Synthesis of methoxy-substituted diazirinyl phenylalanine - A novel photoreactive aspartame derivative for functional analysis of sweet receptors

Article abstract of DOI:10.3987/COM-13-S(S)14

Photoreactive phenylalanine derivatives are well known as functional analysis reagents for target biomolecules. The photophores are commonly introduced at 4-position on benzene. Aspartame, which consists of dipeptide L-Asp-L-Phe-OMe, is one of the most ut

Full text of DOI:10.3987/COM-13-S(S)14

HETEROCYCLES, Vol. 88, No. 1, 2014, pp. -. © 2014 The Japan Institute of Heterocyclic Chemistry
Received, 17th May, 2013, Accepted, 7th June, 2013, Published online, 12th June, 2013
DOI: 10.3987/COM-13-S(S)14
SYNTHESIS OF METHOXY-SUBSTITUTED DIAZIRINYL
PHENYLALANINE –A NOVEL PHOTOREACTIVE ASPARTAME
DERIVATIVE FOR FUNCTIONAL ANALYSIS OF SWEET RECEPTORS
Munenori Sakurai,a Katsuyoshi Masuda,b Lei Wang,a Yuta Murai,a Yasuko
Sakihama,a Yasuyuki Hashidoko,a Yasumaru Hatanaka,c and Makoto
Hashimotoa,*
a
Graduate School of Agriculture, Hokkaido University, Kita 9, Nishi 9, Kita-ku,
b
Sapporo 060-8589, Japan Suntory Institute for Bioorganic Research, 1-1-1
Wakayamadai, Shimamoto-cho, Mishima-gun, Osaka 618-8503, Japan c Graduate
School of Medicine and Pharmaceutical Sciences, University of Toyama, 2360
Sugitani, Toyama, 930-0194 Japan. E-mail: hasimoto@abs.agr.hokudai.ac.jp
Abstract – Photoreactive phenylalanine derivatives are well known as functional
analysis reagents for target biomolecules. The photophores are commonly
introduced at 4-position on benzene. Aspartame, which consists of dipeptide
L-Asp-L-Phe-OMe, is one of the most utilized artificial sweeteners, and
substitution effects on its benzene ring have been reported. Substitution at the
4-position, however, does not maintain its sweetness properties. Trifluoromethyl-
diazirine, which is one of the most reliable photophores, was introduced to a
different site on phenylalanine and the new photoreactive phenylalanine was
converted to aspartame derivatives. The new aspartame derivative had slightly
higher sweetness potency than sucrose standard solution.
Elucidation of protein functions on the basis of structure–activity relationships can reveal the mechanisms
of homeostasis functions in vivo and is of great interests to scientists. In the human body, many proteins
are activated and/or inactivated by ligands to maintain homeostasis. Understanding the mechanism of
molecular interactions between small bioactive ligands and proteins is an important step in rational drug
design and discovery. Photoaffinity labeling is one of the most familiar approaches for chemical biology
analysis. 1 Photoaffinity labeling is also applied to functional analysis of !-amino acid, which represent
fundamental compounds of life. It seems suitable to introduce photophores at the 4-position of
This paper is dedicated to Prof. Dr. Victor Snieckus on the occasion of his 77th birthday.

phenylalanine, because tyrosine, hydroxyl substituted at this position, is one of natural !-amino acids.
On this basis many attempts have been made to construct of photophore-substituted phenylalanine rings
by targeting the 4-position. Trifluoromethylphenyldiazirine is one of the most promising photophores for
photoaffinity labeling, but the need to construct the 3-(trifluoromethyl)diazirinyl moiety each time
hampered researchers for further applications of diazirinyl derivatives. Pioneer work for the preparation
of diazirinyl phenylalanine derivatives also used substitutions at 4-position of phenylalanine.2 Aspartame
(L-Asp-L-Phe-OMe) was one of the most famous artificial sweetener and a relationship between the aryl
substitution on phenylalanine and sweet taste has been reported, but it has not been reported the details of
binding of aspartame to sweet receptors. Methoxy substitution at the 2-position did not reduce the
sweetness and 3-substitution also less influence for sweet potency compared with aspartame.3 On the
other hand, substitution at 4-position dramatically decreased the sweetness, and 4-diazirinyl aspartame
derivative
had
no
sweet
taste.2a
These
results
encouraged
us
to
prepare
2-methoxy-5-trifluoromethyldiazirinyl phenylalanine to maintain sweet potency. In this study, synthesis
of a novel methoxy-substituted diazirinyl phenylalanine derivative and its introduction to aspartame
skeleton is described (Figure 1).
