Straightforward preparation of labeled potassium cyanate by ozonation and application to the synthesis of [13C] or [14C] ureidocarboxylic acids

Article abstract of DOI:10.1002/jlcr.3041

The development of new efficient syntheses of labeled reagents is a great challenge. Avoidance of overcomplicated procedures, availability and cost of starting materials are important considerations in choosing the synthetic route. In this report, we desc

Full text of DOI:10.1002/jlcr.3041

Research Article
Received 13 November 2012,
Revised 8 February 2013,
Accepted 13 February 2013
Published online 2 April 2013 in Wiley Online Library
(wileyonlinelibrary.com) DOI: 10.1002/jlcr.3041
Straightforward preparation of labeled
potassium cyanate by ozonation and
application to the synthesis of [13C] or [14C]
ureidocarboxylic acids
a
b
*
Olivier Loreau and Philippe Marlière
The development of new efficient syntheses of labeled reagents is a great challenge. Avoidance of overcomplicated procedures,
availability and cost of starting materials are important considerations in choosing the synthetic route. In this report, we
describe a facile and rapid preparation of labeled cyanate by ozonation of cyanide, a basic precursor. The crude cyanate
was used without purification for the synthesis of various [13C] or [14C]ureidocarboxylic acids (20-68% yield from potassium
cyanide). According to these results, cyanide ozonation may prove to be a promising alternative to traditional preparations
of labeled cyanate.
Keywords: carbon-13; cyanate; cyanide; ozonation; ureidocarboxylic acid
(d) are given in ppm. Mass spectra were recorded on a Waters liquid
chromatography coupled mass spectrometry (LC/MS) consisting on a
Introduction
Labeled cyanate is a useful reagent for the synthesis of Waters 2545 binary gradient module, a Waters 2996 photodiode array
detector and a Micromass Quattro LC instrument. Infra-red spectra were
obtained on a Perkin Elmer system 2000 FT-IR spectrophotometer. Liquid
scintillation analysis was performed on a Wallac 1409 counter using Ultima
Gold cocktail from Perkin-Elmer.
radiopharmaceuticals involved in pharmacokinetic, metabolism
or transport studies.1–5 In the literature, several methods based
on cyanide oxidation by potassium permanganate or fusion of
[14C]urea have been reported for its preparation.6–9 Unfortunately,
they require the synthesis of a labeled precursor or laborious
isolating procedures for purification. Ratusky and Tykva have also
described the synthesis of potassium [14C]cyanate by exchange
reaction with 14CO2, but the main drawback of this methodology
is the random distribution of 14C-radioactivity between cyanate
Procedure for the preparation of labeled potassium cyanate
Water of 30 mL (pH = 7.45) was placed in a 50-mL multi-necked flask.
The flask was equipped with a magnetic stir bar, a pH electrode and a
and carbon dioxide.10 Investigating alternative methods for the pyrex gas dispersion tube with fritted disk. Labeled potassium cyanide
(0.2–4 mmol, 13–268 mg) was added under stirring. The solution pH
increased to a value between 10 and 11 depending on the quantity of
potassium cyanide. Under stirring, the oxygen/ozone mixture was
allowed to bubble via the gas dispersion tube. After a few minutes,
bubbling was stopped when the pH value has decreased of 1 unit
(ΔpH = À1). Water was removed under vacuum to give crude-labeled
potassium cyanate as a white solid contaminated with a small amount
of bicarbonate. The purity of labeled cyanate was estimated by
subsequent transformation to ureidocarboxylic acids and comparison
with syntheses using unlabeled KCNO as starting material. The purity of
13CNOÀ was found to be around 80–85%. In the case of 14CNOÀ, the
purity was found to be around 65–70%.
preparation of labeled cyanate, we turned our attention to the
ozonation process developed for cyanide detoxification of mining
wastes.11,12 As this methodology is fast and simple, its application
to the synthesis of labeled cyanate would be of great interest. In
this report, we describe the millimole-scale preparation of [13C]
or [14C]cyanate using cyanide oxidation by ozone and the use of
prepared cyanate as chemical reagent for the synthesis of labeled
ureidocarboxylic acids.
Experimental
Potassium [13C]cyanide (99% isotopic enrichment) and NMR solvents were
from Euriso-Top (Saint Aubin, France). Potassium [14C]cyanide (specific
activity: 30.23MBqmmolÀ1) was prepared from unlabeled cyanide and
potassium [14C]cyanide (specific activity: 2.14 GBq mmolÀ1) from Quotient
Bioresearch (Cardiff, United Kingdom). Amino acids were from Sigma-
Aldrich (Saint Quentin Fallavier, France). Ozone was produced from
pure oxygen with a CMG 10–3 ozone generator (A.C.W.), and online pH
monitoring was performed using a pH meter CG 837 (Schott). Microwave
irradiations were conducted using a CEM Discover synthesizer. NMR spectra
were measured on a Bruker Avance 400 spectrometer, and chemical shifts
aCEA – iBiTecS, Service de Chimie Bioorganique et de Marquage, Gif sur Yvette,
France
bIsthmus S.A.R.L. Paris, France
*Correspondence to: Olivier Loreau; CEA – iBiTecS; Service de Chimie Bioorganique
et de Marquage; Gif sur Yvette, France.
E-mail: olivier.loreau@cea.fr
J. Label Compd. Radiopharm 2013, 56 347–350
Copyright © 2013 John Wiley & Sons, Ltd.

