J . Am. Chem. SOC.1984, 106, 1801-1808
1801
A Crown Ether Flavin Mimic: Synthesis and Properties of a Flavin Bearing a Crown Ring as a Recognition Site’ Seiji Shinkai,* Yuichi Ishikawa, Hiroko Shinkai, Takaharu Tsuno, Hiroki Makishima, Kaori Ueda, and Osamu Manabe Contribution from the Department of Industrial Chemistry, Faculty of Engineering, Nagasaki University, Nagasaki 852, Japan. Received September 9, 1983
Abstract: A crown ether flavin mimic (3,10-dimethyl-l’,4’,7’,10’,13’,16’-hexaoxacyclooctadec-2’-eno[2’,3’-i]isoalloxazine, crF1) which has within a molecule both flavin as a catalytic site and crown ring as a recognition site was synthesized. The absorption band and the fluorescence intensity of crFl decreased with increasing alkali metal concentrations. From plots of OD/OD, (or Z/Zo) vs. metal ion concentrations were determined the association constants ( K ) , which were in the order K+ > Rb’ > Cs+ = Na’. The fluorescence was efficiently quenched by tryptamine hydrochloride (Ill,, = 0.224 at IO-, M), suggesting the pseudointramolecular fluorescence quenching due to recognition of the ammonium group by the crown ring. The fluorescence study using a series of benzimidazole derivatives with 2-(CH2)nNH3’ showed that the maximum fluorescence quenching occurs at n = 3. The fluorescence intensity in the presence of these additives increased with increasing K+ concentration, indicating the competitive binding of K’ and additives to the crown ring. The second-order rate constants ( k , ) for the reaction with 1-benzyl-1,4-dihydronicotinamide were enhanced by 1.4- to 2.4-fold by added alkali metal and ammonium cations. Furthermore, the reaction with N3-dodecyl-1-@-( ammoniomethy1)benzyl)- 1,4-dihydronicotinamide proceeded according to Michaelis-Menten kinetics, and a rate enhancement of 29-fold was attained. This is also due to binding of the ammonium group to the crown ring. These results show that crFl is capable of mimicking several important properties of flavoenzymes owing to the attached crown ring as a recognition site.
Coenzymes are prosthetic groups in enzymes and catalyze the enzyme-mediated reactions in the active sites. The catalytic actions have attracted much attention of bioorganic chemists because some of them are capable of catalyzing the reactions even in the absence of apoenzyme^.^-^ Thus, the investigations on the coenzyme catalyses in the model system have provided many clues to elucidate the enzymatic reaction mechanisms reasonably.2-6 In contrast to holoenzymes having both a catalytic site and a recognition site, however, coenzymes themselves consist only of a catalytic site. One may thus expect that the coenzymes bearing an intramolecular recognition site would behave as more attractive enzyme model systems.’ The ability of crown ethers, as well as that of c y c l o d e ~ t r i n sto , ~associate ~~ with a variety of charged and uncharged substrates bears resemblance to early reaction steps in enzyme-mediated reactions. Therefore, a crown ether family may be. a useful candidate for the recognition sites. In fact, there have been several crown ether mimics of prosthetic groups reported, but to the best of our knowledge the precedents are rather limited: sulfhydryl coenzyme N A D H model^,'^^'^ and heme porphyrins.’”I6 Here we report the first example of a crown e t h e r mimic of flavin coenzymes, 3 , l O-dimethyl1’,4’,7’,1CY,13’, 16’-hexaoxacyclooctadec-2’-eno[2’,3’-i]isoalloxazine (crFI). One can expect for the crown ether cavity to recognize not only spherical metal cations but also ammonium cations and others through hydrogen bonding, and the complexation would (1) Coenzyme Models. 