Structure-CaSR-activity relation of kokumi γ-glutamyl peptides

Article abstract of DOI:10.1248/cpb.c16-00293

Modulation of the calcium sensing receptor (CaSR) is one of the physiological activities of γ-glutamyl peptides such as glutathione (γ-glutamylcysteinylglycine). γ-Glutamyl peptides also possess a flavoring effect, i.e., sensory activity of kokumi substances, which modifies the five basic tastes when added to food. These activities have been shown to be positively correlated, suggesting that kokumi γ-glutamyl peptides are perceived through CaSRs in humans. Our research is based on the hypothesis that the discovery of highly active CaSR agonist peptides will lead to the creation of practical kokumi peptides. Through continuous study of the structure-CaSR-activity relation of a large number of γ-glutamyl peptides, we have determined that the structural requirements for intense CaSR activity of γ-glutamyl peptides are as follows: existence of an N-terminal γ-L-glutamyl residue; existence of a moderately sized, aliphatic, neutral substituent at the second residue in an L-configuration; and existence of a C-terminal carboxylic acid, preferably with the existence of glycine as the third constituent. By the sensory analysis of γ-glutamyl peptides selected by screening using the CaSR activity assay, γ-glutamylvalylglycine was found to be a potent kokumi peptide. Furthermore, norvaline-containing γ-glutamyl peptides, i.e., γ-glutamylnorvalylglycine and γ-glutamylnorvaline, possessed excellent sensory activity of kokumi substances. A novel, practical industrial synthesis of regiospecific γ-glutamyl peptides is also required for their commercialization, which was achieved through the ring opening reaction of N-α-carbobenzoxy-L-glutamic anhydride and amino acids or peptides in the presence of N-hydroxysuccinimide.

Full text of DOI:10.1248/cpb.c16-00293

Vol. 64, No. 8
Chem. Pharm. Bull. 64, 1181–1189 (2016)
1181
Regular Article
Structure–CaSR–Activity Relation of Kokumi γ-Glutamyl Peptides
,a
Yusuke Amino,* Masakazu Nakazawa,b Megumi Kaneko,a Takashi Miyaki,c
Naohiro Miyamura,c Yutaka Maruyama,a and Yuzuru Etoa
b
a Institute for Innovation, Ajinomoto Co., Inc.; 1–1 Suzuki-cho, Kawasaki-ku, Kawasaki 210–8681, Japan: Research
Institute for Bioscience Products & Fine Chemicals, Ajinomoto Co., Inc.; 1–1 Suzuki-cho, Kawasaki-ku, Kawasaki
c
210–8681, Japan: and Institute of Food Research and Technologies, Ajinomoto Co., Inc.; 1–1 Suzuki-cho, Kawasaki-ku,
Kawasaki 210–8681, Japan.
Received March 28, 2016; accepted April 28, 2016
Modulation of the calcium sensing receptor (CaSR) is one of the physiological activities of γ-glutamyl
peptides such as glutathione (γ-glutamylcysteinylglycine). γ-Glutamyl peptides also possess a flavoring effect,
i.e., sensory activity of kokumi substances, which modifies the five basic tastes when added to food. These
activities have been shown to be positively correlated, suggesting that kokumi γ-glutamyl peptides are per-
ceived through CaSRs in humans. Our research is based on the hypothesis that the discovery of highly ac-
tive CaSR agonist peptides will lead to the creation of practical kokumi peptides. Through continuous study
of the structure–CaSR–activity relation of a large number of γ-glutamyl peptides, we have determined that
the structural requirements for intense CaSR activity of γ-glutamyl peptides are as follows: existence of an
N-terminal γ-L-glutamyl residue; existence of a moderately sized, aliphatic, neutral substituent at the second
residue in an L-configuration; and existence of a C-terminal carboxylic acid, preferably with the existence of
glycine as the third constituent. By the sensory analysis of γ-glutamyl peptides selected by screening using
the CaSR activity assay, γ-glutamylvalylglycine was found to be a potent kokumi peptide. Furthermore,
norvaline-containing γ-glutamyl peptides, i.e., γ-glutamylnorvalylglycine and γ-glutamylnorvaline, pos-
sessed excellent sensory activity of kokumi substances. A novel, practical industrial synthesis of regiospecific
γ-glutamyl peptides is also required for their commercialization, which was achieved through the ring open-
ing reaction of N-α-carbobenzoxy-L-glutamic anhydride and amino acids or peptides in the presence of N-
hydroxysuccinimide.
Key words calcium sensing receptor; γ-glutamyl peptide; kokumi substance; glutathion; glutamic anhydride
γ-Glutamyl peptides, particularly glutathione (γ-glutamyl- of γ-glutamyl dipeptides and γ-glutamyl tripeptides com-
cysteinylglycine, γ-Glu-Cys-Gly, 36), possess various physio- prising proteinogenic amino acids, we identified various
logical activities, including activation of the calcium sensing γ-glutamyl peptides, including 36 and γ-glutamylvalylglycine
receptor (CaSR) and sensory activity of kokumi substances. (γ-Glu-Val-Gly, 35), as CaSR agonists.7) As the agonist pep-
Figure 1 shows the structures of the γ-glutamyl peptides tides contain an N-terminal γ-L-glutamyl residue comprising
described in this study.
α-amino and α-carboxyl groups, as with amino acids, it is
The extracellular CaSR plays a central role in extracel- reasonable to assume that peptides containing such a unit will
lular calcium homeostasis in mammals.1) Increased blood bind to and activate the CaSR.
calcium levels detected by the CaSR result in suppression of
Another physiological activity of γ-glutamyl peptides is
parathyroid hormone secretion, stimulation of calcitonin se- sensory activity of kokumi substances. Ueda et al. investi-
cretion, and induction of urinary calcium excretion to reduce gated the flavoring effects of diluted extracts of garlic and
the blood calcium to normal levels. It has become apparent onion, which have only minimal aroma in water, but when
that the CaSR is expressed not only in the parathyroid glands added to an umami solution or other types of food, they sig-
and kidneys but also in many other tissues, such as the liver, nificantly enhanced the thickness, continuity, and mouthful-
heart, lungs, gastrointestinal tract, lymphocytes, pancreas, ness of the food to which they had been added. Furthermore,
and the central and peripheral nervous systems, suggesting they isolated and identified 36, γ-glutamyl-S-allyl-L-cysteine,
that it is involved in a wide range of biological functions.2) γ-glutamyl-S-allyl-L-cysteine sulfoxide, and γ-glutamyl-trans-
Several types of compounds have been reported to possess S-propenyl-L-cysteine sulfoxide as some of the key com-
CaSR agonist activity. Amino acids have been reported to pounds responsible for this flavoring effect.8–10) They proposed
bind to the large extracellular Venus flytrap domain (VFD) of that substances with these properties should be referred to as
CaSR, which is a structure common to all class C members kokumi substances. The characteristic of kokumi substances
of the G protein-coupled receptors (GPCRs). CaSR is strongly is that they modify the five basic tastes (especially sweet,
activated by the aromatic amino acids His, Trp, Phe, and Tyr salty, and umami) when added to basic tasting solutions or
but weakly activated by other amino acids, such as Arg, Lys, food; however, at the concentrations tested they have no taste
Val, or Gly. It has been suggested that all amino acids bind themselves.11) More recently, it has been reported that many
to the VFD binding pocket through their amino and carboxyl γ-glutamyl peptides such as γ-Glu-Val (2), γ-Glu-Leu (5),
groups.3,4) γ-Glutamyl peptides have also been reported to bind γ-Glu-Cys-βAla (91), γ-Glu-Gly (11), γ-Glu-Met (9), and 35 are
to the large extracellular VFD of CaSR.5,6)
found in foods, such as edible beans, Gouda cheese, and soy
Previously, in a preliminary hCaSR functional assay sauces, and they enhance the intensity of mouthfulness.12–19)
*To whom correspondence should be addressed. e-mail: yusuke_amino@ajinomoto.com
© 2016 The Pharmaceutical Society of Japan

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Chem. Pharm. Bull.
