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A simple synthesis of radiolabelled bromoacetic acid
0883-2889/89 $3.00+ 0.00 Copyright ~ 1989PergamonPress plc
Appl. Radiat. lsot. Vol. 40, No. 3, pp. 251-255, 1989 Int. J. Radiat. Appl. lnstrum. Part A
Printed in Great Britain. All rights reserved
A Simple Synthesis of Radiolabelled Bromoacetic Acid D O U G L A S N. A B R A M S , ~* R E N E C . - G A U D R E A U L T 2 a n d A N T O I N E A. N O U J A I M 3 ~Department of Nuclear Medicine, Health Sciences Centre, 700 William Avenue, Winnipeg, Manitoba, Canada R3E 0Z3, 2Department of Pharmacology, Faculty of Medicine, Laval University, Quebec City, Quebec, Canada G IK 7P4 and 3Facultyof Pharmacy and Pharmaceutical Sciences, University of Alberta, Edmonton, Alberta, Canada T6G 2N8 (Received 15 Februao' 1988)
A facile procedure for the radiosynthesis of 3H- or 14C-labelledbromoacetate by reaction of sodium acetate with bromine in the presence of elemental sulfur is described. The effects of reaction time and temperature as well as bromine and sulfur concentration were investigated. Optimum conditions in which I mg sodium acetate (mass equivalent to 1 mCi (37 MBq) ~4C)was allowed to react with 10/~L bromine and 0.1 mg sulfur at 105C for 60rain afforded [~4C]bromoacetate in greater than 90% radiochemical yield and [3H]bromoacetate in greater than 60% radiochemical yield based on initial t4C- or 3H-labelledsodium acetate concentration.
Introduction Bromoacetic acid is a useful intermediate in the synthesis of substituted carboxylic acids (Ropp, 1950), biologically active compounds (Ehrensvard and Liljekvist, 1959; Brunsberg et al., 1965) and bifunctional chelating agents (Yeh et al., 1979) among others. Our interest in the study of chelating agents using dual label techniques led us to investigate the synthesis of 3H- and ~4C-labelled bromoacetic acid. A large number of radiosyntheses of ~-halocarboxylic acids have been described. Methods used for the synthesis of [3H]acetic acid such as base catalyzed exchange with acetic acid (Atkinson et al., 1966) or decarboxylation of malonic acid (Fields et al., 1952) are not feasible using bromoacetic acid directly. Two methods for the synthesis of ~4C-labelled bromoacetic acid have been described. Preparation of[~4C]bromoacetic acid from barium carbonate (Ehrensvard and Liljekvist, 1959; Brunsberg et al., 1965) is a lengthy, multistep procedure involving volatile intermediates, and specialized glassware. An alternative synthesis is the much more convenient single step conversion of acetic acid to bromoacetic acid by bromine in the presence of phosphorus (Gidez and Karnoutsky, 1952; Bloch, 1949; Ostwald, 1948). The latter synthetic procedures resulted in near quantitative radiochemical yields under the conditions described. However, the resultant specific activity was very low. Modification of these procedures to give a product with increased specific activity resulted in *Author for correspondence.
unacceptably low radiochemical yields. We therefore attempted the synthesis using bromine in the presence of elemental sulfur (Genvresse, 1892) instead of phosphorus. This procedure proved to be a facile preparation of both [3H] and [J4C]bromoacetate directly from the appropriate radiolabelled sodium acetate. This procedure was not well documented and we undertook to optimize the reaction conditions.
Experimental Materials
All chemicals were of reagent grade quality and used without further purification unless otherwise noted. Bromine was dried with concentrated sulfuric acid and distilled from potassium bromine prior to use. Chromatographic solvents were high pressure liquid chromatography (HPLC) grade and were filtered and degassed when necessary before use. [~4C]Sodium acetate (Amersham) had a specific activity of 2.0 GBq mmol-I (55 mCi mmol-~). Radiochemical purity (fraction of total t"C radioactivity co-chromatographing with a sodium acetate standard) determined by thin layer chromatography (TLC) and reversed phase-high pressure liquid chromatography (RP-HPLC) was greater than 99%. [3H]Sodium acetate (New England Nuclear) had a specific activity of 1 2 6 G B q m m o l (3.4 Ci mmol -~). Radiochemical purity was greater than 98% as determined by co-chromatography with a sodium acetate standard using RP-HPLC and TLC.
251
252
DOUGLAS N. ABRAMSet al.
