A supramolecular enzyme model catalyzing the central cleavage of carotenoids

Journal of Inorganic Biochemistry 88 (2002) 295–304 www.elsevier.com / locate / jinorgbio

A supramolecular enzyme model catalyzing the central cleavage of carotenoids Richard R. French, Philipp Holzer, Michele Leuenberger, Mathias C. Nold, Wolf-D. Woggon* Institute of Organic Chemistry, University of Basel, St. Johanns-Ring 19, CH-4056 Basel, Switzerland Received 21 May 2001; received in revised form 28 August 2001; accepted 10 September 2001

Abstract Several bis-b-cyclodextrin porphyrins have been prepared as supramolecular receptors of carotenoids. The binding constants of carotenoids to receptors were determined by quenching the fluorescence of the porphyrins on hydrophobic binding of carotenoids within the cavities of cyclodextrins. Ka 58.3310 6 M 21 was calculated for binding of b,b-carotene to bis-b-cyclodextrin Zn porphyrin. The corresponding Ru complex catalyzes the central cleavage of carotenoids in the presence of tert-butyl hydroperoxide in a biphasic system.  2002 Elsevier Science Inc. All rights reserved. Keywords: Carotenoids; b-Cyclodextrin; Enzyme model; Ru porphyrins; Vitamin A

1. Introduction Carotenoids, the ‘orange pigments of life’, are biosynthesized de novo in higher plants, algae, fungi and bacteria. Animals and humans, however rely on extraction from their diet [1–3]. The general significance of carotenoids to animal and human nutrition is undisputed, in particular two biological functions are well established: (i) the ability of carotenoids to quench 1 O 2 and (ii) its antioxidant activity depending on oxygen partial pressure [4]. Thus it seems that carotenoids are complementary to tocopherols (vitamin E) preventing lipid peroxidation by a radical chain-braking mechanism [5,6]. The other major function of b,b-carotene 1 derives from its metabolites which are produced by oxidative cleavage providing retinal 2 (provitamin A) and retinoic acid 3. Though it has been known since 1930 that retinol 4 (vitamin A) derives in vivo from 1, the enzymatic origin of central cleavage of b,bcarotene was only shown in 1965 when Olson and Hayaishi reported the identification of in vitro activity of an enzyme from rat intestine [7]. Later an alternative pathway, the excentric cleavage of 1, was discovered yielding apocarotenals, such as 5 which are subsequently degraded to 2 [8–11] (Fig. 1). *Corresponding author. Fax: 141-26-617-1102. E-mail address: [email protected] (W.-D. Woggon).

During the last 35 years many groups have tried unsuccessfully to purify the enzyme catalyzing the central cleavage of 1 and quite a number of investigations failed to provide a conclusive answer on the mechanism of this enzymatic transformation, i.e. it has been impossible to distinguish between a dioxygenase and a monooxygenase mechanism. Nevertheless the enzyme was termed b,bcarotene 15,159-dioxygenase (EC 1.13.11.21) and was often referred to as being an iron dioxygenase. Recently, however, we have been able for the first time to identify the protein which catalyzes the central cleavage of 1 [12]. We have developed a purification protocol for the enzyme from chicken intestinal mucosa (M. Leuenberger, C. Engeloch-Jarret, W.-D. Woggon, unpublished results) and overexpressed the functional 60.3-kDa protein in Escherichia coli and BHK (baby hamster kidney) cell lines [13]. At about the same time another research group published on the expression of the enzyme derived from Drosophila melanogaster [14]. We also investigated the substrate specificity [15] and the reaction mechanism employing a non-symmetric carotenoid and highly enriched 17 O 2 and H 218 O [16]. Accordingly the enzyme is not a dioxygenase but operates by monooxygenase mechanism in which the first step is an epoxidation of the central 15,159 double bond. The nature of the metal complex involved in O 2 cleavage remains to be elucidated by X-ray (A. Wyss, U. Baumann, W.-D. Woggon, unpublished re-

0162-0134 / 02 / $ – see front matter  2002 Elsevier Science Inc. All rights reserved. PII: S0162-0134( 01 )00363-4

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Fig. 1. Metabolism of b,b-carotene 1.

sults). A metal porphyrin can be excluded with certainty because the corresponding chromophore is absent. Parallel to our investigations of the native protein we designed and synthesized an enzyme model for the regioselective cleavage of carotenoids; details of this work are reported here.