37. Preliminary communication: Shinkai, S.; Ishikawa, Y.; Shinkai, H.; Tsuno, T.; Manabe, 0. TefrahedronLeft. 1983, 24, 1539. (2) Walsh, C. In “Enzymatic Reaction Mechanism”; W. H. Freeman and Co.: San Francisco, 1979. (3) Scrimgmur, K. G. In “Chemistry and Control of Enzyme Reactions”; Academic Press: London, 1977. (4) Jencks, W. P. In “Catalysis in Chemistry and Enzymology”; McGraw-Hill: New York, 1969. (5) Bruice, T. C.; Benkovic, S . J. In “Bioorganic Mechanisms”; Benjamin: New York, 1966. (6) Breslow, R. J . Am. Chem. Soc. 1957, 79, 1762. (7) For a comprehensive review see Shinkai, S . Prog. Polym. Sci. 1982, 8, 1. (8) Breslow, R. Acc. Chem. Res. 1980, 13, 170. (9) Tabushi, I. Acc. Chem. Res. 1982, 15, 66. (10) Chao, Y.; Cram, D. J. J . Am. Chem. SOC.1976, 98, 1015. (1 1) Matsui, T.; Koga, K. Tefrahedron L e f f .1978, 1115. (12) (a) van Bergen, T. J.; Kellog, R. W. J . Am. Chem. SOC.1977, 99, 3882. (b) de Vries, J. G.; Kellog, R. M. Zbid. 1979, 101, 2759. (13) Behr, J.-P.; Lehn, J.-M. J . Chem. SOC.,Chem. Commun. 1978, 143. (14) Chang, C. K. J . Am. Chem. SOC.1977, 99, 2820. (15) Thanabal, V.; Krishnan, V. J . Am. Chem. SOC.1982, 104, 3643. (16) Kruse, R.; Breitmaier, E. Chem. Ber. 1981, 114, 832.
0002-7863/84/ 1506-1801$01.50/0
CrFl Me
I
L1 1
induce a change in the flavin reactivities. Furthermore, absorption and fluorescence spectra of the isoalloxazine would be affected by bound guest species. In order to differentiate the contribution of the crown ring in crF1, we used 3-methyllumiflavin (LFl) as a reference compound.
Experimental Section Materials. crFl was synthesized according to Scheme I. 4’-(Tosylamino)benzo- 18-crown-6 (1) was prepared from 4’-aminobenzo-18-crown-6 and tosyl chloride in the presence of excess triethylamine: mp 81-84 O C ; yield 83.8%. 1 (15.0 g, 31 mmol) was dissolved in 380 mL of water containing 5.4 g of NaOH, and dimethyl sulfate (3.91 g, 31 mmol) was added dropwise to the stirred solution. The reaction was continued at 40 O C for 5 h. The progress of the reaction was followed by a TLC method. In order to complete the reaction, NaOH and dimethyl sulfate (one-third of the above amounts) were added after 0.5, 1.0, and 2.5 h. After cooling, the oily layer that separated was recovered by decantation. The aqueous layer was extracted with n-butyl alcohol, the n-butyl alcohol layer being combined with the oil. Concentration of the solution gave brown crystals. The crystals were dissolved once in hot NaOH solution and treated with active charcoal. After filtration, the solution was neutralized by dilute 18-crown-6 HCI. We thus obtained 4’-(N-methyl-N-tosylamino)benzo(2) as an almost colorless oil: yield 48.4%; single spot on TLC; IR (neat) vso2 1150, 1350 cm-’, vc1070 cm-I. 2 (7.54 g, 15 mmol) was nitrated in chloroform (50 mL)-acetic acid (45 mL) at rmm temperature by 18.8 mL of 70% nitric acid. The small amount of the reaction mixture was withdrawn, neutralized with Na,CO,, extracted with chloroform, and subjected to TLC analysis to follow the progress of the reaction. It took about 24 h to complete the 0 1984 American Chemical Society
1802 J . Am. Chem. SOC.,Vol. 106, No. 6, 1984
Shinkai et al.
