Doubly Deuterium-labeled Patchouli Alcohol from Cyclization of Singly Labeled [2- SUPPORTING INFORMATION

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1 Doubly Deuterium-labeled Patchouli Alcohol from Cyclization of Singly Labeled [2-2 H 1 ]Farnesyl Diphosphate Catalyzed by Recombinant Patchoulol Synthase. By Juan A. Faraldos, Shuiqin Wu, Joe Chappell, and Robert M. Coates* Contribution from Department of Chemistry, University of Illinois, 600 South Mathews Avenue, Urbana, IL 61801, And Department of Plant and Soil Sciences, University of Kentucky, Lexington, KY , USA SUPPORTING INFORMATION TABLE OF CONTENTS General Methods and Instrumentation...S1-S2 Detailed Preparative Procedures and Complete Characterization Data S2-S5 Expression and Purification of Recombinant Patchouli Alcohol Synthase..S5 Literature References S5-S6 Reproductions of 1 H, 2 H, 13 C, and 31 P NMR Spectra..S7-S30 Reproductions of Mass Spectra..S1-S37 General Methods and Instrumentation. All chemicals and solvents were obtained from commercial vendors and used without further purification unless otherwise noted. THF, Et 2 O, CH 2 Cl 2, CH 3 CN and toluene were dried by means of a solvent filtration system. In some cases THF and diethyl ether were distilled from sodium/benzophenone ketyl, and CH 2 Cl 2 and benzene were distilled from CaH 2 prior use. DMF was dried by storage over molecular sieves prior to use. GC analyses were conducted on a Rtx-5 30-m fused silica capillary column (split ratio ca 100:1). The following program was used: initial temp 50 o C for 0.5 min, ramp 4 o C/min to 120 or 150 o C at an injector temp of 220 o C. GC/MS (FI) analyses were performed at the School of Chemical Sciences Mass Spectrometry Facility at the University of Illinois using narrow bore ( mm ID) fused silica capillary column for GC/MS on a 70-VSE S1

2 mass spectrometer. 1 H, 13 C, HMQS and 2 H NMR spectra were recorded in CDCl 3 ( 1 H, 7.26; 13 C, 77.0), C 6 D 6, CD 3 OD ( 1 H, 3.30) or CCl 4 ( 2 H NMR) with VXR500, UI500NB or UI600 Varian spectrometers in the School of Chemical Sciences NMR Spectroscopy Facility. Chemical shifts are in ppm and coupling constants are in Hertz. 31 P NMR spectra were not referenced. CDCl 3 from Aldrich Chemical Co. was purified by filtering through basic alumina (Brockmann I, standard grade, 150 mesh, 58 Å and dried overnight over molecular sieves (4 Å) in the dark. C 6 D 6 and CD 3 OD (Aldrich Chemical Co.) were used without purification. TLC analyses were performed on silica gel 60 F254 precoated-plates (250 m). TLC visualizations were performed with 5% phosphomolybdic acid (0.2 M in 2.5% conc. H 2 SO 4 /EtOH (v/v)), I 2 vapor, 0.1 % berberine HCl/EtOH or UV light. Preparative incubations of [2-2 H 1 ]FPP with rpts were carried out as previously reported by Deguerry et al. 1 at the University of Kentucky. Enzymatically-generated hydrocarbons and alcohols were purified by preparative TLC on silica gel with Merck 60F-254 plates with 0.25-mm thickness. Ethyl (2Z, 6E)-[2-2 H 1 ] and (2E,6E)-[2-2 H 1 ]Farnesoates (4). The procedure of Anastasia et al was followed for this olefination reaction. 2b To a cold (-70 o C) solution of ethoxyacetylene (1.0 g, 5.7 mmol, 40% w/w solution in hexane) in dry THF (3 ml) was added a hexane solution of n-buli (1.6 M, 3.4 ml, 5.4 mmol) under N 2. After 1 h, geranyl acetone (446 mg, 2.3 mmol) was added via syringe. The temperature was increased slowly (1 h) to -40 o C and then 3.0 ml of 10 % D 2 SO 4 in D 2 O (99.5 atom % D) was added. The temperature was slowly increased to room temp, and the solution was stirred under N 2. After 12 h, satd NaHCO 3 (15.0 ml) was added, and the mixture was extracted with Et 2 O (15.0 ml x 3). The ethereal solution was washed with brine, dried (MgSO 4 ), and evaporated under reduced pressure. Purification by silica gel flash chromatography using pentane gave ethyl ester 4 (585 mg, 96 %) as a 1:1 mixture of trans and cis isomers with an isotopic purity (deuterium incorporation) >99% ( 1 H NMR analysis) Data for trans ester 4: 1 H NMR (500 MHz, CDCl 3 ) 5.16 (t, J = 7.1 Hz, 1H), 5.08 (s, 1H), 4.14 (q, J = 7.1 Hz, 2H), 2.63 (t, J = 7.5 Hz, 2H), 2.16 (m, 4H), 1.98 (m, 4H), 1.68 (s, 3H), 1.60 (s, 6H), 1.27 (t, J = 4.4 Hz, 3H); data for cis ester 4: 1 H NMR (500 MHz, CDCl 3 ) 5.08 (s, 2H), 4.13 (q, J = 7.1 Hz, S2