2. Experimental

2.1. General All non-aqueous reactions were carried out under an inert argon atmosphere using flame dried glassware. Reagents were used as received from Fluka (Buchs, Switzerland) and Aldrich Chemie (Buchs, Switzerland) unless otherwise stated. Retinal 2 and apocarotenals for HPLC reference were obtained as a gift from F. Hoffman-La Roche. Solvents were dried under standard conditions and freshly distilled prior to use. Thin layer chromatography (TLC) was performed on precoated glass plates (silica gel 60 F 254 ), obtained from Merck (Darmstadt, Germany), using distilled solvents. Normal phase column chromatography was performed on silica gel, 0.040–0.063 mm, 230–400 mesh, obtained from Merck (Darmstadt, Germany), with freshly distilled or HPLC grade solvents. Reversed phase column chromatography was performed on LiChroprep  RP-8 silica gel obtained from Merck (Darmstadt, Germany), with nanopure water and freshly distilled ethanol or HPLC grade acetonitrile. The 300-MHz NMR spectra were recorded on a Varian Gemini 300 spectrometer, 500-MHz NMR spectra were recorded on a Bruker

DRX-500 spectrometer, 600-MHz NMR spectra were recorded on a Bruker DRX-600 spectrometer, d in ppm reported relative to TMS or residual solvent peaks and coupling constants, J, are in Hertz. NMR solvents were obtained from Dr Glaser (Basel, Switzerland), Cambridge Isotope Laboratories (Andover, MA, USA) and Armar ¨ (Dottingen, Switzerland); CDCl 3 was filtered through basic alumina prior to use. MALDI–TOF-MS was recorded on a Perspective Biosystems Vestec Mass Spectrometry Products VoyagerE BiospectrometryE Workstation or a Perspective Biosystems Vestec Mass Spectrometry Products VoyagerE Elite BiospectrometryE Research Station. UV–Vis spectra were recorded on a Hewlett-Packard 8452A Diode Array Spectrophotometer. IR spectra were recorded on a Perkin-Elmer1600 series FTIR spectrometer. Fluorescence spectra were recorded on a ISA Jobin Yvon– Spex FluoroMax-2  spectrometer which was fitted with a thermostatic cell housing and magnetic stirrer and using 10-mm path length quartz cuvettes which were fitted with an appropriate magnetic stir bar. Analytical HPLC (synthesis) and semi-preparative HPLC was performed on a Merck Hitachi LaChrom system (solvent degasser L-7612, pump L-7100, UV detector L-7400, interface D-7000). Preparative HPLC was performed on a Merck Hitachi NovaPrep  200 attached to a Merck Hitachi UV detector L-7400 which was fitted with a preparative UV cell. Analytical HPLC (enzyme mimic reactions) was performed on a Hewlett-Packard Series 1100 HPLC system (solvent degasser G1322A, pump G1312A, autosampler G1313A, thermostatic column housing G1316A, diode array UV detector G1315A). Column types and dimensions, solvent gradients, and retention times are given for individual

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compounds below. Molecular modelling was performed using the MOLOC program on a Silicon Graphics Indy workstation.

2.2. Syntheses 2.2.1. Bis(49 -hydroxyphenyl)-10,20 -bis(40 -methylphenyl)porphyrin 9 a This was obtained according to standard procedures [17,18] as an HPLC-pure compound. Analytical data of 9a: lmax (CH 2 Cl 2 )5420 nm. 1 H NMR (CDCl 3 11% NEt 3 ): 8.84 (d, J54.88 4H-C(b) (pyr)); 8.82 (d, J54.88, 4H-C(b) (pyr)), 4H; 8.08 (d, J57.82, 2H-C(29), 2H-C(69)); 8.01 (d, J58.73, 2H-C(29), 2H-C(69)); 7.52 (d, J57.84, 2H-C(39), 2H-C(59)); 7.12 (d, J58.73, 2H-C(39), 2H-C(59)); 4.9–4.1 (br s, 2 ArOH ); 2.69 (s, 2 ArCH3 ). MS (MALDI-TOF): m /z 674.8; calc. 674.802. 2.2.2. 6 a ,6 9 a -O-h[10,20 -Bis (4 -methylphenyl) porphyrin5,15 -diyl] di-4,1 -phenylenej bis[b -cyclodextrin] 6 a To a solution of 15 mg (22.23 mmol) of diagonal bis-phenol porphyrin 9a in 5 ml of DMF was added 72 mg (0.222 mmol, 10 eq.) of anhydrous caesium carbonate. The reaction was stirred at RT for 2 h after which 286 mg (0.222 mmol, 10 eq.) of tosyl-b-cyclodextrin 8 was added in one portion. The reaction was then heated to 808C for 8 h. Another 286-mg portion of 8 was then added as a solution in 1 ml of DMF and the reaction stirred at 808C overnight. A subsequent portion of 8 was then added in 1 ml DMF and stirring continued for a further 24 h. Then 5 ml of water and 250 mg of ammonium chloride were added to the reaction followed by removal of volatiles under vacuum. The crude residue was purified by preparative HPLC (LiChroprep  100 RP-18 (10 mm) 200-50), 20–60% MeCN in 20 min, 60–100% MeCN in 5 min, then at 100% MeCN until 35 min, 140 ml / min ( ldet 5420 nm) to give 20 mg (31%) of the title compound as a brown amorphous solid. HPLC (LiChrospher  100 RP-18 (5 mm) 250-4), 20–60% MeCN in 20 min, 1.5 ml / min, ldet 5420 nm, R t 510.52 min, lmax (H 2 O)5420 nm. 1 H NMR (DMSO-d 6 ): 8.88 (br d, J54.46, 4H-C(b) (pyr)); 8.81 (br d, 4H-C(b) (pyr)); 8.09 (d, J58.24, 2H-C(29), 2H-C(69), 2H-C(29), 2H-C(69)); 7.63 (d, J58.24, 2H-C(39), 2HC(59); 7.39 (d, J58.06, 2H-C(39), 2H-C(59)); 5.8 (br s, 28H, 28OHs); 5.1–4.7 (m, 14H, anomeric Hs); 4.7–4.3 (m, 12H, 18OHs); 3.9–3.1 (m, 84H, 14H-C(4), 14H-C(5), 14H-C(6), 14H-C(69), 14H-C(2), 14H-C(3)). MS (MALDI-TOF): m /z 2944; calc. 2942.8. 2.2.3. 5,15 -Bis(49 -hydroxyphenyl)-10,20 -bis(40 methylphenyl)-porphyrinato zinc( II) 9 To a solution of 10 mg (14.82 mmol) of diagonal bis-phenol porphyrin 9a in 2 ml of CH 2 Cl 2 was added 1 ml of a saturated methanolic solution of Zn(OAc) 2 . The reaction was heated to 408C for 1 h and then poured into NaHCO 3(sat. aq.) . Extraction with two portions of CH 2 Cl 2