MeO
OR
>
=
L-Asp N
CO2Me
N
L-Asp N
H
CO2Me
CF3
L-Asp N
H
CO2Me
H
MeO
N
N
N
CF3
L-Asp N
H
CO2Me
L-Asp N
H
CO2Me
1 (not sweet)
2
Figure 1. Substitution effects on aromatic in aspartame
Our synthetic methodologies are based on the initial construction of 4-methoxydiazirine,4 followed by
deriving5 to unknown 2-methoxy-5-trifluoromethyldiazirinyl benzyl bromide derivatives and asymmetric
synthesis with ! -amino acid skeleton. 3-(4-Methoxyphenyl)-3-(trifluoromethyl)-3H-diazirine was
synthesized according to our previous paper with minor modifications. 4-Trifluoromethyl anisole (3) was
converted to N-hydroxy oxime with hydroxylamine hydrochloride, tosyloxime with tosyl chloride in
acetone - TEA, then diaziridine (4) with liquid ammonia. These three steps were achieved without
purification, and this produced same yield compared with previous report.4 Activated manganese oxide
was utilized oxidation of diaziridine (4) to diazirine (5) with an improvement in the yield. The aldehyde
was introduced by a Friedel-Crafts reaction with dichloromethyl methyl ether in the presence of titanium
chloride at 0 °C, followed by reduction of aldehyde (6) with sodium borohydride in ethanol with good

yield. The hydroxyl group (7) was converted to benzyl bromide (8) with carbon tetrabromide and
triphenylphosphine (Scheme 1).
F3C
N
N
F3C
O
F3C
i), ii), iii)
NH
NH
F3C
v)
N
N
F3C
N
N
F3C
N
N
iv)
vi)
CHO
vii)
CH2OH
CH2Br
OMe
OMe
OMe
OMe
OMe
OMe
3
4
5
6
7
8
Scheme 1. Synthesis of diazirinyl benzyl bromide derivatives (8). i) NH2OH HCl, pyridine, 80 °C, 3 h,
ii) TsCl, TEA, acetone, 0 °C, 1 h, iii) NH3 (l), Et2O, rt, 8 h, 85% (3 steps), iv) MnO2, Et2O, rt, 6 h, 93%, v)
Cl2CHOMe, TiCl4, 0 °C, 1�h, 98%, vi) NaBH4, EtOH, rt, 4 h, 92%, vii) CBr4, Ph3P, rt, 4 h, 95%
The diazirinyl benzyl bromide (8) was subjected asymmetric synthesis with glycine derivative (9) in the
presence of phosphazene base BTPP (10) and catalytic amounts of chinchonidinium bromide (11) at
-78 °C.6 The condensed product (12) was slightly decomposed during silica gel-column chromatography,
so the reaction mixture was directly subjected to deprotection with TFA to afford new photoreactive
L-phenylalanine derivative (13) (Scheme 2).