Scheme I
4 P 0 - l
6 reaction. The reaction mixture was poured into ice water and extracted with chloroform. The chloroform solution was washed with water and aqueous Na2C03solution and evaporated to dryness. We thus obtained 4’-(N-methyl-N-tosylamino)-5’-nitrobenzo-l8-crown-6 (3): mp 143-146 “C; yield 86.7%; IR (KBr disk) uNo2 1340, 1520 cm-I, uSol 1150, 1350 cm-’, uCQc 1100 cm-I; N M R (CDCIJ 6 2.44 (3 H , s, CH, in Ts), 3.24 (3 H , s, NCH3) 3.63-4.31 (20 H , m, crown protons), 6.64 (1 H, s, 3’-H), 7.30 (2 H , d, H ortho to methyl), 7.47 (1 H, s, 6’-H), 7.63 (2 H, d, H meta to methyl). Anal. (C24H,zN2S0,0)C, H, N . 3 (6.42 g, 1 1.9 mmol) was hydrolyzed to 4’-methylamino)-5’-nitrobenzo-18-crown-6 (4) in 50 mL of 40% H z S 0 4 at 80-100 “C for 1 h. After neutralization with Na2C03, the solution was extracted with chloroform. The chloroform solution was treated with active charcoal and then evaporated to dryness. The solid residue (4) (4.19 g, 90.8% yield) was dissolved in 90 mL of methanol and reduced by hydrogen in the presence of 0.39 g of Pd on carbon. The reaction mixture was filtrated in a nitrogen stream. The filtrate containing 4’-(methylamino)-5’-aminobenzo-l8-crown-6 (5), which was sensitive to air oxidation, was used directly for the following reaction. The completion of the reaction was confirmed by TLC. To 20 mL of a methanol solution containing 1.73 g (10.8 mmol) of alloxane was added 1 g of concentrated HzS04. The methanol solution obtained from catalytic hydrogenation of 4 was mixed with the alloxane solution, and the mixture was refluxed for 1.5 h under a nitrogen stream. After cooling, the orange precipitate was collected by filtration and recrystallized from acetic acid: mp 323-328 O C ; yield of 10-methyl1’,4’,7’, l0’,13’, 16’-hexaoxacyclooctadec-2’-eno[2’,3’-i]isoalloxazine( 6 ) from 3, 28.0%. 6 (0.26 g, 0.56 mmol) was dissolved in 250 mL of N,N-dimethylformamide (DMF) containing 0.78 g (5.6 mmol) of powdered K2C03 and methylated with methyl iodide (0.80 g, 5.6 mmol) at 30-40 OC. After 6 h, the additional amount of methyl iodide (1.45 g, 10.2 mmol) was added. T L C analysis showed that the reaction was over after 12 h. The reaction mixture was concentrated in vacuo, the residue being dissolved in chloroform. The chloroform layer was washed with 0.1 N N a O H and water and evaporated to dryness. The residue (crF1) was recrystallized from ethanol-isopropyl ether: mp 280-284 O C ; yield 89.7%; IR (KBr disk) Y1650 cm-I, uc1110 cm-’; mass spectrum, m / e 476 (M’). Anal. (C22H28N,0a)C, H, N . The N M R spectrum could not be measured because of its poor solubility. Among N A D H model compounds, preparations of l-benzyl-l,4-dihydronicotinamide (BzlNAH) and N3-dodecyl-l-benzyl-l,4-dihydronicotinamide (DodBzlNAH) were described previously.17J8 The potassium salt of N3-carboxymethyl-1-benzyl- 1,4-dihydronicotinamide (BzlNAHCOOK) was synthesized as shown in Scheme 11. (17) Shinkai, S.;Yamada, S.; Kunitake, T. Macromolecules 1978, l J , 65. (1 8) Shinkai, S.; Tsuno, T.; Asatani, Y.; Manabe, 0.J . Chem. Soc., Perkin Trans. 2 1983, 1533.
Crb 1
Scheme I1
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,.
, ~.
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