Synthesis of 13C- and 14C-labelled catechol

Article abstract of DOI:10.1002/jlcr.581

¹³C- and ¹⁴C-uniformly labelled catechol was synthesized from phenol in three steps. Phenol was derivatized with 2-chloro-5-nitrobenzophenone in THF containing NaH, followed by ortho-hydroxylation with 35% aqueous H₂O

Full text of DOI:10.1002/jlcr.581

JOURNAL OF LABELLED COMPOUNDS AND RADIOPHARMACEUTICALS
J Label Compd Radiopharm 2002; 45: 551–558.
Published online in Wiley InterScience (www.interscience.wiley.com). DOI: 10.1002/jlcr.581
Research Article
13 14
Synthesis of C- and C-labelled catechol
.
Rong Ji* and Andreas Schaffer
Biologie V-Umweltanalytik, RWTH Aachen, 52056 Aachen, Germany
Summary
13C- and 14C-uniformly labelled catechol was synthesized from phenol in three
steps. Phenol was derivatized with 2-chloro-5-nitrobenzophenone in THF
containing NaH, followed by ortho-hydroxylation with 35% aqueous H2O2 in
sulphuric acid/glacial acetic acid solution, and by cleavage with piperidine,
giving an overall 80% yield with respect to phenol. Copyright # 2002 John
Wiley & Sons, Ltd.
Key Words: preparation; U-13C-catechol; U-14C-catechol; isotope labelling
Introduction
Catechol and catechol derivatives are ubiquitous in the environment, as
metabolic intermediates in the microbial degradation of many naturally
occurring and anthropogenic aromatic substances.1–3 Though being a
constituent unit of many plant polyphenols, however, catechol is
generally not found in free form in nature.4 As one of the precursors of
soil humic substances, catechol is preferentially used for the synthesis of
model humic substances, especially when radioactive labelling is
involved.5 Experiments with 14C-labelled catechol have shown that
catechol is to a great extent immobilized in soil and incorporated into
soil humic substances.3,4 However, it is still not known how catechol is
stabilized and in what chemical form. While 14C-labelled substances
*Correspondence to: R. Ji, Biologie V-Umweltanalytik, RWTH Aachen 52056 Aachen, Germany.
E-mail: rong.ji@bio5.rwth-aachen.de
Contract/grant sponsor: Deutsche Forschungsgemeinschaft; contract/grant number: SPP 1090
Copyright # 2002 John Wiley & Sons, Ltd.
Received 18 December 2001
Revised 12 February 2002
Accepted 25 February 2002

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R. JI AND A. SCHAFFER
552
serve well for studying the rate and extent of transformation and
stabilization, stable isotope-labelled substances are a prerequisite to
elucidate the stabilization mechanisms.6 Commercial 13C-labelled
chemicals are highly expensive and the unavailability of 13C-catechol
is a big obstacle in elucidating the stabilization mechanisms. To
facilitate such studies, a convenient and effective preparation of
isotopically labelled catechol is very important.
Catechol is generally synthesized from phenol by hydroxylation with
H2O2, mostly in the presence of a catalyst, e.g. titanosilicates,7,8 by
oxidation of phenol with O2,9 or by light irradiation.10 These one step
syntheses not only convert phenol incompletely but also produce a
mixture of dihydroxy compounds with very low ortho-stereoselectivity.
Chew and Heys11 used a three-step pathway to prepare 14C- and
13C-labelled catechol from phenol, which gave 14C- and 13C-catechol in
3.1 and 7% overall yield, respectively. Haider12 used a five-step
preparation for 14C-catechol, with a higher yield (44%). Loudon and
Scott13 described a pathway for ortho-hydroxylation of phenols with
almost quantitative stereoselectivity and Kratzl and Vierhapper14
modified the first derivatizing step for the preparation of 14C-guaiacol
in 70% yield.
In this study, we modified and improved the method of Loudon and
Scott,13 and prepared 14C- and 13C-labelled catechol from phenol in
good yield.
Results and discussion
The preparation of catechol consisted of three steps (Figure 1): Phenol
was firstly derivatized to 2-phenoxy-5-nitro-benzophenone (2) with
2-chloro-5-nitrobenzophenone in tetrahydrofuran (THF) under reflux,
followed by quantitative hydroxylation in the ortho-position to 2-(2-
hydroxyphenoxy)-5-nitrobenzophenone (3) with H2O2 after dissolution
Figure 1. Scheme for the synthesis of catechol
Copyright # 2002 John Wiley & Sons, Ltd.