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followed by drying of the combined organic phases and concentration under vacuum gave the crude product. Purification by column chromatography on silica gel (chloroform11% NEt 3 ) gave 8 mg (74%) of the title compound as a purple solid. R f 50.13 (chloroform11% NEt 3 ); lmax (CH 2 Cl 2 )5430 nm. MS (MALDI-TOF): m /z 738.4; calc. 738.18.

2.2.4. Bis[b -cyclodextrin] zinc ( II) complex 6 To a solution of 7 mg (9.48 mmol) of porphyrinato zinc complex 9 in 2.5 ml of DMF was added 31 mg (94.8 mmol, 10 eq.) of caesium carbonate. The reaction was stirred at RT for 2 h after which 122 mg (94.8 mmol, 10 eq.) of tosyl-b-cyclodextrin 8 was added in one portion. The reaction was then heated to 808C for 8 h. Another 122-mg portion of tosyl-b-cyclodextrin 8 was then added as a solution in 0.5 ml of DMF and the reaction stirred at 808C overnight. A subsequent portion of 8 was then added in 0.5 ml DMF and stirring continued for a further 24 h. HPLC analysis of the reaction showed less than 10% product formation. A further 122-mg portion of 8 was then added in 0.5 ml DMF and stirring continued for a further 24 h. HPLC analysis revealed no further product formation and thus the reaction was concentrated to dryness and the crude product purified by semi-preparative HPLC (LiChrospher  100 RP-18 (10 mm) 250-10), 10% MeCN for 10 min, 10–100% MeCN in 50 min then at 100% MeCN to 65 min, 6 ml / min, ldet 5420 nm, title compound elution at 27 min) to give 1.9 mg (7%) of the title compound as a dark green amorphous solid. lmax (H 2 O)5 426 nm. MS (MALDI-TOF): m /z 3006; calc. 3006.18. 2.2.5. Monobridged porphyrin 24 This was obtained according to standard procedures [17,18] as an HPLC-pure compound. Analytical data of 24: UV–Vis: lmax 5420 nm. 1 H NMR (CDCl 3 ): 8.82 (d, J5 5.0, 4H-C(b) (pyr)); 8.78 (d, J55.0, 4H-C(b) (pyr)); 8.17 (dd, J59.1, 2.2, 2H-C(60)); 8.15 (dd, J59.1, 2.2, 2HC(69)); 7.95 (dd, J59.1, 2.2, 2H-C(20)); 7.74 (td, J59.1, 2.2, H-C(49)); 7.38 (td, J59.1, 2.2, 2H-C(59)); 7.30 (d, J59.1 4H-C(30, 50)); 7.20 (d, J59.1, 2H-C(39)); 3.82 (t, J55.2, 4H-C(a)); 0.78 (m, 4H-C(b)); 20.23 (m, 4HC(g)); 20.53 (m, 4H-C(d)); 21.13 (m, 4H-C(e)); 21.24 (m, 4H-C(f)). MS (MALDI-TOF): m /z 830; calc. 831.03. 2.2.6. Ruthenium( II)porphyrin 25 To a suspension of 30 mg (36 mmol) of bridged freebase porphyrin 24 in 30 ml of decaline was added 30 mg (47 mmol) of Ru 3 (CO) 12 . The reaction was heated at reflux for 4 h, after which decaline was removed under high vacuum. The crude product was purified by column chromatography on silica gel (2:1 hexane:ethyl acetate) to give 10 mg (29%) of 25 as an orange amorphous solid. lmax (CH 2 Cl 2 )5414 nm. HPLC (LiChrospher  100 RP18 (5 mm) 250-4), 90–100% MeOH in 10 min, 1.5 ml / min, ldet 5420 nm, R t 55.6 and 7.1 min (25 (MeOH