Vol. 64, No. 8 (2016)
Fig. 1. Structures of γ-Glutamyl Peptides
Previously, we revealed that kokumi peptides such as 36 tion solvent. They demonstrated that the nucleophilic attack
are perceived through CaSRs in humans and that the CaSR preferentially occurs at the γ-carbonyl in polar aprotic solvents
activity of previously discovered γ-glutamyl peptides com- such as dimethyl sulfoxide (DMSO), whereas it occurs at the
prising proteinogenic amino acids is positively correlated α-carbonyl in nonpolar solvents such as benzene.24,25) King
with the sensory activity of kokumi peptides.7) A previous and Kidd reported that the reaction of N-phthalyl-DL-glutamic
report also indicated that CaSR-expressing taste cells are the anhydride with glycine exclusively afforded N-phthaloyl-γ-
primary detectors of kokumi substances, and that the kokumi DL-glutamylglycine in hot acetic acid in 48% isolated yield.26)
substance-responding taste cells might involve enhancement Their methods have also been applied to the regiospecific syn-
of basic tastes such as umami and sweet taste.20) In other thesis of γ-glutamyl dipeptides.27,28)
words, kokumi γ-glutamyl peptides can be easily identified via
Quesne and Young reported that in the reaction of N-α-
sensory evaluation of the γ-glutamyl peptides selected by pri- carbobenzoxy-glutamic anhydride (Cbz-Glu anhydride, 107)
mary high-throughput screening using the CaSR activity assay with valine ethyl ester in a two phase system of ethyl acetate
as an index. Thus, through further searching for active CaSR and aqueous potassium hydrogen carbonate, N-carbobenzoxy-
agonist peptides, the discovery of other practical kokumi α-glutamylvaline ethyl ester (Cbz-α-Glu-Val-OEt) was exclu-
γ-glutamyl peptides can be achieved.
sively obtained in 32% yield.29) Upon the reaction of 107 with
In this study, to ascertain the structural requirements of Glu in the same solvent, Shiba and Imai obtained the α-isomer
γ-glutamyl peptides for activation of the CaSR, 36 was divided in 34% yield and the γ-isomer in 1.8% yield.30) Manesis and
into three parts, N-terminus, second residue, and C-terminus, Goodman reported that the α- and γ-isomers were obtained
and each part was modified according to previous studies on in a ratio of 2:3 upon the reaction of 107 with ε-N-tert-
the structure–activity relation of 36.21–23) Modified γ-glutamyl butoxycarbonyl-lysine methyl ester in tetrahydrofuran in 42%
peptides composed of non-proteinogenic amino acids, D- total yield.31)
amino acids, α-hydoxy acids, etc. were then screened using
Although there have been many reports on the reaction of
the CaSR assay as an index.
N-protected-glutamic anhydride, to the best of our knowledge,
During the course of this study, 35 was filed in Flavor a regiospecific synthesis of γ-glutamyl peptides through the
and Extract Manufacturers Association of the United States reaction of 107 and an amino acid or peptide has not yet been
(FEMA) as FEMA-GRAS™ No. 4709, filed in the Euro- developed. Based on these previous reports, we have studied
pean Union list of flavoring substance, and approved as a the reaction of 107 and an amino acid or peptide and devel-
food additive in Japan (GRAS: generally recognized as safe). oped a large-scale synthetic procedure of γ-glutamyl peptides,
Therefore, development of a practical industrial production wherein the key step is a regiospecific γ-glutamyl bond forma-
method toward γ-glutamyl peptides is necessary for their com- tion. This was successfully achieved through the reaction of
mercialization. However, selective formation of the γ-glutamyl 107 and an amino acid or peptide in the presence of 0.5–1.0eq
linkage occurs during their synthesis, which is a problem that of N-hydroxysuccinimide (NHS).
must be overcome. Protected glutamic acids, such as N-α-
carbobenzoxy-L-glutamic acid α-benzyl ester, are useful for
small-scale syntheses of γ-glutamyl peptides, as shown in Ex-
Results and Discussion
Determination of CaSR Agonist Activity The theory
perimental; however, such conventional syntheses are difficult behind this experimental technique was previously described
to scale up.
in detail7) and is described briefly in this paper.
The regiospecific ring opening of N-protected-glutamic
A two-electrode voltage clamp assay was performed using
anhydride with an amino acid or peptide is an attractive ap- Xenopus oocytes microinjected with human CaSR cRNA at
proach for the construction of γ-glutamyl peptides. There have −70mV. Peptides were added at various concentrations (1000,
been many reports regarding the regioselectivity of the reac- 500, 300, 100, 50, 30, 10, 3, 1, 0.3, 0.1µM) and the minimum
tion of N-protected-glutamic anhydride with various amines. effective concentration at which a current could be detected
Huang et al. studied the nucleophilic addition of aniline to was a measure of the strength of CaSR activation. The results
N-protected-glutamic anhydride and reported that the regiose- demonstrated that many γ-glutamyl peptides display CaSR ac-
lectivity of the reaction was controlled by the choice of reac- tivity. No response was observed in oocytes injected with dis-

Vol. 64, No. 8 (2016)
Chem. Pharm. Bull.
1183
tilled water as a control. For comparison, EC50 values (µM) for some γ-glutamyl dipeptides are able to activate the CaSR.
γ-glutamyl peptides were also determined using a HEK-293
The CaSR activity of γ-glutamyl dipeptides is summarized
assay, which provided higher sensitivity than the oocyte assay. in Table 1. Among the γ-glutamyl dipeptides composed of
Activities of γ-Glutamyl Dipeptides (γ-Glu-X) The proteinogenic amino acids, γ-Glu-Cys (1) showed the most
structure–CaSR–activity relation of γ-glutamyl peptides was intense activity, while γ-Glu-Val (2) showed subsequent mod-
briefly discussed previously.7) The CaSR activity of γ-glutamyl erate activity. Conversely, some γ-glutamyl dipeptides, such
peptides, including previously reported results, is summarized as γ-Glu-Asp, γ-Glu-Asp, γ-Glu-Gln, γ-Glu-Glu, γ-Glu-Lys,
in Tables 1–3.