O. Loreau and P. Marlière
13C NMR (100 MHz, D2O): d = 128.6 (t, J = 10.8 Hz, 13CNOÀ), 160.6 cooling, the mixture was acidified to pH 2 with 6N HCl. The obtained
(traces of H13CO3À). IR (KBr): 1194, 1275, 2106 cmÀ1
.
precipitate was filtered, washed with cold water and dried under vacuum
to give [14C]ureidocarboxylic acids.
Procedure for the synthesis of [13C]ureidocarboxylic acids
(R)-3-Phenyl-2-[14C]ureidopropanoic acid [14C]2a
The amino acid (1 mmol) was placed in a CEM designed 10-mL reaction
vial. Crude potassium [13C]cyanate was freshly prepared from K13CN
(1.1 mmol) as described previously and dissolved in H2O (2 mL). The
aqueous solution of K13CNO was added, and the resulting mixture was
irradiated to 80ꢀC for 1 h. After cooling, the mixture was acidified to pH
2 with 6N HCl. The obtained precipitate was filtered, washed with cold
water and dried under vacuum to give [13C]ureidocarboxylic acids.
White solid; 103 mg; 15.43 MBq (46% radiochemical yield from K14CN);
specific activity: 31.16 MBq mmolÀ1
.
1H NMR (400 MHz, DMSO-d6): d = 2.83 (dd, J = 7.9, 13.7 Hz, 1H), 2.98 (dd,
J = 5.1, 13.7 Hz, 1H), 4.31 (m, 1H), 5.61 (s, 2H), 6.13 (br d, J = 7.6 Hz, 1H),
7.15–7.35 (m, 5H), 12.63 (br s, 1H). 13C NMR (100 MHz, DMSO-d6): d = 38,
54.1, 126.8, 128.5, 129.6, 137.9, 158.5, 174.3.
(2S,3S)-3-Methyl-2-[14C]ureidopentanoic acid [14C]2b
(R)-3-Phenyl-2-[13C]ureidopropanoic acid [13C]2a
White solid; 76 mg; 13.36 MBq (40% radiochemical yield from K14CN);
White solid; 142 mg (62% yield from K13CN).
specific activity: 30.76 MBq mmolÀ1
.
1H NMR (400 MHz, DMSO-d6): d = 2.84 (dd, J = 7.9, 13.7 Hz, 1H), 2.99 (dd,
J = 5.1, 13.7 Hz, 1H), 4.32 (m, 1H), 5.63 (s, 2H), 6.16 (br d, J = 7.6 Hz, 1H),
7.15–7.35 (m, 5H), 12.65 (br s, 1H). 13C NMR (100 MHz, DMSO-d6): d = 38,
54.1, 126.8, 128.5, 129.6, 137.9, 158.6 (13C-enriched), 174.4. MS: m/z = 210
(M+ 1)+.
1H NMR (400 MHz, DMSO-d6): d = 0.75–0.9 (m, 6H), 1.09 (m, 1H), 1.35
(m, 1H), 1.69 (m, 1H), 4.02 (m,1H), 5.57 (s, 2H), 6.16 (br d, J = 8.6 Hz),
12.47 (br s, 1H). 13C NMR (100 MHz, DMSO-d6): d = 11.9, 16.1, 25, 37.5,
56.9, 158.8, 174.5.
(2S,3S)-3-Methyl-2-[13C]ureidopentanoic acid [13C]2b
Synthesis of unlabeled ureidocarboxylic acids (2a and 2b)
using conventional heating and purity of [14C]cyanate
White solid; 130 mg (68% yield from K13CN).
1H NMR (400 MHz, DMSO-d6): d = 0.75–0.9 (m, 6H), 1.09 (m, 1H), 1.35
(m, 1H), 1.69 (m, 1H), 4.02 (m,1H), 5.59 (s, 2H), 6.16 (br d, J = 8.6 Hz),
12.48 (br s, 1H). 13C NMR (100 MHz, DMSO-d6): d = 11.8, 16.1, 25, 37.4,
56.9, 158.8 (13C-enriched), 174.5.
The amino acid (1 mmol) was placed in an 8-mL Wheaton vial with white
rubber lined cap. KCNO (1.1 mmol) dissolved in H2O (2 mL) was added,
and the resulting mixture was refluxed for 2 h. After cooling, the mixture
was acidified to pH 2 with 6N HCl. The obtained precipitate was filtered,
washed with cold water and dried under vacuum to give unlabeled
ureidocarboxylic acids. (R)-3-Phenyl-2-ureidopropanoic acid 2a (156mg;
75% yield) and (2S,3S)-3-methyl-2-ureidopentanoic acid 2b (113mg; 65%
yield) were obtained as white solids.
MS: m/z = 176 (M + 1)+.
(S)-4-(Methylthio)-2-[13C]ureidobutanoic acid [13C]2c
According to these results, the purity of prepared [14C]cyanate was