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anti / CO syn to bridge) and 25 (MeOH syn / CO anti to bridge). 1 H NMR: 8.61 (d, J55.1, 4H, H-C(3), H-C(7), H-C(13), H-C(17)); 8.52 (d, J55.1, 4H, H-C(2), H-C(8), H-C(12), H-C(18)); 8.25–7.35 (m, 16H, 2H-C(29), 2HC(39), 2H-C(69), 2H-C(59), 2H-C(39), 2H-C(49), 2HC(59), 2H-C(69)); 3.79 (t, J55.1 (4H-C(a)); 0.69 (br, 4H-C(b)); 0.12 (br, 2H-C(f)); 0.07 (br, 4H-C(g)); 0.01 (br, 4H-C(d)); 20.38 (br, 4H-C(e)). MS (MALDI-TOF): m /z 930; calc. 930.08. IR (KBr) 1940.8 cm 21 (nCO).

2.2.7. Bis[b -cyclodextrin] ruthenium ( II) porphyrin 19 To a solution of 7.1 mg (7.6 mmol) of bridged bisphenol ruthenium porphyrin 25 in 3 ml of DMF was added 25 mg (76 mmol, 10 eq.) of anhydrous caesium carbonate. The reaction was stirred at RT for 2 h after which 98 mg (76 mmol, 10 eq.) of tosyl-b-cyclodextrin 8 was added in one portion. The reaction was then heated to 808C for 6 h. Another 98-mg portion of 8 was then added as a solution in 0.5 ml of DMF and the reaction stirred at 808C overnight. Subsequent 98-mg portions of 8 were then added in 0.5 ml DMF at 24, 30 and 48 h. After 56-h reaction time, volatiles were removed under vacuum. The crude residue was purified by semi-preparative HPLC (LiChrospher  100 RP-18 (10 mm) 250-10), 20–60% MeCN in 20 min, 60–100% MeCN in 5 min, then →100% MeCN until 30 min, 6 ml / min, ldet 5420 nm) to give 4 mg (16%) of the title compound 19 as an orange amorphous solid. HPLC (LiChrospher  100 RP-18 (5 mm) 250-4), 20–60% MeCN in 20 min, 1.5 ml / min, ldet 5420 nm, R t 511.3 min, lmax (H 2 O)5412 nm. 1 H NMR (DMSO-d 6 ): 8.88 (d, J54.46, 4H, H-C(2), H-C(8), HC(12), H-C(18); 8.81 (d, 4H, H-3, H-7, H-13, H-17), 5.9–5.6 (m, 28H, 28OHs); 5.1–4.7 (m, 14H, anomeric Hs); 4.7–4.3 (m, 12H, 18OHs,); 3.9–3.1 (m, 88H, 14H-C(4), 14H-C(5), 14H-C(6), 14H-C(69), 14H-C(2), 14H-C(3), 4H-C(a)); 0.65 (br, 4H-C(b)); 0.15→ 20.05 (m, 10HC(f,g,d)), 20.45 (br, 4H-C(e)). MS (MALDI-TOF): m /z 3196; calc. 3196.07. 2.2.8. 179 -Nor-b,f -carotene 26 n-BuLi (0.07 ml, 1.6 M in hexane, 0.11 mmol) was added at 08C to a suspension of triphenyl(2,6-dimethylbenzyl)phosphonium bromide 27 (46 mg, 0.1 mmol) in diethylether (3 ml). The red solution was stirred at RT for 5 min, before solid b-apo-89-carotenal 5 (42 mg, 0.1 mmol) was added in one portion. The mixture was stirred at RT for 1 h, and saturated aq. ammonium chloride solution was added to neutralize the base. The organic phase was separated and the aq. layer extracted with CH 2 Cl 2 . The organic phases were collected, dried (NaSO 4 ), and evaporated. Chromatography (silica gel, hexane / toluene 5:1 (10.5% Et 3 N), R f 50.38) afforded 15 mg (29%) of pure 179-nor-b,f-carotene 26 (HPLC 85% all-(E), three other isomers |5% each). UV–Vis (EtOH) lmax 5452 nm. 1 H NMR: 7.05 (s, 3H, H-C(29), H-C(39), H-C(49)); 6.68 (m, 1H, H-C(159)), 6.67 (dd, J515.0 and