tBu
Br
F3C
N
N
N
P
N
N
H
MeO
N
N
N
N
O
8
N
N
+
N
,
ii)
Ph
Ph
10
11
Ph
Ph
F3C
CO2tBu
CO2tBu
9
OMe
i)
H2N CO2H
12
13
Scheme 2. Synthesis of diazirinyl L-phenylalanine derivative (13). i) CH2Cl2, -78 °C, 8 h, ii) TFA, 0 °C,
2h, 65% (2 steps)
MeO
MeO
N
N
i)
ii)
16 R1 = tBu, R2 = Boc
13
N
N
O
iii)
CF3
CF3
R1O
N
H
CO2Me
H2N
CO2Me
14 (85%)
2 R1 = OH, R2 = H
O
NHR2
Scheme 3. Synthesis of diazirinyl L-phenylalanine derivatives (13). i) SOCl2, MeOH, rt, 8 h, 85%,
ii) N-Boc-L-Asp(OtBu)OSu (15), (i-Pr)2NEt, THF, rt, 8 h, 86%, iii) TFA, 0 °C, 3 h, 97%
The chirality of the synthetic 13 was checked with chiral HPLC to reveal over 96% ee. The racemate 13
was obtained when the reaction temperature proceeded at 0 °C. The diazirinyl phenylalanine was
converted to methyl ester (14) with thionyl chloride in methanol. The methyl ester was reacted with

N-Boc-L-Asp(OtBu)-OSu (15) in the presence of diisopropylethyl amine at room temperature, followed
by deprotection of tBu ester and Boc groups with TFA to afford new photoreactive aspartame derivative
(2) in good yield (Scheme 3). The synthetic compound 2 was subjected to photoirradiation experiment
with black light to ensure the photoreactivity. The decay around 370 nm was measured in a
time-dependent manner (Scheme 4). Analysis with semi-logalism plot calculated half-life of compound 2
as 1.3 min. The result indicated the compound 2 has enough reactivity for photoaffinity label.
Scheme 4. Photolysis of photoreactive aspartame (2) in methanol (1 mM) with black light (100W). UV
spectra of the photolysis reaction were recorded every 1 min until 15 min, then every 5min until 30 min.
The synthesized photoreactive aspartame derivative 2 was subjected to preliminary sensory evaluations
using a filter-paper disk.6 The synthetic compound had less sweet potency than mother compound
aspartame, but a slightly higher potency than sucrose, which is most popular standard for sweet test. It is
well known high concentration of sweet compound can also produced bitter taste. (Table 1).
Concentration
(mg/mL)
Compound
Aspartame Sucrose
2
[a]
-
5
3
1
40
10
1
No test
9
0
0
0
No test
9
6
0.1
Table 1. Preliminary sensory evaluations by filter-paper disk assay (n=9). Numbers indicated that the
recognition of sweetness at the indicated concentration. [a] all test person recognized as bitter.
We have developed synthesis of substituted diazirinyl compounds to produce a photoreactive aspartame
derivative to improve sweetness potency. The phenyl ring of phenylalanine was substituted with a
methoxy and diazirinyl group at 2- and 5- position, respectively. The phenylalanine derivative was
prepared by asymmetric synthesis in the presence of a catalytic amount of chinchonidinium salt without
decomposition of the diazirinyl ring. The photoreative phenylalanine derivative was condensed with the
aspartic acid derivative to construct the aspartame skeleton. The synthetic aspartame derivatives easily

generated carbene and had a sweetness potency that was higher than standard sucrose solution. Further
functional analysis of sweet receptor by the synthetic photoreactive reagent is underway.
EXPERIMANTALS
Genaral methods. NMR spectra were measured by JEOL EX-270 and ECA-500 spectrometers.
ESI-TOF-MS data were obtained with a Waters UPLC ESI-TOF mass spectrometer. Optical rotation data
were obtained with a JASCO DIP-370 polarimeter at 23 °C.
