WO2008119197A1

WO2008119197A1 - 4-(2, 2, 3-trimethylcyclopentyl) butanoic acid used as an off-note masking agent in consumables

Info

Publication number: WO2008119197A1
Application number: PCT/CH2008/000136
Authority: WO
Inventors: Ioana Maria Ungureanu; Nicole Erna Irene Brune; Christopher Todd Simons; Kimberley Gray; Jay Patrick Slack
Original assignee: Givaudan Sa
Priority date: 2007-03-30
Filing date: 2008-03-27
Publication date: 2008-10-09
Also published as: CN101652342A; CN101652342B

Abstract

Disclosed is a Compound that blocks off-notes in consumables and methods of blocking off-notes in consumables including off-notes provided by artificial and natural sweeteners including aspartame, Saccharin, acesulfame K (Acesulfame potassium), sucralose and cyclamate; and including stevioside, swingle extract, glyccerhizin, perillartine, naringin dihydrochalcone, neohesperidine dihydrochalcon mogroside V, rubusoside, rubus extract, and rebaudioside A.

Description

-(2, 2, 3-TRIMETHYLCYCLOPENTYL) BUTANOIC ACID USED AS AN OFF-NOTE

MASKING AGENT IN CONSUMABLES

TECHNICAL FIELD Disclosed is a novel compound that allows to mask or block undesirable off-notes in consumables and the method of blocking off-notes employing said compound in consumables.

SUMMARY Provided is the compound 4-(2,2,3-trimethylcyclopentyl)butanoic acid.

Provided is a flavor composition comprising an off-note providing consumable ingredient and 4-(2,2,3-trimethylcyclopentyl)butanoic acid.

Provided is a consumable comprising a) at least one ingredient in a concentration sufficient to provide an off- note, b) 4-(2,2,3-trimethylcyclopentyl)butanoic acid.

Provided is a consumable as herein described wherein the off-note providing ingredient is selected from the group consisting of sweetener, artificial sweetener, beverage, chewing gum, nutraceutical, and pharmaceutical.

Provided is a consumable of as herein described wherein the off-note providing ingredient is an artificial sweetener selected from the group consisting of aspartame, Ace K, saccharin, sucralose, and sodium cyclamate.

Provided is a consumable as herein described wherein the off-note providing ingredient is a sweetener selected from the group consisting of including stevioside, swingle extract, glyccerhizin, perillartine, naringin dihydrochalcone, neohesperidine dihydrochalcone, mogroside V, rubusoside, rubus extract, and rebaudioside A Provided is a consumable as herein described wherein the off-note providing ingredient is a consumable selected from the group consisting of cocoa, coffee, caffeine, theobromine, diketopiperazines, vitamins, amino acids, vitamin B, casein, soy protein, ibuprofen, salicylic acid, glucoronolactone, acetaminophen, dextromethorphan, naringin, taurin, macrolide (including bioxin and erythomycin), paracetamol, acetylsalicylic acid, cimetidine, ranitidine, amoxicillin, acetominophen, cephalosporines, quassia, propylene glycol, triacetin, potassium, zinc, loperamide, limonin, flavonoides, isoflavones (including genistein and diadzein), polyphenol (including catechin and epicatechin), mint oil, D-menthol, hydrolysed vegetable protein, bitter peptides, preservatives (including benzoic acid, potassium sorbate, polysorbate 80, sodium and potassium lactate, sodium benzoate), citric acid, quinine, urea (contained in chewing gums), essential oils (inclluding thyme, sage, basil, mint), Maillard reaction products (including cyclic amines made from pyrrolidine/glucose, alanine/xylose, proline/sucrose or alanine/xylose, for example diketopiperazines), beer, hops, humulone, trans-isohumulone, lupulone, and hulupone.

Provided is a method of blocking off-notes in consumables wherein a) at least one off-note providing ingredient in a concentration sufficient to provide an off-note, and b) 4-(2,2,3-trimethylcyclopentyl)butanoic acid, are admixed to the consumable.

Provided is a method as herein described wherein the off-note providing ingredient is selected from the group consisting of sweetener, artificial sweetener, beverage, chewing gum, nutraceutical, and pharmaceutical.

Provided is a method as herein described wherein the off-note providing ingredient is an artificial sweetener selected from the group consisting of aspartame, Ace K, saccharin, sucralose, and sodium cyclamate.

