A selective preparation of highly deuterated hydroxybenzoic acids

Article abstract of DOI:10.2174/157017811795685036

A practical method to prepare selected deuterated hydroxybenzoic acids was developed. A hydroxybenzoic acid was dissolved or suspended in deuterium oxide (D₂O), then deuterium chloride (DCl) was added to adjust the pD to 0.32, and the mixture w

Full text of DOI:10.2174/157017811795685036

332
Letters in Organic Chemistry, 2011, 8, 332-336
A Selective Preparation of Highly Deuterated Hydroxybenzoic Acids
Kozo Nakamura*, Kazuki Morozumi and Daisuke Amano
Department of Bioscience and Biotechnology, Faculty of Agriculture, Shinshu University, Nagano 399-4598, Japan
Received November 03, 2010: Revised March 31, 2011: Accepted March 31, 2011
Abstract: A practical method to prepare selected deuterated hydroxybenzoic acids was developed. A hydroxybenzoic
acid was dissolved or suspended in deuterium oxide (D2O), then deuterium chloride (DCl) was added to adjust the pD to
0.32, and the mixture was refluxed under nitrogen for one to six hours. After removing D2O and DCl by lyophilization,
2,6-dihydroxybenzoic-3,5-d2 acid, 3,5-dihydroxybenzoic-2,4,6-d3 acid, 3,5-dimethoxy-4-hydroxybezoic-2,6-d2 acid, and
3,4,5-trihydroxybenzoic-2,6-d2 acid were obtained quantitatively. Aromatic carbons with double substituent effects of
hydroxy or methoxy groups were selectively deuterated. It was shown that the deuterium exchange reactions, which were
electrophilic substitution reactions of deuterium cations to the aromatic carbons on the hydroxybenzoic acids, were
regulated by the substituent effects. Deuteriums on all aromatic carbons were stable under a physiological condition and
were detected in plasma by mass spectrometry analysis after oral administration to rats.
Keywords: Hydroxybenzoic acid, Deuteration, Selective preparation, Substituent effect.
Hydroxybenzoic acids are classified as a category of
plant phenols. They are contained in common foods and
have dietary functionality such as antioxidant activity [1],
antiobesity effects [2], and antitumoral activity [3].
Mechanisms of most hydroxbenzoic acids are still not clear
because they are altered by a variety of chemicals by
metabolic biotransformation in vivo. In addition, it is
impossible to distinguish the same metabolite originating
from foreign substrates or in vivo. Therefore, kinetic analysis
of hydroxybenzoic acids has been extremely difficult. Using
isotope-labeled compounds for kinetic analysis is an efficient
method. An isotope-labeled compound has been utilized as a
kinetic reagent because it is metabolized in the same way as
unlabeled natural compounds. Two types of isotopes exist: a
radio isotope and a stable isotope. A radioisotope-labeled
compound has high sensitivity and exceptionally small
quantities of a sample are detectable, however the biological
activity of radioisotope-labeled compounds is fraught with
peril. On the other hand, a stable isotope-labeled compound
has relatively lower sensitivity but is much safer. Stable
isotopes exist at absolute ratio in nature and are treated as
general reagents. Kinetic analyses of natural products and
drugs can be accomplished by using stable isotope-labeled
compounds [4] and can be put to practical use in the
diagnosis of infection by Helicobacter pylori [5]. Deuterium
is a type of stable isotope of hydrogen composed of a proton
and a neutron. A compound labeled with deuterium was
prepared by exchange of hydrogen with deuterium. Phenols
were partially deuterated with treatment of Raney alloy
catalyst in the presence of alkali or alkaline-earth metal
carbonate in deuterium oxide (D2O) [6]. Flavonoids were
deuterated by using D3PO4/BF3 as the deuteration reagent in
D2O [7]. In sub- and supercritical water, an aromatic ring
containing phenols was deuterated without using a catalyst
[8]. Preparation of deuterated phenols in efficient H-D
exchange with Pt/C was reported by Sajiki et al. [9]. A
deuterated phenol was utilized as a reagent for kinetic
analysis and the effectiveness of this method was shown
[10]. However,
a
selective deuteration method for
hydroxybenzoic acids has not been reported. We developed a
simple and efficient selective preparation method for highly
deuterated hydroxybenzoic acids without using an expensive
reagent. Stability of the deuterated hydroxybenzoic acids
was investigated under a physiological condition and a single
oral administration test in rats was performed to validate
evidence of deuterated hydroxybenzoic acids as reagents for
kinetic analysis of functional food ingredients. The
deuteration mechanism was considered based on substituent
effects by functional groups and estimated electron densities
of the hydroxybenzoic acids.
