sheath gas (482 MPa) and as auxiliary gas. HPLCwas carried out on an Eurospher 100 C-18 column (100ð2 mm i.d., 5µm) (Knauer) with a water (5 mM ammonium

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52 G. TOTH ET AL.

Scheme 1. Formation of reaction products 2–4 from the LOX-catalysed dioxygenation of curcumin (1) and relativeconfiguration of the tetrahydrofuran ring protons of isomers 2–4. The ˛ and ˇ symbols indicate the positions of H-1, H-2,H-4 and H-5 below and above the reference plane of the tetrahydrofuran ring, respectively.

acetate, 0.05% trifluoroacetic acid)–methanol gradient at aflow-rate of 0.2 ml min�1 from 10 to 100% methanol withthe following gradient: 0 min 10% methanol! 18 min100% methanol! 20 min 100% methanol! 23 min10% methanol.

NMR spectroscopy. NMR spectra were measured onBruker Avance DMX-600 and DRX-500 spectrometersat room temperature in acetone-d6. Chemical shifts aregiven on theυ scale:1H NMR spectra were referencedto internal TMS and13C NMR spectra to the solvent.υMe2CO D 29.8 ppm/. In the 1D measurements 64Kdata points were acquired. The FIDs were zero-filled to128K data points. The pulse programs were taken from theBruker software library and the parameters were appliedas reported previously.6 The HMBC measurements wereoptimized for 7 Hz long-range couplings; in the phase-sensitive NOESY experiments, a 500 ms mixing time wasapplied.

RESULTS AND DISCUSSION

HPLC–ESI-MS analysis in the positive-ion mode gaveidentical data for3 and 4, i.e. [MCMeOHC Na]C m/z455, the methanol adduct ion [MCMeOHC H]C m/z 433as the base peak, the ammonium adduct ion [MC NH4]C

m/z 418 and the molecular adduct ion [MC H]C m/z 401.The main product2 showed the same mass spectrum;thus, products2–4 exhibited the same molecular mass of400. MS/MS studies of3 and4 (product ions ofm/z 401,collision energy�21 eV, 0.24 Pa argon) yieldedm/z 249

[C13H13O5]C as the base peak, matching the fragmentationpattern of product2 and affirming structural analogiesbetween2–4. HPLC analysis of3 and 4 employing thechiral phase Chiralcel OD-H resulted in two peaks ofidentical area for each isomer, clearly indicating racemicmixtures of both3 and4.

Despite extended efforts to isolate pure products on apreparative scale,3 and4 could not be separated. There-fore structural elucidation of3 and4 and the assignment oftheir 1H and13C NMR spectra were achieved by analysisof the mixture. Because the NMR spectra of the mixturecontained two sets of signals with different intensities,assignment of the chemical shifts to each isomer was pos-sible. Both sets of13C NMR signals consisted of 21 peaks(seven CH, four CH, two CH3O and eight quaternarysp2 carbons), which indicated the presence of isomers.Unequivocal1H and13C signal and structure assignmentswere achieved by extensive application of one- and two-dimensional NMR spectroscopy utilizing the advantage ofgradient selection and linear prediction. DEPT, gs-COSY,gs-HMQC and gs-HMBC measurements indicating1H, 1Hand1H, 13C one-bond and1H, 13C long-range correlationswere run. Spatial proximities were determined by phase-sensitive NOESY experiments. The characteristic1H and13C NMR data are summarized in Table 1.

Hydroperoxides were the major products formed byLOX-catalysed dioxygenation of (Z,Z)-1,4-pentadienoicfatty acids;7 however, in the case of curcimin wecould not detect hydroperoxide products. Conversionof curcumin with LOX resulted in the exclusiveformation of 2-[(40-hydroxy-30-methoxy)phenoxy]-4-(400-hydroxy-300-methoxyphenyl)-8-hydroxy-6-oxo-3-oxabicy-

Copyright 2000 John Wiley & Sons, Ltd. Magn. Reson. Chem.2000;38: 51–54

STRUCTURES OF DIOXYGENATION ON PRODUCTS OF CURCUMIN 53

Table 1. 1H and 13C chemical shifts and characteristic HMBC and NOESY responses of 3 and 4 in acetone-d6