Provided is a method of as herein described wherein the off-note providing ingredient is a sweetener selected from the group consisting of including stevioside, swingle extract, glyccerhizin, perillartine, naringin dihydrochalcone, neohesperidine dihydrochalcone, mogroside V, rubusoside, rubus extract, and rebaudioside A. Provided is a method as herein described wherein the off-note providing ingredient is a consumable selected from the group consisting of cocoa, coffee, caffeine, theobromine, diketopiperazines, vitamins, amino acids, vitamin B, casein, soy protein, ibuprofen, salicylic acid, glucoronolactone, acetaminophen, dextromethorphan, naringin, taurin, macrolide (including bioxin and erythomycin), paracetamol, acetolsalicilic acid, cimetidine, ranitidine, amoxicillin, acetominophen, cephalosporines, quassia, propylene glycol, triacetin, potassium, zinc, loperamide, limonin, flavonoides, isoflavones (including genistein and diadzein), polyphenol (including catechin and epicatechin), mint oil, D-menthol, hydrolysed vegetable protein, bitter peptides, preservatives (including benzoic acid, potassium sorbate, polysorbate 80, sodium and potassium lactate, sodium benzoate), citric acid, quinine, urea (contained in chewing gums), essential oils (inclluding thyme, sage, basil, mint), Maillard reaction products (including cyclic amines made from pyrrolidine/glucose, alanine/xylose, proline/sucrose or alanine/xylose, for example diketopiperazines), beer, hops, humulone, trans-isohumulone, lupulone, and hulupone.

DETAILED DESCRIPTION

The off-note blocking compound provided herein is 4-(2,2,3- trimethylcyclopentyl)butanoic acid, its structural formula is shown below.

A concentration-response analysis was performed and from results inhibitory concentration (IC) IC₅₀ values can be calculated by nonlinear regression using the function f(x) = (a-d)/(1+(x/C)^nh)+ d; with a = minimum signal, d = maximum signal, nh

= hill coefficient, C = IC_5O. and x = concentration of antagonist.

IC₅₀ is the molar concentration of an antagonist which produces 50% of the maximum possible inhibitory response for that antagonist. A more potent antagonist will have a lower IC₅₀ value.

4-(2,2,3-trimethylcyclopentyl)butanoic acid has an IC₅₀ of about 6 micromolar when tested with the TAS2R44 bitter taste receptor. For most food applications, a low IC₅₀ [micro molar] of 0.05 to 10 is desirable, however, IC₅₀ of 10 to 25 are still good and above 25 may also still acceptable depending on the application.

Various food ingredients (including ingredients naturally contained in food or additives admixed to food including flavor ingredients) provide undesirable off-notes. Particularly undesirable off-notes are the bitter off-notes, metallic off-notes, lingering, licorice-type and astringent off-notes. A particular off-note includes an unpleasant after taste that develops over time after consumption of consumables.

Other particular examples are the bitter and/or metallic and/or astringent and/or "artificial" off-notes and/or a cloyingly sweet off-note (as opposed to the "cleaner" taste of sugar) that are associated with a number of artificial sweeteners including aspartame, Ace K, saccharin, sucralose, and sodium cyclamate. Sometimes these off-notes of artificial sweeteners are described collectively as bitter off-notes.

Further examples of off-note providing ingredients are naturally occurring sweeteners including stevioside, swingle extract, glyccerhizin, perillartine, naringin dihydrochalcone, neohesperidine dihydrochalcone, mogroside V, rubusoside, rubus extract, and rebaudioside A

Still further examples of off-note providing ingredients include cocoa, coffee, caffeine, theobromine, diketopiperazines, vitamins, amino acids, vitamin B, casein, soy protein, ibuprofen, salicylic acid, glucoronolactone, acetaminophen, dextromethorphan, naringin, taurin, macrolide (including bioxin and erythomycin), paracetamol, acetolsalicilic acid, cimetidine, ranitidine, amoxicillin, acetominophen, cephalosporines, quassia, propylene glycol, triacetin, potassium, zinc, loperamide, limonin, flavonoides, isoflavones (including genistein and diadzein), polyphenol (including catechin and epicatechin), mint oil, D-menthol, hydrolysed vegetable protein, bitter peptides, preservatives (including benzoic acid, potassium sorbate, polysorbate 80, sodium and potassium lactate, sodium benzoate), citric acid, quinine, urea (contained in chewing gums), essential oils (inclluding thyme, sage, basil, mint), Maillard reaction products (including cyclic amines made from pyrrolidine/glucose, alanine/xylose, proline/sucrose or alanine/xylose, for example diketopiperazines), beer, hops, humulone, trans-isohumulone, lupulone, hulupone. The addition of off-note blockers will block or mask the off-notes and make them less apparent or unnoticeable. Artificial sweeteners will thereby loose their bitter/metallic taste and/or their cloyingly sweet lingering sweetness and instead taste more like actual sugar (sucrose).

Aspartame is the name for aspartyl-phenylalanine-1 -methyl ester, a dipeptide. It is known under various trademark names including Equal®, and Canderel®. In the European Union, it is also known under the E number (additive code) E951.

Acesulfame potassium (AceK) is the potassium salt of 6-methyl-1 ,2,3- oxathiazine- 4(3H)-one 2,2-dioxide, an N-sulfonylamide. It is also known as Acesulfame K or AceK, or under various trademark names including Sunett® and Sweet One®. In the European Union it is also known under the E number (additive code) E950.

Saccharin is the Na salt of 1 ,2-Benzisothiazol-3(2H)-one, 1 ,1 -dioxide, an N- sulfonamide. It is also known under various trademark names including Sweet'n low®.