3 2H), 2.19 (s, 7H), 2.05 (m, 4H), 1.68 (s, 3H), 1.60 (s, 6H), 1.26 (t, J = 3.4 Hz, 3H); 13 C NMR (125 MHz, CDCl 3 ) 136.1, 135.7, 124.3, 124.2, 123.5, 122.9, 59.41, 59.38, 40.9, 39.7, 39.6, 33.4, 26.7, 26.3, 25.9, 25.7, 25.3, 18.7, 17.7, 16.0, 15.9, MS (EI) m/z (rel. int.) 266 (M + + 1, 4), 265 (M +, 19), 222 (16), 150 (20), 137 (20), 129 (56), 122 (33), 69 (100); HRMS (EI) m/z calcd for C 17 H 27 D 1 O 2 (M + ) Found: The 1 H and 13 C NMR data agree with the literature values reported for unlabelled trans 3a and cis 3b ethyl esters 4. [2-2 H 1 ](2Z, 6E)- and [2-2 H 1 ](2E, 6E)-Farnesols (5). To a cold (-78 o C) solution of a 1:1 mixture of cis,trans and trans,trans esters 4 (560 mg, 2.11 mmol) in dry toluene (5.0 ml) was added a hexane solution of i-bu 2 AlH (1 M, 4.60 ml, 4.60 mmol) under N 2. The resulting solution was stirred for 3 h at - 78 o C and then water (5.0 ml) and 10% aq HCl (2.0 ml) were added. The mixture was extracted with 20 % EtOAc/hexane (10 ml x 3). The organic layer was washed with brine, dried (Na 2 SO 4 ), and evaporated under reduced pressure. Purification by silica gel flash chromatography using 5 % EtOAc/hexane as eluent gave pure cis [2-2 H 1 ]farnesol 5 (188 mg, 38 %), a mixture of cis and trans [2-2 H 1 ]farnesols (126 mg, 25 %) and pure trans [2-2 H 1 ]farnesol 5 (168 mg, 34 %). Data for [2-2 H 1 ]cis, trans-farnesol 5: 1 H NMR (500 MHz, CDCl 3 ) δ (m, 2H), 4.10 (s, 2H,), (m, 8H), 1.75 (s, 3H), 1.68 (s, 3H), 1.60 (s, 3H), 1.17 (br s, 1H, OH); 13 C NMR (125 MHz, CDCl 3 ) δ 140.4, 140.0, 135.9, 131.4, 124.2, 123.5, 58.9, 39.7, 31.9, 26.6, 26.5, 25.7, 23.4, 17.7, 16.0; MS (EI) m/z (rel. int.) 224 (M + + 1, 13), 223 (M +, 66), 206 (M H 2 O, 13), 205 (M + - H 2 O, 55), 192 (48), 190 (47), 180 (43), 162 (100); HRMS (EI) m/z calcd for C 15 H 25 D 1 O (M + ) Found: Data for [2-2 H 1 ]trans, trans-farnesol 5: 1 H NMR (500 MHz, CDCl 3 ) δ 5.10 (m, 2H), 4.14 (s, 2H), (m, 8H), 1.68 (s, 6H), 1.59 (s, 6H), 1.25 (br s, 1H, OH); 13 C NMR (125 MHz, CDCl 3 ) δ 139.7, 135.3, 131.3, 124.3, 123.7, 59.3, 39.7, 39.5, 26.7, 26.3, 25.7, 17.7, 16.2, Noticeable was the apparent absence of the olefinic resonance of C2 (=CD-); 2 H NMR (77 MHz, CHCl 3 ) δ 5.47 (broad s); S3