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SYNTHESIS OF 13C– AND 14C
553
in H2SO4. Catechol (4) was finally obtained by cleavage of the
derivatizing group with piperidine under reflux in ca. 80% yield with
respect to phenol (1). Table 1 summarizes the yields for the various
synthetic steps, together with the yields and the recoveries of
radioactivity in the case of U-14C-catechol synthesis, showing that the
reactions in steps 1 and 2 could be carried out almost quantitatively.
Because the intermediates and the product have strong fluorescent
properties, all synthetic reactions are easily monitored by thin layer
chromatography (TLC).
The derivatization of phenol to 2 can be carried out in THF in 8.5 h
or overnight, or instead in N,N-dimethylformamide (DMF) under reflux
(1108C) for 30 min. Oily brown substances were formed in the reaction
mixture in the case of the DMF-reflux method, probably reaction
products of some mineral oil, which is commercially used for the
dispersion of NaH. These contaminants had to be removed before
sublimation of catechol (4) in step 3, e.g. by using column
chromatography on silica gel at the expense of the yield (ca. 10%).
When using THF as solvent for phenol derivatization, a purification
procedure for the intermediate products (2) and (3) was not necessary.
It is important for the yield of step 2 that 2 is carefully dried and
completely dissolved in H2SO4 before oxidation with H2O2. After
oxidation, the product (3) becomes sticky, if it is not immediately
separated from the reaction mixture by precipitation with H2O.
In the case of step 3, the amount of piperidine should not be too high,
usually about 0.8–1.5 ml for 100 mg of 3, because at the high reaction
temperature piperidine forms dark brown substances, which contami-
nated further the crude catechol (4). Pure catechol could be obtained
either by column chromatography or by sublimation in vacuo. Using
sublimation, we obtained white crystals of catechol in 99.7% purity as
analysed by high-performance liquid chromatography (HPLC) and gas
chromatography mass spectrometry (GC-MS). Purification by TLC
should be conducted under an O2-free atmosphere.
Table 1. Recovery and yields for the synthetic steps
Synthetic step
14C recovery
Yield (%)
(%)
14C
Mass
1–2
2–3
3–4
Total
97.6
96.7
100
94.3
96.6
88.2
80.3
102
96.1
82.3
80.7
94.4
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554
Because there are no data of nuclear magnetic resonance (NMR)
spectroscopy and MS for 2 and 3 found in literature, non-labelled 2 and
3 (purified by recrystallization in acetic acid and methanol, respectively)
were analysed by NMR and GC-MS to facilitate the control of the
syntheses (data see section Experiment). The results of MS analysis of
13C-labelled catechol are similar to natural catechol in the mass
distribution pattern (data not shown). 13C-NMR of 13C-labelled
catechol showed that the signals for 2- and 4-C of the catechol ring
are highly irregular multi-peaks in a broad range of 116.9–114.1 ppm,
while the signals for the other C atoms are relatively orderly multi-peaks
(data not shown). HPLC and TLC results showed that the synthetic
14C-catechol was 99.7% radiochemically pure.
Experimental
TLC was performed on silica gel 60 with fluorescence indicator (Sil G-
.
25 UV254, 0.25 mm; Macherey-Nagel, Duren, Germany) and viewed
under UV light (254 nm). Preparative column chromatography was
conducted with silica gel 60 (particle diameter 0.064–0.200 mm, Merck).
For autoradiography of the TLC plate, a bioimaging analyzer (Fujifilm
BAS-1000; Tokyo, Japan) was used. Quantitative determination of
radioactivity was performed with a liquid scintillation counter LS-5000
TD (Beckman Instrument; CA, USA) using the cocktail Lumasafe Plus
(Lumac LSC; Groningen, The Netherlands).
NMR was performed on an AC-300 (Bruker; Rheinstetten, Ger-
many) equipped with a 10 mm type broad-band probe head or on a
DPX-300 (Brucker) equipped with a 5 mm QNP probe head using
CDCl3 or DMSO-d6 as solvents. For 13C-NMR, proton broad band
decoupling was used, and line broadening was 1.0 and 3.0 Hz for
unlabelled substances and 13C-catechol, respectively.
The GC-MS analysis was conducted on a Hewlett-Packard 5890
Series II gas chromatograph (Agilent Technologies; Waldbronn,
Germany)
equipped
with
an
FS-SE-54-NB-0.5
column
(25 m  0.25 mm, 0.46 mm film thickness; CS Chromatographie Service;
Langerwehe, Germany) and connected to a Hewlett-Packard 5971 A
mass selective detector (MSD), which was operated in the scan mode
(mass range m/z 50–600) with an electron energy of 70 eV. The
temperature program of the GC was 508C for 5 min, 108C/min to
2808C, then 2808C for 5 min. The injection volume was 1 ml. Carrier gas
Copyright # 2002 John Wiley & Sons, Ltd.
