The Odorant (R)-Citronellal Attenuates Caffeine Bitterness by Inhibiting the Bitter Receptors TAS2R43 and TAS2R46

Article abstract of DOI:10.1021/acs.jafc.6b03554

Sensory studies showed the volatile fraction of lemon grass and its main constituent, the odor-active citronellal, to significantly decrease the perceived bitterness of a black tea infusion as well as caffeine solutions. Seven citronellal-related derivatives were synthesized and shown to inhibit the perceived bitterness of caffeine in a structure-dependent manner. The aldehyde function at carbon 1, the (R)-configuration of the methyl-branched carbon 3, and a hydrophobic carbon chain were found to favor the bitter inhibitory activity of citronellal; for example, even low concentrations of 25 ppm were observed to reduce bitterness perception of caffeine solution (6 mmol/L) by 32%, whereas (R)-citronellic acid (100 pm) showed a reduction of only 21% and (R)-citronellol (100 pm) was completely inactive. Cell-based functional experiments, conducted with the human bitter taste receptors TAS2R7, TAS2R10, TAS2R14, TAS2R43, and TAS2R46 reported to be sensitive to caffeine, revealed (R)-citronellal to completely block caffeine-induced calcium signals in TAS2R43-expressing cells, and, to a lesser extent, in TAS2R46-expressing cells. Stimulation of TAS2R43-expressing cells with structurally different bitter agonists identified (R)-citronellal as a general allosteric inhibitor of TAS2R43. Further structure/activity studies indicated 3-methyl-branched aliphatic aldehydes with a carbon chain of ≥4 C atoms as best TAS2R43 antagonists. Whereas odor-taste interactions have been mainly interpreted in the literature to be caused by a central neuronal integration of odors and tastes, rather than by peripheral events at the level of reception, the findings of this study open up a new dimension regarding the interaction of the two chemical senses.

Full text of DOI:10.1021/acs.jafc.6b03554

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Article
The Odorant (R)-Citronellal Attenuates Caffeine Bitterness
by Inhibiting the Bitter Receptors TAS2R43 and TAS2R46
Barbara Suess, Anne Brockhoff, Wolfgang Meyerhof, and Thomas Hofmann
J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.6b03554 • Publication Date (Web): 27 Aug 2016
Downloaded from http://pubs.acs.org on August 27, 2016
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The Odorant (R)ꢀCitronellal Attenuates Caffeine Bitterness
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by Inhibiting the Bitter Receptors TAS2R43 and TAS2R46
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BARBARA SUESS1,#, ANNE BROCKHOFF2,#, WOLFGANG MEYERHOF2, and
THOMAS HOFMANN1*
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1Chair of Food Chemistry and Molecular Sensory Science, Technische Universität
München, LiseꢀMeitnerꢀStraße 34, Dꢀ85354 FreisingꢀWeihenstephan, Germany, and
2Department of Molecular Genetics, German Institute of Human Nutrition (DIFE)
PotsdamꢀRehbrücke, ArthurꢀScheunertꢀAllee 114ꢀ116, 14558 Nuthetal, Germany
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# Both authors contributed equally
*Corresponding author
Phone
Fax
+49 8161ꢀ71ꢀ2902
+49 8161ꢀ71ꢀ2949
Eꢀmail
thomas.hofmann@tum.de
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ABSTRACT
Sensory studies showed the volatile fraction of lemon grass and its main constituent,
the odorꢀactive citronellal, to significantly decrease the perceived bitterness of a black
tea infusion as well as caffeine solutions. Seven citronellalꢀrelated derivatives were
synthesized and shown to inhibit the perceived bitterness of caffeine in a structureꢀ
dependent manner. The aldehyde function at carbon 1, the (R)ꢀconfiguration of the
methylꢀbranched carbon 3, and a hydrophobic carbon chain was found to favor the
bitter inhibitory activity of citronellal, e.g. even low concentrations of 25 ppm were
observed to reduce bitterness perception of caffeine solution (6 mmol/L) by 32%,
whereas (R)ꢀcitronellic acid (100 pm) showed a reduction of only 21%, and (R)ꢀ
citronellol (100 pm) was completely inactive. Cellꢀbased functional experiments,
conducted with the human bitter taste receptors TAS2R7, TAS2R10, TAS2R14,
TAS2R43, and TAS2R46 reported to be sensitive to caffeine, revealed (R)ꢀcitronellal
to completely block caffeineꢀinduced calcium signals in TAS2R43ꢀexpressing cells,
and to a lesser extent in TAS2R46ꢀexpressing cells. Stimulation of TAS2R43ꢀ
expressing cells with structurally different bitter agonists identified (R)ꢀcitronellal as a
general allosteric inhibitor of TAS2R43. Further structure/activity studies indicated 3ꢀ
methylꢀbranched aliphatic aldehydes with a carbon chain of ≥4 Cꢀatoms as best
TAS2R43 antagonists. While odorꢀtaste interactions have been mainly interpreted in
literature to be caused by a central neuronal integration of odors and tastes, rather
than by peripheral events at the level of reception, the findings of this study open up
a new dimension regarding the interaction of the two chemical senses.
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Key words: bitterness inhibition, citronellal, caffeine, monoterpenes, taste
modulation, TAS2R
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INTRODUCTION
Whereas in humans sweet and umami tastes are mediated by only one receptor of
the taste receptor family 1,1 perception of bitterness involves ~25 members of the
taste family 2 (TAS2R) of G proteinꢀcoupled receptors.2ꢀ6 Different subsets of the
entire TAS2R repertoire are expressed by the oral taste receptor cells that are
specifically dedicated to detect bitter compounds.7 The TAS2Rs differ in their tuning
breadth; so called generalists recognize numerous chemically diverse compounds
with substantial overlap,2,4ꢀ6 whereas specialist receptors detect only single or a very
few bitter agonists.2,8,9 The bitter receptors hTAS2R14 and hTAS2R39 were found to
be activated by a series of structurally similar (iso)flavonoids, like homoeriodictyol
and eriodictyol.10 Moreover, two TAS2Rs recognize molecules that share common
structural properties, e.g. TAS2R16 was found to respond specifically to bitter tasting
ßꢀglucopyranosides such as salicin and amygdalin, while molecules with a NꢀC═S
moiety as found in phenylthiocarbamide and propylthiouracil can activate
TAS2R38.11,12 It is well accepted that the combined agonist spectra of the entire
TAS2R repertoire determine the bitter perceptual space of humans.13 Clearly, the
facts that a single TAS2R can recognize multiple bitter compounds and that vice
versa a single bitter compound can activate several TAS2Rs makes it a challenging
task to identify receptor selective bitter antagonists.
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Human sensory experiments has led to a substantial progress in the discovery
of bitter taste inhibitors, both of synthetic and natural origin. Systematic studies on
the bitter inhibiting potential of a series of hydroxylated benzoic acid amides revealed
2,4ꢀdihydroxybenzoic acid Nꢀ(4ꢀhydroxyꢀ3ꢀmethoxyꢀbenzyl) amide (1, Figure 1) as a
potent target molecule reducing the perceived bitterness of caffeine, quinine, salicin,
and Nꢀ
L
ꢀleucylꢀ
L
ꢀtryptophan.14 Moreover, synthetic vanillyl derivatives revealed that
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[2]ꢀgingerdione (2, Figure 1), [2]ꢀdehydroisogingerdione (3), and [3]ꢀgingerdione (4)
decreases the perceived bitterness of caffeine by 35, 23, and 27%, respectively.15
Apart from synthetic bitter receptor antagonists, some naturally occurring bitter
inhibitors have been reported, all belonging to the group of flavonoids. Already in
1976, neodiosmin (5) was observed to increase the bitter taste threshold
concentrations of naringin and limonin solutions.16 Several bitter inhibiting
flavonones, namely homoeriodictyol (6), sterubin (7), and eriodictyol (8), have been
isolated from the medicinal plant Herba Santa, Eriodictyon californicum (H. & A.)
