An efficient laboratory-scale preparative method for [1- 13C]glycocholic acid

Article abstract of DOI:10.1002/jlcr.3072

A breath test using [1-¹³C]glycocholic acid as a substrate is a potential diagnostic method for small intestinal bacterial overgrowth syndrome. [1-¹³C]Glycocholic acid has been thus synthesized in an excellent yield from ethyl [1-¹³C]glycinate hydrochloride in a one-pot reaction. This method is suitable for the preparation of the labeled compound on a laboratory scale, which helps to perform extensive clinical studies of the breath test. Copyright

Full text of DOI:10.1002/jlcr.3072

Protocols and Methods
Received 01 May 2013,
Accepted 16 May 2013
Published online 28 June 2013 in Wiley Online Library
(wileyonlinelibrary.com) DOI: 10.1002/jlcr.3072
An efficient laboratory-scale preparative
13
method for [1- C]glycocholic acid
a
a
b
*
Hidemichi Mitome, Naoko Tokumasu, Yusuke Tando,
Teruo Nakamura,c and Kazuki Akiraa
A breath test using [1-13C]glycocholic acid as a substrate is a potential diagnostic method for small intestinal bacterial
overgrowth syndrome. [1-13C]Glycocholic acid has been thus synthesized in an excellent yield from ethyl [1-13C]glycinate
hydrochloride in a one-pot reaction. This method is suitable for the preparation of the labeled compound on a laboratory
scale, which helps to perform extensive clinical studies of the breath test.
Keywords: stable labeled synthesis; carbon-13; glycocholic acid; laboratory scale; one-pot reaction; breath test
to prepare large amounts of the labeled compound for clinical
Introduction
studies. In this paper, we present a more efficient method suitable
for laboratory-scale preparation of [1-13C]glycocholic acid.
Small intestinal bacterial overgrowth (SIBO) occurs as a complication
of a postgastrectomic state, chronic pancreatitis, liver cirrhosis,
Crohn’s disease, gastric achlorhydria, blind loop syndrome, small
Results and discussion
intestinal diverticulosis, and so on.1 This syndrome causes
At first, we tried to prepare [1-13C]glycocholic acid by using
abdominal pain, chronic diarrhea, nausea, anemia, abdominal
bloating, and constipation. Although the gold standard for the
diagnosis of SIBO is to culture aspirates from the small bowel, it is
Tserng’s procedure. Ethyl [1-13C]glycinate hydrochloride was
prepared quantitatively from [1-13C]glycine by treatment with a
solution of two equivalent moles of hydrogen chloride in
ethanol–ethyl acetate followed by concentrating under reduced
pressure. The hydrogen chloride solution was prepared by the
addition of acetyl chloride into ethanol at room temperature. In
our experiments using Tserng’s method, a gummy material
appeared during the reaction work-up, and it interfered with
the crystallization of the product. This result made it difficult to
obtain the product in a constant yield, unfortunately. This
problem seemed to be due to the low solubility of the reagents
and/or reaction products in ethyl acetate. 4-(4,6-Dimethoxy-
1,3,5-triazin-2-yl)-4-methyl-morpholinium chloride (DMT-MM) is
an effective reagent for the condensation of carboxylic acids
with amines to give the corresponding amide, and it can work
in alcoholic solvents.7 When ethyl [1-13C]glycinate hydrochloride
(1.0 eq.), cholic acid (1.0 eq.), and DMT-MM (1.1 eq.) were simply
mixed in ethanol in the presence of potassium carbonate
invasive, troublesome, and time-consuming. Fromm et al. and Sherr
et al. have independently reported on a noninvasive diagnostic
procedure for SIBO by using a [1-14C]glycocholic acid breath test.2
This method is based on the fact that the ingested [1-14C]
glycocholic acid can be deconjugated by the action of the intestinal
bacteria to produce [1-14C]glycine, which is rapidly absorbed and
converted into 14CO2 and then excreted in exhaled breath. The
serious drawback of this method is that the long half-life of
radioactive 14C makes it undesirable for use in infants, children,
and pregnant women.
To solve this problem associated with the use of 14C, a breath test
using [1-13C] or [1,2-13C2]glycocholic acid was reported by Solomons
et al.,3 where the labeled compounds (250–600 mg) were ingested
and carbon dioxide in exhaled breath was analyzed by mass
spectrometer. Recently, breath tests using 13C-labeled substrates,
which are noninvasive and convenient, and applicable to a wide
range of clinical diagnostic procedures were widely employed.4
These tests are based on 13CO2/12CO2 ratio analysis in exhaled
breath, using a mass spectrometer or infrared spectrometer.
Compact, easily accessible, and inexpensive devices for 13CO2/12CO2
ratio analysis by infrared spectrometry are now available, which can
contribute to the utilization promotion of the breath tests.
aCollege of Pharmaceutical Sciences, Matsuyama University, 4-2 Bunkyo-cho,
Matsuyama, Ehime 790-8578, Japan
bThe Third Department of Internal Medicine, Hirosaki University School of
Medicine, 66-1 Hon-cho, Hirosaki, Aomori 036-8564, Japan
In the previous report by Solomons et al., the labeled compounds
were prepared according to the method reported by Lack et al.,5
where 13C-labeled ethyl glycinate and cholic acid were condensed cHirosaki University Graduate School of Health Sciences, 66-1 Hon-cho, Hirosaki,
Aomori 036-8564, Japan
using N-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline (EEDQ) as a
coupling reagent. Although the EEDQ method was subsequently
*Correspondence to: Hidemichi Mitome, College of Pharmaceutical Sciences,
Matsuyama University, 4-2 Bunkyo-cho, Matsuyama, Ehime 790-8578, Japan.
improved by Tserng et al.,6 it is still time-consuming and requires a
complicated reaction work-up. Thus, the EEDQ method is insufficient E-mail: mitome@cc.matsuyama-u.ac.jp
J. Label Compd. Radiopharm 2013, 56 587–588
Copyright © 2013 John Wiley & Sons, Ltd.