Instrumentation
Reversed phase high pressure liquid chromatographic separations were performed using either a single C-8 Radial Pak (Waters) column or two columns in tandem. Column eluant was 0.01 M KHzPO4 and 0.01 M tetrabutylammonium phosphate (one-half vial of Waters PIC Reagent-A) in HPLC grade water. Column effluent was monitored for ultraviolct (u.v.) absorbance at 214 nm (Tracor 980) and radioactivity (Fto-One DR flow detector using Aquasol-2 (New England Nuclear) as scintillant), The chromatograms were analyzed by integration of both the u.v. (Hewlett Packard 3390A Integrator) and radioactive peaks (Flo-One DR). In addition, 30-s fractions (0.5 mL) of the Flo-One DR effluent were collected (Pharmacia Frac-100), further diluted with Aquasol-2 (5 mL) and analyzed by liquid scintillation counting (LSC) on a Beckman LS9000 scintillation counter. Liquid scintillation results were analyzed using the Beckman Digital Integration program. Samples were quench corrected using the "H number" against the appropriate [3H] or [~4C]n-hexadecane calibrated standards (Amersham). Silica gel (Whatman MK6F) thin layer chromatograms were developed with ethanol (95%):ammonium hydroxide (3:1 v,v) as the solvent. Sodium acetate was detected by spraying the plate with an indicator consisting of methyl red ( 100 mg) : bromothymol blue ( 100 mg) : formaldehyde (50mL):ethanol (200mL) adjusted to pH 5.2 with 0.1 M sodium hydroxide. Sodium acetate gave blue spots on yellow background upon exposure to ammonium hydroxide vapor while bromoacetate yielded pink spots. The distribution of radioactivity on the TLC plates was determined on a Berthold LB2832 TLC Linear Analyzer. Yield o p t i m i : a t i o n
The effect of reaction time, sulfur concentration, bromine concentration and solvent on the reaction rate and chemical yield of bromoacetate was determined. Elemental sulfur was dissolved in chloroform ( 1 0 m g m L ~) and sodium acetate was dissolved in anhydrous methanol (10 mg mL ~) to facilitate accurate addition of the small quantities required for each reaction. All reactions were performed using 1.0 mg of sodium acetate. This is the mass of sodium acetate present in l mCi (37 MBq) of [~4C]sodium acetate. (1 mCi is representative of the amount of radioactivity commonly used in preparative scale radiochemical synthesis.) The appropriate amount of sulfur in chloroform was added to 100,uL (1 mg, 0.012mmol) of sodium acetate in methanol in a 300 i~ L Reactivial. The solvent was carefully removed in t,acuo by rotation of the vial to allow uniform coating of the reagents on the vial wall. The vial was then dried in vacuo (1 mmHg) for 60 min at ambient temperature. The required volume of freshly distilled elemental bromine was added to the vial which was
then sealed and immersed upside down in a 105 C oil bath. The bromine was not added through the septum (Teflon-laminated rubber) to avoid loss of reagents through the septum break due to high pressures during reaction. The reaction was terminated at the designated times by the addition of 500 I~L of water to the reaction mixture. The reaction product was quantitatively transferred and diluted to 25 mL with HPLC solvent to be analyzed directly via RP HPLC. Radiolabelled h r o m o a c e t a t e
Both 'H- and HC-labelled bromoacetate were synthesized using the optimum reaction conditions described previously. Accordingly, 10 i t L (370 kBq) of [~C]sodium acetate in methanol were added to a mixture of carrier sodium acetate (I,02mg, 0.012retool) and sulfur (0.1nag, 0.003mmol) and coated on the reaction vessel wall as described above, Elemental bromine (10/~L) was added and the reaction was heated at 105 C for 60 rain. Similarly, 10 I~L (3.TMBq) or 5001tL (185MBq) of [3H]sodium acetate in water were treated as described above. Radiochemical yields were determined b~ RP HPLC.
Results and Discussion Initial attemps to synthesize [~C]bromoacetate by direct phosphorus catalyzed bromination of" [14C]acetate (Gidez and Kernovsley, 1952; Block, 1949: Ostwald, 1948) were cumbersome. The majority of the published procedures used acetic acid, acetyl chloride or acetic anhydride as both the reagent and the solvent simultaneously. This was impractical for the small-scale syntheses which afforded low radiochemical yields (approximately 5%) at the low acetate concentrations employed (1 mg). Different solvents including benzene, pyridine, dimethylformamide, hexamethylphosphoric triamide and dimethyl sulfoxide were evaluated, but they either competed with the acetate t\~r bromination or simply afforded low radiochemical yields. However, the facile bromination of acetic acid with bromine (Scheme 1), in the presence of elemental sulfur, as reported by Genvresse (1892) offered a simple solution to the problem in that the bromine could act as the solvent. In addition, purification of the product was simplified by the use of fewer Br 2 / S (a)
~4CH3CO2Na
Brt4CH2CO2Na
+ HBr
105 C
Br2/S
~'~
Br3HCHCOzNa
+ HBr
~
BrCHzCO2Na * 3HBr
(b) 3HC H2 CO: Na ~ . ~ 105"C
Scheme 1. The s.,,nthesis of ~H- and ~4C-labelled bromoacetk acid.
Radiosynthesis of bromoacetic acid 100908070-
9 LI,J
6050-
0
4O
2O
11
i
t
0 5 10
20
30
40
50
60
TIME (min.)