Torr., Hydrophyllaceae. While tasteless on their own, all three flavonones were
shown to decrease bitterness when added to caffeine solutions. For the sodium salt
of 8, bitter inhibiting potential was reported for various bitter taste compounds,
namely guaifenesin, paracetamol, quinine, denatonium benzoate, salicin and
amarogentin.17 Further evaluation of structural homologues revealed that phloretin
(9), even though exhibiting intrinsic bitter taste at higher concentrations, can reduce
the caffeine bitterness when applied in subthreshold concentrations.18 Moreover, the
Maillard reaction product 1ꢀcarboxymethylꢀ5ꢀhydroxyꢀ2ꢀhydroxymethylpyridinium
inner salt (10),19 structurally related to the sweetness enhancer alapyridaine formed
upon beef meat processing,20 as well as the ribonucleotide adenosine 5‘ꢀ
monophosphate (11) have been described to show bitterness reducing activity.21
However, in all of these cases the mode of action of how the observed reduction in
bitterness is achieved remained unknown. Particularly, the involvement of specific
bitter taste receptors has not been investigated.
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Yet, only few bitter inhibitors have been discovered that block bitter substanceꢀ
evoked TAS2Rꢀactivation in heterologous receptor expression assays. The first
attempt aimed at identifying antagonists of the bitter taste receptor TAS2R31, which
mediates the bitter offꢀtaste of sulfonamide sweeteners.22 This study successfully
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identified 4ꢀ(2,2,3ꢀtrimethylcyclopentyl) butanoic acid (12, Figure 1) as reversible
insurmountable inhibitor of TAS2R31 and demonstrated that the inhibitor effectively
reduced the perceived bitterness but not the sweetness of sulfonamide sweeteners.
This inhibitor did not only block TAS2R31 responses but also reduced the bitter
agonistꢀmediated responses of five additional TAS2Rs, i.e., TAS2R4, TAS2R7,
TAS2R40, TAS2R43, and TAS2R49.23 Similar observations have been made in the
case of two natural sesquiterpene lactones occurring in various edible plants; these
also blocked the activation of not only one but several TAS2Rs.24 Moreover, 6ꢀ
methoxyflavones have been identified as reversible insurmountable blockers of
TAS2R39.25 Another group of flavonoids, namely sakuranetin (13), 6ꢀ
methoxysakuranetin (14) and jacoesidin (15) were isolated from Eriodictyon
californicum and demonstrated to decrease the responses of the bitter receptor
TAS2R31.26 The sulfonamide probenecid (16) was described as negative allosteric
modulator of TAS2R16.27 Guided by molecular docking experiments, the amino acid
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derivatives γꢀaminobutyric acid, Nα,Nαꢀbis(carboxymethyl)ꢀLꢀlysine, and abscisic acid
were recently revealed as inhibitors of quinineꢀinduced responses from TAS2R4ꢀ
expressing cells.28,29 These previous studies clearly indicate that bitter antagonists
share the ability of bitter agonists to bind to more than one type of TAS2R, and the
set of TAS2Rs activated or inhibited is strictly compound specific.23,24,30
In the present study, we report on the identification of natural compounds
which reduce the bitterness of caffeine by attenuating the caffeineꢀmediated
activation of cognate TAS2Rs. Such compounds may be promising agents to
optimize the taste of foods and beverages.
MATERIALS AND METHODS
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Chemicals. The following chemicals were obtained commercially: (R)ꢀ(+)ꢀ
citronellal ((R)ꢀ17; 95% ee by chiral GC), (S)ꢀ(ꢀ)ꢀcitronellal ((S)ꢀ17; 96% ee by chiral
GC), (R)ꢀ(+)ꢀcitronellol ((R)ꢀ18), (R)ꢀ(+)ꢀcitronellic acid ((R)ꢀ19), DessꢀMartin
periodinane, hydrogen peroxide, and pꢀtoluensulfonic acid monohydrate from Sigmaꢀ
Aldrich (Steinheim, Germany), acetone, Amberlyst 15, caffeine, citronellal (17),
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dichloromethane, ethyl acetate, formic acid, monosodium Lꢀglutamate monohydrate,
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sodium chloride, and sulfuric acid (95ꢀ97%) from Merck KGaA (Darmstadt,
Germany), diethyl ether and ethanol absolute from J.T.Baker (Deventer,
Netherlands), sodium sulfate from Grüssing (Filsum, Germany). HPLC grade
solvents were purchased from J.T.Baker (Deventer, Netherlands), deionized water
used for the chromatography was purified by means of MilliꢀQGradient A10 system
(Millipore, Molsheim, France). Deuterated solvents were supplied by EurisoꢀTop (Gifꢀ
SurꢀYvette, France). For medium pressure liquid chromatography LiChroprep RPꢀ18,
25–40 ꢁm, bulk material was used (Merck KGaA, Darmstadt, Germany). Sensory
analyses were performed with bottled water (Evian). Samples of black tea (Assam),
sugar, organic lemon balm, and organic lemon grass were purchased in a local
supermarket.
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Preparation of Tea Infusions. A portion (2 g) of black tea was infused with
boiling tap water (100 mL) for 4 min, followed by filtration using a cellulose filter. To
evaluate the impact of additives on the bitter taste of the fresh tea infusion, organic
lemon balm (4 g) or organic lemon grass (4 g), respectively, were added to the black
tea drug and the infusion process was carried out as described above.
Isolation of Volatile Fraction from Lemon Grass. A lemon grass infusion
was prepared as follows: 4 g lemon grass were infused with 100mL boiling tap water
for 4 min, followed by filtration using a cellulose filter. Thereafter, the volatile fraction
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was carefully isolated from a freshly prepared, aqueous lemon grass infusion by
means of solventꢀassisted flavor evaporation using a SAFE apparatus.31
Sensory Experiments. Precautions taken for Sensory Analysis. Prior to
sensory analysis, the test (20ꢀ26) were suspended in water, and, after removing the
solvent in high vacuum (< 5mPa), were freeze dried three times and analytically
confirmed to be essentially free of solvents and buffer compounds used. The more
volatile compounds (20ꢀ22, 25, 26) were dissolved in ethanol and the solvent was
carefully removed by flushing with nitrogen. This procedure was repeated three times
and analytically confirmed to be essentially free of solvents and buffer compounds
10 used. Formic acid, which is considered as ‘‘Generally Recognized as Safe” (GRAS)
11 as a flavoring agent for food and feed applications, was used to adjust the pH value
12 of solutions which should be sensorially analysed to pH 6.0, because trace amounts
13 of this acid do not influence the sensory profile of the test solution. In order to
14 minimize the ingesting of test compounds, the “sip and spit” method was applied,
15 meaning that the test solutions were expectorated. To prevent the impact of olfactory
16 cues, each sensory experiment was performed using noseꢀclips. During the sensory
17 analyses, the oral cavity was frequently rinsed with water.
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Training of the Sensory Panel. Thirteen subjects (8 women and 5 men, aged
19 22ꢀ30 years), who gave informed consent to participate in the sensory tests of the
20 present investigation and have no history of known taste disorders, were trained to
21 evaluate the taste of aqueous solutions of the following standard taste compounds in
22 bottled water (pH 6.0):32 sucrose (50 mmol/L) for sweet taste, lactic acid (20 mmol/L)
23 for sour taste, NaCl (20 mmol/L) for salty taste, caffeine (1 mmol/L) for bitter taste,
24 monosodium
Lꢀglutamate (3 mmol/L) for umami taste. The assessors had
25 participated earlier at regular intervals for at least 18 months in sensory experiments
26 (triangle tests, isoꢀintensity testings, scale training) and were, therefore, familiar with
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the techniques applied. To prevent any crossꢀmodal interactions with olfactory inputs,
the panelists wore nose clips.