Deuteration of ten hydroxybenzoic acids was investi-
gated, including 1; 4-hydroxybenzoic acid, 2; 2,3-dihydroxy-
benzoic acid (pyrocatechuic acid), 3; 2,4-dihydroxybenzoic
acid (ꢀ-resorcylic acid), 4; 2,5-dihydroxybenzoic acid
(gentisic acid), 5; 2,6-dihydroxybenzoic acid (ꢁ-resorcylic
acid), 6; 3,4-dihydroxybenzoic acid (protocatechuic acid), 7;
3,5-dihydroxybenzoic acid (ꢂ-resorcylic acid), 8; 3-methoxy-
4-hydroxybenzoic acid (vanillic acid), 9; 3,5-dimethoxy-4-
hydroxybenzoic acid (syringic acid), and 10; 3,4,5-
trihydroxybenzoic acid (gallic acid). Each hydroxybenzoic
acid was dissolved or suspended in D2O and the mixture was
refluxed under nitrogen for 48 hours. After removing D2O,
all partially deuterated hydroxybenzoic acids except for
compound 3 (ꢀ-resorcylic acid) were obtained [11]. It seems
that 3 was first converted to resorcinol by decarbonation and
then decomposed to CO2 and H2O in the reflux process [12].
Hydrogen on 2,6 positions of 10 were relatively highly
exchanged by deuterium, with an exchange ratio of 79.3%
by NMR determination [13, 14]. The deuteration, which is
an acid-catalized hydrogen-exchange reaction, was caused
by electrophilic substitution reaction of the deuterium cation
to the aromatic carbons on the hydroxybenzoic acids [6]. To
*Address correspondence to this author at the Department of Bioscience and
Biotechnology, Faculty of Agriculture, Shinshu University, Nagano 399-
4598, Japan; Tel: +81-265-77-1638; Fax: +81-265-77-5259;
E-mail: knakamu@shinshu-u.ac.jp
1570-1786/11 $58.00+.00
© 2011 Bentham Science Publishers Ltd.

A Selective Preparation of Highly Deuterated Hydroxybenzoic Acids
Letters in Organic Chemistry, 2011, Vol. 8, No. 5 333
determine a more efficient deuteration condition, deuteration
of 10 was investigated under several concentrations of
deuterium cation. In this report, the concentration of
deuterium cation is shown as pD (power of deuterium) in the
same sense as pH [15]. By adding deuterium chloride (DCl)
or sodium deuteroxide (NaOD) to the D2O solvent of 10,
which was originally pD 3.5, the pD was adjusted to 7.0, 2.0,
1.0, 0.56, and 0.32, respectively. And the deuterated ratio on
2,6 positions of 10 was determined after the reflux condition
during 24 hours in each pD [16]. The results are shown in
Fig. (1). At the original pD of 3.5, the deuterated ratio was
increased linearly depending on reaction time and the ratio
was 61.4% after 24-hour reaction. At pD 7.0, the color of the
reaction mixture turned brown immediately after starting the
reflux. Deuteration ratio of the brown product could not be
determined by NMR because of degradation of 10. In
contrast, in the reaction at low pD, the deuterated ratios
increased dramatically. An exchange ratio of over 97% was
achieved in 24, 12, 2, and 0.5 hours at pD 2, 1, 0.56, and
0.32, respectively. Compound 10 was stable in the strongest
acidic condition and the product was obtained quantitatively.
Reactions at lower pD achieved a more efficient deuterated
exchange reaction. At pD 0.32, 3,4,5-trihydroxybenzoic-2,6-
d2 acid (11), whose deuterated ratio at 2,6 positions was 99.3
%, was obtained quantitatively in 1 hour as white powder.