Sucralose is the name for 6-dichloro-1 ,6-dideoxy-β-D-fructo-furanosyl 4-chloro-4- deoxy-α-D-galactopyranoside, which is a chlorodeoxysugar. It is also known by the trade name Splenda®. In the European Union, it is also known under the E number (additive code) E955. Sucralose has an off-note (also designated "aftertaste") that is a lingering liquorice-like off-note sometimes also described as bitter.

The off-note blockers can be added to consumables to block the undesirable off- notes of ingredients present in said consumables or added to such consumables.

Flavor compositions for addition to consumables can be formed that provide the off- note blockers and an off-note providing ingredient for addition to consumables, and optionally food grade excipients. Alternatively, the off note blockers can be directly added to consumables.

In particular, the off-note blockers can be added to flavor compositions or directly to consumables to block the undesirable off-notes of off-note providing ingredients including natural and artificial sweeteners added to such consumables. Consumables include all food products, food additives, nutraceuticals, pharmaceuticals and any product placed in the mouth inluding chewing gum, oral care products, and oral hygiene products including but not limited to, cereal products, rice products, tapioca products, sago products, baker's products, biscuit products, pastry products, bread products, confectionery products, desert products, gums, chewing gums, mouthwash, dental floss, flavored or flavor-coated straws, flavor or flavor-coated food/beverage containers, chocolates, ices, honey products, treacle products, yeast products, baking-powder, salt and spice products, savory products, mustard products, vinegar products, sauces (condiments), tobacco products, cigars, cigarettes, processed foods, cooked fruits and vegetable products, meat and meat products, jellies, jams, fruit sauces, egg products, milk and dairy products, yoghurts, cheese products, butter and butter substitute products, milk substitute products, soy products, edible oils and fat products, medicaments, beverages, carbonated beverages, alcoholic drinks, beers, soft drinks, mineral and aerated waters and other non-alcoholic drinks, fruit drinks, fruit juices, coffee, artificial coffee, tea, cocoa, including forms requiring reconstitution, food extracts, plant extracts, meat extracts, condiments, nutraceuticals, gelatins, pharmaceutical and non-pharmaceutical gums, tablets, lozenges, drops, emulsions, elixirs, syrups and other preparations for making beverages, and combinations thereof.

For example, in consumables containing potassium, the off-note blocker may be added to suppress the bitterness and the metallic off-note associated with potassium.

In coffee and cocoa products, the off-note blocker may be added to suppress the bitterness associated with caffeine, theobromine, and/or diketopiperazines present in said products.

In cheese products, in particular in enzyme-modified cheese products, the off-note blocker may be added to suppress the bitterness associated with bitter peptides present in said cheese products.

In soy products, the off-note blocker may be added to suppress the bitterness and beany off-notes associated with peptides, isoflavones such as genistein and diadzein present in said products.

In HVP (hydrolysed vegetable protein) products, the off-note blocker may be added to suppress the bitterness associated with bitter peptides present in said products. In functional ingredients used in fortified foods, the off-note blocker may be added to suppress the bitterness associated with vitamins and amino acids present in said products.

In pharmaceuticals, the off-note blocker may be added to suppress the bitterness associated with actives or bitter additives present in said products.

In consumables containing solvents, the off-note blocker may be added to suppress the bitterness associated with propylene glycol, triacetin, or ethanol present in said products.

In citrus products, the off-note blocker may be added to suppress the bitterness associated with naringin present in said products.

In neutraceuticals and herb medicines, the off-note blocker may be added to suppress the bitterness associated with actives or additives present in said products.

In consumables containing polyphenols such as catechin and epicatechin, the off- note blocker may be added to suppress the bitterness associated with these ingredients.

In consumables containing preservatives such as potassium sorbate, polysorbate 80, sodium and potassium lactate, sodium benzoate, the off-note blocker may be added to suppress the bitterness associated with said preservatives.

In consumables containing zinc and other mineral supplements, the off-note blocker may be added to suppress the bitterness and metallic off-notes associated with said these mineral supplements. In consumables containing mint oil or menthol (e.g. D-menthol) and citric acid of above 7%, the off-note blocker may be added to suppress the bitterness associated with this combination of ingredients.

In consumables containing quinine, the off-note blocker may be added to suppress the bitterness associated with quinine. In consumables containing artificial sweeteners (e.g. aspartame, saccharin, acesulfameK, sucralose, cyclamate), for example beverages, a off-note blocker may be added to suppress the bitterness associated with artificial sweeteners.

In chewing gums, particular dental-type chewing gums, the off-note blocker may be added to suppress the bitterness associated with urea contained in chewing gums.

In consumables containing essential oils (e.g. thyme, sage, basil, mint), the off-note blocker may be added to suppress the bitterness associated with these essential oils.

In consumables containing vegetables or herbs or their extracts, the off-note blocker may be added to suppress the bitterness associated with these ingredients.

