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, Carston R Wagner

Research output: Contribution to journalArticle

26 Citations (Scopus)

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.

Original languageEnglish (US)
Pages (from-to)102-111
Number of pages10
JournalMolecular pharmaceutics
Volume1
Issue number2
StatePublished - Jan 1 2004

Fingerprint

Isotope Labeling
Nucleosides
Blood Cells
Amino Acids
5'-Nucleotidase
Electrospray Ionization Mass Spectrometry
Zidovudine
Prodrugs
Reverse-Phase Chromatography
Antineoplastic Agents
Antiviral Agents
phosphoramidic acid
Leukemia
High Pressure Liquid Chromatography
Ions
Cell Line

Cite this

Direct measurement of nucleoside monophosphate delivery from a phosphoramidate pronucleotide by stable isotope labeling and LC-ESI(-)-MS/MS. / Kim, Jisook; Chou, Tsui fen; Griesgraber, George W.; Wagner, Carston R.

In: Molecular pharmaceutics, Vol. 1, No. 2, 01.01.2004, p. 102-111.

Research output: Contribution to journalArticle

@article{3a3163798fd74de8b5ce56be5f3a6614,
title = "Direct measurement of nucleoside monophosphate delivery from a phosphoramidate pronucleotide by stable isotope labeling and LC-ESI(-)-MS/MS.",
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.",
author = "Jisook Kim and Chou, {Tsui fen} and Griesgraber, {George W.} and Wagner, {Carston R}",
year = "2004",
month = "1",
day = "1",
language = "English (US)",
volume = "1",
pages = "102--111",
journal = "Molecular Pharmaceutics",
issn = "1543-8384",
publisher = "American Chemical Society",
number = "2",

}

TY - JOUR

T1 - Direct measurement of nucleoside monophosphate delivery from a phosphoramidate pronucleotide by stable isotope labeling and LC-ESI(-)-MS/MS.

AU - Kim, Jisook

AU - Chou, Tsui fen

AU - Griesgraber, George W.

AU - Wagner, Carston R

PY - 2004/1/1

Y1 - 2004/1/1

N2 - 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.

AB - 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.

UR - http://www.scopus.com/inward/record.url?scp=15444363853&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=15444363853&partnerID=8YFLogxK

M3 - Article

C2 - 15832506

AN - SCOPUS:15444363853

VL - 1

SP - 102

EP - 111

JO - Molecular Pharmaceutics

JF - Molecular Pharmaceutics

SN - 1543-8384

IS - 2

ER -