found to be 66% (2a) and 67% (2b).
White solid; 61 mg (29% yield from K13CN).
1H NMR (400 MHz, DMSO-d6): d = 1.76 (m, 1H), 1.89 (m, 1H), 2.02 (s, 3H),
2.44 (t, J = 7.5 Hz, 2H), 4.13 (m, 1H), 5.58 (s, 2H), 6.28 (br d, J= 7.5 Hz, 1H),
12.57 (br s, 1H). 13C NMR (100 MHz, DMSO-d6): d = 15, 29.9, 32.1, 51.82,
158.8 (13C-enriched), 174.8. MS: m/z= 194 (M + 1)+.
Results and discussion
(S)-1-[13C]Carbamoylpyrrolidine-2-carboxylic acid [13C]2d
A few years ago, Carillo-Pedroza and Soria-Aguilar studied the
kinetics and mechanisms of cyanide oxidation by ozone
(Scheme 1).11,12 They observed that, in alkaline aqueous solution,
the complete oxidation of cyanide results in an increase of the
potential redox and a decrease of the pH value. Consequently,
they suggested cyanide oxidation may be followed by these
parameters.
White solid; 35 mg (20% yield from K13CN).
1H NMR (400MHz, DMSO-d6): d = 1.7–1.9 (m, 3H), 2.05 (m, 1H), 3.15–3.35
(m, 2H), 4.13 (m, 1H), 5.88 (s, 2H), 12.41 (br s, 1H). 13C NMR (100 MHz,
DMSO-d6): d = 24.5, 29.6, 46.4, 58.8, 157.7 (13C-enriched), 174.9. MS:
m/z = 160 (M + 1)+.
As we were recently interested in the synthesis of labeled
cyanate for research projects, we decided to test this
methodology. In our case, the optimization of ozonation was
conducted using online monitoring of the solution pH and
[13C]cyanide for rapid 13C NMR analysis of the crude product.
Various amounts of potassium [13C]cyanide (0.2–4 mmol) were
dissolved in water (cyanide concentration: 0.18–3.6 mg mLÀ1).
The initial pH value was between 10 and 11 depending on the
quantity of cyanide. Then, a mixture of oxygen/ozone was
allowed to bubble in the aqueous solution, and the pH value
started to decrease. The bubbling was stopped at different pH
values and, after removal of water, the crude residue was
analyzed by 13C NMR (Figure 1).
Synthesis of unlabeled ureidocarboxylic acids (2a and 2b)
using microwave irradiation and purity of [14C]cyanate
The amino acid (1 mmol) was placed in a CEM designed 10-mL reaction
vial. KCNO (1.1 mmol) dissolved in H2O (2 mL) was added, and the
resulting mixture was irradiated to 80ꢀC for 1 h. After cooling, the mixture
was acidified to pH 2 with 6N HCl. The obtained precipitate was filtered,
washed with cold water and dried under vacuum to give unlabeled
ureidocarboxylic acids. (R)-3-Phenyl-2-ureidopropanoic acid 2a (160 mg;
77% yield) and (2S,3S)-3-methyl-2-ureidopentanoic acid 2b (140 mg;
80% yield) were obtained as white solids.
According to these results, the purity of prepared [13C]cyanate was
found to be 80% (2a) and 85% (2b).
Typically, after a decrease of 0.3 pH unit (ΔpH = À0.3), we
Procedure for the synthesis of [14C]ureidocarboxylic acids
observed the formation of cyanate (13CNOÀ, 128.6 ppm), but
The amino acid (1 mmol) was placed in a 8-mL Wheaton vial with white
rubber lined cap. Crude potassium [14C]cyanate was freshly prepared
from K14CN (specific activity: 30.23 MBq mmolÀ1, 1.1 mmol, 33.22 MBq)
as described previously and dissolved in H2O (2 mL). The aqueous solution
of K14CNO was added, and the resulting mixture was refluxed for 2 h. After
Scheme 1. Cyanide oxidation by ozone.
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J. Label Compd. Radiopharm 2013, 56 347–350