3-(4-Methoxyphenyl)-3-(trifluoromethyl)-diaziridine (4)
2, 2, 2-trifluoro-4’-methoxyacetophenone (3) (5.00 g, 24.5 mmol) and hydroxylamine hydrochloride
(2.78 g, 40.0 mmol) were dissolved in pyridine (50 mL) at room temperature. The reaction mixture was
stirred at 70 °C for 3 h and concentrated. The residue was partitioned between AcOEt and 1N HCl. The
organic layer was washed with H2O and brine, dried over MgSO4, filtrated, and concentrated. The crude
oxime was dissolved in acetone (170 mL) and cooled to 0 !. Triethylamine (8.5 mL, 26.8 mmol) and
p-toluenesulfonyl chloride (5.10 g, 26.8 mmol) were added in succession. The reaction mixture was
stirred for 1 h at the same temperature, filtrated, and then concentrated. In a pressure tube, the Et2O
solution of the crude tosyl oxime was added to excess liquid ammonia at -78 !. The reaction mixture
was warmed to room temperature and then stirred for 8 h. After excess ammonium gas was removed in a
draft chamber, the reaction mixture was concentrated. The residue was purified by column
chromatography (CH2Cl2) to afford 4 (4.55 g, 85%) as colorless amorphous mass. The analytical data is
identical to a previous report.4
3-(4-Methoxyphenyl)-3-(trifluoromethyl)-3H-diazirine (5)
The diaziridine 4 (1.88 g, 8.6 mmol) was dissolved in Et2O (80 mL). Excess MnO2 (activated) was added
until the starting material was consumed completely. The insoluble material was filtrated with Celite. The
filtrate was concentrated, and the residue was purified by column chromatography (hexane/CH2Cl2 1/1) to
afford diazirine 5 (1.74 g, 94%) as yellow oil. The analytical data is identical to a previous report.4
2-Methoxy-5-[3-(trifluoromethyl)-3H-diazirin-3-yl]benzaldehyde (6)
To a mixture of 5 (479 mg, 2.22 mmol) and dichloromethyl methyl ether (2.6 mL, 28.86 mmol), TiCl4
(1.1 mL, 9.99 mmol) was added at 0 °C dropwise. The reaction mixture was stirred at the same
temperature for 1 hour and poured into cold water/AcOEt. The organic layer was washed with sat.
NaHCO3, 1 M HCl, and brine, dried over MgSO4, filtrated, and concentrated. The residue was purified by
1
column chromatography (hexane/AcOEt 8/1) to afford 6 (531 mg, 98%) as yellow solid. H-NMR
(CDCl3) !: 10.43 (1H, s), 7.66 (1H, d, J = 2.3 Hz), 7.47 (1H, dd, J = 2.6, 8.9 Hz), 7.04 (1H, d, J = 8.6
13
Hz), 3.96 (3H, s); C-NMR (CDCl3) !: 188.47, 162.43, 133.91, 127.13, 124.88, 122.01 (q, JCF = 274.4
1
2
19
Hz), 121.46, 112.45, 56.00, 27.97 (q, JCF = 40.2 Hz); F-NMR (CDCl3) !: -65.56; FD-MS: [M]+

calculated for C10H7F3N2O2 244.0460, found 244.0433.
2-Methoxy-5-[3-(trifluoromethyl)-3H-diazirin-3-yl]benzyl alcohol (7)
5-Diazirinyl-2-methoxybenzaldehyde 6 (113 mg, 0.46 mmol) was dissolved in EtOH and cooled to 0 °C.
NaBH4 (19 mg, 0.51 mmol) was added to the reaction mixture at same temperature. The reaction mixture
was stirred at room temperature for 4 h. After acidification of the reaction mixture with 1 M HCl at 0 °C,
the product was extracted with Et2O. The organic solution was dried over MgSO4, filtrated, and
concentrated. The residue was purified by column chromatography (CH2Cl2) to afford 7 (105 mg, 92%) as
yellow solid. 1H-NMR (CDCl3) !: 7.18-7.15 (2H, m), 6.88 (1H, dd, J = 2.0, 7.3 Hz), 4.67 (2H, d, J = 6.3
Hz), 3.88 (3H, s), 2.12 (1H, t, J = 6.4 Hz); 13C-NMR (CDCl3) !: 158.33, 129.98, 127.66, 126.79, 122.26
1
2
19
(q, JCF = 276.6 Hz), 120.97, 110.47, 61.38, 55.55, 30.80 (q, JCF = 40.1 Hz); F-NMR (CDCl3) !:
-65.55; FD-MS: [M]+ calculated for C10H9F3N2O2 246.0616, found 246.0619.