γ-Glu-His, γ-Glu-Phe, and γ-Glu-Tyr, were inactive (data not
Initially, we performed a CaSR agonist activity assay on a shown). Incorporation of a large side chain or a positively or
dipeptide library and found that most of the active dipeptides negatively charged side chain abolished the observed activity.
included a “γ-glutamyl” structure (data not shown). The pres- Among the γ-glutamyl dipeptides containing non-proteinogen-
ence of a γ-peptidic bond between the N-terminal glutamic ic amino acids, γ-Glu-Nva (Nva; norvaline) (18), γ-Glu-Cle
acid and the second residue is the most distinct structural (Cle; cycloleucine) (19), and γ-Glu-Abu (Abu; (S)-2-amino-
feature of active dipeptides. It has been suggested that a com- butanoic acid) (20) showed nearly the same range of activity
mon feature of class C G-protein-coupled receptors (GPCRs) as 1. This indicated that the sulfhydryl group of the cysteine
is the presence of an amino acid binding site. As γ-glutamyl residue is not essential for CaSR activity; however, the incor-
dipeptides have an α-amino acid structure, it is reasonable that poration of a small-to-medium-sized neutral alkyl side chain
is necessary for high activity. Incorporation of D-amino acids
(i.e., γ-Glu-D-Val, 29) produced inactive dipeptides. Analogs
Table 1. Determination of the Calcium Sensing Receptor (CaSR) Activ-
without an α-carboxyl (i.e., N-γ-aminobutyryl-Val) or α-amino
group (i.e., N-glutaryl-Val) were found to be inactive (data
not shown). Moreover, neither α-Glu-Val (31) nor γ-D-Glu-Val
(30) displayed any activity, demonstrating that the γ-L-
glutamyl linkage is required for CaSR activity. Replacement
of the carboxyl group at the C-terminus with an amide group
ity of γ-Glu-X Peptides Using Xenopus Oocytes and HEK-293 Cells
CaSR Activity (µM)
No.
γ-Glutamyl peptides
γ-Glu-Cys
Xenopus oocytes (a) HEK-293 (b)
1
2
3
4
5
6
7
8
9
3
10
0.16–0.46
1.03
γ-Glu-Val
γ-Glu-Ala
γ-Glu-Thr
γ-Glu-Leu
γ-Glu-Ile
50
1.24
50
6.97
Table 2. Determination of the Calcium Sensing Receptor (CaSR) Activ-
ity of γ-Glu-X-Gly Peptides Using Xenopus Oocytes and HEK-293 Cells
100
100
300
500
500
5.07
9.91
CaSR Activity (µM)
γ-Glu-Ser
γ-Glu-Orn
γ-Glu-Met
11
No.
γ-Glutamyl peptides
70.4
Xenopus oocytes (a) HEK-293 (b)
35 γ-Glu-Val-Gly
36 γ-Glu-Cys-Gly
37 γ-Glu-Ala-Gly
38 γ-Glu-Ser-Gly
39 γ-Glu-Gly-Gly
40 γ-Glu-Ile-Gly
0.1
3
0.030–0.075
0.7
10 γ-Glu-Asn
159
200
300
409
11 γ-Glu-Gly
1000
10
0.016
12 γ-Glu-Trp
10
30
13 γ-Glu-Pro
14 γ-Glu-Cys(Me)
15 γ-Glu-Tau
30
300
100
300
300
300
3
41 γ-Glu-Thr-Gly
42 γ-Glu-Leu-Gly
43 γ-Glu-Pro-Gly
44 γ-Glu-Cys(Me)-Gly
45 γ-Glu-Abu-Gly
46 γ-Glu-Algly-Gly
47 γ-Glu-Ser(Me)-Gly
48 γ-Glu-Nva-Gly
49 γ-Glu-Tle-Gly
50 γ-Glu-Pen-Gly
51 γ-Glu-Aib-Gly
52 γ-Glu-Cle-Gly
53 γ-Glu-allo-Ile-Gly
54 γ-Glu-allo-Thr-Gly
55 γ-Glu-Hse-Gly
56 γ-Glu-Cys(n-Butyl)-Gly
57 γ-Glu-Nle-Gly
2.8
16 γ-Glu-Cys(Me)(O)
17 γ-Glu-Met(O)
18 γ-Glu-Nva
300
1000
0.12
0.15
0.21
0.49
1.69
3.06
3.19
4.41
5.53
8.87
15.4
19 γ-Glu-Cle
3
0.025
0.011
20 γ-Glu-Abu
21 γ-Glu-Ape
0.037
22 γ-Glu-Cys(Carboxymethyl)
23 γ-Glu-Tle
0.052–0.055
0.043–0.090
0.16–0.23
0.35–1.17
0.5–1.5
0.63
24 γ-Glu-Ser(Me)
25 γ-Glu-Nle
26 γ-Glu-β-homoAla
27 γ-Glu-allo-Ile
28 γ-Glu-Aib
0.7
29 γ-Glu-D-Val
30 γ-D-Glu-Val
31 α-Glu-Val
Inactive
Inactive
Inactive
1000
1.18
4
5
32 γ-Glu-Val-NH2
33 γ-Glu-Val-OMe
34 γ-Glu-Val-ol
58 γ-Glu-Cys(1,2-Dicarboxy-
5–10
1000
ethyl)-Gly
1000
59 γ-Glu-Cys(Allyl)-Gly
100
(a) Minimum effective concentration. (b) EC50 values. Abbreviations: Cys(Me), S-
(methyl)-cysteine; Tau, taurine; Cys(Me)(O), S-(methyl)-cysteine sulfoxide; Met(O),
methionine sulfoxide; Nva, norvaline; Cle, cycloleucine; Abu, (S)-2-aminobutanoic
acid; Ape, (S)-3-aminopentanoic acid; Cys(Carboxymethyl), S-(carboxymethyl)-
cysteine; Tle, tert-leucine; Ser(Me), O-(methyl)-serine; Nle, norleucine; β-homoAla,
(S)-3-aminobutanoic acid; Aib, α-methylalanine; Val-ol, valinol.
60 γ-Glu-Cys(n-Propyl)-Gly
117
(a) Minimum effective concentration. (b) EC50 values. Abbreviations: Algly,
α-allylglycine; Pen, penicillamine; Hse, homoserine; Cys(n-Butyl), S-(n-butyl)-cys-
teine; Cys(1,2-dicarboxyethyl), S-(2,3-dicarboxymethyl)-cysteine; Cys(Allyl), S-
(allyl)-cysteine; Cys(n-Propyl), S-(n-propyl)-cysteine.

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Chem. Pharm. Bull.