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Sensory Evaluation of Black Tea Infusions. (i) Fresh infusions prepared from
black tea (2.0 g/100 mL) mixed with lemon balm (4.0 g/100 mL) or lemon grass (4.0
g/100 mL), respectively, (ii) fresh black tea infusions (50 mL, 4 g/100 mL) spiked with
the volatile SAFE distillate (50 mL) obtained from a fresh lemon grass infusion (4
g/100 mL), and (iii) fresh black tea infusions (100 mL, 2 g/100 mL) spiked with
citronellal (17; 25, 50, and 100 ppm) were presented to panellists at room
temperature in 3ꢀalternative forced choice (3ꢀAFC) tests in comparison to the
standard tea infusion (control, without any additive) in randomized order. First, the
panellists were asked to identify the odd sample and, then, to describe the sensory
difference subsequently.
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Sensory Evaluation of the Bitterness Inhibiting Potential of Oxygenated
Monoterpenes. Following a protocol reported earlier,32ꢀ35 the target monoterpenes
were dissolved in an aqueous caffeine solution (6 mmol/L; pH 6.0) containing 5%
ethanol. Using duo tests and randomly coded cups, these solutions were compared
to a series of 5% hydroethanolic reference solutions containing increasing caffeine
concentrations (2.0, 3.5, 5.0, 6.5, and 8 mmol/L). Assessors were asked to identify
the solution with the weaker bitter taste, sensory data were transferred into the
percentage of answers considering the monoterpene containing solution to have a
less intense bitter taste. After Probit transformation, the caffeine concentration at
which 50% panelists consider the test solution to be less bitter is calculated and
considered to be the isointensity concentration of the monoterpene/caffeine solution
in comparison to the pure caffeine reference solution. To minimize carryꢀover effects,
panelists rinsed their mouth with a 5% hydroethanolic solution between the tests.
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Synthesis
of
(R)-7-Hydroxycitronellol
((R)-20)
and
(R)-7-
Hydroxycitronellic acid ((R)-21). For the synthesis of (R)-20 and (R)-21, (R)ꢀ
citronellol (R)-18 (1.6 mmol) or (R)ꢀcitronellic acid (R)-19 (1.6 mmol), respectively,
were added drop wise to a solution of aqueous sulfuric acid (50/50, v/v) at 0°C. After
stirring for 1 h, the ice bath was removed and stirring continued for additional 2 h.
The reaction mixture was poured into ice, neutralized with sodium hydroxide solution
(10%, w/v) and the reaction products isolated by means of MPLC on RPꢀ18 material.
After removing the solvent under reduced pressure and lyophilisation, the structure of
the target compounds (R)-20 and (R)-21 (purity >98%, HPLCꢀDAD/ELSD) were
confirmed by UPLCꢀTOFꢀMS and NMR experiments. While the (R)ꢀconfiguration of
the carbon atom 3 of the educt is expected to be retained, the nonꢀenantioselective
synthetic procedure used is expected to lead to a racemic (R/S)ꢀmixture at carbon
atom 7 of the products.
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(3R)-7-Hydroxycitronellol, (R)-20, Figure 2: UPLCꢀTOFꢀMS (ESIꢀ): m/z
173.1553 ([MꢀH]ꢀ, measured), m/z 173.1547 ([MꢀH]ꢀ, calculated for C10H21O2). H
1
NMR (400 MHz, CDCl3, COSY): δ [ppm] 0.90 [d, 3H, J = 6.6 Hz, HꢀC(10)], 1.16ꢀ1.19
[m, 1H, HꢀC(4a)], 1.22 [s, 6H, HꢀC(8), HꢀC(9)], 1.26ꢀ1.41 [m, 4H, HꢀC(2a), HꢀC(4b),
HꢀC(5)], 1.41ꢀ1.48 [m, 2H, HꢀC(6)], 1.52ꢀ1.67 [m, 2H, HꢀC(2b), HꢀC(3)], 3.61ꢀ3.75 [m,
13
2H, HꢀC(1)]. C NMR (100 MHz, CDCl3, HSQC, HMBC): δ [ppm] 19.7 [CH3, C(10)],
21.6 [CH2, C(5)], 29.1, 29.2 [CH3, C(8), C(9)], 29.4 [CH, C(3)], 37.5 [CH2, C(4)], 39.7
[CH2, C(2)], 43.9 [CH2, C(6)], 61.0 [CH2, C(1)], 71.4 [C, C(7)].
(R)-7-Hydroxycitronellic acid, (R)-21, Figure 2: UPLCꢀTOFꢀMS (ESIꢀ): m/z
187.1336 ([MꢀH]ꢀ, measured), m/z 187.134 ([MꢀH]ꢀ, calculated for C10H19O3). 1H NMR
(400 MHz, CD3OD, COSY): δ [ppm] 0.96 [d, 3H, J = 6.6 Hz, HꢀC(10)], 1.17 [s, 6H, Hꢀ
C(8), HꢀC(9)], 1.18ꢀ1.26 [m, 1H, HꢀC(4a)], 1.27ꢀ1.39 [m, 2H, HꢀC(4b), HꢀC(5a)], 1.39ꢀ
1.49 [m, 3H, HꢀC(5b), HꢀC(6)], 1.87ꢀ2.00 [m, 1H, HꢀC(3)], 2.08 [dd, 1H, J = 8.0, 14.7
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Hz, HꢀC(2a)], 2.29 [dd, 1H, J = 6.1, 14.7 Hz, HꢀC(2b)]. C NMR (100 MHz, CD3OD,
HSQC, HMBC): δ [ppm] 20.1 [CH3, C(10)], 22.8 [CH2, C(5)], 29.18, 29.22 [CH3, C(8),
C(9)], 31.5 [CH, C(3)], 38.5 [CH2, C(4)], 42.7 [CH2, C(2)], 44.9 [CH2, C(6)], 71.4 [C,
C(7)], 177.2 [C, C(1)].
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Synthesis of (3R)-3,7-Dimethyloctane-1,6,7-triol ((3R)-22) and (3R)-6,7-
Dihydroxycitronellic Acid ((3R)-23). (R)ꢀCitronellol ((R)-18, 1 mmol) or (R)ꢀ
citronellic acid (R)-19 (1 mmol), respectively, was added drop wise to a solution of
hydrogen peroxide (1.2 mmol) in formic acid (10 mL) and, then stirred at 70°C for 2 h.
After separating the solvent under reduced pressure, sodium hydroxide solution (1
mL; 20%, w/w) was added. The reaction products were isolated by means of RPꢀ18
MPLC. After removing the solvent under reduced pressure and lyophilisation, the
reaction product (3R)ꢀ6,7ꢀdihydroxycitronellic acid ((3R)-23) was obtained in a purity
of >98% (HPLCꢀDAD/ELSD) and its structure confirmed by UPLCꢀTOFꢀMS and
NMR. To isolate (3R)ꢀ3,7ꢀdimethyloctaneꢀ1,6,7ꢀtriol ((3R)-22), methanol was
removed under reduced pressure and the aqueous extract extracted with ethyl
acetate (3x100 mL). The organic layer was dried over sodium sulfate and the solvent
evaporated in vacuum to afford the target product (3R)-22 in high purity of >98%
(HPLCꢀDAD/ELSD) and its structure confirmed by UPLCꢀTOFꢀMS and NMR. While
the (R)ꢀconfiguration of the carbon atom 3 of the educt is expected to be retained, the
nonꢀenantioselective synthetic procedure used is expected to lead to a racemic
(R/S)ꢀmixture at carbon atoms 6 and 7 of the products.