2-Methoxy-5-[3-(trifluoromethyl)-3H-diazirin-3-yl]benzyl bromide (8)
To a solution of 7 (91 mg, 0.37 mmol) and CBr4 (246 mg, 0.74 mmol) in dry CH2Cl2, Ph3P (194 mg, 0.74
mmol) was slowly added at 0 !. The reaction mixture was stirred at room temperature for 4 h and
concentrated. The residue was purified by column chromatography (hexane/CH2Cl2 1/1) to afford 8 (108
1
mg, 95%) as yellow oil. H-NMR (CDCl3) !: 7.19-7.17 (2H, m), 6.89 (1H, d, J = 8.6 Hz), 4.50 (2H, s),
13
1
3.91 (3H, s); C-NMR (CDCl3) !: 158.46, 129.14, 128.79, 127.05, 122.16 (q, JCF = 274.9 Hz), 121.06,
2
19
111.29, 55.86, 28.07 (q, JCF = 39.7 Hz), 27.64; F-NMR (CDCl3) !: -65.50; ESI-TOF-MS: [M+H]+
calculated for C10H8BrF3N2O 307.9772, found 307.9743.
L-2-Methoxy-5-[3-(trifluoromethyl)-3H-diazirin-3-yl]phenylalanine (13)
The diazirinyl benzyl bromide derivative 8 (97 mg, 0.32 mmol), tert-butylglycinate benzophenone imine
9 (102 mg, 0.35 mmol), and O-allyl-N-9-anthracenylmethylcinchonidium bromide 11 (30 mg, 0.050
mmol) was dissolved in dry CH2Cl2 and cooled to -78 °C under N2. To the solution was slowly added
phosphazene base BTPP (1-tert-butyl-2,2,2-tri(1-pyrrolidinyl)phosphazene) 10 (125 "L, 0.47 mmol)
followed by stirring for 8 h. After removal of the solvent, the residue was dissolved in Et2O. The organic
layer was washed with water twice and brine, dried over MgSO4, and concentrated to afford crude 12.
The crude 12 was dissolved in TFA (2 mL) at 0 °C. The reaction mixture was stirred at room temperature
for 2 h. After concentration, the residue was partitioned between Et2O and 1 M HCl, and the organic layer
was extracted with 1 M HCl three times. The aqueous solutions were combined and concentrated. The
residue was purified by column chromatography on washed silica gel (MeCN/MeOH/H2O 8/1/1) to afford
diazirinyl phenylalanine 13 (86 mg, 65% for 2 steps) as white solid. 1H-NMR (CD3OD) !:7.28 (1H, dd, J
= 1.5, 8.7 Hz), 7.16-7.03 (2H, m), 4.24 (1H, t, J = 6.4 Hz), 3.91 (3H, s), 3.40-3.33 (2H, m), 3.12 (1H, dd,
J = 7.3, 14.2 Hz); 13C-NMR (CD3OD) !: 171.08, 160.68, 130.80, 129.52, 125.01, 123.70 (q, 1JCF = 273.2
2 19
Hz), 121.86, 112.67, 56.27, 53.86, 32.57, 29.23 (q, JCF = 40.2 Hz); F-NMR (CD3OD) !: -67.29 ppm.

ESI-TOF-MS: [M+H]+ calculated for C12H13F3N3O3 304.0909, found 304.0894; [!]D ꢀ9.1 (c 1.0 MeOH)
L-2-Methoxy-5-[3-(trifluoromethyl)-3H-diazirin-3-yl]phenylalanine methyl ester (14)
The diazirinyl phenylalanine 13 (56 mg, 0.13 mmol) was dissolved in MeOH and cooled to 0 °C. To the
solution SOCl2 (91 "L, 1.04 mmol) was slowly added. The reaction mixture was stirred at room
temperature for 8 h and concentrated. The residue was dissolved in AcOEt, washed with sat. NaHCO3,
and dried over MgSO4. After removal of the solvent, the residue was purified by column chromatography
(CH2Cl2/MeOH 19/1) to afford 14 (34 mg, 85%) as colorless amorphous mass. 1H-NMR (CDCl3) !: 7.09
(1H, dd, J = 1.6, 8.2 Hz), 6.96 (1H, d, J = 1.6 Hz), 6.85 (1H, d, J = 8.6 Hz), 3.83 (3H, s), 3.77 (1H, t, J =
13
6.6 Hz), 3.69 (3H, s) 3.06 (1H, dd, J = 5.9, 13.2 Hz) 2.87 (1H, dd, J = 7.9, 13.5 Hz); C-NMR (CDCl3)
1
!: 175.37, 160.59, 130.46, 128.60, 127.34, 123.74 (q, JCF = 273.8 Hz), 121.49, 112.26, 56.20, 54.88,
2
19
52.47, 36.02, 29.25 (q, JCF = 40.2 Hz); F-NMR (CDCl3) !: -65.49; ESI-TOF-MS: [M+H]+ calculated
for C13H15F3N3O3 318.1066, found 318.1086. [!]D + 22.3 (c 1.0 MeOH).
