Selectively Fluorinated Citronellol Analogues Support a Hydrogen Bonding Donor Interaction with the Human OR1A1 Olfactory Receptor

C-2 fluorinated and methylated stereoisomers of the fragrance citronellol 1 and its oxalate esters were prepared from (R)-pulegone 11 and explored as agonists of the human olfactory receptor OR1A1 and assayed also against site-specific mutants. There were clear isomer preferences and C-2 difluorination as in 18 led to the most active compound suggesting an important hydrogen bond donor role for citronellol 1. C-2 methylation and the corresponding oxalate ester analogues were less active.

The gas was dried by passing through concentrated H2SO4 and anhydrous CaCl2, and then bubbled through 11 for 5 h. After the addition was complete, the reaction could be followed by TLC (hexane : ethyl acetate = 6 : 1) to completion. The solution of the two isomers could be used directly without isolation.

S-4
A solution of 12 (8.95 g, 0.053 mol) in dry THF (45 mL) was gradually added to a suspension of LiAlH4 solution (2.5 g, 33 mL of 2M, 0.066 mol) in a 250 mL. The flask that was kept under N2 at 0 °C with stirring for 5 h. Water (5 mL) was added cautiously and the whole was diluted with 30 mL ethyl acetate and then filtered with the aid of celite with suction. The filtercake was washed with ethyl acetate and the filtrate was dried over anhydrous Na2SO4 and concentrated under reduced pressure to give the product as a light yellow oil 1 (8 g, 99%). 1 1.17 (dddd,J = 13.4,9.4,7.6,6.0 Hz,1H),0.90 (d,J = 6.6 Hz,3H

S-5
A solution of methylamine in ethanol (33%) (7 eq.) was added to a round bottom flask equipped with a magnetic stir bar and charged with ethyl 2-amino-3-phenylpropionate hydrochloride salt (14a, 1 eq.). The mixture was stirred for 48 h at rt and was then concentrated under reduced pressure. The resulting oil was dissolved in chloroform (50 mL), washed with saturated K2CO3 (50 mL) and extracted into chloroform (3 x 50 mL).
The organics were dried over anhydrous Na2SO4, filtered and concentrated under reduced pressure. This material was used without further purification.
The reaction was cooled to rt and concentrated under reduced pressure. The product was purified over silica gel to give 14c. 14c was then 1:1 mixed with dichloroacetic acid (DCA) and recrystalised to give 14.

Synthesis of fluorinated (R)-citronellol (General procedure B)
N-Fluorobenzenesulfonimide (NFSI, 5 eq.) and 14 (20 mol%) were added to a solution of 13 in THF(13.5 mL) and isopropanol (1.5 mL) at -15 °C and the mixture was stirred for 16 h. The reaction was diluted with Et2O (10 mL) and filtered through a pad of Davisil® Silica Gel, eluting with Et2O. Me2S (10 mL) was added forming a white precipitate. The resulting mixture was washed with saturated NaHCO3 solution (3 x 150 mL) and brine (1 x 150 mL) and dried over anhydrous Na2SO4, filtered and concentrated under reduced pressure. The resulting oil was dissolved in DCM (12 mL) and ethanol (8 mL) and then NaBH4 (2.5 eq.) was added at 0 °C and the reaction was left to warm to rt with stirring. After 1 h, saturated NH4Cl solution (150 mL) was added at 0 °C. The mixture was warmed to rt and stirred vigorously 1 h. The cloudy suspension was allowed to separate and 75 mL of DCM was added. The solution was extracted into DCM (3 x 80 mL) and the combined organics were washed with saturated NaHCO3 solution (3 x 100 mL) and brine (1 x 150 mL) and dried over anhydrous Na2SO4. The product was concentrated under reduced pressure and purified over silica gel.

S-7
Alcohol 3 was prepared according to General Procedure B from 13 (210 mg, 1.36 mmol), NFSI (2.15 g, 6.  (1 x 50 mL) and then dried over anhydrous Na2SO4, filtered and concentrated under reduced pressure. The resulting oil was dissolved in DCM (12 mL) and ethanol (8 mL) and NaBH4 (189.2 mg, 5 mmol) was added at 0 °C and the reaction warmed to rt. After
The product was purified over silica gel.

Synthesis of methylated oxazolidinone (General procedure D)
Sodium hexamethyldisilamide (NaHMDS, 3.6 eq.) was added to a flask containing a solution of 20 or 21 (1 eq.) in dry THF (60 mL) at -78 °C. After 1 h, iodomethane (MeI, 14 eq.) was added dropwise and the solution was stirred for 6 h at -78 °C. The reaction mixture was quenched with glacial acetic acid (AcOH, 2.5 mL) and aqueous NH4Cl solution (25 ml) at -78 °C and then brought to 0 °C. The aqueous layer was extracted with ethyl acetate (3 x 40 mL). The organic layers were combined and washed with aqueous Na2S2O3 solution (50 ml) and brine (50 mL), dried over anhydrous Na2SO4, filtered and concentrated under reduced pressure. It was purified over silica gel to give the product.

Synthesis of methylated (R)-citronellol (General procedure E)
NaBH4 (3 eq.) was added to a solution of 22 or 23 (1 eq.) in dry THF (20 mL) at 0 ºC and then water (8 mL) was added. The solution was warmed to rt and stirred for 6 h.
The reaction mixture was quenched with aqueous HCl (1 M 35 mL) and the aqueous layer was extracted with ethyl acetate (3 x 30 mL). The organics were washed with brine (80 ml), dried over anhydrous Na2SO4, filtered and concentrated under reduced pressure. The product was purified over silica gel.

Additional experimental procedures Clones and Mutagenesis
OR1A1 and an N-terminal rhodopsin tag were cloned into the pCI mammalian expression vector, as described previously. 8 For OR1A1 active site mutants, sitedirected mutagenesis was carried out using overlap extension PCR. The identities of all constructs were confirmed by sequencing.

Luciferase assay
HEK293T-derived Hana3A cell line was grown in minimum essential medium containing 10% FBS at 37 °C with 5% CO2 and were plated onto 96-well plates (Corning) for experiments. After 18-24 h, OR1A1, mRTP1S, CRE-Luciferase, and pRL-SV40 were transiently transfected into cells using Lipofectamine 2000 (Invitrogen). Twenty-four hours after transfection, the cells were stimulated with odorants dissolved in CD293 medium (Gibco). We used the Dual-Glo Luciferase kit (Promega) and followed the manufacturer's protocol for measuring chemiluminescence using a Synergy H1 plate reader (BioTek). All responses were normalised to the activity of wild type OR1A1 to (2S,3R)-monofluoro-citronellol 3. The results were analysed with GraphPad Prism 8.