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SYNTHESIS OF 13C– AND 14C
555
was helium (1 ml/min). For 3 and 4, samples (5 mg) were derivatized with
100 ml N,O-bis-(trimethylsilyl)-trifluoracetamide (BSTFA, 98% purity,
Merck, Darmstadt, Germany) at 608C for 30 min in closed crimp cap
vials.
HPLC was conducted on a RP-18 column (Nucleosil 100-5 C18; CS
Chromatographie Service) using a Hewlett-Packard Series 1100
chromatograph system equipped with a diode array UV-detector and
an on-line radioactivity detector (Ramona 2000; Raytest, Strauben-
hardt, Germany) with a 2420 quartz cell (0.37 ml; Raytest). For elution
(1 ml/min), the following gradient was applied: solution A (1% acetic
acid in water) and B (1% acetic acid in methanol): A:B (60:40/v:v) for
5 min, linear gradient to 100% B for 20 min, isocratic B (100%) for
5 min, return to initial conditions within 5 min.
The U-14C-phenol (1), with a specific activity of 1.62 Â 1011 Bq/mmol,
was supplied in petrol ether by Hartmann Analytic (Braunschweig,
Germany). The U-13C-phenol with 99% atom 13C was supplied as a
.
solid by Euriso-Top (Saarbrucken, Germany). 2-Chloro-5-nitrobenzo-
phenone (99% purity) was purchased from Acros (Geel, Belgium). The
remaining chemicals were purchased from commercial sources.
14C-labelled 2-phenoxy-5-nitro-benzophenone (2)
U-14C-phenol (4.35 Â 109 Bq, 2.53 mg) diluted with unlabelled phenol
(91.5 mg) (1) in dry petrol ether (boiling point 60–958C, 5 ml) was added
at room temperature to a suspension of NaH (60%, 50 mg) in dry petrol
ether (3 ml) in a 50-ml round bottom flask, which was connected to a
reflux condenser. Then 2-chloro-5-nitrobenzophenone (277 mg, in 1 ml
dry THF) was added dropwise to the flask with stirring. The mixture
turned dark brown and H2 was released. The reaction mixture was
heated under reflux at 708C and samples monitored by GC-MS and
TLC on silica gel with pentane:ethyl acetate (5:1/v:v) as eluent (Rf of 1,
2-chloro-5-nitrobenzophenone, and 2: 0.39, 0.49, and 0.41, respectively).
During the reaction, oily ‘precipitates’ with brown-red colour were
formed in the bottom of the flask. After 8.5 h reaction, GC-MS and
TLC showed that all the phenol and 2-chloro-5-nitrobenzophenone has
reacted. Autoradiography indicated that the product (2) contained
94.5% of the total radioactivity. The reaction was terminated by adding
1 M NaOH (6 ml) to the flask, resulting in a yellow organic phase and a
red-brown aqueous phase. The aqueous phase was extracted with
CH2Cl2 four times (first 10 ml, then 6–7 ml). The extract and the organic
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J Label Compd Radiopharm 2002; 45: 551–558

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R. JI AND A. SCHAFFER
556
phase were combined and dried over Na2SO4 overnight. Yellow crude
product (2) of 0.326 g was obtained after rotary evaporation of the
organic solvents and drying over P2O5 in a desiccator overnight,
containing 6.87 Â 107 Bq of radioactivity. NMR and MS data of non-
1
labelled 2: H-NMR (d, CDCl3): 8.40, 8.39, 8.29, 8.26, 8.25, 7.89, 7.86,
7.62, 7.51, 7.49, 7.46, 7.39, 7.37, 7.34, 7.21, 6.98, 6.95, 6.94, 6.91; 13C-
NMR (d, CDCl3): 193.23, 160.61, 154.24, 142.40, 136.53, 133.94, 130.37,
130.31, 129.76, 128.64, 127.21, 125.92, 125.70, 120.36, 116.45; MS (m/z,
(%)): 319 (M+, 17.1), 302 (15.5), 272 (11.5), 242 (8.0), 226 (5.9), 196
(18.0), 180 (2.5), 168 (10.2), 139 (16.8), 126 (1.5), 105 (100), 77 (93.8), 51
(28.2).