Fig. 1. Effect of reaction time on the chemical yield of bromoacetic acid. Sodium acetate (1.02mg) was reacted with 10pL bromine and 0.1 mg sulfur at 105'C for various lengths of time. Chemical yields were determined by RP HPLC analysis.
reagents. Since there was no mention of using this technique for the radiochemical synthesis of either ~H- or ~4C-labelled bromoacetate in the literature, we investigated the reaction further. The effects of temperature, time, bromine concentration and sulfur concentration on the reaction rate and yield of bromoacetate by direct bromination of sodium acetate with bromine in the presence of elemental sulfur were determined. The initial reaction condition were derived from the scaled-down synthesis originally reported by Genvresse (1892). The reaction was studied on a 1 mg scale, which conveniently represented the mass of sodium acetate present in a commercial sample of l mCi (37 MBq) of [t4C]sodium acetate. Figure 1 gives the time course of the reaction for the above conditions. The reaction proceeded slowly. The maximal chemical yield reached a plateau at 40 rain ap.d neared completion by 60 min. With 60 min as the optimal reaction time, the effect of the volume of bromine used in the reaction was investigated. An initial larger scale reaction using a 10 M excess was evaluated to determine if a large excess of bromine would result in polybrominated side products. Analysis of the product by R P - H P L C did not reveal the presence of products other than starting material and monobromoacetate. The effect of bromine volume on the reaction with 1 mg of sodium acetate is given in Fig. 2. The chemical yield increased dramatically with
253
bromine volumes corresponding to 2 M equivalents and greater. It was noted that after immersion of the reaction vessel in the oil bath, the majority of the bromine collected on the top of the reaction vessel which was cooler relative to the immersed portion of the vial containing the reagents coated on the vial wall. This was remedied by simply inverting and completely immersing the reaction vessel in the oil bath so that, upon heating, the bromine concentrated itself in the cone of the reaction vessel. This procedure reduced the variability in the results considerably. Further experiments with 50 and 100/~L volumes of bromine decreased the recovery of monobrominated acetate without apparent formation of the di- and tribromo derivatives. Figure 3 demonstrates that although the concentration of sulfur has some influence on chemical yield, it probably serves as a catalyst since nearly quantitative yields were obtained using approximately 0.25 equivalents (0.003 mmol), with respect to acetate (0.012 mmol), of sulfur. The poorer yields at lower sulfur concentrations may be related to the difficulty in evenly and reproducibly distributing the small amounts of sulfur on the vessel walls. This would result in areas having insufficient sulfur to effectively promote the reaction between bromine and acetate. The minimum amount appeared to be between 0.05 and 0.1 mg. The order of layering the sulfur and
1009080706050"
30-
u
r
0
I
5
110
BROMINE VOLUME (pL)
Fig. 2. The effect of bromine volume on the chemical yield of bromoacetic acid. Sodium acetate (1.02 mg) was reacted with 0.1 mg of sulfur and various amounts of bromine at 105C for 60min. Chemical yields were determined by RP HPLC analysis.
254
DOUGLAS N. ABRAMSeta/. 100-
/
90-
80-
70-
60-
50-
40-
30-
2010ba
0
I
I
I
I
0.05
0.10
0.15
0.20
SULFUR CONCENTRATION (mg)
Fig. 3, The effect of sulfur concentration on the chemical yield of bromoacetic acid. Sodium acetate (1.02 mg) was reacted with 10 uL bromine and various amounts of sulfur at 105C for 60 min. Chemical yields were determined by RP HPLC analysis.
acetate did not have an appreciable effect on the reaction yield. The significantly different solubilities of sulfur and acetate did not allow c o n c u r r e n t application of the reagents in the same solvent. The presence of water in the reaction decreased the yield. The vials were preheated to 105 C for 10 min, before addition of the bromine, to drive off the water. G o o d yields could be o b t a i n e d without this step but the results were less reproducible. Bromoacetic acid was observed to slowly decompose at temperatures greater than 110 C in the presence of b r o m i n e and sulfur. However, decomposition was less than 5% alter 6 0 m i n at temperatures between 100 and 110 C. At temperatures below 100 C, the reaction proceeded much more slowly. The optimal reaction conditions l\~r the conversion of 1 mg of sodium acetate to b r o m o a c e t a t e were to react the sodium acetate at 105 C for 60 mira in the presence of 0.01 mE b r o m i n e and 0.1 mg of elemental sulfur. The results from six ExperimEnts using [~4C]sodium acetatc tracer added to the reaction are given in Table 1. The radiochemical yields were determined by RP H P L C with a radioactivity flow detector. In addition, 3 0 s eluatE fractions were counted by liquid scintillation counting. The chemical yields were determined from the area under the u.v. a b s o r p t i o n peak on the RP H P L C trace of the reaction aliquot as c o m p a r e d to a reference standard. The resultant r a d i o c h r o m a t o g r a m overlaid with a u.v. a b s o r p t i o n trace from the analysis of reaction 5 is reproduced in Fig. 4. The m a j o r u.v. peaks present
W 1
<
_o
,x-
~3 <
o
co rn ,< >
1
B___z< INJECTION
ACETATE
U V ( 2 1 4 nm)
Br -'
BROMOACETATE
TIME
Fig. 4. Radio high pressure liquid chromatogram of [ ~4C]bromoacetate. Column: Waters Radial Pak C-8. Solvent: 0.01M KH2PO4:0.01 M tetrabutylammonium phosphate: flow rate: I m L m i n : u.v: 214nm: radioactivity detector: Flo-One liquid scintillation mode. Trace A: t4C radioactivity. Trace B: u.v absorption.