In consumables containing Maillard reaction products (i.e. cyclic amines made from proline/sucrose or alanine/xylose, e.g. diketopiperazines), the off-note blocker may be added to suppress the bitterness associated with Maillard reaction products.

In beer and consumables containing beer or hops, the off-note blocker may be added to suppress the bitterness associated with hops.

EXAMPLES Example 1

Sensorial evaluation in various consumables

The following products were tested by panels of 6 to 10 bitter sensitive panelists.

Panelists were asked to describe the differences in off-notes and bitter notes between the product with 0.001% (wt/wt) off-note blocker unless otherwise stated and a control without off-note blocker.

A) Aspartame/Acesulfame-K containing Diet Energy Drink

The diet energy drink contained taurin, acesulfame K, aspartame, sucralose, glucuronolacton, caffeine, B-group vitamins (Niacin, pantothenic acid, B6, B12), aroma, sucrose, glucose, colours The sample containing the off-note blocker was found to be less bitter compared to the control.

B) Sucrose/glucose-sweetened energy drink

The diet energy drink contained taurin, glucuronolacton, caffeine, B-group vitamins (Niacin, pantothenic acid, B6, B 12), aroma, sucrose, glucose, colours.

The sample containing the off-note blocker was found to have less off-notes, to be less bitter, and less astringent compared to the control.

C) Iced low-sugar coffee

The sample containing the off-note blocker was found to be less bitter, and less astringent compared to the control.

D) Commercial Vanilla Flavored Nutritional Drink

Vanilla flavored nutritional drink containing calcium caseinate, soy protein isolate, sodium caseinate, vitamins and minerals.

The sample containing the off-note blocker was found to be less chalky, to have reduced protein/vitamin induced off-notes notes, and to be less astringent compared to control.

E) Saccharin sweetened cola soft drink

The sample containing the off-note blocker was found to be less bitter and to have a reduced after taste compared to the control.

F) Loperamide containing mint-flavored pharmaceutical syrup The syrup contained 1 mg loperamide HCI per 7.5 ml serving. The off-note blocker was used in a concentration of 0.004% (wt/wt).

The sample containing the off-note blocker was found to be less bitter with especially the lingering bitter after taste reduced.

G) Daytime cough syrup

The daytime cough syrup contained 325 mg acetaminophen, 10 mg dextromethorphan HBr, 5 mg phenylephrine HCI per 15 ml serving.

The sample containing the off-note blocker was found to be less bitter.

H) Dark chocolate

The sample containing the off-note blocker was found to be less bitter.

I) Baking Chocolate (100% Cocoa, Unsweetened)

The off-note blocker was used in a concentration of 0.002% (wt/wt).

The sample containing the off-note blocker was found to be less metallic, less bitter, especially the alkaloid/caffeine-like bitterness was reduced while the upfront, warm, woody bitterness was retained.

J) Overcooked coffee

Coffee was brewed and cooked on a burner for 3 hours. The off-note blocker was used in a concentration of 0.0005% (wt/wt).

The sample containing the off-note blocker was found to be less bitter.

K) Aspartame/Acesulfame-K sweetened plain nonfat yoghurt The yoghurt contained 0.0193% (wt/wt) and acesulfame-K 0.0083% (wt/wt). The off-note blocker was used in a concentration of 0.00175 % (wt/wt).

The sample containing the off-note blocker was found to have less off-notes compared to control.

L) Aspartame/Acesulfame-K sweetened cola soft drink

The off-note blocker was used in a concentration of 0.0063% (wt/wt).

The sample containing the off-note blocker was found to have less off-notes than the control.

M) Sucralose sweetened cola soft drink

The off-note blocker was used in a concentration of 0.0035% (wt/wt).

The sample containing the off-note blocker was found to have less off-notes and be less bitter than the control.

Example 2

Sensory evaluation of 4-(2.2,3-trirnethylcyclopentyl)butanoic acid in yoghurt sweetened with sucralose

4-(2,2,3-trimethylcyclopentyl)butanoic acid was synthesized as described in example 4. The sample, control and reference were prepared and stored in refrigerated conditions for 6 days. The sample was tasted and compared to the negative control.

Control/Reference:

Ingredient % (by weight)

Sucralose 0.0173

Plain Fat Free Yoghurt ad 100

Sample

Ingredient % (by weight)

Sucralose 0.0173

4-(2,2,3-trimethylcyclopentyl)butanoic 0.000875 acid

Plain Fat Free Yoghurt ad 100

Sample and control were compared to the reference and evaluated for the presence of off-notes as described in example 1.

The sample was rated to have less off-notes than the control.

Example 3

Sensory evaluation of 4-(2,2,3-trimethylcyclopentyl)butanoic acid in water sweetened with saccharin

Tested samples were 1 mM, 3 mM, and 7 mM saccharin in water, and the same samples with an added off-note blocker, 4-(2,2,3-trimethylcyclopentyl)butanoic acid (36 ppm). All samples were presented blindly so that panelists could not identify which was which.