12.0 H-C(119)); 6.65 (dd, J515.0 and 11.5 H-C(11)); 6.64 (m, H-C(15)); 6.58 (d, J516.2, H-C(79)); 6.40 (d, J5 15.0, H-C(129)), 6.37 (d, J516.2, H-C(89)); 6.36 (d, J5 15.0, H-C(12)); 6.25 (d, J512.3, H-C(14)); 6.24 (d, J5 12.3, H-C(149)), 6.23 (d, J512.01, H-C(109)); 6.17 (d, J516H, H-C(7)); 6.15 (d, J511.5, H-C(10)); 6.13 (d, J516.0, H-C(8)); 2.33 (s, 6H, H3 -C(169), H3 -C(189)); 2.07 (s, H3 -C(199)); 2.01 (t, J56.9, 2H-C(4)); 1.98 (br s, 6H, H3 -C(20), H3 -C(209)); 1.97 (br s, H3 -C(19)); 1.72 (s, H3 -C(18)); 1.62 (br t, J56.0, 2H-C(3)); 1.47 (td, J56.0 and 2.0, 2H-C(2)); 1.03 (s, 6 H, H3 -C(16), H3 -C(17)). 13 C NMR: 139.2 (C(89)), 138.5 (C(129)), 138.3 (C(6)), 138.1 (C(8)), 137.7 (C(69)), 137.6 (C(12)), 137.1 (C(139)), 136.7 (C(13)), 136.5 (C(19), C(59)), 136.4 (C(9)), 135.8 (C(99)), 133.3 (C(14)), 132.8 (C(109)), 132.7 (C(149)), 131.2 (C(10)), 130.7 (C(159)), 130.2 (C(15)), 129.8 (C(5)), 128.2 (C(29), C(49)), 127.1 (C(7)), 126.7 (C(39)), 126.0 (C(79)), 125.5 (C(11)), 125.1 (C(119), C(159)), 40.0 (C(2)), 34.7 (C(1)), 33.5 (C(4)), 29.3 (C(16), C(17)), 22.1 (C(18)), 21.5 (C(169), C(189)), 19.6 (C(3)), 13.17, 13.12, 13.10 (C(19), C(199), C(20), C(209)). MS (HR-EI): (C 39 H 50 ) m /z 518.3906; calc. m /z 518.3912. (EI): m /z 518 (51, [M] 1 ), 516 (11), 426 (14, [M-92] 1 ), 412 (8, [M-106] 1 ), 360 (11, [M-158] 1 ), 119 (100).

2.3. Binding experiments 2.3.1. Determination of I0 To a 3-ml aliquot of 0.25 mM cyclodextrin dimer 6 in water in a quartz cuvette which had been fitted with a magnetic stirring bar, THF (60 ml) was added. The solution was equilibrated at 258C with stirring for 5 min and was then allowed to stand at 258C for a further 5 min. The emission spectrum of 6 ( lexc 5426 nm) was measured to afford a value for I0 ( lem 5610 nm). 2.3.2. Binding of b,b -carotene 1 to receptors Samples containing increasing concentrations of b,bcarotene 1 (or 26) (0.2, 0.5, 1, 2, 3, and 4 eq.) were added as a solution in THF (60 ml) to 3-ml aliquots of 0.25 mM 6 (or 6a) in water. After addition, each sample was subjected to identical equilibration conditions as those used for the I0 measurement. Emission spectra were then recorded ( lexc 5 426 nm, lem 5610 nm) to give values for If . All experiments were repeated three times. Analysis of the data gave a value of Ka 58.3310 6 M 21 . Binding of b,b-carotene 1 and nor-b,f-carotene 26 to the Zn-free cyclodextrin dimer 6a gave values of Ka 5 2.4310 6 M 21 and Ka 55.0310 6 M 21 , respectively. 2.4. Enzyme mimic reactions 2.4.1. General procedure for cleavage reactions A round bottomed flask which had been purged with argon and fitted with an appropriately sized egg shaped

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magnetic stir bar was charged with a solution of dimers 13 or 19 (10 mol%) in 1 ml H 2 O. tert-Butylhydroperoxide (TBHP, 70% in water) (300 equiv. 13 or 19) was added. b,b-Carotene 1 or synthetic carotenoid 26 (1 mmol) was added to the reaction flask as a solution in 9:1 hexane:chloroform (1 ml) to produce a biphasic system. The reaction was closed and stirred vigorously to ensure good mixing of the two phases. At time points during the reaction, stirring was stopped to allow phase separation. Aliquots (20 ml) of the organic phase were taken and subjected to HPLC analysis (LiChrospher 100 Rp-18 5 mm, length3ID5125 mm34.6 mm, 258C, 1 ml / min, gradient: acetonitrile:1% NH 4 OAc (aq) 1:1 (100%)→acetonitrile:iPrOH 1:1 (100%) in 10 min, →acetonitrile:iPrOH 1:1 (100%) for 5 min, then acetonitrile:iPrOH 1:1 (100%)→acetonitrile:1% NH 4 OAc (aq) 1:1 (100%) in 2 min). Detection was by diode array detector. R t 2858.8 min, R t 2510.4 min, R t 15512.1 min, R t 165

Fig. 2. Synthesis of supramolecular receptors of b,b-carotene 1.

