Supporting Information

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1 Supporting Information Wiley-VCH Weinheim, Germany

2 Supporting Information Wiley-VCH Weinheim, Germany

3 Prediction of Perception: Probing the hor17-4 Olfactory Receptor Model with Sila-Analogs of Bourgeonal and Lilial Leszek Doszczak, Philip Kraft,* Hans-Peter Weber, Rüdiger Bertermann, Annika Triller, Hanns Hatt,* and Reinhold Tacke* Universität Würzburg, Institut für Anorganische Chemie, Am Hubland, Würzburg, Germany, Fax: (+49) Givaudan Schweiz AG, Fragrance Research, Überlandstrasse 138, 8600 Dübendorf, Switzerland, Fax: (+41) Lehrstuhl für Zellphysiologie, Ruhr-Universität Bochum, Universitätsstraße 150, Bochum,Germany, Fax: (+49) Table 1. The full molecular-modeling data set in kcal/mol The enthalpic interaction energy E inter is corrected by the ligand protein stress energy E Ligand, stress, which takes into account the energy difference between the bound status versus the relaxed status of protein and ligand, providing a corrected enthalpic binding energy E inter,corr. E inter = sum of (vdw inter, attractive and repulsive, HB inter) E Ligand, stress = difference of (E Ligand, docked, E Ligand, vacuum) E Protein, stress = difference of (E protein, docked, E Protein, vacuum) E L-P, stress E inter,corr. Threshold = sum of (E Ligand, stress, E Protein, stress) = difference of (E inter, E L-P, stress) = limiting concentration of nasal perception (in ng/l) hor17-4 Ligand vdw inter, vdw inter, HB inter E inter E Ligand, E Ligand, E Ligand, attractive repulsive docked vacuum stress (S)-Lilial [(S)-1a] (R)-Lilial [(R)-1a] (S)-Sila-lilial [(S)-1b] (R)-Sila-lilial [(R)-1b] Bourgeonal (2a) Sila-bourgeonal (2b)

4 hor17-4 Ligand E Protein, E Protein, E Protein, E L-P, stress E inter,corr E int,corr Threshold docked vacuum stress rac rac [ng/l air] (S)-Lilial [(S)-1a] (R)-Lilial [(R)-1a] (S)-Sila-lilial [(S)-1b] (R)-Sila-lilial [(R)-1b] Bourgeonal (2a) Sila-bourgeonal (2b) Table 2. Alignment of hs-or1e1, hs-or17-4 to bt-rhodopsin (1U19) (based on alignment of Man et al. [12]) 1U19 : #1 bold/black: AA in LBP, X.50 OR1E1: #2 bold/red: AA conserved OR17-4: #3 red: conserved in OR1E1 and OR17-4 N' #1 YV...PFSNKTGVVRSPFEAPQYYLAEPWQ #2 IS...DFLLL.GLPI...QPEQ #3 MDGGNQSEGSEFLLL.GMSES...PEQ FlLL G PEQ TM #1 FSMLAAYMFLLIMLGFPINFLTLYVTV #2 QNLCYALFLAMYTTLLIGNLLIIVLIR #3 QQILFWMFLSMYLVTVVGNVLIILAIS Q FL Y GN LII I IL1. 73 #1 QHKKLRTPLN #2 LDSHLHTPMY #3 SDSRLHTPVY DS LHTP Y TM #1 YILLNLAVADLFMVFGGFTTTLY #2 LFLSNLSFSDLCFSSVTIPKLLQ #3 FFLANLSFTDLFFVTNTIPKMLV FL NLSF DL F TIPK L EL #1 TSLHGYFVFGPTGC #2 NMQNQDPSIPYADC #3 NLQSHNKAISYAGC N Q I YA C 2