15 ml of each sample was presented in random order to 15 bitter sensitive panelists. In two replications for each saccharin concentration, panelists were asked to select the most bitter sample. Panelists directly compared each concentration pair of sample with and without off-note blocker.

The majority of panelists (23 of 30; p=0.003, d = 1.030± 0.13) selected 7 mM saccharin without off-note blocker as tasting more bitter than the sample with off-note blocker. The data was subjected to a beta-binomial d' analysis to exclude over- dispersion (gamma=0.00; p=0.396). _.

The 1 mM and 3 mM samples without off-note blocker were also selected as tasting more bitter than the sample with off-note blocker by a majority of panelists.

4-(2,2,3-trimethylcyclopentyl)butanoic acid (36 ppm) significantly reduced the bitterness of 7 mM saccharin.

Example 4

Synthesis of 4-(2,2,3-trimethylcvclopentyl)butanoic acid All reactions were carried out under an inert atmosphere of nitrogen in dry, clean, glassware unless otherwise specified. Air and moisture sensitive liquids were added via syringe. Flash column chromatography was completed using the Teledyne lsco CombiFlash Companion/TS system. Analytical TLC (thin layer chromatography) was performed on EMD Science 60 P254 silica gel plates. ¹H and ¹³C NMR were recorded on a Bruker 300 MHz instrument. Gas chromatography/mass spectrometry experiments were conducted on an Agilent Technologies 6890N Network GC and 5975 Inert XL Mass Selective Detector Instruments.

4-(2,2,3-trimethylcyclopentyl)butanoic acid was synthesized in three steps starting with campholenic aldehyde.

In a first step 1), campholenic aldehyde was reacted with

(carbethoxymethylene)triphenylphosphorane using Methyl tert-butyl ether (MTBE) as solvent under standard Wittig conditions to form the corresponding ester (see figure below). In a second step 2), ethyl 4~(2,2,3-trimethylcyclopent-3-enyl)but~2-enoate is hydrogenated using Pd/C as catalyst , yielding ethyl 4-(2,2,3- trimethylcyclopentyl)butanoate.

In a third step 3), the hydrogenated product (ethyl 4-(2,2,3- trimethylcyclopentyl)butanoate) is saponified with aqueous NaOH in THF to form 4- (2,2,3-trimethylcyclopentyl)butanoic acid.

campholenic phosphorane aldehyde compound ethyl 4-(2,2,3-trimethylcyclopent-3-enyl)but-2-enoate

ethyl 4-(2,2,3-trimethyloyclopent-3-enyl)but-2-enoate ethyl 4-(2,2,3-trimethylcyclopentyl)butanoate

ethyl 4-(2,2,3-trimethylcyclopeπtyl)butanoate 4-(2,2,3-trimethylcyclopentyl)butanoic acid

1) Synthesis of ethyl 4-(2,2,3-trimethylcyclopent-3-enyl)but-2-enoate from 2- (2,2,3-trimethylcyclopent-3-enyl)acetaldehycie (campholenic aldehyde):

Campholenic aldehyde (10.0 g, 65.7 mmol) and MTBE (100 ml) were added to a flame-dried 250 ml round bottom flask and the flask was slowly cooled to 0° C in an ice water bath.

(Carbethoxymethylene)triphenyl-phosphorane (22.8 g, 66.0 mmol) was slowly added to the solution. The reaction was stirred at 0° C for 20 minutes then stirred at room temperature for 24 h. After the reaction was completed (as tested by TLC), the reaction mixture was reduced to half its volume by solvent evaporation in vacuo and 100 ml of hexanes were added. The flask containing the reaction mixture was then placed in an ice bath for 30 minutes. The concentrated reaction mixture with hexanes was then filtered through a plug of celite, silica, and sand and washed with hexanes, then washed with 9:1 and 1:1 (v/v) Hexanes/MTBE.

The crude was concentrated in vacuo and purifed via flash column chromatography using 1:8 (v/v) Hexanes/Ethyl acetate (EtOAc) as solvent to give ethyl 4-(2,2,3- trimethylcyc!opent-3-enyl)but-2-enoate (13.1 g, 89% yield based on starting material), which is an viscous liquid at room temperature.

Analytical results: ¹H NMR (300 MHz, CDCI3) δ 6.92 (dd, J = 10.5, 7.5 Hz, 1 H), 5.79 (d, J = 15.6 Hz, 1 H), 5.13 (s, 1 H)₁ 4.13 (m, 2H), 2.3 (m, 2H), 2.1 (m, 1H), 1.8 (m, 2H), 1.52 (s, 3H)₁ 1.24 (t, J = 15, 3H), 0.91 (s, 3H), 0.71 (s, 3H) ;

¹³C NMR (75 MHz, CDCI3) δ 166.1 , 148.6, 147.8, 121.5, 121.3, 59.6, 48.9, 46.2, 35.2, 32.9, 25.5, 19.5, 14.0, 12.2; MS m/z 222.