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(3R)-3,7-Dimethyloctane-1,6,7-triol ((3R)-6,7-dihydroxycitronellol), (3R)-22,
Figure 3: LCꢀ MS (ESI+): m/z (%) 124 (50, [M+Na]+), 191.0 (100, [M+H]+), 175 (80,
1
[MꢀOH]+), 155 (80, [MꢀH3O2]+). H NMR (400 MHz, CDCl3, COSY): δ [ppm] 0.90 [d,
3H, J = 6.2 Hz, HꢀC(10a)], 0.92 [d, 3H, J = 6.2 Hz, HꢀC(10b)], 1.15 [s, 6H, HꢀC(8)],
1.20 [s, 6H, HꢀC(9)], 1.22ꢀ1.28 [m, 1H, HꢀC(5a)], 1.28ꢀ1.49 [m, 4H, HꢀC(2a), HꢀC(5b),
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HꢀC(5c)], 1.50ꢀ1.62 [m, 5H, HꢀC(2b), HꢀC(3a), HꢀC(5d)], 1.62ꢀ1.73 [m, 2H, HꢀC(3b)],
3.31 [dd, 1H, J = 1.7, 10.2 Hz, HꢀC(6a)], 3.35 [dd, 1H, J = 1.7, 10.2 Hz, HꢀC(6b)],
3.58ꢀ3.78 [m, 4H, HꢀC(1)]. 13C NMR (100 MHz, CDCl3, HSQC, HMBC): δ [ppm] 19.6
[CH3, C(10a)], 19.9 [CH3, C(10b)], 23.1 [CH3, C(9a)], 23.2 [CH3, C(9b)], 26.4 [CH3,
C(8)], 28.4 [CH2, C(5a)], 28.7 [CH, C(3a)], 29.2 [CH2, C(5b)], 29.7 [CH, C(3b)], 33.2
[CH2, C(4a)], 33.9 [CH2, C(4b)], 39.5 [CH2, C(2a)], 39.8 [CH2, C(2b)], 60.6 [CH2,
C(1a)], 60.6 [CH2, C(1b)], 73.25 [C, C(7a)], 73.26 [C, C(7b)], 78.2 [CH, C(6a)], 79.0
[CH, C(6b)].
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4
5
6
7
8
9
(3R)-6,7-Dihydroxycitronellic acid, (3R)-23, Figure 3: UPLCꢀTOFꢀMS (ESIꢀ):
m/z 203.1283 ([MꢀH]ꢀ, measured), m/z 203.1291 ([MꢀH]ꢀ, calculated for C10H19O4). 1H
NMR (500 MHz, DMSOꢀd6, COSY): δ [ppm] 0.86 [d, 3H, J = 7.0 Hz, HꢀC(10a)], 0.88
[d, 3H, J = 7.0 Hz,. HꢀC(10b)], 0.98 [s, 6H, HꢀC(8)], 1.03 [s, 6H, HꢀC(9)], 1.04ꢀ1.11
[m, 2H, HꢀC(4a), HꢀC(5a)], 1.11ꢀ1.18 [m, 1H, HꢀC(5b)], 1.21ꢀ1.29 [m, 1H, HꢀC(4b)],
1.31ꢀ1.40 [m, 1H, HꢀC(4c)], 1.43ꢀ1.50 [m, 1H, HꢀC(5c)], 1.50ꢀ1.59 [m, 2H, HꢀC(4d),
HꢀC(5d)], 1.75ꢀ1.86 [m, 2H, HꢀC(3)], 1.93 [dd, 1H, J = 8.3, 14.5 Hz, HꢀC(2a)], 1.96
[dd, 1H, J = 8.3, 14.5 Hz, HꢀC(2b)], 2.15 [dd, 1H, J = 5.7, 11.4 Hz, HꢀC(2c)], 2.18 [dd,
1H, J = 5.8, 11.4 Hz, HꢀC(2d)], 2.95ꢀ3.08 [m, 2H, HꢀC(6)]. 13C NMR (125 MHz,
DMSOꢀd6, HSQC, HMBC): δ [ppm] 19.6 [CH3, C(10a)], 20.0 [CH3, C(10b)], 24.49
[CH3, C(9a)], 24.51 [CH3, C(9b)], 26.3 [CH3, C(8)], 28.23 [CH2, C(5a)], 28.24 [CH2,
C(5b)], 29.9 [CH, C(3a)], 30.1 [CH, C(3b)], 33.7 [CH2, C(4a)], 33.9 [CH2, C(4b)], 42.0
[CH2, C(2a)], 42.5 [CH2, C(2b)], 71.61 [C, C(7a)], 71.64 [C, C(7b)], 77.5 [CH, C(6a)],
77.7 [CH, C(6b)], 174.5 [C, C(1)].
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25
26
Synthesis
of
(3R)-3-Methyl-5-(2,2,5,5-tetramethyl-1,3-dioxolan-4-yl)
pentan-1-ol ((3R)-24). Using a literature procedure with some modifications,36 (3R)ꢀ
3,7ꢀdimethyloctaneꢀ1,6,7ꢀtriol ((3R)-22, 0.7 mmol) and pꢀtoluensulfonic acid (0.7
mmol) are dissolved in dried acetone (5 mL). After stirring at room temperature for 24
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h, the crude reaction mixture was added to an aqueous ammonium hydroxide
solution (25%, v/v; 20 mL), and the product was isolated by extraction with diethyl
ether (3x50 mL). The combined organic layers were washed with water (2x150 mL)
and brine (1x150 mL) and, after drying over sodium sulfate, the solvent was removed
under reduced pressure to afford the target compound in a purity of >98% (HPLCꢀ
DAD/ELSD) and the structure confirmed by NMR experiments.
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6
7
(3R)-3-Methyl-5-(2,2,5,5-tetramethyl-1,3-dioxolan-4-yl)pentan-1-ol,
(3R)-24,
1
8
Figure 3: H NMR (400 MHz, benzeneꢀd6, COSY): δ [ppm]. 0.82 [d, 3H, J = 6.6 Hz,
HꢀC(10a)], 0.82 [d, 3H, J = 6.6 Hz, HꢀC(10b)], 1.079 [s, 3H, HꢀC(8/9)], 1.082 [s, 3H,
HꢀC(8/9)], 1.156 [s, 3H, HꢀC(8/9)], 1.163 [s, 3H, HꢀC(8/9)], 1.14ꢀ1.27 [m, 4H, HꢀC(5a),
HꢀC(2a)], 1.27ꢀ1.35 [m, 3H, HꢀC(5b)], 1.365 [s, 3H, HꢀC(12/13)], 1.367 [s, 3H, Hꢀ
C(12/13)], 1.40ꢀ1.47 [m, 3H, HꢀC(5c), HꢀC2b)], 1.48 [s, 6H, HꢀC(12/13)], 1.47ꢀ1.70
[m, 9H, HꢀC(5d), HꢀC(3), HꢀC(4)], 3.33ꢀ3.47 [m, 4H, HꢀC(1)], 3.62 [dd, 1H, J = 1.9,
3.4 Hz, HꢀC(6a)], 3.64 [dd, 1H, J = 2.1, 3.3 Hz, HꢀC(6b)]. 13C NMR (101 MHz,
benzeneꢀd6, HSQC, HMBC): δ [ppm] 19.8 [CH3, C(10)], 23.2 [CH3, C(8/9)], 26.3
[CH3, C(8/9)], 26.4 [CH3, C(8/9)], 27.2 [CH2, C(4)], 27.4 [CH3, C(12)], 29.0 [CH3,
C(13)], 29.7 [CH, C(3)], 30.0, 34.7 [CH2, C(5a)], 34.8 [CH2, C(5b)], 40.0 [CH2, C(2a)],
40.1 [CH2, C(2b)], 60.7 [CH2, C(1a)], 60.6 [CH2, C(1b)], 80.1 [C, C(7)], 84.0 [CH,
C(6a)], 84.2 [CH, C(6b)], 106.6 [C, C(11a)], 106.7 [C, C(11b)].