14C-labelled 2-(2-hydroxyphenoxy)-5-nitrobenzophenone (3)
All of the 14C-labelled 2, without purification, was completely dissolved
in 1.2 ml of concentrated H2SO4 in a 50-ml round bottom flask. Glacial
acetic acid (4 ml) was then added to dilute the solution. H2O2 (1.0 ml,
35%) was added dropwise to the solution within 1 min with strong
stirring. The reaction mixture was stirred for 60 min at room
temperature. Then the mixture was poured onto ice (150 g), forming a
fine precipitate, which was separated by filtration under vacuum and
washed thoroughly with H2O. The filter pellet was completely dissolved
in ethyl acetate (EtOAc). Crude product (3) was obtained by
evaporation of the EtOAc dried over Na2SO4 (0.329 g, containing
6.63 Â 107 Bq of radioactivity). TLC (pentane:EtOAc=3:1/v:v, Rf of 2
and 3: 0.72 and 0.44, respectively) showed 97.5% of 14C was located on
the spot of 3. NMR and MS data of non-labelled 3: 1H-NMR
(d,CDCl3): 8.32, 8.31, 8.29, 8.26, 8.25, 7.96, 7.94, 7.93, 7.70, 7.57, 7.55,
7.52, 7.16, 7.14, 7.13, 7.11, 7.06, 7.05, 7.03, 6.91; 13C-NMR (d, CDCl3):
194.39, 161.40, 148.83, 142.24, 141.61, 135.66, 134.75, 130.46, 129.00,
127.84, 127.53, 125.92, 122.27, 120.38, 118.24, 116.94; MS (m/z (%)),
derivatized with BSTFA: 407 (M+, 13.7), 392 (35.4), 346 (3.5), 318 (1.8),
300 (0.9), 253 (4.8), 223 (1.8), 207 (3.0), 166 (24.2), 151 (39.2), 136 (13.8),
105 (100), 77 (68.7), 73 (67.6), 51 (11.6).
U-14C-labelled catechol (4)
14C-labelled 3 (88.1 mg, 1.78 Â 107 Bq), without purification, was
dissolved in dry piperidine (1 ml) in a 50-ml pear-shaped flask connected
to a water condenser. Under the protection of argon, the solution was
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SYNTHESIS OF 13C– AND 14C
557
heated at 1108C in an oil bath with stirring for 90 min. The reaction
mixture was cooled to room temperature, diluted with CH2Cl2 (3 ml),
then cooled in an ice bath, and HCl (2 M, 12 ml) was added dropwise
with stirring. The aqueous phase was extracted with a mixture of
CH2Cl2 and EtOAc (1:1/v:v) five times each with 6 ml. The extract and
the organic phase were combined and dried over Na2SO4. TLC
(CHCl3:EtOAC=5:1/v:v) showed a yellow spot (Rf: 0.94) as a
contaminant; 92.9% radioactivity was located in the catechol spot
(Rf: 0.41). Solvents were removed by rotary evaporation. The remaining
liquid was dissolved in CH2Cl2 (14 ml) and extracted with 1 mM HCl
(6 Â 8 ml). Catechol (4) was extracted from the HCl solution with a
mixture of EtOAc:CH2Cl2 (1:1/v:v) (7 Â 6 ml). The extract was dried
over Na2SO4. TLC (CHCl3:EtOAc=5:1/v:v; Rf of 3 and 4: 0.86 and
0.41, respectively) and autoradiography showed 98.6% radioactivity
purity. After evaporation of the solvents, 4 (Rf: 0.94) was obtained. The
crude product was sublimed under 0.05 mbar at 558C for 3 h, cooled at
08C, resulting in 23.8 mg of crystalline catechol (4) with a specific
radioactivity of 7.28 Â 107 Bq/mmol.
Synthesis of U-13C-catechol (4)
The synthesis of U-13C-catechol was conducted under the same reaction
conditions as for U-14C-catechol except that phenol was derivatized in
dry DMF as solvent at 1108C for 30 min.. The product of the first
synthetic step, 13C-labelled 2-phenoxy-5-nitro-benzophenone (2), was
separated from the reaction mixture by precipitation with H2O. Before
sublimation of crude 13C-catechol (4), it was purified using column
chromatography with silica gel 60 (CHCl3:EtOAc=5:1/v:v). For 500 mg
U-13C-phenol, 297 mg U-13C-catechol was obtained (65% yield with
respect to phenol).
Conclusion
U-13C- and U-14C-catechol were successfully synthesized from phenol in
an overall 80% yield, considerably higher than previously reported
in the literature. This method uses temperate reaction conditions and
the product intermediates are sufficiently pure for the next synthetic
steps.
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R. JI AND A. SCHAFFER
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Acknowledgements
This study was supported by the Deutsche Forschungsgemeinschaft
(project SPP 1090). We are grateful to the following for assistance:
.
Dieter Enders, Zai-Xin Chen, Konrad Haider, and Bernhard Blumich.
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