Development of an assay for the estimation of N10-propargyl-5,8-dideazafolic acid polyglutamates in tumor cells

A method is described herein for the isolation and quantitation of polyglutamates of the thymidylate synthase (TS) inhibitor N 10-propargyl-5,8-dideazafolic acid (CB3717) in tumor cells exposed to the drug in vitro. Cells were incubated with 50 μ m 3H-CB3717 for 12 h and then disrupted by sonication...

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Veröffentlicht in:Analytical biochemistry 1988-08, Vol.172 (2), p.344-355
Hauptverfasser: Sikora, Ewa, Newell, David R., Jackman, Ann L., Simmonds, Alan J., Jones, Terence R., Calvert, A.Hilary
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container_end_page 355
container_issue 2
container_start_page 344
container_title Analytical biochemistry
container_volume 172
creator Sikora, Ewa
Newell, David R.
Jackman, Ann L.
Simmonds, Alan J.
Jones, Terence R.
Calvert, A.Hilary
description A method is described herein for the isolation and quantitation of polyglutamates of the thymidylate synthase (TS) inhibitor N 10-propargyl-5,8-dideazafolic acid (CB3717) in tumor cells exposed to the drug in vitro. Cells were incubated with 50 μ m 3H-CB3717 for 12 h and then disrupted by sonication. CB3717 and its polyglutamates were extracted by boiling in 0.01 m Tris-HCl pH 10. The extract was concentrated by lyophilization and analyzed by reverse phase HPLC (10 × 0.46-cm Polygosil 5-μm C 18 column) using linear gradient elution (5–16% acetonitrile in 0.1 m sodium acetate, pH 5, over 15 min, 2 ml/min). Recovery of radioactivity at each stage of the method was >70%. CB3717 and its polyglutamates were identified by co-chromatography with synthetic standards and by inhibition of partially purified TS. Quantitation was by means of radiochemical analysis. The 3H-CB3717 used in these studies was prepared by catalytic tritiation of diethyl-(2-chloro-4-nitrobenzoyl)- l-glutamate followed by consecutive alkylation with propargyl bromide and 2-amino-6-bromomethyl-3,4-dihydro-4-oxoquinazoline hydrobromide. The free diacid was prepared as required by hydrolysis in sodium hydroxide and purified by HPLC. Tritiation in only one position was confirmed by 3H NMR. Following the exposure of L1210 leukemia cells to 50 μ m 3H-CB3717 for 12 h the total cellular radioactivity level was approximately 7 μ m, of which 27% was present as polyglutamated metabolites with four and five glutamate residues.
doi_str_mv 10.1016/0003-2697(88)90454-X
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Cells were incubated with 50 μ m 3H-CB3717 for 12 h and then disrupted by sonication. CB3717 and its polyglutamates were extracted by boiling in 0.01 m Tris-HCl pH 10. The extract was concentrated by lyophilization and analyzed by reverse phase HPLC (10 × 0.46-cm Polygosil 5-μm C 18 column) using linear gradient elution (5–16% acetonitrile in 0.1 m sodium acetate, pH 5, over 15 min, 2 ml/min). Recovery of radioactivity at each stage of the method was &gt;70%. CB3717 and its polyglutamates were identified by co-chromatography with synthetic standards and by inhibition of partially purified TS. Quantitation was by means of radiochemical analysis. The 3H-CB3717 used in these studies was prepared by catalytic tritiation of diethyl-(2-chloro-4-nitrobenzoyl)- l-glutamate followed by consecutive alkylation with propargyl bromide and 2-amino-6-bromomethyl-3,4-dihydro-4-oxoquinazoline hydrobromide. The free diacid was prepared as required by hydrolysis in sodium hydroxide and purified by HPLC. Tritiation in only one position was confirmed by 3H NMR. 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Cells were incubated with 50 μ m 3H-CB3717 for 12 h and then disrupted by sonication. CB3717 and its polyglutamates were extracted by boiling in 0.01 m Tris-HCl pH 10. The extract was concentrated by lyophilization and analyzed by reverse phase HPLC (10 × 0.46-cm Polygosil 5-μm C 18 column) using linear gradient elution (5–16% acetonitrile in 0.1 m sodium acetate, pH 5, over 15 min, 2 ml/min). Recovery of radioactivity at each stage of the method was &gt;70%. CB3717 and its polyglutamates were identified by co-chromatography with synthetic standards and by inhibition of partially purified TS. Quantitation was by means of radiochemical analysis. The 3H-CB3717 used in these studies was prepared by catalytic tritiation of diethyl-(2-chloro-4-nitrobenzoyl)- l-glutamate followed by consecutive alkylation with propargyl bromide and 2-amino-6-bromomethyl-3,4-dihydro-4-oxoquinazoline hydrobromide. The free diacid was prepared as required by hydrolysis in sodium hydroxide and purified by HPLC. Tritiation in only one position was confirmed by 3H NMR. Following the exposure of L1210 leukemia cells to 50 μ m 3H-CB3717 for 12 h the total cellular radioactivity level was approximately 7 μ m, of which 27% was present as polyglutamated metabolites with four and five glutamate residues.