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24
25
26
Synthesis
of
(R)-7-Hydroxycitronellal
((R)-25)
and
(3R)-6,7-
Hydroxycitronellal ((3R)-26). Using
a
literature procedure with some
modifications,37 a solution of (R)ꢀ20 and (3R)ꢀ24 (1 mmol), respectively, in dried
dichloromethane (0.8 mL) was added dropwise to a solution of DessꢀMartin
periodinane (1.2 mmol) in dried dichloromethane (4 mL). After 1 h, the solution was
diluted with diethyl ether (50 mL) and extracted with an aqueous sodium thiosulfate
solution (50 mL; 2 M, saturated with sodium hydrogencarbonate). The aqueous
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phase was extracted twice with diethylether (50 mL), the combined organic phases
were extracted with of aqueous sodium hydrogencarbonate solution (200 mL),
followed by water (200 mL). After drying the organic phase over sodium sulfate, the
solvent was separated in vacuum and (R)ꢀ25 (purity: >98%, HPLCꢀELSD) isolated by
means of RPꢀ18 MPLC. To obtain (3R)-26, the acetonide was hydrolyzed as reported
earlier.38 The product was dissolved in a mixture of THF and water (4+1, v/v) in
presence of Amberlystꢀ15 (300 mg) and is stirred at room temperature for 16 h. After
filtration, the reaction product (3R)ꢀ26 was isolated by RPꢀ18 MPLC and its structure
confirmed by means of UPLCꢀTOFꢀMS and NMR experiments. While the (R)ꢀ
configuration of the carbon atom 3 of the educt is expected to be retained, the nonꢀ
enantioselective synthetic procedure used is expected to lead to a racemic (R/S)ꢀ
mixture at carbon atoms 6 and 7 of the product.
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25
26
(3R)-7-Hydroxycitronellal, (R)-25, Figure 3: UPLCꢀTOFꢀMS (ESI+): m/z
1
173.1520 ([M+H]+, measured), m/z 173.1536 ([M+H]+, calculated for C10H21O2). H
NMR (400 MHz, DMSOꢀd6, COSY): δ [ppm] 0.89 [d, 3H, J = 6.7 Hz, HꢀC(10)], 1.06 [s,
6H, HꢀC(8), HꢀC(9)], 1.17ꢀ1.26 [m, 1H, HꢀC(4a)], 1.27ꢀ1.36 [m, 5H, HꢀC(4b), HꢀC(5),
HꢀC(6)], 1.91ꢀ2.04 [m, 1H, HꢀC(3)], 2.21 [ddd, 1H, J = 2.5, 7.8, 16.3 Hz, HꢀC(2a], 2.39
[ddd, 1H, J = 2.3, 5.7, 16.2 Hz, HꢀC(2b)], 9.66 [t, J = 2.3 Hz, 1H, HꢀC(1)]. 13C NMR
(100MHz, DMSOꢀd6, HSQC, HMBC): δ [ppm] 19.0 [CH3, C(10)], 21.7 [CH2, C(5)],
27.9 [CH, C(3)], 29.7, 29.8 [CH3, C(8), C(9)], 37.5 [CH2, C(4)], 44.2 [CH2, C(6)], 50.8
[CH2, C(2)], 69.2 [C, C(7)], 204.2[C, C(1)].
(3R)-6,7-Hydroxycitronellal, (3R)-26, Figure 3: UPLCꢀTOFꢀMS (ESI+): m/z
1
189.1502 ([M+H]+, measured), m/z 189.1485 ([M+H]+, calculated for C10H21O3). H
NMR (500 MHz, CDCl3, COSY): δ [ppm] 0.88 [d, 3H, J = 6.5 Hz, HꢀC(10a)], 0.92 [d,
3H, J = 6.5 Hz,. HꢀC(10b)], 1.16 [m, 14H, HꢀC(8), HꢀC(9), HꢀC(5a), HꢀC(5b)], 1.35ꢀ
1.58 [m, 1H, HꢀC(4a)], 1.39ꢀ1.51 [m, 1H, HꢀC(4b)], 1.40ꢀ1.58 [m, 2H, HꢀC(4c), Hꢀ
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C(5c)], 1.59ꢀ1.68 [m, 3H, HꢀC(5c), HꢀC(3)], 2.23ꢀ2.39 [m, 2H, HꢀC(2a)], 2.40ꢀ2.48 [m,
2H, HꢀC(2b)], 3.33ꢀ3.40 [m, 2H, HꢀC(6)], 9.77 [t, 2H, J = 2.0 Hz, HꢀC(1)]. 13C NMR
(126 MHz, CDCl3, HSQC, HMBC) δ [ppm] 19.6 [CH3, C(10a)], 19.9 [CH3, C(10b)],
23.19 [CH3, C(9a)], 23.24[CH3, C(9b)], 26.5 [CH3, C(8)], 28.70 [CH2, C(4a)], 29.1
[CH2, C(4b)], 29.77 [CH, C(3a)], 29.81 [CH, C(3b)], 33.7 [CH2, C(5a)], 34.1 [CH2,
C(5b)], 39.56 [CH2, C(2a)], 39.83 [CH2, C(2b)], 73.06 [C, C(7a)], 73.11 [C, C(7b)],
78.57 [CH, C(6b)], 79.10 [CH, C(6b)], 202.85 [CH, C(1)].
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Cell-based Bitter Taste Receptor Experiments. To assess whether
oxygenated monoterpenes directly target human bitter taste receptors to reduce
caffeine bitterness, we implemented functional expression of TAS2Rs in HEK293T
Gα16gust44 cells as described previously.8,39 As five members of the family of
human bitter taste receptors, namely TAS2R7, TAS2R10, TAS2R14, TAS2R43, and
TAS2R46, have been reported to be sensitive to caffeine,2 cells were transfected with
expression constructs for TAS2R7, TAS2R10, TAS2R14, TAS2R43, and TAS2R46
using Lipofectamine2000 (Invitrogen). After 22 h, cells were loaded with the calciumꢀ
sensitive fluorescent dye Fluo4ꢀAM and incubated for 1.5 h at 37°C. Excess dye was
removed by subsequent washing with bath solution (130 mM NaCl, 5 mM KCl, 10
mM Hepes, 2 mM CaCl2, and 10 mM glucose (pH 7.4)). Test compounds were
dissolved in bath solution, and preꢀmixed for coꢀapplication. Changes in cytosolic
calcium levels to automated application of test compounds were monitored with a
FLIPR Tetra ICCD (Molecular Devices). Fluorescence signals were corrected for
responses of mockꢀtransfected cells and normalized to background fluorescence.
Concentrationꢀresponse relations and values for halfꢀmaximal receptor activation
(EC50) were calculated in SigmaPlot (SPSS) by nonlinear regression using the
function: f(y) = min + (max-min)/(1 + (x/EC50)-Hillslope). Data were collected from at
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least three independent experiments carried out in duplicates.