Radiosynthesis of bromoacetic acid Table I. Radiochemical yield of [14Clbromoacetate % Yield* Reaction 1 2 3 4 5 6
Chemical (u.v.)+ 84.7 35.8 70.2 77.0 99.1 93.0
(LSC)++ 98.7 44.1 78.5 94.2 93.3 --
Radiochemical (flow)§ ---94.2 94.9 91.7
*[~4C]Sodium acetate (1.0 mg) was reacted with 10 # L bromine and 0.01 mg sulfur at 105 C for 60rain. +Product absorption peak integration compared to known standard. +Liquid scintillation analysis of 30s (0.5mL) eluate fractions. §Product radioactivity peak integration compared to known standard. are acetate (1) bromide ion (2) and bromoacetate (3). The concentration o f acetate after the reaction was too low to detect under these conditions, therefore the acetate (peak 1) has been overlaid from a trace of a sodium acetate standard. Only two i m p o r t a n t peaks appear in the radioactive ~4C trace and these corres p o n d to acetate and bromoacetate. These two peaks represented more than 95% o f the injected radioactivity. There was no indication of formation of either the di- or tribrominated reaction products. Similar analysis o f the 3H reaction product resulted in a third radioactive peak (3H) which had a retention time corresponding to the column void volume (data not included). This extra peak probably corresponds to [3H]water, although this has not been confirmed. This would be an expected by-product due to exchange between the water added to quench the reaction and [3H]HBr formed in the bromination of [3H]acetate (Scheme 1). Statistically, one would expect to lose approximatley one-third o f the 3H activity. This is supported by the data given in Table 2. Reaction 4 in which only 0.01 mg o f sodium acetate Table 2. Radiochemical yield of [~H]bromoacetate Radiochemical yield* % [~H]acetatet Reaction 1 2 3 4~
Consumed 98,9 91.1 98.4 60.8
% Initial ~H concentration+ + Observed 73.7 60.0 67.2 37.7
Theoretical (65.9) (60.7) (65.6) (40.5)
*[~H]Sodium acetate (1.0 mg) was reacted with l0 # L bromine and 0. Img sulfur at 105C for 60rain. *Determined as 100 % 3H in acetate peak on RP HPLC analysis and liquid scintillation counting. ,+Determined by liquid scintillation counting of bromoacetate peak on RP-HPLC analysis. §[3HlSodium acetate (0.01 rag) was reacted with 10 ~L bromine and 0.1 mg sulfur at 105 C for 60rain.
255
was used (no carrier acetate was added to the reaction) did not yield as well as the carrier added reactions. This may be due to the need to evenly distribute the reagents on the vessel wall. This process was made more difficult by the small acetate concentration used.
Conclusions The radiochemical synthesis o f bromoacetic acid labelled with either 3H or ~4C can be accomplished in high radiochemical yields via the sulfur catalyzed b r o m i n a t i o n o f appropriately labelled sodium acetate. The reaction proceeds readily at acetate concentrations o f approximately 1 mg; however, the radiochemical yields decreased when the a m o u n t o f acetate was decreased to 0.01 mg. The reaction product was not contaminated with either the di- or tribromoacetic acid derivatives under the conditions employed. Acknowledgements--The authors would like to thank Ms Peggy Schon and Susan Joy for their patient handling of the manuscript.
References Atkinson J. G., Gsakvary J. J., Morse A. T. and Stuart R. S. (1966) Base catalyzed deuterium exchange reactions. A simple synthesis of ct-deutero carboxylic acids. In Proc. 2nd Int. Conf. on Methods o f Preparing and Storing Labelled Compounds (Ed. Sirchis J.). European Atomic Energy Community, Brussels. Bloch K. (1949) The synthesis of glutathione in isolated liver. J. Biol. Chem. 179, 1245. Brunsberg U., Bunte O. and Lindskoug I. (1965) Synthesis of bromoacetic acid-l,2-C14 and glycolic acid-l,2-C14. Acta Chem. Scand. 19, 246. Ehrensvard G. and Liljekvist J. (1959) Synthesis of uracil2,4,5,6-C 14 with high specific activity. Studies in microsynthesis I. Acta Chem. Scand. 13, 2070. Fields M., Rothschild S. and Leoffere M. A. (1952) Synthesis of 2,4-dichlorophenoxyacetic acid labeled with isotopic carbon. J. Am. Chem. Soc. 74, 2435. Gidez L. I. and Karnovsky M. L. (1952). A synthesis of ~tor fl-CI4-1abeled glycerol. J. Am. Chem. Soc. 74, 2413. Genvresse M. P. (1892) Nouvelle pr6paration des acides gras bromes. Bull Soc. Chim. Ft. 7, 364. Ostwald R. (1948) Synthesis of chloroacetic acid and glycine labeled with radioactive carbon in the carboxyl group. J. Biol. Chem. 173, 207. Ropp G. A. (1950) C ~4 Tracer studies in the synthesis o f malonic acid-2-C ~4 and diethyl malonate-2-C TM. J. Am. Chem. Soc. 72, 4459. Yeh S. M., Sherman D. G. and Meares C. F. (1979) A new route to "bifunctional" chelating agents: conversion of amino acids to analogs of ethylenedinitrilotetracetic acid. Anal. Biochem. 100, 152.