L-N-tert-Butoxycarbonyl-!!-tert-butylaspartyl-2-methoxy-5-[3-(trifluoromethyl)-3H-diazirin-3-yl]-L-
phenylalanine methyl ester (16)
The diazirinyl phenylalanine methyl ester 14 (51 mg, 0.16 mmol) and L-N-Boc-!-tert-butyl-aspartic acid
succinimide ester 15 (73 mg, 0.19 mmol) was dissolved in THF and cooled to 0 #. To the mixture,
DIPEA (56 "L, 0.32 mmol) was added, followed by stirring at room temperature for overnight. After the
reaction, the solutions were concentrated in vacuo. The residue was purified by column chromatography
(hexane/AcOEt 2/1) to afford protected diairinyl aspartame 16 (81 mg, 86%) as colorless amorphous
mass. 1H-NMR (CDCl3) !: 7.22 (1H, d, J = 7.3 Hz), 7.14 (1H, dd, J = 2.0, 8.6 Hz,), 6.93 (1H, d, J = 8.9
Hz), 5.58 (1H, d, J = 6.9 Hz), 4.71 (1H, dd, J = 6.3, 13.5 Hz), 4.43 (1H, br s), 3.90 (3H, s), 3.67 (3H, s),
3.09 (2H, d, J = 6.6 Hz), 2.83 (1H, dd, J = 4.3, 17.1 Hz), 2.53 (1H, dd, J = 6.9, 17.1 Hz), 1.43 (9H, s),
13
1.39 (9H, s); C-NMR (CDCl3) !: 171.34, 171.18, 170.74, 158.68, 155.23, 129.46, 129.29, 125.55,
122.19 (q, 1JCF = 274.3 Hz), 120.95, 110.78, 81.58, 80.19, 55.68, 53.08, 52.10, 50.67, 37.67, 32.14, 28.23,
2
19
27.90 (q, JCF = 36.9 Hz); F-NMR (CD3OD) !: -65.52 ppm; ESI-TOF-MS: [M+H]+ calculated for
C26H36F3N4O8 589.2485, found 589.2470. [!]D -7.5 (c 1.0 MeOH).
L-Aspartyl-2-methoxy-5-[3-(trifluoromethyl)-3H-diazirin-3-yl]-L-phenylalanine methyl ester (2)
The Boc- and tBu- protected diazirinyl aspartame 16 (40 mg, 0.068 mmol) was dissolved in TFA (2 mL)
at 0 °C. The mixture was stirred at room temperature for 3 h. After concentration, the residue was
partitioned between diethyl ether and 1 M HCl, and the organic layer was extracted with 1 M HCl three
times. After concentration of aqueous layer, the residue was purified by column chromatography on
washed silica gel (MeCN/MeOH/H2O 8/1/1) to afford diazirinyl aspartame 2 (36 mg, 97%) as a colorless
amorphous mass. 1H-NMR (CD3OD) !: 7.15 (1H, dd, J = 2.3, 8.6 Hz), 7.01 (1H, d, J = 8.9 Hz), 6.94 (1H,
d, J = 2.0 Hz), 4.69 (1H, dd, J = 6.4, 8.7 Hz), 4.07 (1H, dd, J = 4.6, 8.6 Hz), 3.84 (3H, s), 3.61 (3H, s),

13
3.18 (1H, dd, J = 6.3, 13.5 Hz), 2.98-2.86 (2H, m), 2.76 (1H, dd, J = 8.6, 18.1 Hz); C-NMR (CD3OD)
1
!: 172.91, 169.37, 160.52, 130.29, 128.72, 127.22, 123.70 (q, JCF = 273.8 Hz), 121.50, 112.32, 64.21,
2
19
56.30, 53.85, 52.77, 50.89, 36.22, 33.28, 29.23 (q, JCF = 40.8 Hz); F-NMR (CD3OD) !: -67.26 ;
ESI-TOF-MS: [M+H]+ calculated for C17H20F3N4O6 433.1335, found 433.1342; ["]D +2.9 (c 1.0 MeOH).