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12.2 min. In all reactions, carotenoids 1 (R t 515.1 min) and 26 (R t 513.8 min) were completely consumed after 24 h. The cleavage of b,b-carotene 1 gave aldehydes 2, 15, and 16 in 30% total yield, whereas the cleavage of 26 furnished retinal 2 and Ph-retinal 28 in 14% yield each.

3. Results and discussion The fact that b,b-carotene 15,159-monooxygenase controls the regiospecific cleavage of one C=C bond out of a possible six within the substrate is an intriguing and challenging one. In order to mimic such a regioselective system the following strategy was employed: (i) synthesis of a receptor for 1 in which the binding constant, Ka , for 1 is orders of magnitude greater than that for retinal 2, in order to prevent product inhibition; (ii) introduction of a reactive metal complex which is capable of cleaving (E)configured, conjugated double bonds to aldehydes; (iii) use of a co-oxidant which is inert towards 1 in the absence of the metal complex. The supramolecular construct 6 (Fig. 2) consisting of two b-cyclodextrin moieties linked by a porphyrin spacer, was designed by means of the MOLOC program to be an ideal candidate for the binding of 1. Each of the cyclodextrins was shown to be capable of binding one of the

Fig. 3. Quenching of the fluorescence of the porphyrin receptor 6 on binding the substrate 1.

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Fig. 4. Cleavage of (E)-configured, conjugated double bonds catalyzed by ruthenium tetraphenylporphyrin (0.2%) and tert-butyl hydroperoxide (TBHP).

cyclohexenoid endgroups of b,b-carotene, leaving the porphyrin to span the polyene chain. In this complex approximately half of 1 would be included in the cyclodextrin cavities and the C(15)–C(159) double bond would be directly under any metal that could be subsequently inserted into the porphyrin. In the absence of 1 several unproductive conformations of 6 are possible due to rotation about the ether linkages; in the presence of 1, however, an induced fit should be observed yielding the inclusion complex 7. As well as having the role of a spacer and potential metal ligand, the porphyrin in 7 is also useful for the determination of the binding constant, Ka , of 1 to 6. Porphyrins display a characteristic fluorescence at |600– 650 nm and the ability of carotenoids to quench this fluorescence was envisaged as a sensitive probe for the binding interaction of the two entities in an aqueous medium. It could be reasonably postulated that a cyclodextrin dimer such as 6 should be capable of providing a Ka for 1 in the region of 10 5 –10 7 M 21 .

Following established procedures in our laboratory [17] the synthesis of the carotene receptor 6 was pursued as shown in Fig. 2. Commercially available 6-O-( p-tosyl)-bcyclodextrin 8 was coupled with the Zn porphyrin 9 obtained after Zn insertion into corresponding free ligand 9a [16]. The product 6 was purified by reversed-phase HPLC and Ka for 1 was determined by fluorescence quenching (Fig. 3). The graphs in Fig. 3 revealed a binding constant Ka 5 8.3310 6 M 21 [19,20]. The corresponding free-base porphyrin 6a displays a binding constant Ka 52.4310 6 M 21 . The 3.5 times higher Ka of 6 is due increased planarity of this Zn porphyrin as compared to the saddle-shaped conformation of 6a. This satisfied the first of our strategic criteria for mimicking the biological system as the binding constant for retinal 2 to b-cyclodextrin has been reported to be 3.6310 3 M 21 [21], accordingly product inhibition should not be observed if cleavage of the central double bond of 1 could be accomplished. With regard to the choice of a metalloporphyrin capable of cleaving double bonds, there was only one precedent in the literature: reaction of a-methyl styrene to give acetophenone in the presence of tert-butyl hydroperoxide (TBHP) [22]. We have systematically studied the reactivity of open face as well as face protected ruthenium porphyrins towards substrates containing conjugated (E)-configured double bonds in the presence of TBHP. A representative example is the cleavage of (E,E)-1,4-

Fig. 5. Synthesis of supramolecular catalyst 13.

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Fig. 6. Cleavage of b,b-carotene 1 by 13 / TBHP.

Fig. 7. Binding modes of b,b-carotene 1 to the catalyst.