Appl. Radiat. lsot. Vol. 40, No. 3, pp. 251-255, 1989 Int. J. Radiat. Appl. lnstrum. Part A
Printed in Great Britain. All rights reserved
A Simple Synthesis of Radiolabelled Bromoacetic Acid D O U G L A S N. A B R A M S , ~* R E N E C . - G A U D R E A U L T 2 a n d A N T O I N E A. N O U J A I M 3 ~Department of Nuclear Medicine, Health Sciences Centre, 700 William Avenue, Winnipeg, Manitoba, Canada R3E 0Z3, 2Department of Pharmacology, Faculty of Medicine, Laval University, Quebec City, Quebec, Canada G IK 7P4 and 3Facultyof Pharmacy and Pharmaceutical Sciences, University of Alberta, Edmonton, Alberta, Canada T6G 2N8 (Received 15 Februao' 1988)
A facile procedure for the radiosynthesis of 3H- or 14C-labelledbromoacetate by reaction of sodium acetate with bromine in the presence of elemental sulfur is described. The effects of reaction time and temperature as well as bromine and sulfur concentration were investigated. Optimum conditions in which I mg sodium acetate (mass equivalent to 1 mCi (37 MBq) ~4C)was allowed to react with 10/~L bromine and 0.1 mg sulfur at 105C for 60rain afforded [~4C]bromoacetate in greater than 90% radiochemical yield and [3H]bromoacetate in greater than 60% radiochemical yield based on initial t4C- or 3H-labelledsodium acetate concentration.
Introduction Bromoacetic acid is a useful intermediate in the synthesis of substituted carboxylic acids (Ropp, 1950), biologically active compounds (Ehrensvard and Liljekvist, 1959; Brunsberg et al., 1965) and bifunctional chelating agents (Yeh et al., 1979) among others. Our interest in the study of chelating agents using dual label techniques led us to investigate the synthesis of 3H- and ~4C-labelled bromoacetic acid. A large number of radiosyntheses of ~-halocarboxylic acids have been described. Methods used for the synthesis of [3H]acetic acid such as base catalyzed exchange with acetic acid (Atkinson et al., 1966) or decarboxylation of malonic acid (Fields et al., 1952) are not feasible using bromoacetic acid directly. Two methods for the synthesis of ~4C-labelled bromoacetic acid have been described. Preparation of[~4C]bromoacetic acid from barium carbonate (Ehrensvard and Liljekvist, 1959; Brunsberg et al., 1965) is a lengthy, multistep procedure involving volatile intermediates, and specialized glassware. An alternative synthesis is the much more convenient single step conversion of acetic acid to bromoacetic acid by bromine in the presence of phosphorus (Gidez and Karnoutsky, 1952; Bloch, 1949; Ostwald, 1948). The latter synthetic procedures resulted in near quantitative radiochemical yields under the conditions described. However, the resultant specific activity was very low. Modification of these procedures to give a product with increased specific activity resulted in *Author for correspondence.
unacceptably low radiochemical yields. We therefore attempted the synthesis using bromine in the presence of elemental sulfur (Genvresse, 1892) instead of phosphorus. This procedure proved to be a facile preparation of both [3H] and [J4C]bromoacetate directly from the appropriate radiolabelled sodium acetate. This procedure was not well documented and we undertook to optimize the reaction conditions.
Experimental Materials
All chemicals were of reagent grade quality and used without further purification unless otherwise noted. Bromine was dried with concentrated sulfuric acid and distilled from potassium bromine prior to use. Chromatographic solvents were high pressure liquid chromatography (HPLC) grade and were filtered and degassed when necessary before use. [~4C]Sodium acetate (Amersham) had a specific activity of 2.0 GBq mmol-I (55 mCi mmol-~). Radiochemical purity (fraction of total t"C radioactivity co-chromatographing with a sodium acetate standard) determined by thin layer chromatography (TLC) and reversed phase-high pressure liquid chromatography (RP-HPLC) was greater than 99%. [3H]Sodium acetate (New England Nuclear) had a specific activity of 1 2 6 G B q m m o l (3.4 Ci mmol -~). Radiochemical purity was greater than 98% as determined by co-chromatography with a sodium acetate standard using RP-HPLC and TLC.
251
252
DOUGLAS N. ABRAMSet al.