Photolysis of compound 2
A 1 mM methanolic solution of the diazirinyl aspartame 2 was placed in a quartz cuvette. Photolysis was
performed with black-light (100 W). Spectra were measured after each minute, and then the half-life was
calculated from the decrements of the absorbance around 370 nm.
Preliminary sensory evaluations by filter-paper disk
Nine healthy test persons aged between 20 and 28 years were recruited from authors’ laboratory
members. Onto filter-paper discs with a width of 5 mm, 10 "L solutions were adsorbed, each at 40, 10, 1
and 0.1 mg/mL concentrations, and the discs were put on the same area of the tongue. The lowest
concentration (0.1 mg/mL) was applied first, followed by the next level of concentration, and so on, until
the test person recognized a sweet taste. Forty milligrams per milliliter of sucrose is well known as
standard for sweet flavor.
ACKNOWLEDGEMENTS
MH thanks the Fugaku Foundation for financial support. Part of this work was performed under the
Cooperative Research Program of “Network Joint Research Center for Materials and Devices”.
REFERENCES
1. a) J. Brunner, Annu. Rev. Biochem., 1993, 62, 483; b) Y. Hatanaka, H. Nakayama, and Y. Kanaoka,
Rev. Heteroatom Chem., 1996, 14, 213; c) G. Dormán and G. D. Prestwich, Trends Biotechnol.,
2000, 18, 64; d) T. Tomohiro, M. Hashimoto, and Y. Hatanaka, Chem. Records, 2005, 5, 385; e) M.
Hashimoto and Y. Hatanaka, Eur. J. Org. Chem., 2008, 2513; f) L. Dubinsky, B. P. Krom, and M. M.
Meijler, Bioorg. Med. Chem., 2012, 20, 554.
2. a) M. Nassal, J. Am. Chem. Soc., 1984, 106, 7540; b) L. B. Shih and H. Bayley, Anal. Biochem.,
1985, 144, 132; c) G. Baldini, B. Martoglio, A. Schachenmann, C. Zugliani, and J. Brunner,
Biochemistry, 1988, 27, 7951; d) C. W. G. Fishwick, J. M. Sanderson, and J. B. C. Findlay,
Tetrahedron Lett., 1994, 35, 4611; e) H. Nakashima, M. Hashimoto, Y. Sadakane, T. Tomohiro, and
Y. Hatanaka, J. Am. Chem. Soc., 2006, 128, 15092; f) K. Masuda, A. Koizumi, T. Misaka, Y.
Hatanaka, K. Abe, T. Tanaka, M. Ishiguro, and M. Hashimoto, Bioorg. Med. Chem. Lett., 2010, 20,
1081.
3. M. Kawai, M. Chorev, J. Marin-Rose, and M. Goodman, J. Med. Chem., 1980, 23, 420.

4. a) Y. Hatanaka, M. Hashimoto, H. Kurihara, H. Nakayama, and Y. Kanaoka, J. Org. Chem., 1994,
59, 383; b) M. Hashimoto, Y. Kato, and Y. Hatanaka, Tetrahedron Lett., 2006, 47, 3391.
5. M. Hashimoto, Y. Kanaoka, and Y. Hatanaka, Heterocycles, 1997, 46, 119.
6. E. J. Corey, F. Xu, and M. C. Noe, J. Am. Chem. Soc., 1997, 119, 12414.
7. K. Berling, J. Knutsson, A. Rosenblad, and M. Unge, Acta Oto-Laryngologica, 2011, 131, 488.