This is the ideal condition in which to prepare 11 from 10 in
lab-scale production.
and 95.5%, respectively. Elongating the reaction times, 3,5-
dimethoxy-4-hydroxybezoic-2,6-d2 acid (14) with 92.6% of
deuterated ratio was quantitatively obtained in 6-hour
reaction. It was estimated that the deuterated ratio at 4
position of 12 decreased slightly by re-hydrogenation after
the almost complete deuteration. As determined later, 15.8%
of the deuterium at the 4 position on 12 was exchanged with
hydrogen under a physiological condition in 7 days and this
actual example supported the re-hydrogenation. At pD 0.32,
compounds 1, 4, 6-8 were only scarcely deuterated in 24
hours, and 2 was partially deuterated in 1 hour but half of it
decomposed. It was impossible to obtain highly deuterated
compounds by elongating the reaction time.
To examine stability of the deuterated hydroxybenzoic
acids under a physical condition, changes of the deuterated
ratio on 11 and 12 were determined during 7 days [17]. As
shown in Table 2, deuterium on 2,6 positions of 11 and 12
were just barely re-hydrogenated and 94.8% and 99.9% of
deuterium were maintained in saline for 7 days. The
deuterated ratio of the 4 position on 12 was 84.2% after 7
days and the deuterium was slightly re-hydrogenated. After
oral administration of compound 11 to rats, an mass
spectrometry (MS) peak of protonated 11 at 173.1 m/z was
detected in the plasma [18]. Compounds 11 and 12 were
stable under the physiological condition during 7 days and
deuterium isotopes on both were maintained sufficiently. In
addition, they were absorbed through the digestive system of
living bodies and were detected from analysis of body tissue.
The conclusion is that deuterated hydroxybenzoic acids are
able to be utilized as reagents for kinetic analysis.
100
The deuterium exchange reaction in this study was an
electrophilic substitution reaction of deuterium cations to the
aromatic carbons on hydroxybenzoic acids. Aromatic
carbons with a high density of electrons with ꢃ-bonds
generally react with an electrophilic reagent. The reactivity
was directly affected by substituent effect. Hydroxybenzoic
acids in this research possess hydroxy group(s), methoxy
group(s) and a carboxyl group as substituted groups in the
molecule. Because negative charges on ortho/para positions
to the hydroxy group increase in the resonance forms of
phenol, electrophilic substitution reactions have an
ortho/para directing effect. A methoxy group also has a
similar, and weaker, effect. On the other hand, because
positive charges on ortho/para positions to the carboxyl
group increase in the resonance forms of benzoic acid,
electrophilic substitution reaction have a meta directing
effect. Electrophilic sensitivities of aromatic carbons on
hydroxybenzoic acids were estimated to be integrated in both
directing effects. We calculated electron densities of the
aromatic carbons as an indicator of the electrophilic
sensitivities using CAChe work system [19]. It was
estimated that a carbon atom with a greater negative charge
was more easily attacked by deuterium cations.
80
pD 0.32
60
pD 0.56
pD 1.0
40
20
0
pD 2.0
pD 3.5
0.5
1
2
3
6
12
24
Time (h, log-scale)
Fig. (1). Time Course of Deuterated Ratio at 2,6 Positions of 10 in
D2O at Various pD Values. ꢀ: pD 0.32, ꢁ: pD 0.56, ꢀ: pD 1.0, ꢁ:
pD 2.0, ꢂ: pD 3.5.
Subsequently, compounds 1, 2, 4-9 were deuterated by
the reflux condition at pD 0.32. Preparation of highly
deuterated hydroxybenzoic acids in this study is summarized
in Table 1. Additionally, 3,5-dihydroxybenzoic-2,4,6-d3 acid
(12) and 2,6-dihydroxybenzoic-3,5-d2 acid (13) were
prepared quantitatively in the same ideal condition as
preparation of compound 11. Deuterated ratio at 2,6 and 4
positions of 12, and 3,5 positions of 13 were 99.0%, 94.3%,
Table 3 shows deuterium exchange ratios in the same
reaction conditions (pD 0.32, reflux, 1 hour); substituent
effects with hydroxy, methoxy and carboxyl groups; and
electron densities of the aromatic carbons on 1, 2, 4-10. Only
aromatic carbons with double substituent effects of the
hydroxy groups were highly deuterated. Deuterium exchange
ratios of 2,6 positions on 10, 2,6 and 4 positions on 7, and

334 Letters in Organic Chemistry, 2011, Vol. 8, No. 5
Nakamura et al.