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Medium Pressure Liquid Chromatography (MPLC). Medium pressure liquid
chromatography was performed on a Sepacore chromatography system (Buechi,
Flawil, Switzerland) consisting of two Cꢀ605 pumps with a Cꢀ615 pump manager, a
manual rheodyne injection port (20 mL loop), a Cꢀ635 UV/Vis detector, and a Cꢀ660
fraction collector. Before the eluent was directed into the fraction collector, an aliquot
(1 mL) of the eluent was split into a Sedex LTꢀELSD Model 85 detector (Sedere,
Alfortville, France). Büchi Sepacore Record 1.0 (Büchi Labortechnik AG, Flawil,
Switzerland) software was used for data acquisition. Chromatography was performed
with a flow rate of 40 mL/min on a selfꢀpacked 150 x 40 mm i.d. polypropylene
cartridge filled with 25ꢀ40 ꢁm LiChroprep RPꢀ18 Material (Merck KGaA, Darmstadt,
Germany), monitoring the eluent on the ELS detector and using 0.1% aqueous formic
acid (solvent A) and methanol (solvent B) as eluents. After sample injection (5 mL),
isocratic elution with 40% solvent A was performed for 3 min. Subsequently, the
solvent B was increased linearly to 75% within 20 min, then to 100% within 3min and
maintained at 100% for 5 min. After separating the organic solvent under reduced
pressure, fractions containing the product were extracted three times with ethyl
acetate (100 mL) and, after drying over sodium sulfate, the solvent was removed
under reduced pressure.
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High-Performance Liquid Chromatography (HPLC). Analytical HPLC was
carried out with an HPLC apparatus (Jasco, GroßꢀUmstadt, Germany), consisting of
two PUꢀ2087 Plus Pumps, a M800 gradient mixer, a DGꢀ2080ꢀ53 degasser, an auto
sampler (ASꢀ2055 Plus), a diode array detector (MDꢀ2010 Plus) and a Sedex LTꢀ
ELSD Model 85 (Sedere, Alfortville, France). Chrompass Software was used for data
acquisition. With a flow rate of 0.8 mL/min, chromatography was performed on a 250
x 4.6mm i.d., 5 ꢁm, MicrosorbꢀMW 100ꢀ5 C18 column (Varian, Darmstadt, Germany),
monitoring the eluent at 220 and 260 nm and using 1% aqueous formic acid (solvent
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A) and methanol (solvent B) as eluents. After sample injection (10 ꢁL), isocratic
elution with 100% solvent A was performed for 5 min. Subsequently, the content of
organic solvent was linearly increased to 100% within 25 min, maintaining 100%
solvent B for additional 5 min.
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4
5
High-Performance Liquid Chromatography/Mass Spectroscopy (HPLC-
MS/MS). For HPLCꢀESIꢀMS/MS analysis, a Dionex UHPLC UltiMate® 3000 HPLC
(Dionex, Idstein), consisting of a binary pump (HPGꢀ3400SD), a degasser (SRDꢀ
3400), an autosampler (WSPꢀ3000TSL), a column compartment (TCCꢀ3000SD), and
operating with the DC MS Link 2.8.0.2633 software, was linked to an API 4000
QTRAP mass spectrometer (AB Sciex Instruments, Darmstadt, Germany), which was
equipped with an electrospray ionization (ESI) source with direct flow infusion.
Nitrogen served as nebulizer gas (3.4 bar), and, heated at 400°C, as turbo gas for
solvent drying (4.1 bar). Nitrogen also served as curtain (1.3 bar) and collision gas
(0.06 bar). The ion spray voltage was set at ꢀ4500 V in the negative mode and at
5500 V in the positive mode. The mass spectrometer was operated in the fullꢀscan
mode detecting positive or negative ions.
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UPLC-Time-of-Flight Mass Spectrometry (UPLC/TOF-MS). Mass spectra of
the compounds were measured on a Waters Synapt G2 HDMS mass spectrometer
(Waters, Manchester, UK) coupled to an Acquity UPLC core system (Waters)
consisting of a binary solvent manager, sample manager and column oven. The
compounds were dissolved in methanol (1 mL) and aliquots (1ꢀ5 ꢁL) were injected
into the UPLCꢀTOFꢀMS system equipped with a 150 x 2 mm i.d., 1.7ꢁm, BEH C18
column (Waters). Operated with a flow rate of 0.3 mL/min at a temperature of 40°C,
the following gradient was used for chromatography: starting with a mixture (5/95,
v/v) of acetonitrile and aqueous formic acid (0.1%, pH 2.5), the acetonitrile content
was increased to 95% within 3 min and held at 95% for 1 min. Calibration of the
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Synapt G2 was performed using a solution of sodium formate (0.5 mmol/L) in 2ꢀ
propanol/water (9/1, v/v). Data was processed using MassLynx 4.1 (Waters) and the
tool elemental composition. All data was lock mass (leucine enkephalin) corrected
([M+H]+, m/z 556.2771, [MꢀH]ꢀ, m/z 554.2615). Measurements were performed using
negative ESI and the resolution modus consisting of the following parameters:
capillary voltage (+2.5 or ꢀ3.0 kV), sampling cone (30 V), extraction cone (4.0 V),
source temperature (150°C), desolvation temperature (450°C), cone gas (30 L/h),
and desolvation gas (850 L/h).
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9
Nuclear Magnetic Resonance Spectroscopy (NMR). The H, 13C, and 2Dꢀ
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NMRꢀspectroscopic experiments were performed on a 500 MHz Avance III NMR
spectrometer from Bruker (Rheinstetten, Germany), equipped with a a triple res. cryo
probe (TCI) and and a DRX or a AvanceꢀIIIꢀ400 (400 MHz) spectrometer equipped
with a QNP or a Broadband Observe BBFOplus probe (Bruker, Rheinstetten,
Germany). Unless stated otherwise, all experiments were performed at 300 K.
Samples were dissolved in deuterated chloroform (CDCl3), deuterated
dimethylsulfoxide (d6ꢀDMSO), deuterated benzene (benzeneꢀd6) or deuterated
methanol (CD3OD). For accurate NMR signal assignment, COSY, HSQC, and HMBC
experiments were carried out using pulse sequences of the Bruker software library.
While data processing was performed using Topspin Version 1.3 (Bruker,
Rheinstetten, Germany), the individual data interpretation was done with
MestReNova 5.1.0ꢀ2940 (Mestrelab Research S.L., Santiago de Compostela, Spain).
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RESULTS AND DISCUSSION
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To reduce the typical bitter taste of black tea infusions, many consumers add milk
thus decreasing the concentration of “free” bitter compounds available for taste
receptor activation by nonꢀcovalent binding to milk proteins.40 In our search for foodꢀ
derived molecules that inhibit the bitter taste of foods and beverages like tea,
preliminary sensory studies recently gave indication that lemon grass extract is able
to reduce the perceived bitter taste of tea (data not published). To further
substantiate this observation and to identify the active principle in lemon grass, first,
more detailed sensory studies were performed in the following.
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Lemon Balm and Lemon Grass Attenuate Perceived Bitterness of a Black
Tea Infusion. Black tea beverages were spiked with lemon balm or lemon grass
extract, respectively, and were sensorially compared to a beverage without any
additive (control) by means of a trained panel wearing nose clips to exclude any
olfactory cues. Using a threeꢀalternative forced choice (3ꢀAFC) test, the panelists
were asked to identify the least bitter tasting sample. Both tea infusions containing
either lemon balm, or lemon grass were identified as significantly (α = 0.001) less
bitter when compared to the standard tea infusion (Table 1). To study the potential
impact of volatiles in inducing the perceived bitterness reduction, the volatile fraction
was isolated from the lemon grass extract by means of SAFE distillation. Spiking this
volatile fraction to the standard tea infusion still induced a decrease in bitterness (α =
0.05). Confirming literature knowledge,41 GC/MS analysis revealed (R)ꢀcitronellal (17;
Figure 2) as a major constituent in the volatile fraction isolated from lemon grass
(data not shown). Therefore, another sensory experiment aimed at evaluating the
bitterness inhibitory potential of this monoterpene aldehyde. Indeed, the perceived
bitterness of the tea infusion was significantly decreased in the presence of racemic
citronellal (17) and (R)ꢀcitronellal ((R)ꢀ17) even at 50 ppm (α = 0.01), whereas the
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corresponding (S)ꢀcitronellal ((S)ꢀ17) did not show any significant effects (α = 0.2;
Table 1).