2) Hydrogenation reaction forming ethyl 4-(2,2,3-trimethylcyclopentyl)butanoate from ethyl 4-(2,2,3-trimethylcyclopent-3-enyl)but-2-enoate:

The catalyst, Degussa type Pd/C (1.5 g, 10% Pd), was added to a three necked round bottom flask with a stir bar, flushed with nitrogen. The catalyst was covered with distilled water (8 ml) and ethyl 4-(2,2,3~trimethylcyclopent-3-enyl)but-2-enoate (10.0 g, 44.9 mmol) in AcOEt (200 ml) was added to the reaction flask . The flask was flushed with hydrogen and the reaction was run under hydrogen balloons. After 17 h at room temperature the reaction was found to be completed by gas chromatography/mass spectrometry (GCMS). The resulting reaction mixture was filtered through a celite plug. The filtrate was concentrated in vacuo and purified via flash column chromatography EtOAc/ Hexanes (gradient 0 to 40 % v/v) to give purified ethyl 4-(2,2,3-trimethylcyclopentyl)butanoate, which is a colorless viscous liquid (7.48 g, 74% yield based on starting material).

Analytical results::

¹H NMR (300 MHz, CDCI3) δ 4.18 (q, J = 7.14, 2H), 2.34 (m, 2H), 1.82 (m, 3H), 1.56 (m, 5H), 1.30 (t, J =7.2, 3H), 0. (m, 2H), 0.99 (s, 3H), 0.86 (d, J = 5.4, 2H), 0.52 (s,

3H);

¹³C NMR (75 MHz, CDCI3) δ 173.8, 60.1 , 50.6, 45.2, 42.2, 34.8, 30.1 , 30.0, 28.1,

25.6, 24.3, 14.3, 14.2, 13.8;

MS m/z 226.

3) Synthesis of 4-(2,2,3-trimethylcyclopentyl)butanoic acid from ethyl 4-(2,2,3- trimethylcyclopentyl)butanoate by saponification with NaOH:

Ethyl 4-(2,2,3-trimethylcyclopentyl)butanoate (5 g, 22 mmol) was added to THF (25 ml) into a round bottom flask. Aqueous 1 N NaOH (25 ml) was then added to the flask and the reaction mixture was refluxed for 8 h. Upon completion of the reaction as tested using thin layer chromatography (TLC), the refluxed reaction mixture was diluted with 1 N NaOH (25 ml) and the aqueous phase washed with MTBE twice (50 ml each). The aqueous phase was treated with aqueous 1.0 N HCI until it reached pH 3, then thrice extracted with EtOAc (50 ml each). The AcOEt extracts were combined and concentrated in vacuo and the crude purified via flash column chromatography with hexanes/ EtOAc (0-40% gradient) to give 4-(2,2,3- trimethylcyclopentyl)butanoic acid, which is a colorless viscous liquid (3.82 g, 87% yield based on starting material).

Analytical results::

¹H NMR (300 MHz, CDCI3) δ 11.22 (br s, 1 H), 2.37 (m, 2H), 1.75 (m, 3H), 1.49 (m, 4H), 1.17 (m, 3H), 0.86 (s, 3H), 0.84 (d, J = 6.9 Hz, 3H), 0.52 (s, 3H); 1³C NMR (75 MHz, CDCI3) δ 179.9, 50.6, 45.2, 42.2, 34.4, 30.1 , 30.0, 28.1 , 25.0, 24.0, 14.3, 13.8; MS m/z 198.

Example 5 TAS2R44 bitter taste receptor assay for IC 50 determination

1 ) Generation of human TAS2R44 expression vector

The full length gene of human TAS2R44 was amplified by polymerase chain reaction (PCR) using gene-specific primers that span the entire coding region as described in WO 2004/029087.

The TAS2R44 cDNA was subcloned into an expression cassette based on either of the following plasmids/expression vectors: pcDNA3.1Zeo (Invitrogen). These vectors contain within their multiple cloning sites the nucleotide sequence coding for the first 45 amino acids of the rat somatostatin receptor subtype 3 (RSS tag) to facilitate cell surface targeting of the transgene (SEQ ID #4) and the nucleotide sequence coding for the herpes simplex virus (HSV) glycoprotein D epitope (HSV epitope in aminoterminal to carboxyterminal direction, HSV tag). (SEQ ID #3) for facilitating immunocytochemical detection. The TAS2R44 construct contains RSS tag, TAS2R44, and the HSV tag which are fused in frame to allow translation into the receptor protein and the resulting receptor cDNA.

This transfected expression vector is called pcDNA3.1Zeo-TAS2R44 (SEQ ID # 1) and allows for expression of the TAS2R44 protein (SEQ ID #2).

2) Generation of a cell line stably expressing Gα16-gustducin44 and TAS2R44 A cell line that stably expresses the human bitter taste receptor (TAS2R44) was generated by transfecting pcDNA3.1 Zeo-TAS2R44 into HEK293T/Gα16-gustducin 44 cells (both formed as described in under 1) above). The host cell line HEK-293T is commercially available from the American Tissue Culture Collection (catalog #CRL- 1573).

