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Direct Measurement of Nucleoside Monophosphate Delivery from a Phosphoramidate Pronucleotide by Stable Isotope Labeling and LC−ESI⁻-MS/MS

Kim, Jisook and Chou, Tsui-fen and Griesgraber, George W. and Wagner, Carston R. (2004) Direct Measurement of Nucleoside Monophosphate Delivery from a Phosphoramidate Pronucleotide by Stable Isotope Labeling and LC−ESI⁻-MS/MS. Molecular Pharmaceutics, 1 (2). pp. 102-111. ISSN 1543-8384. doi:10.1021/mp0340338. https://resolver.caltech.edu/CaltechAUTHORS:20220323-565463000

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Abstract

Amino acid phosphoramidates of nucleosides have been shown to be potent antiviral and anticancer agents with the potential to act as nucleoside monophosphate prodrugs. To access their ability to deliver 3‘-azido-3‘-deoxythymidine (AZT) 5‘-monophosphate to cells, the decomposition pathway of an ¹⁸O-labeled AZT amino acid phosphoramidate was investigated by capillary reverse-phase high-performance liquid chromatography interfaced with negative ion electrospray ionization mass spectrometry (LC−ESI--MS/MS). ¹⁸O-labeled l-AZT tryptophan phosphoramidate methyl ester ([¹⁸O]2) was synthesized with an ¹⁸O/¹⁶O relative ratio of 1.22 ± 0.18. For CEM cells, a human T-lymphoblast leukemia cell line, incubated with [18O]2, values of 1.55 ± 0.37, 0.34, and 0.13 were found for the ¹⁸O/¹⁶O relative ratio of intracellular AZT-MP for time intervals of 0.5, 4, and 20 h, respectively. The decrease in the level of labeled AZT-MP in CEM cells corresponded to a rapid increase in the amount of intracellular AZT presumably by dephosphorylation of AZT-MP. In contrast, for peripheral blood mononuclear cells (PBMCs), the ¹⁸O/¹⁶O relative ratio values of intracellular AZT-MP were 1.43, 1.06, and 0.61 for time intervals of 0.5, 4, and 20 h, respectively. Intracellular AZT in PBMCs was nearly undetectable for each time interval. Taken together, these results are consistent with the detection of direct P−N bond cleavage by CEM cells and PBMCs. However, AZT phosphoramidates are able to more effectively deliver AZT-MP to PBMCs than to CEM cells. Differential expression of 5‘-nucleotidase in CEM cells relative to PBMCs is likely the reason for this discrepancy. Although applied to a phosphoramidate pronucleotide, the judicious use of ¹⁸O labeling and LC−MS is a general approach that could be applied to the investigation of the intracellular fate of other pronucleotides.


Item Type:Article
Related URLs:
URLURL TypeDescription
https://doi.org/10.1021/mp0340338DOIArticle
ORCID:
AuthorORCID
Chou, Tsui-fen0000-0003-2410-2186
Wagner, Carston R.0000-0001-7927-719X
Additional Information:© 2004 American Chemical Society. Received 24 December 2003. Published online 3 February 2004. Published in issue 1 March 2004. We thank Dr. Balfour's group in ACTU in the Department of Laboratory Medicine and Pathology at the University of Minnesota for providing PBMCs. Also, we thank Dr. Natalia Y. Tretyakova at the University of Minnesota for her assistance in LC−MS. We are grateful to the Unversity of Minnesota Cancer Center for access to the capillary HPLC−ESI-MS instrument. This research is supported by NIH-NCI Grant CA 89615.
Funders:
Funding AgencyGrant Number
NIHCA 89615
Subject Keywords:Prodrugs; antiviral agent; AZT; phosphoramidate
Issue or Number:2
DOI:10.1021/mp0340338
Record Number:CaltechAUTHORS:20220323-565463000
Persistent URL:https://resolver.caltech.edu/CaltechAUTHORS:20220323-565463000
Official Citation:Direct Measurement of Nucleoside Monophosphate Delivery from a Phosphoramidate Pronucleotide by Stable Isotope Labeling and LC−ESI--MS/MS Jisook Kim, Tsui-fen Chou, George W. Griesgraber, and Carston R. Wagner Molecular Pharmaceutics 2004 1 (2), 102-111 DOI: 10.1021/mp0340338
Usage Policy:No commercial reproduction, distribution, display or performance rights in this work are provided.
ID Code:114035
Collection:CaltechAUTHORS
Deposited By: George Porter
Deposited On:25 Mar 2022 16:39
Last Modified:25 Mar 2022 16:39

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