2 3 4

HMBC HMBC1Ha 13C 1Hb 13C 13C partners NOESY 1Hc 13C 13C partners NOESY

1 3.63 55.8 3.72 56.5 2;6;7;8 2;5 4.02 53.5 5;6;8 2;52 5.90 97.3 5.83 100.6 1;4;5;8 1 5.81 96.5 1;4;5 14 5.41 80.2 5.07 84.1 1;2;5;6;100;200;600 5;600 5.12 78.5 6;100;200;600 5;200

5 3.26 54.8 3.33 59.1 4;6;8;100 1;4;200;600 3.16 60.2 1;4;6;8;100 1;4;200

6 — 200.8 — 203.8 — 203.77 4.89 109.4 4.95 106.4 1;5;6 4.90 107.7 1;5;68 — 187.9 — 189.0 — 187.510 — 148.4 — 148.4 — 148.620 6.92 105.4 6.95 105.6 40;60 6.86 105.7 40;60

30 — 149.0 — 149.2 — 149.040 — 145.6 — 145.9 — 145.750 6.87 115.9 6.90 116.0 10;30;40 6.88 115.9 10;30;40

60 6.71 112.9 6.73 113.0 40 6.70 113.3 40

MeO-30 3.87 56.4 3.88 56.6 30 3.86 56.5 30

100 — 129.8 — 135.8 — 134.0200 6.84 111.5 7.23 111.2 4;100;300;400;600 5 7.09 110.7 4;100;300;400;600 4;5300 — 147.6 — 148.0 — 148.2400 — 146.7 — 146.6 — 146.8500 6.69 114.9 6.78 115.3 100;300;400 600 6.80 115.5 100;300;400 600

600 6.74 120.5 6.98 119.8 4;200;400;500 4;5;500 6.93 119.6 4;200;400;500 500

MeO-300 3.76 56.1 3.84 56.2 300 3.84 56.2 300

a 3J.H-1,H-2/ D 0 Hz, 3J.H-4,H-5/ D 8.2 Hz, 3J.H-1,H-5/ D 6.3 Hz.b 3J.H-1,H-2/ D 1.5 Hz, 3J.H-4,H-5/ D 3.9 Hz, 3J.H-1,H-5/ D 7.1 Hz.c 3J.H-1,H-2/ D 5.8 Hz, 3J.H-4,H-5/ D 7.0 Hz, 3J.H-1,H-5/ D 8.5 Hz.

clo[3.3.0]-7-octene, which contains four stereocentres(C-1, C-2, C-4, C-5). We found products3 and 4 to beisomers of2.

In order to differentiate between2–4, H-1, H-2, H-4and H-5 were marked with and ˇ symbols, indicat-ing their positions below and above the reference planeof the tetrahydrofuran ring, respectively (Scheme 1). Wenote that all compounds under study were racemates,although the structural formulae show only one enan-tiomeric series with H-1 in the -position. Determinationof the structures of2–4was rendered difficult by the con-formational flexibility of the tetrahydrofuran ring. Hencedifferent conformational equilibria should be taken intoaccount. The introduction of the, ˇ notation is usefulbecause pseudorotation interconverts the axial and equa-torial positions.

For 2, detailed analysis of the1H NMR spectrumrevealed the envelope conformation of the tetrahydrofu-ran ring (the out-of-plane atom of the ring is O-3) with an˛,ˇ,˛,˛ configuration of H-1, H-2, H-4 and H-5 protons.Considering the nearly planar character of the condensedcyclopentenone ring, the junction of the tetrahydrofuranring can only be ofcis type, i.e. both H-1 and H-5 shouldbe ˛. Therefore, we should consider,ˇ,ˇ,˛, ˛,˛,˛,˛or ˛,˛,ˇ,˛ isomers, respectively, as possible structuresfor 3 and 4. Pseudorotation of the flexible tetrahydro-furan ring can modify the actual ring conformation andsteric positions of the attached substituents at the C-2 andC-4 atoms. For evaluation of the stereochemistry of thetetrahydrofuran moiety, the vicinal3J.H,H/ coupling con-stants and the NOESY responses were utilized. Inspection