Table 1. Preparation of Highly Deuterated Hydroxybenzoic Acids with DCl/D2O
Deuterated hydroxybenzoic acid
D ratio on aromatic carbon (%)
Reaction
time (h)
Yield (%)
2
3
4
5
6
3,4,5-Trihydroxybenzoic-2,6-d2 acid (11)
3,5-Dihydroxybenzoic-2,4,6-d3 acid (12)
2,6-Dihydroxybenzoic-3,5-d2 acid (13)
1
1
1
6
99.3
99.0
-
-
-
94.3
7.4
-
-
99.3
99.0
-
99
99
99
99
-
95.5
-
-
95.5
-
3,5-Dimethoxy-4-hydroxybenzoic-2,6-d2 acid (14)
92.6
92.6
Table 2. Stabilities of D on Compounds 11 and 12 in a Physiological Condition
Days
Compound
C* No.
0
1
3
5
7
3,4,5-Trihydroxybenzoic-2,6-d2 acid (11)
3,5-Dihydroxybenzoic-2,4,6-d3 acid (12)
2,6
4
99.3 (100)**
94.3 (100)
99.0 (100)
96.6 (97.5)
88.8 (94.2)
99.0 (100)
95.8 (96.5)
84.1 (89.2)
99.0 (100)
94.7 (95.4)
82.1 (87.1)
99.0 (100)
94.1 (94.8)
79.4 (84.2)
98.9 (99.9)
2,6
*Position of carbon atom on the deuterated hydroxybenzoic acid. **% of deuterated ratio (relative ratio) on the corresponding carbon(s).
Table 3. Deuterium Exchange Ratios (pD 0.32, reflux, 1 hour), Substituent Effects* and Calculated Electron Densities of 1, 2, 4-10
Carbon No.
2
3
4
5
6
D ratio (%)
substituent effects
electron density
D ratio (%)
0
2.7
-
2.7
0
– / ꢀ
-0.036
18.2
ꢁ/ꢀ
4-Hydroxybenzoic acid (1)
– / ꢀ
ꢁ / –
-
ꢁ / –
-0.035
-0.230
-
-0.197
-
-
19.1
20.8
2,3-Dihydroxybenzoic acid (2)
2,5-Dihydroxybenzoic acid (4)
2,6-Dihydroxybenzoic acid (5)
3,4-Dihydroxybenzoic acid (6)
3,5-Dihydroxybenzoic acid (7)
3-Methoxy-4-hydroxybenzoic acid (8)
3,5-Dimethoxy-4-hydroxybezoic acid (9)
3,4,5-Trihydroxybenzoic acid (10)
substituent effects
electron density
D ratio (%)
-
-
-
ꢁ/ꢀ
ꢁ/–
-
-0.103
-0.105
-0.075
2.9
-
2.5
0
-
substituent effects
electron density
D ratio (%)
-
ꢁ/–
ꢁ/ꢀ
-
ꢁ/ꢀ
-
-0.160
-0.089
-
-0.122
-
-
95.5
7.4
95.5
substituent effects
electron density
D ratio (%)
-
ꢁꢁ/–
–/ꢀ
ꢁꢁ/–
-
-
-0.266
-0.025
-0.232
-
0
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
0
0
substituent effects
electron density
D ratio (%)
ꢁ/ꢀ
-0.108
99.0
ꢁꢁ/ꢀ
-0.194
0
-
ꢁ/–
ꢁ/ꢀ
-
-0.167
-0.069
99.0
ꢁꢁ/ꢀ
-0.151
0
94.3
-
substituent effects
electron density
D ratio (%)
ꢁꢁ/ꢀ
-
-0.216
-
-
-
-
-
-
-
-
-
-
0
substituent effects
electron density
D ratio (%)
ꢂ/ꢀ
-0.131
42.4
ꢂꢂ/ꢀ
-0.107
99.3
ꢁꢁ/ꢀ
-0.144
ꢁ/–
ꢂ/ꢀ
-0.166
-0.070
42.4
ꢂꢂ/ꢀ
-0.107
99.3
ꢁꢁ/ꢀ
-0.144
-
-
-
-
-
-
substituent effects
electron density
D ratio (%)
substituent effects
electron density
*ꢁ, ꢂ, and ꢀ means substituent effects of hydroxy, methoxy and carboxyl groups, respectively.