5 TM #1 NLEGFFATLGGEIALWSLVVLAI #2 LTQMYFFLLFGDLESFLLVAMAY #3 LTQLYFLVSLVALDNLILAVMAY LTQ YF L IL MAY IL #1 ERYVVVCKPMSNF.RFGENH #2 DRYVAICFPLHYTAIMSPML #3 DRYVAICCPLHYTTAMSPKL DRYVAIC PLHYT MSP L TM #1 AIMGVAFTWVMALACAAPPLV #2 CLALVALSWVLGTTFAMLHTL #3 CILLLSLCWVLSVLYGLIHTL C L L WVL HTL EL #1 G...WSRYIPEGMQCSCGIDYYTPHEE...TNNES #2 LMARLCFCADNVIPHFFCDMSALLKLAFSDTRVNEW #3 LMTRVTFCGSRKIHYIFCEMYVLLRMACSNIQINHT LM FC I FC LL S TM #1 FVIYMFVVHFIIPLIVIFFCYG #2 VIFIMGGLILVIPFLLILGSYA #3 VLIATGCFIFLIPFGFVIISYV V G I IPF I SY IL #1 QLVFTVKEAAAQQQESATTQKAEKE #2 RIVSSILK...VPSSKGICKAFST #3 LIIRAILR...IPSVSKKYKAFST I IL PS KAFST TM #1 VTRMVIIMVIAFLICWLPYAGVAFY #2 CGSHLSVVSLFYGTVIGLYLCSSAN #3 CASHLGAVSLFYGTLCMVYLKPLHT C SHL VSLFYGT YL EL #1 IFTHQGSDFGPI #2 SSTLK... #3 YS.VK... S K TM #1 FMTIPAFFAKTSAVYNPVIY #2 DTVMAMMYTVVTPMLNPFIY #3 DSVATVMYAVVTPMMNPFIY D V MY VVTPM NPFIY C' #1 IMMNKQFRNCMVTTLCCGKNPLGDDEASTTVSKTETS #2 SLRNRDMKGALSRVIHQKKTF... 3

6 #3 SLRNKDMHGALGRLLDKHFKR... SLRN DM GAL Table 3. The 23 amino acids that form the ligand-binding pocket (LBP) of the hor17-4 receptor model. TM 2 F86 TM 3 L114 L117 V118 V121 A122 N125 TM 4 Y167 TM 5 T207 I211 F212 TM 6 Y261 C265 M266 Y268 I269 L272 TM 7 V292 A295 V296 T298 EL 2 E188 M189 General Information for the Experimental Section The syntheses were carried out under dry N 2 where necessary, organic solvents were dried and purified according to standard procedures and stored under dry N 2. The NMR spectra were recorded at 23 C in CDCl 3 on a Bruker DRX-300 or Bruker Avance-400 NMR spectrometer. Chemical shifts (ppm) were determined relative to internal CHCl 3 ( 1 H, δ = 7.24), internal CDCl 3 ( 13 C, δ = 77.0), and external SiMe 4 ( 29 Si, δ = 0). The 2 J HH coupling constants reported for the CH 2 groups represent absolute values. The AA XX 1 H spin systems were analyzed by using the program WIN-DAISY 4.05 (Bruker-Franzen GmbH: Bremen, Germany, 1998). Samples for olfactory analysis were additionally purified by bulb-to-bulb distillation. The odor thresholds (geometrical means) were determined by GC olfactometry. Precisely adjusted dilutions of the sample substances were injected splitless in descending order of concentration into a GC until the panellist sniffing in blind failed to detect an odor by pressing a button at the correct time. Synthesis of Sila-lilial (1b) applying the hydrazone methodology Sila-lilial (1b) was synthesized according to Scheme 4. 8 N 10 LDA N Si N N + Si CN 11 71% 12 18% 11 H 2 O CuCl 2 1b 66% Scheme 4. Synthesis of Sila-lilial (1b) applying the hydrazone methodology. 4