O. Loreau and P. Marlière
Table 1. Synthesis of labeled ureidocarboxylic acids
Labeled ureidocarboxylic
Amino acid
acid
(2a–d: yielda)
13CNO-
[13C]2a: 62%
[14C]2a: 46%
pH = -0.3
13CN-
[13C]2b: 68%
[14C]2b: 40%
2-
13CO3
[13C]2c: 29%
[13C]2d: 20%
13CNO-
pH = -1
‘*’ denotes labeling.
aOverall isolated yield from K*CN (two steps).
H13CO3
-
ozone was stopped at ΔpH = À1 to ensure
a complete
consumption of cyanide. After removal of water, the crude cyanate
was used without further purification with various a-amino acids
(Table 1). A range of [13C]ureidocarboxylic acids ([13C]2a–d) was
obtained with moderate to good yields (20–68% from K13CN)
using microwave irradiation (80ꢀC, 1 h). The chemical purity of
K13CNO was estimated to be around 80–85% by comparison with
the synthesis of 2a and 2b using unlabeled KCNO (1.1 eq.) as
starting material. (R)-3-Phenyl-2-[14C]ureidopropanoic acid [14C]
2a and (2S,3S)-3-methyl-2-[14C]ureidopentanoic acid [14C]2b
were also prepared by conventional heating (reflux, 2 h).
The radiochemical yields from potassium [14C]cyanide were,
respectively, 46% ([14C]2a) and 40% ([14C]2b). In this case, the
purity of [14C]cyanate was found to be around 65–70%.
Figure 1. Optimization of cyanide oxidation by ozone (13C NMR analysis in D2O).
cyanide (13CNÀ, 165.08 ppm) was still present in solution. Traces
of carbonate (13CO32À, 168.28 ppm) were also detected. At
ΔpH = À1, only cyanate and bicarbonate (H13CO3À, 160.61 ppm)
were present. This procedure is fast because only a few minutes
are required for the complete consumption of cyanide.
According to the literature, carbonate and bicarbonate are usual
by-products. They are formed with an excess of ozone or by
hydrolysis of cyanate under alkaline conditions.
Ureidocarboxylic acids are closely related to essential
biochemical processes.13,14 They are also useful precursors of
hydantoins and amino acids.15–17 As they can be readily
prepared from a-amino acids and potassium cyanate by
conventional heating18,19 or microwave irradiation,20 we chose
this reaction to test our [13C] or [14C]cyanate as labeling reagent
(Scheme 2).
Conclusions
We have optimized the synthesis of labeled cyanate by cyanide
ozonation using online pH monitoring and 13C NMR analysis.
This oxidative methodology, involving ozone avoids laborious
purification, minimizes the handling of radiochemical materials
and, consequently, is well adapted to radiolabeled work.
Potassium [13C] or [14C]cyanate was readily prepared in a millimole
scale and used without purification as chemical reagent. Its
suitability for the labeling was confirmed by the successful
synthesis of [13C] or [14C]ureidocarboxylic acids.
The preparation of labeled cyanate was carried out with 1.1
equivalent of potassium [13C] or [14C]cyanide. Oxidation by
Acknowledgements
The authors thank Céline Puente and David-Alexandre Buisson
(CEA, iBiTecS, Service de Chimie Bioorganique et de Marquage)
for mass spectrometry and liquid scintillation analyses.
Scheme 2. Synthesis of [13C] or [14C]ureidocarboxylic acids from a-amino acids
and labeled potassium cyanide.
J. Label Compd. Radiopharm 2013, 56 347–350
Copyright © 2013 John Wiley & Sons, Ltd.
www.jlcr.org

O. Loreau and P. Marlière
[10] J. Ratusky, R. Tykva, J. Label. Compd. Radiopharm. 1967; 3,
50–52.
Conflict of Interest
[11] F. R. Carillo-Pedroza, M. J. Soria-Aguilar, Eur. J. Mineral Process.
Environ. Prot. 2001; 1, 55–61.
The authors did not report any conflict of interest.
[12] J. R. Parga, S. S. Shukla, F. R. Carillo-Pedroza, Waste Management
2003; 23, 183–191.
[13] A. B. P. van Kuilenburg, H. van Lenthe, M. Löffler, A. H. van Gennip,
Clin. Chem. 2004; 50, 2117–2124.
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J. Label Compd. Radiopharm 2013, 56 347–350