of the Dreiding stereomodel shows that the dihedral anglebetweencis-arranged H-1 and H-2 bonds can range from�30° to C30°, whereas in case oftrans H-1 and H-2 pro-tons the dihedral angle is between 70 and 150°. The mea-sured3J.H-1,H-2/ D 1.5 Hz value for3 is in accordancewith an H-1/H-2 dihedral angle of 70° calculated from themodified Karplus equation,8 i.e. H-2 should be . For 4the 3J.H-1,H-2/ D 5.8 Hz value proves acis arrangementof H-2 relative to H-1, and a ca�30° angle. The value of3J.H-4,H-5/ coupling is 3.9 Hz for3 and 7.0 Hz for4 andthe calculated H-4/H-5 dihedral angles are 125° and�30°,respectively. Therefore,3 has˛,ˇ,ˇ,˛ and4 ˛,˛,˛,˛ rela-tive configurations. In the former case the predominatingconformation should be an envelope with C-2 and in thelatter case with O-3 as the out-of-plane atom.

Using the distance between the aromatic H-500 and H-600

hydrogens (2.4A) as a reference value, we calculatedthe characteristic intramolecular hydrogen–hydrogen dis-tances of3 and 4 from the volume integrals determinedby phase-sensitive NOESY. The data were compared withthose taken from the Dreiding stereomodel of the preferredconformers of3 and4 (Table 2).

These data give further evidence for the all-cis arrange-ment of protons H-1–H-5 on the tetrahydrofuran ring in4 and for thetrans arrangement of H-1/H-2 and H-4/H-5protons in3, respectively.

To our knowledge,2–4 contain a new type of bicyclicring which is exclusively formed by LOX-catalyseddioxygenation of curcumin. Solely racemic stereoiso-mers were formed. Considering the symmetric structureof curcumin, no enantioselectivity was to be expected.

Copyright 2000 John Wiley & Sons, Ltd. Magn. Reson. Chem.2000;38: 51–54

54 G. TOTH ET AL.

Table 2. Intramolecular hydrogen–hydrogen distances(A) for 3 and 4

3 4

NOESY Model NOESY Model

H-1/H-2 3.2 2.8 2.7 2.4H-1/H-5 2.6 2.5 2.4 2.4H-4/H-5 3.2 3.0 2.7 2.4

Usually, hydroperoxides are the major products formed byLOX-catalysed dioxy-genation of fatty acids.7 In the caseof curcumin none of the products2–4 is a hydroperoxide,but intermediate formation of a curcumin hydroperoxideshould be taken into account. Certainly such an intermedi-ate should be very unstable and easily undergo rearrange-ment reactions. To date we do not know which hydrogen isabstracted in the first step of the enzymatic conversion orin which position insertion of O2 takes place. However,hydrogen abstraction should occur in only one positionof curcumin, otherwise we would expect formation ofstructural isomers as dioxygenation products. From thefact that all products are stereoisomers one can concludethat oxygen insertion occurs for all three products at thesame position of the molecule. Hence2–4 should derivefrom the same precursor via rearrangement and migra-tion of the phenolic ring. We assume that the formationof three products instead of a single product is due torearrangement reactions in the course of the formation ofthe bicyclic ring, which should not be enzyme-catalysed

but chemical. Further work is necessary to elucidate themechanism leading to products2–4.

Acknowledgements

We thank the Deutsche Forschungsgemeinschaft (SFB 347) and theUniversity of Wurzburg (scholarship for M. Roth) for financial support.Gabor Toth thanks the Hungarian Scientific Research Fund (OTKA No.T026264) for financial support. We are grateful to B. Gutsche for herskilful assistance with LC–MS measurements and thank E. Ruckdeschelfor recording the 600 MHz NMR spectra.

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Copyright 2000 John Wiley & Sons, Ltd. Magn. Reson. Chem.2000;38: 51–54