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Synthesis of Citronellal-Related Compounds. To gain some more insight
into the structural requirements of citronellal (17) to affect bitterness perception of
caffeine, a series of monoꢀ and dihydroxylated monoterpenes was synthesized
(Figure 2). Since sensory experiments revealed bitterness inhibitory potential for (R)ꢀ
citronellal (Figure 2A) only but not for (S)ꢀcitronellal (Table 1), the following
syntheses were limited to the (R)ꢀstereoisomer. To introduce an additional hydroxyl
function, water was added to the double bond at position 6 and 7 of (R)ꢀcitronellol
((R)ꢀ18) and (R)ꢀcitronellic acid ((R)ꢀ19) in strongly acidic reaction medium to result in
7ꢀhydroxycitronellol ((R)ꢀ20) and 7ꢀhydroxycitronellic acid ((R)ꢀ21), respectively
(Figure 2B) with a yield of 46 and 66%, respectively. Dihydroxylation of the double
bond of (R)ꢀ18 and (R)ꢀ19, respectively, was accomplished by epoxidation with
hydrogen peroxide and formic acid and subsequent hydrolysis to give (3R)ꢀ6,7ꢀ
dihydroxycitronellol ((3R)ꢀ22)) in a yield of 44% and (3R)ꢀ6,7ꢀdihydroxycitronellic acid
((3R)ꢀ23)) in a yield of 44% (Figure 2C). Furthermore, (R)ꢀ18 was oxidized to (R)ꢀ7ꢀ
hydroxycitronellal ((R)ꢀ25) in a yield of 34% (Figure 2D). To oxidize (3R)ꢀ22 to the
corresponding aldehyde, the vicinal hydroxyl functions were protected as 1,2ꢀ
acetonide, followed by Dess Martin periodinane oxidation, and subsequent hydrolytic
deprotection, thus affording (3R)ꢀ6,7ꢀdihydroxycitronellal ((3R)ꢀ26) in a yield of 12%,
(Figure 2E). In total, a series of nine monoterpene derivatives (17ꢀ22, 25, 26),
differing both in the degree of hydroxylation at position 6 and 7, as well as in the
oxidation status at position 1 were prepared and purified to evaluate their caffeine
bitterness inhibitory activity.
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Human Sensory Evaluation of Citronellal and Related Monoterpenes. To
investigate the bitter inhibitory potential of the monoterpenoids 17ꢀ22, 25, and 26, an
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appropriate sensory assay was developed. Adapting the principles of sensory tests
allowing for the evaluation of umami enhancers,32ꢀ35 aqueous solutions containing
caffeine (6 mmol/L) and a constant concentration of the test compound were
compared to five solutions of increasing caffeine concentration in duo tests applying
forced choice conditions. Panelists were asked to name the sample showing the less
intense bitter taste. The sensory responses were transformed into the percentage of
panelists rating the monoterpene containing sample as less bitter compared to the
caffeine sample. After Probit transformation, the value corresponding to 50% of
positive answers was calculated and considered to be the isoꢀintensity concentration
at which the bitterness of the reference and the test solution could not be
differentiated.
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In order to test the accuracy of this experimental design, a series of pure
caffeine solutions was offered to the sensory panel as pseudoꢀtest solutions. In three
independent sessions, test solutions containing 4.0, 5.0 and 6.0 mmol/L caffeine,
respectively, were assessed with an isointense caffeine concentration of 3.8, 5.0, and
5.9 mmol/L, thus demonstrating the high precision of the sensory test. Therefore, the
sensory test was considered fit for purpose to evaluate the bitter inhibiting potential of
the monoterpenes 17ꢀ22, 25, and 26, respectively.
In a first set of experiments, the impact of the stereochemical configuration in
position 3 of citronellal (17) was evaluated by comparing racemic 17 against the
corresponding (R)ꢀ and (S)ꢀstereoisomers. The bitterness perception of caffeine was
reduced by 18% by 100 ppm racemic 17 and 28% by 100 ppm (R)ꢀ17, whereas the
same concentration of (S)ꢀ17 did only show a marginal effect, e.g. the bitterness of
the 6 mmol/L caffeine solution was perceived to be isointense to a 5.4 mmol/L
caffeine solution (Table 2). A further decrease of the concentration of (R)ꢀ17 from
100 ppm to 50 and 25 ppm did not result in an increase in caffeine’s perceived bitter
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intensity, thus suggesting that the bitter inhibiting potential of (R)ꢀ17 was fully
exploited even at the lowest concentration tested.
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Modification of the oxidation status at carbon atom 1 had a fundamental
impact on the bitter inhibitory activity, e.g. oxidation of the aldehyde (R)ꢀ17 (28%
bitterness reduction) to the carboxylic acid (R)ꢀ19 showed only somewhat lower
bitterness inhibitory activity of 21%, whereas reduction to the alcohol (R)ꢀ18
completely diminished its inhibitor activity (Table 2). Similarly, all other monoterpene
alcohols tested, namely (R)ꢀ18, (R)ꢀ20, and (3R)ꢀ22, did not affect caffeine bitterness
perception. In the group of citronellic acids, the bitter inhibiting activity decreased with
increasing hydrophilicity, e.g. (R)ꢀ19 lacking any hydroxyl group in the carbon chain
showed a 21% bitterness reduction, while introduction of one hydroxyl group ((R)ꢀ20)
induced a decreased inhibitory effect with a 15% reduction and the dihydroxylated
(3R)ꢀ23 did not affect caffeine bitterness at all (Table 2). In comparison, within the
series of aldehydes, 100 ppm of citronellal ((R)ꢀ17) as well as the monoꢀ ((R)ꢀ25) and
dihydroxylated aldehydes ((3R)ꢀ26) showed similar bitter inhibitory activity of 28, 25,
and 28%, respectively (Table 2). Decreasing the concentration of (R)ꢀ17 and (R)ꢀ25
to 50 and 25 ppm revealed a difference between both compounds only at 25 ppm.
While (R)ꢀ17 still showed a high bitter inhibition of 32%, the perceived caffeine
bitterness was reduced by (R)ꢀ25 (25 ppm) only by 17%, thus indicating lower activity
with increasing hydroxylation degree as observed for the citronellic acids.
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Cell-based Bitter Taste Receptor Experiments. In order to identify the
molecular target responsible for the citronellalꢀmediated reduction of the bitterness of
caffeine, cellꢀbased functional experiments were conducted with human bitter taste
receptors. As TAS2R7, TAS2R10, TAS2R14, TAS2R43, and TAS2R46 have been
reported to be sensitive to caffeine,5 these five TAS2Rs were functionally expressed
in heterologous cells, challenged with caffeine (3.0 mmol/L), and the calcium
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responses were monitored in the absence and presence of (R)ꢀ17 and (S)ꢀ17. At a
concentration of 0.50 mmol/L, stereoisomer (S)ꢀ17, which lacked any significant bitter
inhibitory potential (Table 2), induced strong calcium responses in mockꢀtransfected
cells preventing the assessment of its potential to block activation of the five bitter
taste receptors (data not shown). In comparison, (R)ꢀ17 did not generate any
fluorescence signals at 0.5 mmol/L in mockꢀtransfected cells, thus enabling the
examination of its bitter inhibitory activity. Cells transfected with cDNAs for TAS2R7, ꢀ
10, ꢀ14, ꢀ43, or ꢀ46 responded to 3.0 mmol/L caffeine by increasing their intracellular
calcium concentrations, albeit with greatly different signal amplitudes (Figure 3A).