A Selective Preparation of Highly Deuterated Hydroxybenzoic Acids
Letters in Organic Chemistry, 2011, Vol. 8, No. 5
335
dimethylarginine dimethylaminohydrolase (DDAH) activity in
mouse tissue. J. Chromatogr. B, 2007, 851(1-2), 220-8.
Graham, DY; Klein, PD; Evans, DJ Jr; Evans, DG; Alpert, LC;
Opekun, AR; Boutton, TW. Campylobacter pylori detected
noninvasively by the 13C-urea breath test. Lancet, 1987, 1(8543),
1174-7.
Tashiro, M; Iwasaki, A; Fukata, G. Studies on selective preparation
of aromatic compounds. 14. An attempt to prepare all possible
deuterated phenols by the reductive dehalogenation of the
corresponding halophenols with Raney alloys in an alkaline
deuterium oxide solution. J. Org. Chem., 1978, 43, 196-199.
Wähälä, K; Hase, T; Adlercreutz, H. Synthesis and labeling of
isoflavone phytoestrogens, including daidzein and genistein. Proc.
Soc. Exp. Biol. Med., 1995, 208(1), 27-32.
Sajiki, H.; Esaki, H.; Aoki, F.; Maegawa, T.; Hirota. Aromatic ring
favorable and efficient H-D exchange reaction catalyzed by Pt/C.
Tetrahedron Lett., 2005, 46, 6995-6998.
Kubo, M.; Takizawa, T.; Wakai, C.; Matubayasi, N.; Nakahara, M.
Noncatalytic kinetic study on site-selective H/D exchange reaction
of phenol in sub- and supercritical water. J. Chem. Phys., 2004,
121(2), 960-969.
(a) Wähälä K; Rasku S; Parikka K. Deuterated phytoestrogen
flavonoids and isoflavonoids for quantitation. J. Chromatogr. B,
2002, 777(1-2), 111-22. (b) Yamauchi K; Yasuda S; Fukushima K.
Evidence for the biosynthetic pathway from sinapic acid to syringyl
lignin using labeled sinapic acid with stable isotope at both
methoxy groups in Robinia pseudoacacia and Nerium indicum. J.
Agric. Food Chem., 2002, 50(11), 3222-7.
3,5 positions on 5 were 99.3%, 99.0%, 94.3%, and 95.5%,
respectively. Secondly, deuterium exchange ratios of 2,6
positions on 9, with double substituent effects of the
methoxy groups, was 42.4%. All carbons with single
substituent effect of a hydroxy or a methoxy group were just
barely deuterated, except for compound 2. Carbons on 2
were deuterated in relatively large portions compared to
others with single substituent effects. Compound 2 was
unstable in the reaction condition and another excitation
effect, not the substituent effects, could accrete the reaction.
Suppressive substituent effects with a carboxyl group did not
affect all of the deuteration reactions. Results of the semi-
empirical molecular orbital method showed that all aromatic
carbons had negative charges. However, the intensity of the
negative charge did not correlate with the deuterated ratio.
The reason for this may be that the extremely low pD was
not considered in the calculation results. At pD 0.32, every
hydroxy and carboxyl group was almost completely
undissociated. Substituent effects by the functional groups
were supposed to be reduced under the strong acidic
conditions. According to this supposition, under strong
acidic conditions, single substituent effects by hydroxy,
methoxy or carboxyl groups were hardly able to work and
double substituent effects by hydroxy or methoxy groups
were effective in the deuterated reaction of hydroxybenzoic
acids. In acidic conditions, suppression of the substituent
effects decreased the reactivity of aromatic carbons and was
expected to increase the stability of some hydroxybenzoic
acids.