Instrumentation
Reversed phase high pressure liquid chromatographic separations were performed using either a single C-8 Radial Pak (Waters) column or two columns in tandem. Column eluant was 0.01 M KHzPO4 and 0.01 M tetrabutylammonium phosphate (one-half vial of Waters PIC Reagent-A) in HPLC grade water. Column effluent was monitored for ultraviolct (u.v.) absorbance at 214 nm (Tracor 980) and radioactivity (Fto-One DR flow detector using Aquasol-2 (New England Nuclear) as scintillant), The chromatograms were analyzed by integration of both the u.v. (Hewlett Packard 3390A Integrator) and radioactive peaks (Flo-One DR). In addition, 30-s fractions (0.5 mL) of the Flo-One DR effluent were collected (Pharmacia Frac-100), further diluted with Aquasol-2 (5 mL) and analyzed by liquid scintillation counting (LSC) on a Beckman LS9000 scintillation counter. Liquid scintillation results were analyzed using the Beckman Digital Integration program. Samples were quench corrected using the "H number" against the appropriate [3H] or [~4C]n-hexadecane calibrated standards (Amersham). Silica gel (Whatman MK6F) thin layer chromatograms were developed with ethanol (95%):ammonium hydroxide (3:1 v,v) as the solvent. Sodium acetate was detected by spraying the plate with an indicator consisting of methyl red ( 100 mg) : bromothymol blue ( 100 mg) : formaldehyde (50mL):ethanol (200mL) adjusted to pH 5.2 with 0.1 M sodium hydroxide. Sodium acetate gave blue spots on yellow background upon exposure to ammonium hydroxide vapor while bromoacetate yielded pink spots. The distribution of radioactivity on the TLC plates was determined on a Berthold LB2832 TLC Linear Analyzer. Yield o p t i m i : a t i o n
The effect of reaction time, sulfur concentration, bromine concentration and solvent on the reaction rate and chemical yield of bromoacetate was determined. Elemental sulfur was dissolved in chloroform ( 1 0 m g m L ~) and sodium acetate was dissolved in anhydrous methanol (10 mg mL ~) to facilitate accurate addition of the small quantities required for each reaction. All reactions were performed using 1.0 mg of sodium acetate. This is the mass of sodium acetate present in l mCi (37 MBq) of [~4C]sodium acetate. (1 mCi is representative of the amount of radioactivity commonly used in preparative scale radiochemical synthesis.) The appropriate amount of sulfur in chloroform was added to 100,uL (1 mg, 0.012mmol) of sodium acetate in methanol in a 300 i~ L Reactivial. The solvent was carefully removed in t,acuo by rotation of the vial to allow uniform coating of the reagents on the vial wall. The vial was then dried in vacuo (1 mmHg) for 60 min at ambient temperature. The required volume of freshly distilled elemental bromine was added to the vial which was
then sealed and immersed upside down in a 105 C oil bath. The bromine was not added through the septum (Teflon-laminated rubber) to avoid loss of reagents through the septum break due to high pressures during reaction. The reaction was terminated at the designated times by the addition of 500 I~L of water to the reaction mixture. The reaction product was quantitatively transferred and diluted to 25 mL with HPLC solvent to be analyzed directly via RP HPLC. Radiolabelled h r o m o a c e t a t e
Both 'H- and HC-labelled bromoacetate were synthesized using the optimum reaction conditions described previously. Accordingly, 10 i t L (370 kBq) of [~C]sodium acetate in methanol were added to a mixture of carrier sodium acetate (I,02mg, 0.012retool) and sulfur (0.1nag, 0.003mmol) and coated on the reaction vessel wall as described above, Elemental bromine (10/~L) was added and the reaction was heated at 105 C for 60 rain. Similarly, 10 I~L (3.TMBq) or 5001tL (185MBq) of [3H]sodium acetate in water were treated as described above. Radiochemical yields were determined b~ RP HPLC.
Results and Discussion Initial attemps to synthesize [~C]bromoacetate by direct phosphorus catalyzed bromination of" [14C]acetate (Gidez and Kernovsley, 1952; Block, 1949: Ostwald, 1948) were cumbersome. The majority of the published procedures used acetic acid, acetyl chloride or acetic anhydride as both the reagent and the solvent simultaneously. This was impractical for the small-scale syntheses which afforded low radiochemical yields (approximately 5%) at the low acetate concentrations employed (1 mg). Different solvents including benzene, pyridine, dimethylformamide, hexamethylphosphoric triamide and dimethyl sulfoxide were evaluated, but they either competed with the acetate t\~r bromination or simply afforded low radiochemical yields. However, the facile bromination of acetic acid with bromine (Scheme 1), in the presence of elemental sulfur, as reported by Genvresse (1892) offered a simple solution to the problem in that the bromine could act as the solvent. In addition, purification of the product was simplified by the use of fewer Br 2 / S (a)
~4CH3CO2Na
Brt4CH2CO2Na
+ HBr
105 C
Br2/S
~'~
Br3HCHCOzNa
+ HBr
~
BrCHzCO2Na * 3HBr
(b) 3HC H2 CO: Na ~ . ~ 105"C
Scheme 1. The s.,,nthesis of ~H- and ~4C-labelled bromoacetk acid.
Radiosynthesis of bromoacetic acid 100908070-
9 LI,J
6050-
0
4O
2O
11
i
t
0 5 10
20
30
40
50
60
TIME (min.)