7 The alkylation of propanal N,N-dimethylhydrazone (10) in the presence of LDA provided the respective hydrazone 11 only in 71% yield due to a competitive formation of compound 12 (isolated in 18% yield). Compound 11 was prepared analogously to the synthesis of the hydrazone 9 using propanal N,N-dimethylhydrazone [16] (2.40 g, 24.0 mmol). Compound 11 and the byproduct 12 were isolated by column chromatography on silica gel (( m; pentane/et 2 O (5/2 (v/v))) to afford 11 (3.73 g, 71%) and 12 (800 mg, 18%) as colorless liquids. Data for 11: 13 C NMR (75.5 MHz): δ = (CHN), (CHCCH 2 ), (CSi(CH 3 ) 3 ), (2C, SiCCH) (2C, CHCCH 2 ), 43.3 (N(CH 3 ) 2 ), 41.7 (CH 2 ), 38.5 (CHCH 3 ), 18.3 (CHCH 3 ), 1.1 (Si(CH 3 ) 3 ). Data for 12: 1 H NMR (400.1 MHz): δ = 7.47 (δ XX, 2H; SiCCH) and 7.21 (δ AA, 2H; CHCCH 2, AA XX system, 4 J XX = 1.4 Hz, 3 J AX = 3 J A X = 7.6 Hz, 5 J AX = 5 J A X = 0.6 Hz, 4 J AA = 1.8 Hz), (m, 3H; partially overlapping multiplets of CH 2 CHCH 3 ), 1.32 (d, 3 J = 6.8 Hz, 3H; CHCH 3 ), 0.25 (s, 9H; Si(CH 3 ) 3 ); 13 C NMR (100.6 MHz): δ = (CHCCH 2 ), (CSi(CH 3 ) 3 ), (2C, SiCCH), (2C, CH 2 CCH), (CN), 40.0 (CH 2 ), 27.4 (CHCN), 17.7 (CHCH 3 ), 1.2 (Si(CH 3 ) 3 ); 29 Si NMR (79.5 MHz): δ = 4.0; elemental analysis (%) calcd for C 13 H 19 NSi ( g mol 1 ): C 71.83; H 8.81; N 6.44; found: C 71.84; H 8.65; N Sila-lilial (1b) was then prepared analogously to the synthesis of Sila-bourgeonal (2b) using a solution of 11 (3.67 g, 14.0 mmol) in pentane (140 ml) and a solution of CuCl 2 2H 2 O (5.73 g, 33.6 mmol) in water (28 ml). Column chromatography on silica gel (( m; pentane/et 2 O (9/1(v/v))) afforded 1b (2.03 g, 66%) as a colorless liquid. The analytical data of the product were identical with those obtained for the reference material 1b synthesized by Heck coupling. Multigram scale synthesis of Sila-bourgeonal (2b) starting from 4-bromo-4-methylbenzene (6) Preparation of 7: A solution of 6 (171 g, 1.00 mol) in THF (800 ml) was added dropwise with stirring to Mg turnings (24.8 g, 1.02 mol), and the boiling mixture was then stirred for 1 h and then allowed to cool to 20 C, followed by addition of Me 3 SiCl (114 g, 1.05 mol). The mixture was then stirred for 1 h and allowed to cool to 20 C. Subsequently, NEt 3 (10 ml) and ice-cold water (500 ml) were added in turn. The organic layer was separated, the aqueous layer was extracted with pentane (3 200 ml), and the combined organic extracts were washed with saturated aqueous NaCl solution (200 ml) and dried (MgSO 4 ). The solvent was removed under reduced pressure, and the residue was distilled in vacuo to afford 7 (155 g, 94%; C/7 mbar) as a colorless liquid. Preparation of 8: N-Bromosuccinimide (93.4 g, 525 mmol) and dibenzoyl peroxide (484 mg, 2.00 mmol) were added to a solution of 7 (82.2 g, 500 mmol) in CCl 4 (325 ml). The reaction mixture was heated to reflux, and the heating mantle was then removed. When the spontaneous boiling ceased (ca. 5 min), the mixture was refluxed for a further 15 min and then cooled to 0 C (formation of a precipitate). The reaction mixture was then applied to the top of a pad of silica gel ( m) in a glass frit, and the product was washed out of the residue with pentane (200 ml). The solvent was removed under reduced pressure, and the residue was distilled in vacuo to afford 8 (87.2 g, 72%; b.p C/14 mbar) as a colorless liquid. Preparation of 9: A 2.5 M solution of BuLi in hexanes (172 ml, 430 mmol BuLi) was added dropwise at 78 C to a stirred solution of ipr 2 NH (45.5 g, 450 mmol) in THF (400 ml). The cooling bath was removed and the mixture allowed to warm to 20 C, followed by dropwise addition of the ethanal N,N-dimethylhydrazone [16] (37.9 g, 440 mmol) at 78 C with stirring. The reaction mixture was again allowed to warm to 20 C (white precipitate), followed by dropwise addition of 8 [18] (97.3 g, 400 mmol) at 78 C with stirring, and the reaction mixture was then stirred at 20 C for 12 h. Subsequently, saturated aqueous NaCl solution (400 ml) and pentane (400 ml) were added, the organic layer was separated, the aqueous layer was extracted with pentane (3 200 ml), and the combined organic extracts were dried (MgSO 4 ). The solvent was removed under reduced pressure to afford crude 9 (93.3 g, ca. 94%). 5