4 MS (EI) m/z (rel. int.) 224 (M + + 1, 12), 223 (M +, 62), 206 (M H 2 O, 11), 205 (M + - H 2 O, 53), 192 (100), 190 (44), 180 (51); HRMS (EI) m/z calcd for C 15 H 25 D 1 O (M + ) Found: (2E, 6E)- [2-2 H 1 ]Farnesyl Chloride (6). The general procedure of Collington and Meyers 4 was followed. To a cold (0 o C) and well-stirred suspension of trans alcohol 5 (168 mg, 0.75 mmol), LiCl (500 mg, 11.8 mmol), and s-collidine (500 L, 547 mg, 4.5 mmol) in DMF (10.0 ml) was added methanesulfonyl chloride (250 L, 170 mg, 1.5 mmol) via syringe and under N 2. The mixture was stirred for 3 h, poured into ice water (50 ml), and extracted with pentane (20 ml x 3). The combined organic extracts were washed with satd Cu(NO 3 ) 2 (40 ml x 2), deionized water (40 ml), and satd NaHCO 3 (40 ml), dried (Na 2 SO 4 ),and concentrated under reduced pressure to afford 196 mg of a light yellow oil. Purification by rapid flash chromatography on silica gel afforded mg (98 %) of pure deuterated chloride 6: 1 H NMR (400 MHz, CDCl 3 ) δ 5.09 (t, J = 6.8 Hz, 2H), 4.10 (s, 2H), (m, 8H), 1.73 (s, 3H), 1.68 (s, 3H), 1.60 (s, 6H); 13 C NMR (100 MHz, CDCl 3 ) δ 142.7, 135.6, 131.4, 124.3, 123.4, 41.1, 39.6, 39.3, 26.6, 26.0, 25.7, 17.7, 16.1, (2E, 6E)-[2-2 H 1 ]Farnesyl Diphosphate, Trisammonium Salt (1-d 1 ). The procedure by Poulter and coworkers 5 was followed. A suspension of chloride 6 (178.1 mg, 0.74 mmol), 4Å molecular sieve powder (1.5 g), and HOPP(NBu 4 ) 3 (1.5 g, 1.7 mmol) in dry CH 3 CN (15 ml) was stirred at room temp under N 2. After 24 h, the suspension was diluted with additional CH 3 CN (20 ml), and the solids were filtered through a pad of Celite. The filtrate was evaporated under reduced pressure using a 40 C water bath to yield the Bu 4 N + salt of (2E, 6E)-[2-2 H 1 ]FPP. The salt was dissolved in 5 ml of ion-exchange buffer (25 mm NH 4 CO 3 in 2% v/v 1-propanol/ water) and loaded onto a column of BioRad (NH4)+ cation exchange resin (ammonium form). The flask was washed twice with 20 ml of buffer, and both washes were loaded onto the column before elution with 100 ml of ion-exchange buffer. Fractions containing the diphosphate ( 31 P NMR evidence) were combined and lyophilized (18 h) to give a white solid. Solidliquid extraction with MeOH (6 ml x 5) and removal of the solvent under reduced pressure at room S4

5 temp gave 203 mg (63 % yield, 90 % pure by 1 H and 31 P NMR analyses) of the trisammonium salt 1-d 1. Recrystallization from MeOH at 0 o C gave mg (58 %) of essentially pure diphosphate 1-d 1 : 1 H NMR (500 MHz, CD 3 OD) δ 5.09 (broad t, J = 7.5 Hz, 1H), 5.97 (app t, J = 6.0 Hz, 1H), 4.41 (d, J = 5.0 Hz, 2H), (m, 8H), 1.73 (s, 3H), 1.58 (s, 3H), 1.55 (s, 3H), 1.49 (s, 3H), 1.48 (s, 3H); 13 P NMR (202 MHz, CD 3 OD) δ (broad d, J = Hz). Purification of Recombinant Patchouli Alcohol Synthase (rpts). The characterized PTS gene identified by Deguerry et al 1 was initially inserted into the bacterial protein expression vector pet28a. The recombinant vector containing the PTS gene fused to a leader sequence coding for a N-terminal hexahistidine purification tag was transformed into BL21(DE3) (Novagen, Madison, WI) E. coli cells for protein over-expression as described before. 1 Bacterial cultures (100 to 1,000 ml) were then grown at 37 o C until an approximate O.D600 = 0.6. After cooling on ice for 5 min, isopropyl β-d-thiogalactoside (final concentration 0.25 mm) was added to induce synthase gene expression and the cultures continued to incubate at room temp overnight. Cells were harvested by centrifugation, re-suspended in approximately 1/10th of their original volume in lysis buffer (80 mm potassium phosphate, ph 7.0, 10 mm meta-bisulfate, 10 mm 2-mercaptoethanol, 10 mm sodium ascorbate). After sonication and clarification by centrifugation, patchoulol synthase was purified from the culture lysate to near homogeneity using Ni 2+ affinity chromatography. A total of 4 to 12.5 ml of purified protein was dialyzed twice against 2 L of dialysis buffer (20 mm. HEPES, ph 7.5, 5 mm MgCl 2, 1 mm dithiothreitol). The purified recombinant PTS synthase was used immediately for most of the in vitro experiments, or stored at -80 o C after adjustment to 50% (V/V) glycerol. References 1. Deguerry, F.; Pastore, L.; Wu, S.; Clark, A.; Chappell, J.; Schalk, M. Arch. Biochem. Biophys. 2006, 454, S5

6 2. (a) Arigoni, D.; Cane, D. E.; Shim, J. H.; Croteau, R.; Wagschal, K. Pytochemistry 1993, 32, (b) Anastasia, M.; Allevi, P.; Ciuffreda, P.; Fiecchi, A.; Gariboldi, P.; Scala, A. J. Chem. Soc. Perkin Trans. I, 1985, (a) Gibbs, R. A.; Krishnan, U.; Dolence, J. M.; Poulter, C. D. J. Org. Chem. 1995, 60, (b) Xie, H.; Shao, Y.; Becker, J. M.; Naider, F.; Gibbs, R. A. J. Org. Chem. 2000, 65, Collington, E. W.; Meyers, A. I. J. Org. Chem. 1971, 36, Woodside, A. B.; Huang, Z.; Poulter, C. D. Org. Synth. 1993, Coll. Vol. 8, S6

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