Fig. 1. Effect of reaction time on the chemical yield of bromoacetic acid. Sodium acetate (1.02mg) was reacted with 10pL bromine and 0.1 mg sulfur at 105'C for various lengths of time. Chemical yields were determined by RP HPLC analysis.
reagents. Since there was no mention of using this technique for the radiochemical synthesis of either ~H- or ~4C-labelled bromoacetate in the literature, we investigated the reaction further. The effects of temperature, time, bromine concentration and sulfur concentration on the reaction rate and yield of bromoacetate by direct bromination of sodium acetate with bromine in the presence of elemental sulfur were determined. The initial reaction condition were derived from the scaled-down synthesis originally reported by Genvresse (1892). The reaction was studied on a 1 mg scale, which conveniently represented the mass of sodium acetate present in a commercial sample of l mCi (37 MBq) of [t4C]sodium acetate. Figure 1 gives the time course of the reaction for the above conditions. The reaction proceeded slowly. The maximal chemical yield reached a plateau at 40 rain ap.d neared completion by 60 min. With 60 min as the optimal reaction time, the effect of the volume of bromine used in the reaction was investigated. An initial larger scale reaction using a 10 M excess was evaluated to determine if a large excess of bromine would result in polybrominated side products. Analysis of the product by R P - H P L C did not reveal the presence of products other than starting material and monobromoacetate. The effect of bromine volume on the reaction with 1 mg of sodium acetate is given in Fig. 2. The chemical yield increased dramatically with
253
bromine volumes corresponding to 2 M equivalents and greater. It was noted that after immersion of the reaction vessel in the oil bath, the majority of the bromine collected on the top of the reaction vessel which was cooler relative to the immersed portion of the vial containing the reagents coated on the vial wall. This was remedied by simply inverting and completely immersing the reaction vessel in the oil bath so that, upon heating, the bromine concentrated itself in the cone of the reaction vessel. This procedure reduced the variability in the results considerably. Further experiments with 50 and 100/~L volumes of bromine decreased the recovery of monobrominated acetate without apparent formation of the di- and tribromo derivatives. Figure 3 demonstrates that although the concentration of sulfur has some influence on chemical yield, it probably serves as a catalyst since nearly quantitative yields were obtained using approximately 0.25 equivalents (0.003 mmol), with respect to acetate (0.012 mmol), of sulfur. The poorer yields at lower sulfur concentrations may be related to the difficulty in evenly and reproducibly distributing the small amounts of sulfur on the vessel walls. This would result in areas having insufficient sulfur to effectively promote the reaction between bromine and acetate. The minimum amount appeared to be between 0.05 and 0.1 mg. The order of layering the sulfur and
1009080706050"
30-
u
r
0
I
5
110
BROMINE VOLUME (pL)
Fig. 2. The effect of bromine volume on the chemical yield of bromoacetic acid. Sodium acetate (1.02 mg) was reacted with 0.1 mg of sulfur and various amounts of bromine at 105C for 60min. Chemical yields were determined by RP HPLC analysis.
254
DOUGLAS N. ABRAMSeta/. 100-
/
90-
80-
70-
60-
50-
40-
30-
2010ba
0
I
I
I
I
0.05
0.10
0.15
0.20
SULFUR CONCENTRATION (mg)
Fig. 3, The effect of sulfur concentration on the chemical yield of bromoacetic acid. Sodium acetate (1.02 mg) was reacted with 10 uL bromine and various amounts of sulfur at 105C for 60 min. Chemical yields were determined by RP HPLC analysis.
acetate did not have an appreciable effect on the reaction yield. The significantly different solubilities of sulfur and acetate did not allow c o n c u r r e n t application of the reagents in the same solvent. The presence of water in the reaction decreased the yield. The vials were preheated to 105 C for 10 min, before addition of the bromine, to drive off the water. G o o d yields could be o b t a i n e d without this step but the results were less reproducible. Bromoacetic acid was observed to slowly decompose at temperatures greater than 110 C in the presence of b r o m i n e and sulfur. However, decomposition was less than 5% alter 6 0 m i n at temperatures between 100 and 110 C. At temperatures below 100 C, the reaction proceeded much more slowly. The optimal reaction conditions l\~r the conversion of 1 mg of sodium acetate to b r o m o a c e t a t e were to react the sodium acetate at 105 C for 60 mira in the presence of 0.01 mE b r o m i n e and 0.1 mg of elemental sulfur. The results from six ExperimEnts using [~4C]sodium acetatc tracer added to the reaction are given in Table 1. The radiochemical yields were determined by RP H P L C with a radioactivity flow detector. In addition, 3 0 s eluatE fractions were counted by liquid scintillation counting. The chemical yields were determined from the area under the u.v. a b s o r p t i o n peak on the RP H P L C trace of the reaction aliquot as c o m p a r e d to a reference standard. The resultant r a d i o c h r o m a t o g r a m overlaid with a u.v. a b s o r p t i o n trace from the analysis of reaction 5 is reproduced in Fig. 4. The m a j o r u.v. peaks present
W 1
<
_o
,x-
~3 <
o
co rn ,< >
1
B___z< INJECTION
ACETATE
U V ( 2 1 4 nm)
Br -'
BROMOACETATE
TIME
Fig. 4. Radio high pressure liquid chromatogram of [ ~4C]bromoacetate. Column: Waters Radial Pak C-8. Solvent: 0.01M KH2PO4:0.01 M tetrabutylammonium phosphate: flow rate: I m L m i n : u.v: 214nm: radioactivity detector: Flo-One liquid scintillation mode. Trace A: t4C radioactivity. Trace B: u.v absorption.