Transfection was performed as follows:

On day 0, the HEK293T Gα16-gustducin44 cells were seeded in a 6-well plate at a density of 900,000 cells per well and grown over night in selective growth medium (DMEM with 10% (v/v) heat-inactivated fetal bovine serum, 2 mM L-glutamine, 100 units/ml penicillin, 100 μg/ml streptomycin, 200 μg/ml G418 and 200 μg/ml zeocin). On day 1 , the medium was exchanged with 2 ml of antibiotic-free and serum-free growth medium. 10 μl Lipofectamine 2000 was dissolved in 250 μl DMEM and incubated for 5 minutes at room temperature. In parallel, 4 μg TAS2R44 vector DNA were dissolved in 250 μl DMEM. These two resulting solutions are mixed and incubated for 20 minutes at room temperature before they are added to the cells into the cell culture medium. After 4 hours, the medium is replaced with antibiotic-free, serum-containing growth medium.

The cells were incubated in humidified atmosphere (37 ⁰C, 5% CO₂). After 24 hours, the cells were re-plated in selective growth medium and were further incubated in a humidified atmosphere (37 ⁰C₁ 5% CO₂). After 2 to 4 weeks of culture (replacing medium as necessary), zeocin-resistant colonies were selected and expanded. The selected clone was tested successfully for functional expression of TAS2R44.

3) Fluo-4 Calcium Assay Fluo-4_AM (Invitrogen) is a fluorescent indicator of intracellular calcium dynamics (change in concentration) and allows monitoring changes in the calcium concentration, particularly an increase in response to receptor activation occurring after agonist exposure.

At day 0, the HEK293T cell line stably expressing Gα16-gustducin44 and TAS2R44 formed as described under 2) was seeded in antibiotic-free growth medium (standard DMEM with 10% (v/v) heat-inactivated fetal bovine serum, 2 mM L-glutamine standard DMEM with 10% (v/v) heat-inactivated fetal bovine serum, 2 mM L- glutamine, 100 units/ml penicillin, and 100 μg/ml streptomycin) into black wall/clear bottom 96-well plates, coated with poly(ethylenimine) (0.005% v/v) at a concentration of 15,000 cells per well and incubated for 48 hours in humidified atmosphere (37 ⁰C, 5% CO₂).

At the time of the assay, the growth medium was discarded and the cells were further incubated in a humidified atmosphere (37 ⁰C, 5% CO₂) for 1 hour with 50 μl of loading buffer consisting of 1.5 μM Fluo-4 AM and 2.5 μM probenicid (Sigma-Aldrich) in DMEM.

Afterwards, the 96-well plate was washed 5 times with 200 μl of assay buffer (130 mM NaCI, 5 mM KCI, 10 mM HEPES, 2 mM CaCI₂, and 5 mM dextrose, pH 7.4) per well, using an automated plate washer (BioTek). The plate was further incubated for 30 minutes at room temperature in the dark to allow for complete de-esterification of the Fluo-4. Afterwards the plate was washed 5 times with 200 μl of assay buffer per well, and reconstituted with 180 μl of assay buffer per well.

For assay reading, the plate was placed in a Fluorometric Imaging Plate Reader (FLIPR) (FLIPR-TETRA™, Molecular Devices), and receptor activation was initiated by addition of 20 μl of a tenfold concentrated agonist stock solution (to give the desired agonist end concentration when added to the 180 microliter assay buffer volume), which was prepared in assay buffer.

Fluorescence was continuously monitored for 20 seconds to give a signal baseline

(averaged to give F₀) prior to agonist addition and for 120 seconds after agonist addition. The change in signal divided by F₀ gives ΔF/F₀ indicated in the table, with ΔF being the maximum signal occurring within the 120 seconds minus the minimum signal (occurring within the 120 seconds after agonist addition.

All data was collected from at least two independent experiments each carried out in triplicate.

A concentration-response analysis was performed and IC₅₀ values were calculated by nonlinear regression using the function f(x) = (a-d)/(1 +(x/C)^nh)+ d; with a = minimum signal, d = maximum signal, nh = hill coefficient, C = IC₅O., and x= antagonist concentration. IC₅₀ is the molar concentration of an antagonist which produces 50% of the maximum possible effective/inhibitory response for that antagonist. A more potent antagonist will have a lower IC₅₀ value.

The obtained calcium signals were corrected for the response of cells expressing only the G Protein α subunit (Gα16-gustducin44) and normalized to the fluorescence of cells prior to the stimulus using ΔF/F0 (Fmax-Fmin/F0).

Example 6

Determination of IC50 for 4-(2,2,3-trimethylcyclopentyl)butanoic acid

The method was performed as described in example 5, using saccharin as agonist.

The cells are exposed to a constant concentration of saccharin (0.5 mM) and to a set of different concentrations of 4-(2,2,3-trimethylcyclopentyl)butanoic acid. A fluo-4 calcium assay was performed as described above in example 5 and gave an IC₅₀ of 7.36 microM. This means that 4-(2,2,3-trimethylcyclopentyl)butanoic acid inhibits the response of the TAS2R44 bitter taste receptor and will be useful to block bitter taste.