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diphenyl-1,3-butadiene 10 which is first epoxidized and subsequently gives benzaldehyde 11 and cinnamylaldehyde 12 almost quantitatively (Fig. 4) (Ph.D. thesis, P. Holzer). We have also tested the stability of 1 towards various cooxidants, and finally tert-butyl hydroperoxide (TBHP) was chosen, which showed no degradation of 1 within 24 h in the absence of catalyst. With the above prerequisites satisfied, the stage was set to employ the supramolecular system 13 to investigate the catalytic cleavage of b,b-carotene 1 [20]. The ruthenium porphyrin 13 was obtained by reaction of the Ru-porphyrin bis-phenol 14 with the b-cyclodextrin derivative 8 in DMF

for 3 days in the presence of caesium carbonate (Fig. 5). The reaction was followed by analytical HPLC and the desired product purified by preparative HPLC. For the catalytic cleavage of b,b-carotene 1, a biphasic system was established in which 1 is extracted from a 9:1 mixture of hexane and chloroform into a water phase containing 13 (10%) and TBHP. The reaction products, released from the receptor, are then extracted into the organic phase, aliquots of which were subjected to HPLC conditions developed for the analysis of carotene monooxygenase enzyme studies [12,13]. The reaction products were identified by retention time (co-injection with authen-

Fig. 8. Synthesis of the monobridged enzyme model; fifth (CO) and sixth (MeOH) ligand of Ru are not shown, in fact 25 is obtained as a mixture of two diastereoisomers, i.e. CO ligand syn / MeOH anti to bridge and vice versa.

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tic samples) and by their UV spectra. Quantification was by means of external calibration curves. The ratio of reaction products is given in Fig. 6. It is evident that 1 is not only cleaved at the central double bond but also at C(129)=C(119) to give 129-apocarotenal 15 and at C(109)=C(99) to give 109-apocarotenal 16. The combined yield of aldehydes 2, 15 and 16 was 30%, which compares well with the efficiency of b,b-carotene 15,159 monooxygenase which gives retinal 2 in 20–25% [12,14]. Most interestingly, the double bond in closest proximity to the central one, C(149)=C(139), remains untouched (Fig. 6). At this stage we considered two possible setups leading to the cleavage of double bonds other than C(15)–C(159): (i) binding of b,b-carotene in an ‘unproductive’ fashion, see 17, or (ii) lateral movement of the substrate within the two cyclodextrins, see 18 (Fig. 7). To investigate the first possibility we prepared the mono-bridged Ru porphyrin 19. We reasoned that oxidation of the substrate through binding by only one cyclodextrin could be prevented by protection of one porphyrin face by means of an alkane bridge. Note that the substrate 1 cannot approach 19 on the face of the porphyrin that is protected by the alkane bridge; molecular modelling revealed that b,b-carotene cannot slide between the porphyrin plane and the bridge. The synthesis of 19 (Fig. 8) was pursued by condensation of the dipyromethane 20 with 2-pivaloyloxy benzaldehyde 21 according to standard procedures [17,18]. After ester hydrolysis and ether cleavage bridging of the free phenols with 1,11-dibromo undecane 22 gave a mixture of vicinal isomer 23 and diagonal isomer 24 from which the latter could be easily separated. Note that the vicinal regioisomer 23 derives from the well-known acid catalyzed scrambling during the condensation of 20 and 21 [23,24]. Ru was inserted into pure 24 and the mono bridged Ru porphyrin 25 condensed with the b-cyclodextrin monotosylate 8 yielding the target complex 19. Reaction of b,b-carotene 1 with 19 / TBHP under the same conditions as described for 13 gave the same ratio of aldehydes 2, 15 and 16 as shown in Fig. 6. Thus it was concluded that the production of 15 and 16 is not due to substrate binding as shown in 17, but rather due to lateral movement of 1 within the cavities of the cyclodextrins, see 18. Regarding the latter aspect we reasoned that the selectivity of double bond oxidation displayed by catalyst 13 should change if at least one of the endgroups of the substrate 1 is exchanged for an equally hydrophobic substituent displaying different contacts with the interior of the b-cyclodextrin cavity. For this purpose we chose Pheb-carotene 26, prepared from 8-apocarotenal 5 and the Wittig salt 27 in a straightforward manner. The analogue 26 turned out to be an excellent substrate of the enzyme b,b-carotene 15,159-monooxygenase producing the two aldehydes 2 and 28 in equal amounts. Catalytic oxidation of 26 with the enzyme model 13 / TBHP was indeed very regiospecific since only retinal 2 and the corresponding Phe analogue 28 were detected (Fig. 9).

303

Fig. 9. Preparation and cleavage of the substrate analogue 26.