H. Mitome et al.
Scheme 1. Preparation of [1-13C]glycocholic acid.
drying under reduced pressure to obtain crude crystals. The crude crystals
were recrystallized from ethanol–ethyl acetate to afford [1-13C]glycocholic
acid (13.4 g, 95.6% yield): colorless needles (mp 166–7ꢀC); IR (KBr) υmax
3407, 2935, 1655 cmÀ1; 1H-NMR (500 MHz, DMSO-d6) d 12.43 (s, 1H, COOH),
8.07 (t, J=5.8, 1H, NH), 4.30 (d, J= 3.6, 1H, OH), 4.08 (d, J= 3.3, 1H, OH), 3.98
(d, J= 3.3, 1H, OH), 3.78 (br s, 1H), 3.71 (t, J= 5.8, 2H), 3.61 (br s, 1H), 3.19
(m, 1H), 2.25–2.10 (m, 3H), 2.00 (m, 2H), 1.83–1.60 (m, 6H), 1.50–1.13 (m,
11H), 0.97 (m, 1H), 0.93 (d, J= 6.5, 3H), 0.84 (m, 1H), 0.81 (s, 3H), 0.59 (s, 3H);
13C-NMR (DMSO-d6) d 172.9, 171.4 (strong), 70.9, 70.3, 66.2, 46.1, 45.7, 41.4,
41.3, 40.7, 40.2, 35.2, 35.0, 34.8, 34.3, 32.1, 31.5, 30.3, 28.5, 27.2, 26.1, 22.7,
22.5, 17.0, 12.3; ESIMS (negative mode) m/z 465.3032 (calcd for C1253CH42NO6- ,
465.3051).
(1.1 eq.), the reaction smoothly proceeded at room temperature.
The resulting reaction mixture was directly subjected to hydrolysis
by potassium carbonate to constantly give [1-13C]glycocholic acid
in an excellent yield (Scheme 1).
The present simple and convenient method using a one-pot
reaction enabled the laboratory-scale preparation of pure [1-13C]
glycocholic acid with a minimal loss of relatively high cost of
[1-13C]glycine, which contributes to clinical studies of the breath test
for the diagnosing of SIBO.
Experimental
General
Acknowledgement
[1-13C]glycine (enrichment specification 99%) was purchased from Cambridge
Isotope Laboratories, Inc. (Tewksbury, MA, USA) and used as received.
The DMT-MM was prepared from 2-chloro-4,6-dimethoxy-1,3,5-triazine and
4-methylmorpholine according to the method of Kunishima et al.8 Cholic acid,
2-chloro-4,6-dimethoxy-1,3,5-triazine and 4-methylmorpholine were
purchased from Tokyo Chemical Industry Co., Ltd.(Tokyo, Japan) and
was used as received. Other reagents and solvents were obtained from
Wako Pure Chemical Industries, Ltd.(Osaka, Japan).
We are grateful to Professor M. Kawase, Matsuyama University,
for his invaluable suggestions.
Conflict of Interest
The authors did not report any conflict of interest.
Melting point was measured with a Yanako MP-J3 melting point apparatus
and was uncorrected. Infrared (IR) spectrum was recorded with a JASCO FT-IR-
4100 type A spectrometer. 1H and 13C NMR spectra were taken with a Bruker
BioSpin AVANCE 500 spectrometer. Chemical shifts were given on d (ppm)
scale with tetramethylsilane as an internal standard (s, singlet; d, doublet; t,
triplet; m, multiplet; br, broad). High-resolution electrospray ionization mass
spectrum (ESIMS) was obtained with a Bruker Daltonics micro TOF-Q
spectrometer.
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[1-13C]Glycocholic acid
To a solution of ethyl [1-13C]glycinate hydrochloride (4.22 g, 30.0 mmol) in
ethanol (150 mL) were added cholic acid (12.3 g, 30.0 mmol), potassium
carbonate (4.56 g, 33.0 mmol), and DMT-MM (9.13 g, 33.0 mmol), followed
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J. Label Compd. Radiopharm 2013, 56 587–588