[5]
[6]
[7]
[8]
[9]
[10]
[11]
Each hydroxybenzoic acid (100 mg) was dissolved or suspended in
D2O (20 ml, D 99.9%, Cambridge Isotope laboratories, Inc.,
Andover, USA) and the mixture was refluxed on an oil bath at 120
ꢄC under nitrogen for 48 hours. After removal of D2O by
lyophilization, partially deuterated hydroxybenzoic acids except for
3 (ꢀ-resorcylic acid) were quantitatively obtained. The products
were analyzed by NMR and MS to confirm deuterated products.
NMR spectroscopic data were recorded on a Bruker DRX500
spectrometer (Bruker BioSpin Corp., Billerica, MA, USA) at 500
1
MHz for H and 125 MHz for 13C NMR at 25 ꢄC. Chemical shifts
As described above, highly deuterated hydroxybenzoic
acids, 11, 12, 13, and 14 were selectively and quantitatively
prepared only by refluxing hydroxybenzoic acids in acidic
D2O. These deuterated hydroxybenzoic acids were stable
under physiological conditions and were detected by MS
analysis in vivo. Our results showed that these deuterated
compounds can be utilized as reagents for kinetic analysis.
The deuterium exchange reactions on the hydroxybenzoic
acids were induced by double substituent effects of hydroxy
or methoxy groups.
were referenced to the signal of trimethylsilane as an internal
standard. LC-MS analysis was performed with a Waters 2695 (LC)
and Quattro micro API (MS) system (Waters, Milford, MA, USA).
The HPLC separations were performed using a Cadenza HS C-18
reversed phase column (150 mm ꢁ 4.6 mm i.d., Imtakt, Kyoto,
Japan). Elution was performed with 0.1 % (v/v) folic acid in
purified water (Solvent A) and acetonitrile with 0.1 (v/v) folic acid
(Solvent B) as mobile phase: 0-20 min, isocratic Solvent A; 20-30
min, gradient 0-70% Solvent B; 30-40 min, isocratic 70% Solvent
B. Chromatography was performed at 35 ꢄC with a flow rate of 0.8
mL/min (LC) and 0.3 mL/min (MS), injection volume of 10 ꢅL,
and detection at 280 nm. Mass spectra were acquired in
electrospray ionization (ESI) mode using 3500 V capillary voltage,
20 V cone voltage, desolvation gas (N2) flow of 350 L/h, cone gas
(N2) flow of 50 L/h, source temperature of 100 °C, and desolvation
temperature of 350 °C. The mass spectrometer was operated in
positive mode as 80 eV, target m/z = 200 and scanning range m/z
50-1000.
ACKNOWLEDGEMENT
This work was supported by KAKENHI (18688006) of
Japan Society for the Promotion of Science (JSPS).
[12]
[13]
Li J; Sato M; Ohshima T. Degradation of phenol in water using a
gas-liquid phase pulsed discharge plasma reactor. Thin Solid Films,
2007, 515(9), 4283-4288.
Yoshino A; nakashima Y; yasuki A; takahashi K. Kinetic Studies
by Means of the NMR Techniques. I. Acid-Catalyzed Proton-
Exchange Reactions of Some Substituted Phenols. Bull. Chem. Soc.
Jpn., 1988, 61, 3393-3397.
Deuteration ratios in this research were determined by a modified
method of Yoshino et al. [13]. A well-dried deuterated product
(10.0 mg) was dissolved in D2O or CD3OD (1.00 ml each, D 100%,
REFERENCES AND NOTES
[1]
Hsu CL; Yen GC. Effect of gallic acid on high fat diet-induced
dyslipidaemia, hepatosteatosis and oxidative stress in rats. Br. J.
Nutr., 2007. 98(4), 727-35.
[2]
Ng, TB; He, JS; Niu, SM; Zhao, L; Pi, ZF; Shao, W; Liu, F. A
gallic acid derivative and polysaccharides with antioxidative
activity from rose (Rosa rugosa) flowers. J. Pharm. Pharmacol.,
2004, 56(4), 537-45.