Vol. 64, No. 8 (2016)
Table 3. Determination of the Calcium Sensing Receptor (CaSR) Activ-
ity of γ-Glu-Val-Y Peptides Using Xenopus Oocytes and HEK-293 Cells
shown). Incorporation of proteinogenic amino acids with small
hydrophilic side chains or small-to-medium-sized hydropho-
bic side chains generated agonists. Incorporation of valine,
containing a β-branched side chain, as the second residue
(35) produced an agonist with an EC50 value 30 times greater
than that of 36. Moreover, intense agonists were generated
due to the incorporation of non-proteinogenic L-amino acids
with small-to-medium-sized hydrophobic side chains compris-
ing one to three carbon(s) or two carbons and one sulfer or
oxygen atom. Since the activity of γ-Glu-Nva-Gly (48) was
approximately two orders of magnitude higher than that of
γ-Glu-Nle-Gly (Nle; norleucine) (57), the steric requirements
for expression of activity must be strictly controlled. How-
ever, incorporation of a D-amino acid (i.e., γ-Glu-D-Val-Gly,
81) generated inactive compounds (Table 3). These results
suggest that the side chain of the second residue must have
an appropriate size and orientation for activation of CaSR. In
tripeptides, the cysteine thiol group is considered to play an
important role, but was not found to be essential for biological
activity. Thus, the CaSR agonist function of γ-glutamyl tripep-
tides does not depend on the functionality of the thiol group.
Activities of N-Terminal or C-Terminal Modified Ana-
logs of γ-Glu-Val-Y Activities of 35 analogs modified at the
N- or C-terminus are shown in Table 3.
CaSR Activity (µM)
No.
γ-Glutamyl peptides
Xenopus oocytes (a) HEK-293 (b)
35 γ-Glu-Val-Gly
0.1
10
0.030–0.075
0.3–0.5
3.7
60 γ-Glu-Val-Gln
61 γ-Glu-Val-Cys
10
30
62 γ-Glu-Val-Pro
63 γ-Glu-Val-Ser
30
64 γ-Glu-Val-Phe
30
65 γ-Glu-Val-Asn
30
66 γ-Glu-Val-Orn
100
100
300
1000
1000
1000
1000
1000
1000
1000
67 γ-Glu-Val-His
68 γ-Glu-Val-Ala
69 γ-Glu-Val-Thr
70 γ-Glu-Val-Met
71 γ-Glu-Val-Asp
72 γ-Glu-Val-Arg
73 γ-Glu-Val-Lys
74 γ-Glu-Val-Glu
75 γ-Glu-Val-Val
76 γ-Glu-Val-D-Ala
77 γ-Glu-Val-D-Ser
78 γ-Glu-Ala-Leu
1.26
4.27
4.17
Replacement of the γ-glutamyl residue with a β-aspartyl res-
idue (88) or γ-D-glutamyl residue (82) yielded inactive tripep-
tides. α-Glutamyl peptide α-Glu-Val-Gly (84) exhibited weak
activity; however, it is not clear whether the observed activity
was the inherent activity of the α-glutamyl peptide or the ac-
tivity of the γ-glutamyl peptide that remained after synthesis.
Almost all 35 C-terminal modified analogs comprising a
proteinogenic amino acid were active, as opposed to Ile, Leu,
Trp, and Tyr. However, all tripeptide C-terminal modified
analogs were less active than 35. Replacement of Gly with Gln
and Cys yielded tripeptides (60, 61) that were active at concen-
trations three orders of magnitude less than that of 35. Incor-
poration of β-Ala (β-alanine) or D-amino acids, such as D-Ala
and D-Ser, instead of Gly (90–93, 76, 77) generated inactive
peptides or weak agonists. It is interesting that the replace-
ment of Gly with an α-hydroxy acid, such as glycolic acid or
L-lactic acid (96–101), afforded similar intense CaSR activities
to 35. Among the tetrapeptides, wherein another amino acid
was added to the C-terminus of 35, γ-Glu-Val-Gly-Gly (94)
and γ-Glu-Val-Gly-Gln (95) exhibited weak activity.
79 γ-Glu-Abu-Leu
80 γ-Glu-Abu-Pro
2.49
4.59
81 γ-Glu-D-Val-Gly
82 γ-D-Glu-Val-Gly
83 γ-D-Glu-D-Val-Gly
84 α-Glu-Val-Gly
Inactive
Inactive
Inactive
>10
Inactive
Inactive
Inactive
Inactive
Inactive
Inactive
5–10
85 α-D-Glu-Val-Gly
86 α-Glu-D-Val-Gly
87 α-D-Glu-D-Val-Gly
88 β-Asp-Val-Gly
89 β-Asp-Val-βAla
90 γ-Glu-Val-βAla
91 γ-Glu-Cys-βAla
92 γ-Glu-Abu-βAla
93 γ-Glu-Ala-βAla
94 γ-Glu-Val-Gly-Gly
95 γ-Glu-Val-Gly-Gln
96 γ-Glu-Val-Glycolic acid
97 γ-Glu-Abu-Glycolic acid
98 γ-Glu-Tle-Glycolic acid
99 γ-Glu-Val-L-Lactic acid
100 γ-Glu-Abu-L-Lactic acid
101 γ-Glu-Tle-L-Lactic acid
2.45
9.96
4.91
2.03
0.05
0.14
0.065–0.079
0.102
Summary of the CaSR Activities of γ-Glutamyl Pep-
tides With respect to the activities of several dipeptide and
tripeptide analogs, the γ-glutamyl residue is mandatory for
the peptide to be active, with a few exceptions. It has been
indicated that the exact alignment as well as the presence of
the two charged groups on the N-terminal residue are required
0.038
0.057
(a) Minimum effective concentration. (b) EC50 values. Abbreviations: βAla,
β-alanine.
(γ-Glu-Val-NH2, 32), methoxycarbonyl group (γ-Glu-Val-OMe, for binding to the CaSR. A small-to-medium-sized side chain
33), or hydroxymethyl group (γ-Glu-Val-ol, 34) yielded on the second residue in an L-configuration, as found in Val,
γ-glutamyl-amide compounds with very weak activities.
Abu, Nva, α-allylglycine (Algly), O-(methyl)-serine (Ser(Me)),
Activities of γ-Glutamyl Tripeptides (γ-Glu-X-Gly) Ac- and tert-leucine (Tle), most likely enhanced binding to the
tivities of γ-Glu-X-Gly analogs with center position modifica- receptor site in an active orientation. The presence of a third
tion are shown in Table 2. Tripeptides wherein the cysteine residue, especially one with a free carboxy terminus and no
side chain was replaced by a large hydrophobic, basic, or acid- side chain (i.e., glycine), significantly enhanced the activity.
ic side chain were found to be inactive. Thus, γ-Glu-Arg-Gly, However, the existence of this C-terminal third residue is not
γ-Glu-Asn-Gly, γ-Glu-Asp-Gly, γ-Glu-Gln-Gly, γ-Glu-Glu-Gly, essential for activity, but it is preferable. These findings were
γ-Glu-His-Gly, γ-Glu-Lys-Gly, γ-Glu-Met-Gly, γ-Glu-Orn-Gly, consistent with reports by Wang et al. and Broadhead et al.,
γ-Glu-Phe-Gly, and γ-Glu-Tyr-Gly were inactive (data not in which the importance of the interaction between both N-

Vol. 64, No. 8 (2016)
Chem. Pharm. Bull.