We do not know exactly whether substrates 1 and 26 approach the b-cyclodextrin cavities from primary face as indicated in the cartoon-like structures 7, 7a and 18 (Figs. 2 and 7); in fact both the secondary and the primary face are wide enough to allow the entry of the substrates bulky endgroups. Nevertheless our results suggest that stronger hydrophobic interactions between the aromatic endgroup of 26 and the b-cyclodextrin cavity are responsible for stabilizing the 1:1 inclusion complex with the central double bond under the reactive ruthenium center. In contrast 1 slides within the inclusion complex exposing three double bonds rather than one to the reactive Ru=O. Determination of the binding constant of 26 to the receptor 6a supports this interpretation, i.e. Ka 55.0310 6 M 21 of 26 is two times larger than for 1. The supramolecular system, presented here, is one of the few examples which mimic the reactivity and selectivity of an enzymatic reaction using unmodified, original substrates of an enzyme. This construct is distinct from other elegant approaches [25,26] which require substrates having substituents that support binding to the cyclodextrins and are prone to product inhibition. Furthermore the problem to epoxidize / cleave (E)-configured, conjugated double bonds has been successfully solved, and it can be envisaged that this oxo ruthenium porphyrin catalyzed double bond cleavage will be applicable in preparative chemistry (Ph.D. thesis, P. Holzer).

References ¨ [1] H. Pfander (Ed.), Key To Carotenoids, Birkhauser, Basel, 1987. [2] G. Britton, S. Liaaen-Jensen, H. Pfander (Eds.), Isolation and ¨ Analysis, Carotenoids, Vol. 1a, Birkhauser, Basel, 1995.

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[3] G. Britton, S. Liaaen-Jensen, H. Pfander (Eds.), Biosynthesis and ¨ Metabolism, Carotenoids, Vol. 3, Birkhauser, Basel, 1998. [4] R. Edge, D.J. McGarvey, T.G. Truescott, Photochem. Photobiol. B: Biol. 41 (1997) 189–200. [5] G.W. Burton, J. Nutr. 119 (1989) 109–111. [6] A. Mortensen, L.H. Skibsted, T.G. Truescott, Arch. Biochem. Biophys. 385 (2001) 13–19. [7] J.A. Olson, O. Hayaishi, Proc. Natl. Acad. Sci. USA 54 (1965) 1365–1370. [8] S. Hansen, W. Maret, Biochemistry 27 (1988) 200–206. [9] A.A. Dmitrovskii, in: T. Ozawa (Ed.), New Trends in Biological Chemistry, Japan Scientific Soc., Tokyo, 1991, pp. 297–308. [10] X.-D. Wang, G. Tang, J.G. Fox, N.I. Krinsky, R.M. Russell, Arch. Biochem. Biophys. 258 (1995) 8–16. [11] K.-J. Yeum, A.L. Dos Anjos Ferreira, D. Smith, N.I. Krinsky, R.M. Russell, Free Radic. Biol. Med. 29 (2000) 105–114. [12] A. Wyss, G. Wirtz, W.-D. Woggon, R. Brugger, M. Wyss, A. Friedlein, H. Bachmann, W. Hunziker, Biochem. Biophys. Res. Commun. 271 (2000) 334–336. [13] A. Wyss, G. Wirtz, W.-D. Woggon, R. Brugger, M. Wyss, A. Friedlein, H. Bachmann, W. Hunziker, Biochem. J. 354 (2001) 521–529. [14] J. von Lintig, K. Vogt, J. Biol. Chem. 275 (2000) 11915–11920.

¨ [15] G.M. Wirtz, C. Bornemann, A. Giger, R.K. Muller, H. Schneider, G. Schlotterbeck, G. Schiefer, W.-D. Woggon, Helv. Chim. Acta 84 (2001) 2301–2315. [16] M. Leuenberger, C. Engeloch-Jarret, Angew. Chem. Int. Ed. 40 (2001) 2614–2617. [17] H.-A. Wagenknecht, C. Claude, W.-D. Woggon, Helv. Chim. Acta 81 (1998) 1506–1520. ¨ [18] B. Staubli, H. Fretz, U. Piantini, W.-D. Woggon, Helv. Chim. Acta 70 (1987) 1173–1193. [19] R.R. French, J. Wirz, W.-D. Woggon, Helv. Chim. Acta 81 (1998) 1521–1527. [20] R.R. French, P. Holzer, M.G. Leuenberger, W.-D. Woggon, Angew. Chem. Int. Ed. 39 (2000) 1267–1269. [21] Q.-X. Guo, T. Ren, Y.-P. Fang, Y.-C. Liu, J. Incl. Phenom. Mol. Recog. Chem. 22 (1995) 251–256. [22] S. Tagaki, T.K. Miyamoto, Inorg. Chim. Acta 173 (1990) 215–221. [23] J.S. Lindsey, R.W. Wagner, J. Org. Chem. 54 (1989) 828–836. [24] D.M. Wallace, S.H. Leung, M.O. Senge, K.M. Smith, J. Org. Chem. 58 (1993) 7245–7257. [25] R. Breslow, X. Zhang, R. Xu, M. Maletic, R. Merger, J. Am. Chem. Soc. 118 (1996) 11678–11679. [26] R. Breslow, X. Zhang, Y. Huang, J. Am. Chem. Soc. 119 (1997) 4535–4536.