8 Preparation of 2b: A solution of oxalic acid (50.4 g, 560 mmol) in water (750 ml) was added to a solution of crude 9 (93.2 g, ca. 375 mmol) in pentane (1.13 L), and the mixture was stirred vigorously for 3 h until the starting material had been consumed. The layers were separated, and the organic layer was washed twice with a solution of oxalic acid (12.6 g) in water (170 ml). The combined aqueous layers were extracted with pentane (3 100 ml), the combined organic layers were dried (MgSO 4 ), the solvent was removed under reduced pressure, and the residue was distilled in vacuo to afford 2b (55.3 g, 67% starting from 8; C/0.015 mbar) as colorless liquid. The analytical data of the product were identical with those obtained for the reference material 2b synthesized by the copper(ii) assisted cleavage procedure reported in the article. Preparation of (4-iodophenyl)trimethylsilane (4) A 1.6 M solution of BuLi in hexanes (19.0 ml, 30.4 mmol BuLi) was added dropwise at 78 C to a stirred solution of 3 (9.90 g, 30.0 mmol) in Et 2 O (30 ml). The cooling bath was removed, and the reaction mixture was allowed to warm to 0 C within 1.5 h, followed by dropwise addition of Me 3 SiCl (3.59 g, 33.0 mmol) at 78 C. The cooling bath was removed and the mixture stirred at 20 C for 12 h, followed by washing with a saturated aqueous Na 2 CO 3 solution (3 50 ml) and drying (MgSO 4 ). The solvent was removed under reduced pressure, and the residue was applied to the top of a pad of silica gel ( m) in a glass frit, and the product was washed out of the residue with pentane. The solvent was removed under reduced pressure to afford 4 (8.10 g, 98%) as a colorless liquid. 1 H NMR (400.1 MHz): δ = 7.67 (δ XX, 2H; ICCH) and 7.22 (δ AA, 2H; SiCCH, AA XX system, 4 J XX = 1.8 Hz, 3 J AX = 3 J A X = 7.8 Hz, 5 J AX = 5 J A X = 0.5 Hz, 4 J AA = 1.7 Hz), 0.23 (s, 9H; Si(CH 3 ) 3 ); 13 C NMR (100.6 MHz): δ = (CSi(CH 3 ) 3 ), (2C, ICCH), (2C, SiCCH), 95.7 (CI), 1.3 (Si(CH 3 ) 3 ); 29 Si NMR (79.5 MHz): δ = 3.3; elemental analysis (%) calcd for C 9 H 13 ISi ( g mol 1 ): C 39.14; H 4.74; found: C 39.54; H Attempt to synthesize Sila-bourgeonal (2b) via Heck coupling (fi formation of 5) Pd(OAc) 2 (113 mg, 503 mol) was added to a mixture of 4 (2.77 g, 10.0 mmol), prop-2-en-1-ol (697 mg, 12.0 mmol), and NEt 3 (1.21 g, 12.0 mmol). After stirring the reaction mixture for 10 h at C, it was allowed to cool to 20 C, and the product was isolated by column chromatography on silica gel ( m; pentane/et 2 O (3/1 (v/v))) to afford 5 (1.50 g, 76%) as a colorless liquid. 1 H NMR (400.1 MHz): δ = 9.43 (s, 1H; CHO), 7.44 (δ XX, 2H; SiCCH) and 7.09 (δ AA, 2H; CHCCH 2, AA XX system, 4 J XX = 1.5 Hz, 3 J AX = 3 J A X = 7.5 Hz, 5 J AX = 5 J A X = 0.7 Hz, 4 J AA = 1.9 Hz), 7.37 (δ XX, 2H; SiCCH) and 7.12 (δ AA, 2H; CHCCH 2, AA XX system, 4 J XX = 1.5 Hz, 3 J AX = 3 J A X = 7.5 Hz, 5 J AX = 5 J A X = 0.6 Hz, 4 J AA = 1.9 Hz), (m, 1H; CCH), 3.56 (s, 2H; CCH 2 C), (m, 4H; AA BB system, CH 2 CH 2 ), 0.25 (s, 9H; Si(CH 3 ) 3 ), 0.22 (s, 9H; Si(CH 3 ) 3 ); 13 C NMR (100.6 MHz): δ = (CO), (CHCCHO), (CHCCHO), (CCH 2 CH 2 CH), (CCH 2 CCHO), (CSi(CH 3 ) 3 ), (CSi(CH 3 ) 3 ), (2C, SiCCH), (2C, SiCCH), (2C, CH 2 CCH), (2C, CH 2 CCH), 34.4 (CH 2 CH 2 CH), 30.9 (CH 2 CCHO), 29.6 (CH 2 CHCHO), 1.09 (Si(CH 3 ) 3 ), 1.10 (Si(CH 3 ) 3 ); 29 Si NMR (79.5 MHz): δ = 4.2, 4.3; elemental analysis (%) calcd for C 24 H 34 OSi 2 ( g mol 1 ): C 73.03; H 8.68; found: C 73.45; H The expected Sila-bourgeonal (2b) was present in the reaction mixture in trace quantities only. 6