The comparatively high signal amplitudes of TAS2R43 and TAS2R46 to 3.0 mmol/L
caffeine might indicate a major role of these two receptors for the perceptual
sensitivity to caffeine bitterness. Coꢀapplication of (R)ꢀ17 with caffeine completely
blocked the caffeineꢀinduced calcium signals in TAS2R43ꢀexpressing cells, and to a
lesser extent in TAS2R46ꢀexpressing cells (Figures 3 A, B). Surprisingly, two
receptors, namely TAS2R7 and TAS2R14, showed a robust calcium increase upon
sole application of (R)ꢀ17 (Figures 3 A, C). However, only cells transfected with
TAS2R14 elicited the typical immediate and transient fluorescence signal, suggesting
that (R)ꢀ17 acts as an agonist on this bitter taste receptor. Indeed, a similar
ambivalent nature in terms of selective suppression of few TAS2Rs, but contrariwise
activation of other members of this family, has also been revealed for natural
sesquiterpene lactones and flavonones.10,24,25 Since other studies on bitter taste
receptor antagonists lack analyses on their potential agonistic properties, it still
remains to be determined, if such ambivalent effects concern a unique feature of few
compounds or were inherent to all TAS2R inhibitors.
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To examine its inhibitory potential in more detail, inhibition responses to (R)ꢀ17
were recorded next. Cells expressing TAS2R43 or TAS2R46 were challenged with
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(R)ꢀ17 up to 0.60 mmol/L, and a fixed concentration of 3.0 mmol/L caffeine (Figure
3D). Caffeine exhibits similar potency at both receptors, indicated by EC50 values of
about 0.94 ± 0.14 mmol/L (TAS2R43; n=4; Figure 4A) and 1.35 ± 0.32 mmol/L
(TAS2R46; n=3; data not shown). Intriguingly, the cellular calcium responses to
caffeine mediated by TAS2R43 started to be significantly reduced in the presence of
0.03 mmol/L (R)ꢀ17, and were entirely blocked at 0.3 mmol/L (R)ꢀ17 (Figure 3D). The
halfꢀmaximal inhibitory concentration (IC50) amounted to 0.084 ± 0.012 mmol/L (R)ꢀ
17 (n=4). The caffeine response of TAS2R46ꢀexpressing cells was significantly
attenuated only by coꢀapplication of 0.6 mmol/L (R)ꢀ17 (Figure 3D), indicating
different sensitivities of the two sequenceꢀconserved bitter taste receptors for the
monoterpene antagonist. These findings are consistent with previous studies which
demonstrated a partial overlap of the TAS2R43 and TAS2R46 bitter agonist and
antagonist repertoires.2,24,42
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Next, we recorded concentration responses from TAS2R43ꢀexpressing cells to
caffeine in the absence and in the presence of increasing concentrations of (R)ꢀ17 in
order to determine the type of antagonism on hand (Figure 4A). Coꢀapplication of the
monoterpene caused parallel rightꢀshifts of the response function and diminished the
maximum response amplitude Rmax of the agonist caffeine, suggesting that (R)ꢀ17
acts as an allosteric inhibitor on TAS2R43. The changed potency of caffeine can be
explained by cooperative effects, which have been described to occur with allosteric
interactions.43
In order to assess whether the inhibitory effect of (R)ꢀ17 on TAS2R43 is
specific for caffeine, cells were stimulated with EC90ꢀconcentrations of four additional
bitter agonists of distinct chemical classes. Like responses to caffeine, those to the
glucopyranoside helicin, the quaternary ammonium compound denatonium benzoate,
and the benzothiazole saccharin were completely blocked by coꢀapplication of 0.3
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mmol/L (R)ꢀ17 (Figure 4B). Calcium transients elicited by the most potent agonist,
the phenanthrene aristolochic acid I, were only partially attenuated at this antagonist
concentration, but completely suppressed when inhibitor concentration was raised to
the highest applicable concentration of 0.6 mmol/L (Figure 4B). Therefore, the
odorant (R)ꢀcitronellal ((R)ꢀ17) can be concluded to represent a general blocker of
the TAS2R43 bitter taste receptor.
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In order to investigate the TAS2R43 inhibitory activity of some structurally
related odorꢀactive aliphatic aldehyde, the following experiments were expanded to
include the malty smelling propionaldehyde (27), isobutyraldehyde (28),
isovaleraldehyde (29), and 2ꢀmethylbutyraldehyde (30), as well as the green, grassy
smelling hexanal (31), all of which has been recently reported to be among the 230
key food odorants constructing the characteristic scent of foods and beverages
(Figure 5).44 In addition, racemic 3ꢀmethylhexanal (32) was tested as it contains the
3ꢀmethylalkylcarbaldehyde motif as found in citronellal. These test compounds were
coꢀapplied with caffeine (3.0 mmol/L) up to their highest applicable concentration
(Figure 6A). Propionaldehyde, isobutyraldehyde, and hexanal showed no effect on
the caffeineꢀinduced TAS2R43ꢀresponses. Sole application of these compounds did
not trigger calcium transients suggesting that aldehydes per se are incapable of
interacting with TAS2R43 (Figure 6B). In contrast, coꢀapplication of caffeine with the
two methylꢀbranched C4 aldehydes, isovaleraldehyde and 2ꢀmethylbutyraldehyde,
led to significantly increased calcium responses (Figure 6A). This effect can be
attributed to the ability of isovaleraldehyde (29) and, in particular, of 2ꢀ
methylbutyraldehyde (30), to elicit transient calcium signals from TAS2R43ꢀ
expressing cells, and hence, to act as agonists at this bitter receptor (Figure 6B).
While hexanal (31) did not show any significant antagonistic effects, intriguingly, coꢀ
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application of caffeine with 3ꢀmethylhexanal (32) efficiently and comprehensively
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blocked caffeineꢀinduced fluorescence (Figure 6A). With a threshold of 0.03 mmol/L
and an IC50 value of 0.074 ± 0.026 mmol/L (n=3), 3ꢀmethylhexanal (32) turned out to
be an equally potent antagonist of TAS2R43 compared to (R)ꢀ17 (threshold: 0.03
mmol/L; IC50: 0.084 ± 0.012 mmol/L, n=4). These data clearly indicate the 3ꢀmethylꢀ
branched aliphatic aldehydes 3ꢀmethylhexanal and (R)ꢀcitronellal as best TAS2R43
antagonists, while isovaleraldehyde (29) and, in particular, of 2ꢀmethylbutyraldehyde
(30) were found to function as TAS2R43 receptor agonists.
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Taken together, our results support the role of TAS2R43, and, to a lesser
extent, TAS2R46, as the molecular target of the natural monoterpene (R)ꢀcitronellal
for attenuating the bitterness of caffeine. To the best of our knowledge, this study is
the first to disclose the influence of odorants on bitter taste perception at the level of
taste receptors. Previous research on the sensory interaction between odors and
tastes though provided indication of specific mutual interference, mainly by
compounds which are commonly encountered together in food or beverages. Most
previous studies focussed on the tasteꢀodor combination of sucrose and fruity odors
like strawberry odor, caramelꢀlike (e.g. 4ꢀhydroxyꢀ2,4ꢀdimethylꢀ3(2H)ꢀfuranone) and
vanillaꢀlike odors (e.g. vaniline).30,45ꢀ48 Sensory evaluation of such congruent mixtures
suggested that the perceived intensity of the odor can be enhanced by sucrose, and
to a lesser extent, the perceived sweetness of sucrose is selectively enhanced by
some odors.30,45ꢀ48 In comparison, suppression of taste by an odor component of a
mixture has rarely been reported.48 However, the observed effects have been
interpreted as the consequence of a central neuronal integration of odors and tastes
to construct the perceived flavor,46 and have not been considered as peripheral
events at the level of reception. In this context, the findings of this study open up a
new dimension regarding the interaction of the two chemical senses.
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ACKNOWLEDGEMENT
This work was supported in part by the European Union’s Seventh Framework
Programme for research, technological development and demonstration (to WM
#613879; SynSignal). We thank Peggy Grossmann for excellent technical assistance.
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