</description><subject>Animals</subject><subject>Biological and medical sciences</subject><subject>cancer chemotherapy</subject><subject>Chromatography, High Pressure Liquid</subject><subject>drug metabolism</subject><subject>Folic Acid - analogs &amp; derivatives</subject><subject>Folic Acid - analysis</subject><subject>folic acids</subject><subject>HPLC, drug metabolites</subject><subject>isotope analysis</subject><subject>Leukemia L1210</subject><subject>Medical sciences</subject><subject>Mice</subject><subject>Neoplasms - analysis</subject><subject>organic synthesis, radioactive</subject><subject>Peptides - analysis</subject><subject>Polyglutamic Acid - analogs &amp; derivatives</subject><subject>Polyglutamic Acid - analysis</subject><subject>Quinazolines - analysis</subject><subject>Thymidylate Synthase - antagonists &amp; 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Newell, David R. ; Jackman, Ann L. ; Simmonds, Alan J. ; Jones, Terence R. ; Calvert, A.Hilary</author></sort><facets><frbrtype>5</frbrtype><frbrgroupid>cdi_FETCH-LOGICAL-c319t-efe35ae4c9500be5eeea1608dc27e665ef213f16e03d10ccb9567d96361b9a403</frbrgroupid><rsrctype>articles</rsrctype><prefilter>articles</prefilter><language>eng</language><creationdate>1988</creationdate><topic>Animals</topic><topic>Biological and medical sciences</topic><topic>cancer chemotherapy</topic><topic>Chromatography, High Pressure Liquid</topic><topic>drug metabolism</topic><topic>Folic Acid - analogs &amp; derivatives</topic><topic>Folic Acid - analysis</topic><topic>folic acids</topic><topic>HPLC, drug metabolites</topic><topic>isotope analysis</topic><topic>Leukemia L1210</topic><topic>Medical sciences</topic><topic>Mice</topic><topic>Neoplasms - analysis</topic><topic>organic synthesis, radioactive</topic><topic>Peptides - analysis</topic><topic>Polyglutamic Acid - analogs &amp; derivatives</topic><topic>Polyglutamic Acid - analysis</topic><topic>Quinazolines - analysis</topic><topic>Thymidylate Synthase - antagonists &amp; 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Cells were incubated with 50 μ m 3H-CB3717 for 12 h and then disrupted by sonication. CB3717 and its polyglutamates were extracted by boiling in 0.01 m Tris-HCl pH 10. The extract was concentrated by lyophilization and analyzed by reverse phase HPLC (10 × 0.46-cm Polygosil 5-μm C 18 column) using linear gradient elution (5–16% acetonitrile in 0.1 m sodium acetate, pH 5, over 15 min, 2 ml/min). Recovery of radioactivity at each stage of the method was &gt;70%. CB3717 and its polyglutamates were identified by co-chromatography with synthetic standards and by inhibition of partially purified TS. Quantitation was by means of radiochemical analysis. The 3H-CB3717 used in these studies was prepared by catalytic tritiation of diethyl-(2-chloro-4-nitrobenzoyl)- l-glutamate followed by consecutive alkylation with propargyl bromide and 2-amino-6-bromomethyl-3,4-dihydro-4-oxoquinazoline hydrobromide. The free diacid was prepared as required by hydrolysis in sodium hydroxide and purified by HPLC. Tritiation in only one position was confirmed by 3H NMR. Following the exposure of L1210 leukemia cells to 50 μ m 3H-CB3717 for 12 h the total cellular radioactivity level was approximately 7 μ m, of which 27% was present as polyglutamated metabolites with four and five glutamate residues.</abstract><cop>San Diego, CA</cop><pub>Elsevier Inc</pub><pmid>2461114</pmid><doi>10.1016/0003-2697(88)90454-X</doi><tpages>12</tpages></addata></record>
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subjects Animals
Biological and medical sciences
cancer chemotherapy
Chromatography, High Pressure Liquid
drug metabolism
Folic Acid - analogs & derivatives
Folic Acid - analysis
folic acids
HPLC, drug metabolites
isotope analysis
Leukemia L1210
Medical sciences
Mice
Neoplasms - analysis
organic synthesis, radioactive
Peptides - analysis
Polyglutamic Acid - analogs & derivatives
Polyglutamic Acid - analysis
Quinazolines - analysis
Thymidylate Synthase - antagonists & inhibitors
Tumors
title Development of an assay for the estimation of N10-propargyl-5,8-dideazafolic acid polyglutamates in tumor cells
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