[14]
Cambridge
Isotope
Laboratories,
Inc.)
and
3-
[3]
[4]
Faried, A; Kurnia, D; Faried, LS; Usman, N; Miyazaki, T; Kato, H;
Kuwano, H. Anticancer effects of gallic acid isolated from
Indonesian herbal medicine, Phaleria macrocarpa (Scheff.) Boerl,
on human cancer cell lines. Int. J. Oncol., 2007, 30(3), 605-613.
(a) Dainty, JR; Bullock, NR; Hart, DJ; Hewson, AT; Turner, R;
Finglas, PM; Powers, HJ. Quantification of the bioavailability of
riboflavin from foods by use of stable-isotope labels and kinetic
modeling. Am. J. Clin. Nutr., 2007, 85(6), 1557-64. (b) Maas, R;
Tan-Andreesen, J; Schwedhelm, E; Schulze, F; Böger, RH. A
stable-isotope based technique for the determination of
(trimethylsilyl)propionic-2,2,3,3-d4 Acid sodium salt (TSP, 50.0
ꢅl of 10.0 mg/ml in D2O) was added as an internal standard. And
800 ꢅl of the mixture was put into an NMR tube and 1H NMR
spectra was obtained as described previously. The peak area of 1H-
NMR spectrum (ꢂ1H) of deuterated product and standard
compound were obtained based on that of TSP. A deuteration
ration was determined by the following formula:
Deuteration ratio (%) = (ꢂ1H of product/ꢂ1H of standard) x 100
In this calculation, increases of molecular weight of deuterated
products were not considered. So, the maximum error in the

336 Letters in Organic Chemistry, 2011, Vol. 8, No. 5
Nakamura et al.
deuteration ratio was estimated to be 0.72%, in the case of 50%
deuteration of 1, which was composed of 1 (exact mass: 138.03)
and 4-hydroxybenzoic-2,3,5,6-d4 acid (exact mass: 142.06) in the
ratio 1:1. Deuterated ratios of the products from 1-9 were as
follows. compound, position of carbon atom (D%): 1, 2,6 (7.2%),
3,5 (9.7%); 2, 4 (1.8%), 5 (41.2%), 6 (4.6%); 3, N.D.; 4, 3 (11.9%),
4 (15.8%), 6 (10.3%); 5, 3,5 (66.6%), 4 (21.4%); 6, 2 (1.3%), 5
(18.4%), 6 (0.84%); 7, 2,6 (83.8%), 4 (66.9%); 8, 2 (1.3%), 5
(24.7%), 6 (1.3%); 9, 2,6 (2.6%).
ꢇC. Changes in the deuterated ratio were determined by the method
using NMR as described previously. The relative standard
deviation for the nine determinations was 0.15% and the accuracy
of the determination of 11 and 12 were excellent.
Aqueous solution of 11 at pH 7 was orally administrated to rats (n
=3) at the dose of 10.0 mg/kg. After 30 minutes, LC/MS analysis
described previously detected the peak originating from the
administered compound at m/z [M+H]+ 173.1 in every plasma
sample.
CAChe ver. 6.01-MOPAC2002 (Fujitsu Ltd.) was used for the
calculation. At the beginning, energy minimization of each
molecule was performed by using molecular mechanics method
(CONFLEX/MM2). Then the global minimum conformation in
aqueous medium was obtained by the semi-empirical method
(CONFLEX/PM5), in which the solvent effect was modeled with
Conductor-like Screening Model (COSMO). Electron densities of
the aromatic carbons were automatically displayed on the global
minimum conformation.
[18]
[19]
[15]
[16]
A
power of deuterium cation (pD) in this research was
a
measurement value determined with a commercially available pH
meter (IM-22P, DKK-TOA Corporation, Tokyo, Japan) and a pH
meter reading was not corrected.
Original solution of 10 at pD 3.5 was prepared by dissolving 10
(100 mg) into D2O (20 ml). By adding 40 ꢆl of NaOD (40% w/w in
D2O) and 20 ꢆl, 40 ꢆl, 400 ꢆl, and 1000 ꢆl of DCl (35% w/w in
D2O) to the original solution, a series of solutions at pD 7.0, 2.0,
1.0, 0.56, 0.32 were prepared.
[17]
11 and 12 were dissolved in saline (10 ml each) adjusting pH to 7.0
using NaOD and the solutions were then incubated for 7 days at 37