1185
terminal amino acid and the C-terminal carboxyl groups of
A typical profile of the sensory activity of standard kokumi
γ-glutamyl tripeptides and the active site of the CaSR was γ-glutamyl peptide, 36, was previously demonstrated.7) Briefly,
discussed.5,6)
the peak taste intensity of basal solutions containing low con-
Figure 2 shows the putative interaction between 35 and the centrations of umami and salty compounds was doubled by
CaSR, where the VFD mediates 35. The following three inter- adding 0.08% 36. The point of subjective equivalence (PSE)
actions must occur between γ-glutamyl peptides and the VFD of peptide 35 was determined as the concentration that was
of CaSR: γ-L-glutamyl residue with a zwitterionic binding site, judged to produce a sensation equal to that of 36 in umami
which has an amino acid binding site for VFD; hydrophobic and salty taste standard solution [0.05% monosodium gluta-
side chain of Val with a hydrophobic interaction site; and C- mate (MSG), 0.05% inosine monophosphate (IMP), and 0.5%
terminal carboxylic acid of glycine with an ionic binding site.
sodium chloride (NaCl)] with the sensory analysis method (see
Brief Description of the Sensory Activity of Kokumi Experimental). It was found that 0.01% 35 in standard solution
γ-Glutamyl Peptides Sensory analysis of γ-glutamyl peptides produced the sensory activity equivalent to 0.128% 36; there-
selected by screening using the CaSR activity assay was con- fore, we estimated that the sensory activity of kokumi peptide
ducted. EC50 of 35, 48, and 18 in CaSR assay were 0.030–0.075, 35 is 12.8-fold greater than that of 36.7,32–34) Similarly, the sen-
0.052–0.055, and 0.12µM, whereas that of 36 was 0.7µM.
sory activity of Nva-containing tripeptide 48 and dipeptide 18
were approximately 200- and 100-fold greater than that of 36,
i.e., 20- and 10-fold greater than that of 35, respectively. The
taste recognition threshold concentration of 48 (0.028ppm)
and 18 (0.077ppm) were significantly lower than that of 35
(0.47ppm) (see Experimental). Therefore, the strength of sen-
sory activity of kokumi peptides 48 and 18 compared with 35
were demonstrated by two sensory analysis methods. Interest-
ingly, although Nva is a non-proteinogenic amino acid, 48 was
recently detected in human serum samples.35)
The reason why 48 and 18 exhibited the intense sensory
activity of kokumi γ-glutamyl peptides relative to 35 has not
been fully understood yet. Quantitative analysis of the sensory
activity of these peptides is currently under investigation and
will be published elsewhere.
Development of
a
Production Procedure toward
γ-Glutamyl Peptides Based on previously reported stud-
ies,24–31) we attempted to improve upon the γ-selectivity of
the reaction between Cbz-Glu anhydride (107) and an amino
acid or peptide. Although Shiba and Imai reported that the
Fig. 2. Putative Interaction between γ-Glu-Val-Gly (35) and the CaSR
Chart 1. Large-Scale Chemical Synthesis of γ-Glu-Val-Gly (35)

1186
Chem. Pharm. Bull.
Vol. 64, No. 8 (2016)
α-isomer was predominantly obtained upon reaction of 107 of a large number of γ-glutamyl peptides demonstrated the
and Glu,30) we found that Cbz-γ-glutamyl peptides were pre- following structural requirements for the intense CaSR activ-
dominantly obtainable by the reaction between 107 and an ity of γ-glutamyl peptides: existence of an N-terminal γ-L-
amino acid or peptide in the presence of 0.5–1.0eq of NHS. glutamyl residue; existence of a moderately sized, aliphatic,
For example, in the reaction between 107 and Ala in an ethyl neutral substituent at the second residue with an L-configura-
acetate and aqueous sodium bicarbonate two layer system, the tion; and existence of a C-terminal carboxylic acid, preferably
ratio of Cbz-α-Glu-Ala to Cbz-γ-Glu-Ala was 1.0:15.8 in the with the existence of glycine as the third constituent. By the
presence of 0.5eq of NHS in 81% total yield, whereas the ratio sensory analysis of γ-glutamyl peptides selected by screen-
was 1.0:0.7 in the absence of NHS in 68% total yield. When ing using the CaSR activity assay, it was found that 35 and
Val was used instead of Ala in the two layer system, the ratio Nva-containing γ-glutamyl peptides, 48 and 18, possessed ex-
of α- to γ-isomer was 1.0:23.7 in the presence of 1.0eq of cellent sensory activity of kokumi substances. A practical in-
NHS and 1.0:0.7 in the absence of NHS. When reacted in dustrial synthesis of regiospecific γ-glutamyl peptides 35 was
a homogeneous solution such as acetone, the ratio of α- to also developed for their commercialization. This was achieved
γ-isomer was still 1.0:20.2 in the presence of 1.0eq of NHS through the regiospecific ring opening reaction of Cbz-Glu
and 1.0:0.7 in the absence of NHS. When the dipeptide Val- anhydride (107) and an amino acid or peptide in the presence
Gly was used instead of Val, the ratio of α- to γ-isomer was of 0.5–1.0eq of N-hydroxysuccinimide.
1.0:26.8 in the presence of 1.0eq of NHS in 85% total yield
after optimization of the reaction conditions.
Experimental
As an example of this newly developed procedure, an
Materials Chemicals for peptide synthesis were obtained
outline of the synthesis of 35 on a 200mol scale is shown in from either Bachem AG (Switzerland), Watanabe Chemi-
Chart 1. In this process, the NHS used in the preceding reac- cal Industries, Ltd. (Japan), or Kokusan Chemical Co., Ltd.
tion was used in the subsequent reaction. Cbz-Val NHS ester (Japan). Commercially available peptide samples were pur-
(103) was prepared via condensation of Cbz-Val (102) and chased from Sigma-Aldrich (U.S.A.), Dojin Chemical Labora-
NHS with N,N′-dicyclohexylcarbodiimide (DCC). Insoluble tory (Japan), Bachem AG, and Kokusan Chemical (Japan) and
material (i.e., urea) was filtered off to give an ethyl acetate used as received. Dipeptide samples and tripeptides compris-
solution of 103. An aqueous solution of the dipeptide Val-Gly ing proteinogenic amino acids were a contract manufactured
(105) was prepared via the reaction of 103 and glycine in a product (Kokusan Chemical Co., Ltd. and Peptide Institute,
water and ethyl acetate two layer system, followed by the Inc., Japan). Other peptides containing non-proteinogenic
deprotection of the Cbz group by hydrogenation with Pd/C in amino acids and tetrapeptides were synthesized in our labo-
water. Cbz-Glu anhydride (107) was prepared by the reaction ratories using liquid phase synthesis and characterized by
of Cbz-Glu (106) and DCC in ethyl acetate, followed by filtra- 1H-NMR spectroscopy (Bruker AVANCE400 (400MHz)) and
tion of the generated dicyclohexylurea. To the aqueous solu- electrospray ionization (ESI)-MS (Thermo Quest TSQ700).
