KIT – University of the State of Baden-Wuerttemberg and National Research Center of the Helmholtz Association Synthesis and hydrogen isotope labelling of N-acyl-L-homoserine lactones (AHLs) a Karlsruhe Institute of Technology, Institute of Organic Chemistry, Fritz-Haber-Weg 6, 76131 Karlsruhe, Germany; E-mail: dorota.jakubczyk@kit.edu b Karlsruhe Institute of Technology, Institute of Functional Interfaces, Hermann von Helmholtz-Platz 1, 76344 Eggenstein-Leopoldshafen, Germany KIT, Campus South, Institute of Organic Chemistry (IOC) KIT, Campus North, Institute of Functional Interfaces (IFG) Introduction Aim Synthesis and hydrogen isotope labelling of N-acyl-L-homoserine lactones Results and discussion 1.2. Deuterium labelling via catalytic reduction of the double bond (scheme 2) Scheme 3. a) 4-Dimethylaminopyridine (DMAP); 1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC); CH 2 Cl 2 ;RT; b) L-homoserine lactone hydrobromide, Et 3 N; CH 3 CN; RT-80°C; c) Ethylene glycol, p-TsOH, CH(OMe) 3 , PhMe, 110°C-RT; d) Pd(OAc) 2 , THF, CH 3 COOH, MeOH, NaBR 4 , NaOH aq , -196 °C – RT; e) HClO 4 , CH 2 Cl 2 , 0°C-RT; R = H, D or T; Y = yield (see Table 2). 2.1. Synthesis of a highly biologically active, deuterium and tritium labelled N-(3-oxododecanoyl)-L-homoserine lactones (scheme 3) Conclusion The new methods of isotopic labelling of AHL was developed. The method is efficient and enable further biological investigations; Structures of the products were confirmed by TLC, 1 H NMR, 13 C NMR, ESI-TOF MS, HRMS, IR and Raman spectroscopy, TLC / autoradiography and Liquid Scintillation Counter. References [1] Schwarzmann, G. Biochim. Biophys. Acta 1978, 529, 106; [2] Amara, N.; Mashiach, R.; Amar, D.; Krief, P.; Spieser, S. A. H.; Bottomley, M. J.; Aharoni, A.; Meijler, M. M. J. Am. Chem. Soc. 2009, 131, 10610; [3] http://spot.pcc.edu/~jvolpe/b/bi234/lec/8_9defenses/8_hostDefense1_L.htm; accessed 04.07.2011; [4] Chhabra, S. R., C. Harty, et al., Journal of Medicinal Chemistry, 2003, 46(1) 97-104. N-acyl-L-homoserine lactones 1 (AHLs) are natural products which Results and discussion belong to semiochemicals (signal molecules or infochemical compounds). They act as messengers within (pheromones) or between (allomones) species. 1 R – acyl or alkyl chain AHL is so-called quorum sensing molecule, which enables inter-bacterial and inter-kingdom communication (Inter-kingdom Signalling). Inter-kingdom Signalling Interactions with a variety of mammalian cells: Induction of apoptosis Induces the chemotaxis of neutrophils (Fig. 1) Fig. 1. Chemotaxis of neutrophils Elucidating the mechanism of Inter-kingdom Signalling 1.1. Synthesis of terminally unsaturated N-acyl-L-homoserine lactones – substrates for the isotopic labelling (scheme 1) Scheme 1. a) Oxalyl chloride, hexane, RT-45°C, 20-22 h; b) L-homoserine lactone hydrobromide, Et 3 N, CH 2 Cl 2 , 0°C-RT, 4,5-5,5 h; n = 1, 3, 4, 6. Table 1. Results for the deuterium labelling of the terminally unsaturated AHLs; a Determined by 1 HNMR and mass spectrometry; b Determined by ESI-TOF MS. Unconventional conditions of the reaction (Fot. 1, 2, 3) Fot. 1. Reaction starts in the liquid nitrogen Fot. 2. Agitation in the thermomixer Fot. 3. Special control area for radiation Table 2. Results for the synthesis of the deuteriumand tritum labelled N-(3-oxododecanoyl)-L-homoserine lactone ; a Determined by 1 HNMR and mass spectrometry; b Determined by ESI-TOF MS; c Determined by Liquid Scintillation counter. Dorota Jakubczyk a,b , Stefan Bräse a , Gerald Brenner-Weiß b Deuterium and tritium labelled compounds have several important applications in many branches of science e.g. in the investigation of drug metabolism, biological activity, reaction mechanisms or kinetic studies. Contrary to most of the isotopic labelling methods based especially on the use of tritium gas, this procedure does not require sophisticated apparatuses, complicated work-up and the tritium gas is generated in situ from a solid reagent. Moreover the uncommon catalytic reduction of the double bond is presented herein. Detection of AHLs crossing eukaryotic cell membranes Scheme 2. Deuterium labelling of AHLs via catalytic reduction of the double bond (See Table 1). Entry n Time [h] Additives, notes Other deuterated reagents Yield [%] D content a at preterminal C atom [%] D content a at terminal C atom [%] Isomers b 1 1 25 H 2 O - 56 34-56 99 [M+(1,2,3,4)+H + ] [M-(1,2)+H + ] 2 1 22,5 excess of NaBD 4 D 2 O , MeOD, 1M NaOD in D 2 O 94 96 99 [M+(1,2,3,4,5,6)+H + ] [M-(1,2)+H + ] 3 1 17 excess of NaBD 4 MeOD, 30% NaOD in D 2 O diluted in MeOD (1 M NaOD) 89 70 99 [M+(1,2)+H + ] [M-(1,2)+H + ] 4 3 22 H 2 O - 83 54 99 [M+(1,2,3,4)+H + ] [M-(1,2)+H + ] 5 4 18 H 2 O - 74 41 99 [M+(1,2,3,4)+H + ] [M-(1,2)+H + ] 6 6 20 H 2 O - 94 51 99 [M+(1,2,3,4)+H + ] [M-(1,2)+H + ] 7 6 23 excess of NaBD 4 D 2 O, CD 3 COOD, MeOD,1M NaOD in D 2 O 90 69 99 [M+(1,2,3,4,5,6)+H + ] [M-(1,2)+H + ] Step d Entry R Time [h] Additives,n otes Other deuterated reagents Yield [%] D content a at preterminal C atom [%] D content a at terminal C atom [%] Isomers b Specific radioactivity c [mCi/mmol] 1 H 13 NaBH 4 - 81 - - - - 2 D 16 excess of NaBD 4 1M NaOD (30% NaOD in D 2 O diluted in MeOH) 79 90 99 [M+(1,2)+H + ] [M-(1,2)+H + ] - 3 D 20 excess of NaBD 4 MeOD, 30% NaOD in D 2 O diluted in MeOD (1 M NaOD) 89 68 99 [M+(1,2)+H + ] [M-(1,2)+H + ] - 4 D 20 NaBD 4 Pd(PPh 3 ) 4 - Failed - - - - 5 T 16 NaBT 4 - 78 95 99 [M+1+H + ] [M-1+H + ] 588,5