Radiosynthesis of bromoacetic acid Table I. Radiochemical yield of [14Clbromoacetate % Yield* Reaction 1 2 3 4 5 6
Chemical (u.v.)+ 84.7 35.8 70.2 77.0 99.1 93.0
(LSC)++ 98.7 44.1 78.5 94.2 93.3 --
Radiochemical (flow)§ ---94.2 94.9 91.7
*[~4C]Sodium acetate (1.0 mg) was reacted with 10 # L bromine and 0.01 mg sulfur at 105 C for 60rain. +Product absorption peak integration compared to known standard. +Liquid scintillation analysis of 30s (0.5mL) eluate fractions. §Product radioactivity peak integration compared to known standard. are acetate (1) bromide ion (2) and bromoacetate (3). The concentration o f acetate after the reaction was too low to detect under these conditions, therefore the acetate (peak 1) has been overlaid from a trace of a sodium acetate standard. Only two i m p o r t a n t peaks appear in the radioactive ~4C trace and these corres p o n d to acetate and bromoacetate. These two peaks represented more than 95% o f the injected radioactivity. There was no indication of formation of either the di- or tribrominated reaction products. Similar analysis o f the 3H reaction product resulted in a third radioactive peak (3H) which had a retention time corresponding to the column void volume (data not included). This extra peak probably corresponds to [3H]water, although this has not been confirmed. This would be an expected by-product due to exchange between the water added to quench the reaction and [3H]HBr formed in the bromination of [3H]acetate (Scheme 1). Statistically, one would expect to lose approximatley one-third o f the 3H activity. This is supported by the data given in Table 2. Reaction 4 in which only 0.01 mg o f sodium acetate Table 2. Radiochemical yield of [~H]bromoacetate Radiochemical yield* % [~H]acetatet Reaction 1 2 3 4~
Consumed 98,9 91.1 98.4 60.8
% Initial ~H concentration+ + Observed 73.7 60.0 67.2 37.7
Theoretical (65.9) (60.7) (65.6) (40.5)
*[~H]Sodium acetate (1.0 mg) was reacted with l0 # L bromine and 0. Img sulfur at 105C for 60rain. *Determined as 100 % 3H in acetate peak on RP HPLC analysis and liquid scintillation counting. ,+Determined by liquid scintillation counting of bromoacetate peak on RP-HPLC analysis. §[3HlSodium acetate (0.01 rag) was reacted with 10 ~L bromine and 0.1 mg sulfur at 105 C for 60rain.
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was used (no carrier acetate was added to the reaction) did not yield as well as the carrier added reactions. This may be due to the need to evenly distribute the reagents on the vessel wall. This process was made more difficult by the small acetate concentration used.
Conclusions The radiochemical synthesis o f bromoacetic acid labelled with either 3H or ~4C can be accomplished in high radiochemical yields via the sulfur catalyzed b r o m i n a t i o n o f appropriately labelled sodium acetate. The reaction proceeds readily at acetate concentrations o f approximately 1 mg; however, the radiochemical yields decreased when the a m o u n t o f acetate was decreased to 0.01 mg. The reaction product was not contaminated with either the di- or tribromoacetic acid derivatives under the conditions employed. Acknowledgements--The authors would like to thank Ms Peggy Schon and Susan Joy for their patient handling of the manuscript.
References Atkinson J. G., Gsakvary J. J., Morse A. T. and Stuart R. S. (1966) Base catalyzed deuterium exchange reactions. A simple synthesis of ct-deutero carboxylic acids. In Proc. 2nd Int. Conf. on Methods o f Preparing and Storing Labelled Compounds (Ed. Sirchis J.). European Atomic Energy Community, Brussels. Bloch K. (1949) The synthesis of glutathione in isolated liver. J. Biol. Chem. 179, 1245. Brunsberg U., Bunte O. and Lindskoug I. (1965) Synthesis of bromoacetic acid-l,2-C14 and glycolic acid-l,2-C14. Acta Chem. Scand. 19, 246. Ehrensvard G. and Liljekvist J. (1959) Synthesis of uracil2,4,5,6-C 14 with high specific activity. Studies in microsynthesis I. Acta Chem. Scand. 13, 2070. Fields M., Rothschild S. and Leoffere M. A. (1952) Synthesis of 2,4-dichlorophenoxyacetic acid labeled with isotopic carbon. J. Am. Chem. Soc. 74, 2435. Gidez L. I. and Karnovsky M. L. (1952). A synthesis of ~tor fl-CI4-1abeled glycerol. J. Am. Chem. Soc. 74, 2413. Genvresse M. P. (1892) Nouvelle pr6paration des acides gras bromes. Bull Soc. Chim. Ft. 7, 364. Ostwald R. (1948) Synthesis of chloroacetic acid and glycine labeled with radioactive carbon in the carboxyl group. J. Biol. Chem. 173, 207. Ropp G. A. (1950) C ~4 Tracer studies in the synthesis o f malonic acid-2-C ~4 and diethyl malonate-2-C TM. J. Am. Chem. Soc. 72, 4459. Yeh S. M., Sherman D. G. and Meares C. F. (1979) A new route to "bifunctional" chelating agents: conversion of amino acids to analogs of ethylenedinitrilotetracetic acid. Anal. Biochem. 100, 152.