tion of 105 containing 1eq of each NHS used in the previous
Peptides 35, 48, and 18 as well as other peptides used for
reaction and sodium bicarbonate, the solution of 107 in ethyl the sensory evaluation test were synthesized in our labora-
acetate was slowly added at room temperature to produce tories. Glutamine- and cysteine-containing peptides were
Cbz-α-Glu-Val-Gly and Cbz-γ-Glu-Val-Gly (108) in a ratio of prepared immediately before use, while other peptides were
approximately 3:97. Deprotection of the Cbz group via hy- stored at −20°C after preparation. Peptides having a purity of
drogenation with Pd/C was performed in the water and ethyl 95% or higher were used for evaluation of the CaSR activation
acetate two layer system, followed by recrystallization from a activity, while those having a purity of 98% or higher were
mixed solvent system of water and methanol to afford 35 in used for the sensory evaluation test. However, for 1, a purity
approximately 54% overall yield (α/γ<1/99).
of 80% or higher was used. When the solution in which each
The reason for this reversal of selectivity of the reaction sample was dissolved showed an acidic or alkaline pH, the
in the presence or absence of NHS is unclear; however, the solution was adjusted to approximately neutral using a sodium
γ-selectivity can partially be explained by the steric influence hydroxide solution or hydrochloric acid solution.
of the Cbz group on both carbonyls. In glutamic anhydride
modified with a large carbobenzoxy group, nucleophilic ad-
dition at the γ-position is preferred. Replacement of the in-
tramolecular hydrogen bond existing between the hydrogen
on the α-amino nitrogen and the oxygen of the α-carbonyl,
which activates the electrophilicity of the α-carbonyl, with an
intermolecular hydrogen bond between the hydrogen on the
α-amino nitrogen and the oxygen of NHS is also a possible
explanation, as discussed by Huang et al. regarding the role
Liquid Phase Synthesis of γ-
L-Glutamyl-L-valylglycine
(35, γ-Glu-Val-Gly) N-α-tert-Butoxycarbonyl-L-valine (Boc-
Val-OH, 8.69g, 40.0mmol) and glycine benzyl ester hydro-
chloride (Gly-OBzl·HCl, 8.07g, 40.0mmol) were dissolved
in methylene chloride (100mL) and the solution was kept
at 0°C. Triethylamine (6.13mL, 44.0mmol), 1-hydroxy-
benzotriazole (HOBt, 6.74g, 44.0mmol), and 1-ethyl-3-(3-
dimethylaminopropyl)carbodiimide hydrochloride (WSC·HCl,
8.44g, 44.0mmol) were added to the solution and the mixture
of dimethylsulfoxide in γ-selectivity.24)
Another possible ex- was stirred overnight at room temperature. The reaction mix-
planation is activation of the less hindered γ-carboxyl group ture was concentrated under reduced pressure and the result-
upon formation of the NHS γ-ester, which is the short-lived ing residue was dissolved in ethyl acetate (200mL). The solu-
intermediate of the reaction of 107 and NHS. Details on this tion was washed with water (50mL), 5% aqueous citric acid
regiospecific synthesis will be reported in the near future.
solution (50mL×twice), saturated brine (50mL), 5% aqueous
sodium bicarbonate solution (50mL×twice), and saturated
brine (50mL). The organic layer was dried over anhydrous
Conclusion
The present study on the structure–CaSR–activity relation magnesium sulfate, filtered, and the filtrate was concentrated

Vol. 64, No. 8 (2016)
Chem. Pharm. Bull.
1187
under reduced pressure. The residue was recrystallized from (1H, m), 1.82–1.88 (1H, m), 2.04–2.12 (2H, m), 2.45 (2H, t,
ethyl acetate/n-hexane to obtain Boc-Val-Gly-OBzl (13.2g, J=7.40Hz), 3.79 (1H, t, J=6.36Hz), 4.31–4.45 (1H, m), 4.57
36.2mmol) as white crystals.
Boc-Val-Gly-OBzl (5.47g, 15.0mmol) was added to a 4N
(2H, s). ESI-MS m/z: 291.10 (M+H)+.
Large-Scale Synthesis of γ-L-Glutamyl-L-valylglycine
HCl/dioxane solution (40mL) and the mixture was stirred (35)
at room temperature for 50min. Dioxane was removed by
concentration under reduced pressure, n-hexane (30mL) was
[Step 1]
N-Hydroxysuccinimide (NHS; 60.1kg, 522mol, 1.05 equiv.)
added to the residue, and the mixture was concentrated under and N,N′-dicyclohexylcabodiimide (DCC (107.8kg, 522mol,
reduced pressure. This procedure was repeated thrice to quan- 1.05 equiv) in ethyl acetate (147L) were added to Cbz-Val
titatively obtain H-Val-Gly-OBzl·HCl.
(102, 125kg, 498mol) in ethyl acetate (675L) and the mixture
H-Val-Gly-OBzl·HCl (15.0mmol) and N-α-carbobenzoxy- was stirred under ambient conditions. After completion of the
L-glutamic acid α-benzyl ester (Cbz-Glu-OBzl, 5.57g, reaction, insoluble solids (dicyclohexylurea) were removed by
15.0mmol) were dissolved in methylene chloride (50mL) filtration to afford Cbz-Val N-hydroxysuccinimide ester (103)
and the solution was kept at 0°C. Triethylamine (2.30mL, in ethyl acetate. The resultant solution was added to glycine
16.5mmol), HOBt (2.53g, 16.5mmol), and WSC·HCl (3.16g, (39.2kg, 522mol, 1.05 equiv) in water (225L). The mixture
16.5mmol) were added to the solution and the mixture was was stirred at 10°C while maintaining slightly basic conditions
stirred at room temperature for 2d. The reaction mixture was by adding an aqueous sodium hydroxide solution (25%). The
concentrated under reduced pressure and the resulting residue water layer was then separated to afford Cbz-Val-Gly (104) in
was dissolved in heated ethyl acetate (1500mL). The solution water. Ethyl acetate (375L) was added to the 104 solution and
was washed with water (200mL), 5% aqueous citric acid solu- the Cbz group was deprotected via hydrogenation in the pres-
tion (200mL×twice), saturated brine (150mL), 5% aqueous ence of a palladium catalyst (10% Pd/C, 1.3kg). After remov-
sodium bicarbonate solution (200mL×twice), and saturated ing the palladium catalyst by filtration, the water phase was
brine (150mL). The organic layer was dried over anhydrous separated to afford a solution of Val-Glu (105).
magnesium sulfate, filtered, and the filtrate was concentrated
[Step 2]
under reduced pressure. The deposited crystals were collected
DCC (112.9kg, 547mol, 1.10 equiv.) in ethyl acetate (153L)
by filtration and dried under reduced pressure to obtain Cbz- was added to Cbz-Glu (106, 146.9kg, 522mol, 1.05 equiv.)