Example 7

Determination of IC50 for 4-(2,2,3-trimethylcyclopentyl)butanoic acid

The method was performed as described in example 6, exchanging saccharin for Acesulfame K (0.8 mM) as agonist. An ICs₀ of 5.04 microM was determined. This means that 4-(2,2,3-trimethylcyclopentyl)butanoic acid inhibits the response of the TAS2R44 bitter taste receptor and will be useful to block bitter taste.

Claims

Claims:

1. 4-(2,2,3-trimethylcyclopentyl)butanoic acid.

2. A flavor composition comprising an off-note providing consumable ingredient and 4-(2,2,3-trimethylcyclopentyl)butanoic acid.

3. A consumable comprising a) at least one ingredient in a concentration sufficient to provide an off- note, b) 4-(2,2,3-trimethylcyclopentyl)butanoic acid.

4. The consumable of claim 3 wherein the off-note providing ingredient is selected from the group consisting of sweetener, artificial sweetener, beverage, chewing gum, nutraceutical, and pharmaceutical.

5. The consumable of claim 3 wherein the off-note providing ingredient is an artificial sweetener selected from the group consisting of aspartame, Acesulfame K, saccharin, sucralose, and sodium cyclamate.

6. The consumable of claim 3 wherein the off-note providing ingredient is a sweetener selected from the group consisting of including stevioside, swingle extract, glyccerhizin, perillartine, naringin dihydrochalcone, neohesperidine dihydrochalcone, mogroside V, rubusoside, rubus extract, and rebaudioside A

7. The consumable of claim 3 wherein the off-note providing ingredient is a consumable selected from the group consisting of cocoa, coffee, caffeine, theobromine, diketopiperazines, vitamins, amino acids, vitamin B, casein, soy protein, ibuprofen, salicylic acid, glucoronolactone, acetaminophen, dextromethorphan, naringin, taurin, macrolide (including bioxin and erythomycin), paracetamol, acetylsalicylic acid, cimetidine, ranitidine, amoxicillin, acetominophen, cephalosporines, quassia, propylene glycol, triacetin, potassium, zinc, loperamide, limonin, flavonoides, isoflavones (including genistein and diadzein), polyphenol (including catechin and epicatechin), mint oil, D-menthol, hydrolysed vegetable protein, bitter peptides, preservatives (including benzoic acid, potassium sorbate, polysorbate 80, sodium and potassium lactate, sodium benzoate), citric acid, quinine, urea (contained in chewing gums), essential oils (inclluding thyme, sage, basil, mint), Maillard reaction products (including cyclic amines made from pyrrolidine/glucose, alanine/xylose, proline/sucrose or alanine/xylose, for example diketopiperazines), beer, hops, humulone, trans-isohumulone, lupulone, and hulupone.

8. A method of blocking off-notes in consumables wherein a) at least one off-note providing ingredient in a concentration sufficient to provide an off-note, and b) 4-(2,2,3-trimethylcyclopentyl)butanoic acid are admixed to the consumable.

9. The method of claim 8 wherein the off-note providing ingredient is selected from the group consisting of sweetener, artificial sweetener, beverage, chewing gum, nutraceutical, and pharmaceutical.

10. The method of claim 8 wherein the off-note providing ingredient is an artificial sweetener selected from the group consisting of aspartame, Ace K, saccharin, sucralose, and sodium cyclamate.

11. The method of claim 8 wherein the off-note providing ingredient is a sweetener selected from the group consisting of including stevioside, swingle extract, glyccerhizin, perillartine, naringin dihydrochalcone, neohesperidine dihydrochalcone, mogroside V, rubusoside, rubus extract, and rebaudioside A.

12. The method of claim 8 wherein the off-note providing ingredient is a consumable selected from the group consisting of cocoa, coffee, caffeine, theobromine, diketopiperazines, vitamins, amino acids, vitamin B, casein, soy protein, ibuprofen, salicylic acid, glucoronolactone, acetaminophen, dextromethorphan, naringin, taurin, macrolide (including bioxin and erythomycin), paracetamol, acetolsalicilic acid, cimetidine, ranitidine, amoxicillin, acetominophen, cephalosporines, quassia, propylene glycol, triacetin, potassium, zinc, loperamide, limonin, flavonoides, isoflavones (including genistein and diadzein), polyphenol (including catechin and epicatechin), mint oil, D-menthol, hydrolysed vegetable protein, bitter peptides, preservatives (including benzoic acid, potassium sorbate, polysorbate 80, sodium and potassium lactate, sodium benzoate), citric acid, quinine, urea (contained in chewing gums), essential oils (inclluding thyme, sage, basil, mint), Maillard reaction products (including cyclic amines made from pyrrolidine/glucose, alanine/xylose, proline/sucrose or alanine/xylose, for example diketopiperazines), beer, hops, humulone, trans-isohumulone, lupulone, and hulupone.