Glu(Val-Gly-OBzl)-OBzl (6.51g, 10.5mmol) as white crystals. in ethyl acetate (514L) at 10°C. After stirring the solution
Cbz-Glu(Val-Gly-OBzl)-OBzl (6.20g, 10.03mmol) was under ambient conditions overnight, the resultant solids (di-
suspended in ethanol (200mL), to which 10% palladium/ cyclohexylurea) were removed by filtration to afford Cbz-Glu
carbon (1.50g) was added. The reduction reaction was per- anhydride (107) in ethyl acetate. The resultant solution was
formed at 55°C for 5h under a hydrogen atmosphere. During added to the 105 solution obtained in step 1 in the presence
the reaction, a total of 100mL of water was gradually added. of sodium bicarbonate (43.9kg, 522mol, 1.05 equiv.). After
The catalyst was removed by filtration using a Kiriyama fun- completion of the reaction, tetrahydrofuran (625L) was added
nel and the filtrate was concentrated under reduced pressure and the pH of the water layer was acidified with concentrated
to half volume. The reaction mixture was further filtered hydrochloric acid. Peptide 108 was then extracted into the
through a membrane filter and the filtrate was concentrated organic layer (ethyl acetate and tetrahydrofuran) and washed
under reduced pressure. The resulting residue was dissolved with 10% aqueous sodium chloride solution (625L) and 3%
in a small volume of water and ethanol was added to deposit aqueous sodium chloride solution (625L×twice). Water (537L)
the crystals, which were then collected by filtration and dried was then added to the solution, and the Cbz group was depro-
under reduced pressure to obtain 35 (2.85g, 9.40mmol) as a tected via hydrogenation in the presence of a palladium cata-
white powder.
lyst (10% Pd/C, 2.1kg). After removing the palladium catalyst,
the water layer was separated and concentrated. Addition of
methanol (1687L) to the residue afforded 35 as crystals, which
were collected and dried (86kg, 283mol, α/γ <1/99).
1H-NMR (D2O) δ: 0.87 (3H, d, J=6.8Hz), 0.88 (3H, d,
J=6.8Hz), 1.99–2.09 (3H, m), 2.38–2.51 (2H, m), 3.72 (1H, t,
J=6.35Hz), 3.86 (1H, d, J=17.8Hz), 3.90 (1H, d, J=17.8Hz),
4.07 (1H, d, J=6.8Hz). ESI-MS m/z: 304.1 (M+H)+.
1H-NMR (D2O) δ: 0.88 (3H, d, J=6.8Hz), 0.89 (3H, d,
γ-L-Glutamyl-L-α-aminobutyrylglycine (45, γ-Glu-Abu-Gly) J=6.8Hz), 1.99–2.10 (3H, m), 2.40–2.50 (2H, m), 3.72 (1H,
1H-NMR (D2O) δ: 0.89 (3H, t, J=7.48Hz), 1.63–1.69 (1H, m), t, J=6.4Hz), 3.86 (1H, d, J=17.8Hz), 3.91 (1H, d, J=17.8Hz),
1.73–1.79 (1H, m), 2.04–2.10 (2H, m), 2.41–2.47 (2H, m), 4.08 (1H, d, J=6.8Hz). 13C-NMR (D2O) δ: 17.7, 18.7, 26.5,
3.74 (1H, t, J=6.36Hz), 3.87 (1H, d, J=17.8Hz), 3.90 (1H, d, 31.5, 41.7, 54.2, 60.0, 173.9, 174.0, 174.5, 175.3. IR (KBr)
J=17.8Hz), 4.14 (1H, dd, J=5.66 and 8.34Hz). ESI-MS m/z: cm−1: 3321 (N–H), 3282 (N–H), 2940 (C–H), 1712 (C=O),
290.10 (M+H)+.
1654 (C(C=O)–O), 1919 (amide I band), 1541 (amide II band),
γ-L-Glutamyl-L-tert-leucylglycine (49, γ-Glu-Tle-Gly) 1238 (C–C(C=O)–O). ESI-MS m/z: 304.2 (M+H)+. FAB-MS
1H-NMR (D2O) δ: 0.95 (9H, s), 2.04–2.08 (2H, m), 2.45–2.48 m/z: 304.1505 (M+H) (Calcd for C12H22N3O6: 304.1509). mp:
(2H, m), 3.73 (1H, t), 3.87–3.90 (2H, m), 4.07 (1H, s). ESI-MS 225–228°C. [α]D20−29 (c=1.0, H2O).
m/z: 318.10 (M+H)+.
Synthesisof γ-L-Glutamyl-L-norvalylglycine(48) γ-L-Glu-
γ-L-Glutamyl-L-α-aminobutyryl-glycolic Acid (97, γ-Glu- L-Nva-Gly was prepared in the same manner as γ-L-Glu-
Abu-Gly Acid) In the first step of this synthesis, condensa- L-Val-Gly.
tion of Boc-Abu and glycolic acid benzyl ester was carried out
with WSC·HCl (1.1 equiv.) and 4-dimethylaminopyridine (0.3 m), 1.55–1.73 (2H, m), 2.02–2.10 (2H, m), 2.35–2.48 (2H, m)
equiv.). 3.72 (1H, t, J=6.2Hz), 3.84 (1H, d, J=17.6Hz), 3.89 (1H, d,
1H-NMR (D2O) δ: 0.86 (3H, t, J=7.40Hz), 1.60–1.74 J=18.0Hz), 4.20 (1H, dd, J=5.2 and 5.6Hz). 13C-NMR (D2O)
1H-NMR (D2O) δ: 0.81 (3H, t, J=7.4Hz), 1.22–1.38 (2H,

1188
Chem. Pharm. Bull.
Vol. 64, No. 8 (2016)
δ: 12.7, 18.4, 26.0, 31.1, 33.0, 41.3, 53.7, 53.8, 173.5, 174.8, as follows.7) The cDNA was diluted with Opti-MEM medium
175.0. IR (KBr) cm−1: 3376 (N–H), 3350 (N–H), 2940 (C–H), (Invitrogen, U.S.A.), mixed with FuGENE6 (Roche Applied
1679 (C=O), 1642 (amide I band), 1515 (amide II band). Science, Switzerland), and poured onto HEK-293 cells grown
ESI-MS m/z: 304.1 (M+H)+. FAB-MS m/z: 304.1512 (M+H) at a submaximal concentration. After 24h of culture in a 96-
(Calcd for C12H22N3O6: 304.1509). mp: 191–192°C. [α]D20−25 to well plate, the cells were incubated with 5µM Calcium-4 (Cal-
−24 (c=1.0, H2O).
cium-4 Assay Kit, Molecular Devices, U.S.A.) for 45–60min
Synthesis of γ-L-Glutamyl-L-norvaline (18) γ-L-Glu- and measurements were conducted using a calcium-image
L-Nva was prepared in the same manner as γ-L-Glu-L-Val-Gly, analyzer (FlexStation, Molecular Devices) with the associ-
with the exception that the condensation was carried out once. ated software. Activation of CaSR expressed in HEK-293 cells
1H-NMR (D2O) δ: 0.81 (3H, t, J=7.4Hz), 1.22–1.35 (2H, m), leads to an increase in intercellular calcium ions, which was
1.57–1.76 (2H, m), 2.03–2.09 (2H, m), 2.38–2.43 (2H, m) 3.74 determined using Calcium-4, a calcium dye. This dye binds
(1H, t, J=6.4Hz), 4.20 (1H, dd, J=5.2 and 5.2Hz). 13C-NMR free Ca2+, resulting in an increase in dye fluorescence, which
(D2O) δ: 12.7, 18.4, 26.2, 31.2, 32.6, 53.2, 53.8, 173.5, 174.6, is excited at 485nm and emits at 525nm. The concentration
176.8. IR (KBr) cm−1: 3310 (N–H), 2961 (C–H), 1722 (C=O), dependence of the fluorescence intensity was analyzed with
1653 (amide I band), 1633 (C(C=O)–O), 1529 (amide II band), various CaSR agonists. The assay buffer for calcium imaging
1211 (C–C(C=O)–O). ESI-MS m/z: 247.1 (M+H)+. FAB-MS contained 0.75mM CaCl2.
m/z: 247.1298 (M+H) (Calcd for C10H19N2O5: 247.1294). mp:
Evaluation of the Sensory Activity of Kokumi γ-Glutamyl
Peptides Quantitative analyses of the sensory activity of ko-
197–199°C. [α]D20−14 to −13 (c=1.0, H2O).
Preparation of cRNA The preparation of human cDNA kumi peptides with standard 36: A quantitative evaluation of
was described previously.7) cRNA of the hCaSR was prepared the human sensory analyses was performed by a panel of 17
from human kidney cDNA (Clontech) using a PCR method well trained assessors who judged the PSE between sample
as follows. Primer oligonucleotide DNAs (forward primer (N: and reference solution of 36 in umami and salty taste standard
5′-ACT AAT ACG ACT CAC TAT AGG GAC CAT GGC ATT solution. Test samples of 35 (0.01%) were mixed with umami
TTATAGCTGCTGCTGG-3′) and reverse primer (C: 5′-TTA and salty taste standard solutions (0.05% MSG, 0.05% IMP,
TGAATTCACTACGTTTTCTGTAACAG-3′); National Cen- and 0.5% NaCl). A series of 36 concentrations were chosen
ter for Biotechnology Information (NCBI) accession number in logarithmically equal steps at 50% intervals (0.02, 0.03,
NM_000388) were synthesized and PCR was performed using 0.044, 0.07, 0.10, 0.15, 0.23, 0.34% (w/v)). Each test sample
PfuUltra DNA Polymerase (Stratagene, U.S.A.) under the fol- was paired twice with the reference 36 solution, and the as-
lowing conditions. After reacting at 94°C for 3min, a cycle of sessors were required to rate the sensation produced by a
reactions at 94°C for 30s, 55°C for 30s, and 72°C for 2min test sample as either more or less intense than that of refer-
was repeated 35 times, followed by reaction at 72°C for 7min. ence solution 5s after tasting. The PSE was defined as the
The plasmid vector pBR322 (TaKaRa, Japan) was digested concentration that was judged to produce a sensation equal to
with the restriction enzyme EcoRV. The PCR product was that of the standard solution. The sample concentration was
ligated to the EcoRV cleavage site of pBR322 using a ligation determined by a preliminary test. Data were analyzed by the
kit (Promega, U.S.A.). hCaSR cRNA was synthesized using a probit method.
cRNA preparation kit (Ambion, U.S.A.) with this sequence as
a template.
Determination of taste recognition threshold concentrations
of kokumi peptides: Taste recognition threshold concentra-
Determination of CaSR Agonist Activity Using Oocytes tions for sensory activity of kokumi peptides were determined
CaSR agonist-induced currents were characterized using oo- as follows. The minimum peptide concentration at which the
cytes microinjected with hCaSR cRNA as follows.7) Xenopus assessors recognize the increase of taste intensity compared
laevis ovarian lobes were surgically removed, defolliculated, with umami and salty taste standard solution without peptides
and treated with collagenase II. Oocytes were then micro- was defined as the recognition threshold concentration in this
injected with 10–20ng of hCaSR cRNA and incubated for study. Seven concentration sample solution were prepared for
36–48h at 15°C in Barth’s solution. Activation of the CaSR each peptide in an umami and salty taste standard solution:
(Gq class GPCR) expressed in oocytes leads to an increase 35: 0.05, 0.1, 0.2, 0.5, 1.0, 2.0, and 5.0ppm; 48: 0.002, 0.005,
in intercellular calcium ions, which concomitantly activates 0.001, 0.02, 0.05, 0.1, and 0.2ppm; and 18: 0.005, 0.01, 0.02,
oocyte endogenous calcium-dependent chloride channels 0.05, 0.1, 0.2, and 0.5ppm. The tasting concentration level was
with a measurable current. The oocytes were impaled by two determined in a preliminary sensory experiment. Five well
electrodes in a voltage clamp configuration with a Geneclamp trained assessors tasted these samples in these order and in
500 (Axon) and responses were recorded using AxoScope the reverse order and compared them with a standard solution
9.0 recording software (Axon) at a membrane potential of without peptides. The threshold value of the assessors was ap-
−70mV. The oocytes were challenged with a 0.1–1000µM proximated by averaging the threshold values of individuals in
solution of CaSR agonists in a perfusion buffer containing
96mM NaCl, 2mM KCl, 1mM MgCl2, 1.8mM CaCl2, and 5mM
4-(2-hydroxyethyl)-1-piperazineethansulfonic acid (HEPES)
buffer (pH 7.2). The recorded peak current was deemed to be
the strength of receptor activation. Un-injected oocytes did not
respond to calcium or the tested CaSR agonists.
Determination of CaSR Agonist Activity Using HEK-293
Cells hCaSR cDNA was constructed in an expression vec-
tor, pcDNA3.1, and transiently transfected into HEK-293 cells
three independent sessions.
Acknowledgments We would like to thank Kiyoshi
Miwa, Toshihisa Kato, Tohru Kouda, and Hiroaki Takino for
their encouragement and continued support. We are grateful
to Reiko Yasuda, Fumie Futaki, Takeaki Ohsu, Sen Takeshita,
Yuki Tahara, Hiroaki Nagasaki, Takaho Tajima, and Motonaka
Kuroda for their helpful discussions and assistance. We are
also grateful to members of the taste panel at Institute of Food

Vol. 64, No. 8 (2016)
Chem. Pharm. Bull.
yano H., Eto Y., Food Chem., 134, 1640–1644 (2012).
1189
Research and Technologies in Ajinomoto Co., Inc.
16) Kuroda M., Kato Y., Yamazaki J., Kai Y., Mizukoshi T., Miyano H.,
Eto Y., J. Agric. Food Chem., 60, 7291–7296 (2012).
17) Kuroda M., Kato Y., Yamazaki J., Kai Y., Mizukoshi T., Miyano H.,
Eto Y., Food Chem., 141, 823–828 (2013).
18) Miyamura N., Kuroda M., Kato Y., Yamazaki J., Mizukoshi T., Mi-
yano H., Eto Y., Food Sci. Technol. Res., 20, 699–703 (2014).
19) Miyamura N., Iida Y., Kuroda M., Kato Y., Yamazaki J., Mizukoshi
T., Miyano H., J. Biosci. Bioeng., 120, 311–314 (2015).
20) Maruyama Y., Yasuda R., Kuroda K., Eto Y., PLoS ONE, 7, e34489
(2012).
Conflict of Interest All authors were employees of Aji-
nomoto Co., Inc. when this study was conducted and have no
further conflicts of interest to declare.
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