Synthesis of nucleotides with specific radiolabels in ribose. Primary 14C and secondary 3H kinetic isotope effects on acid-catalyzed glycosidic bond hydrolysis of AMP, dAMP, and inosine

D. W. Parkin, H. B. Leung, Vern L. Schramm

Research output: Contribution to journalArticle

83 Citations (Scopus)

Abstract

Adenosine 5'-phosphate was synthesized with 3H or 14C label specifically located as [1'-3H]AMP, [1'-14C] AMP, [5'-3H]AMP, and [5'-14C]AMP. The synthesis was accomplished from adenine and glucose or adenine and ribose using enzymes from the pentose pathway and/or from the purine salvage pathways. Structural analysis of the compounds confirmed the locations of the radiolabels. The methods provide a general scheme for the efficient synthesis of adenine nucleotides of high purity with 3H or 14C at any stable position on the ribose ring. Synthesis of [5'-14C]dAMP and [1'-3H]dAMP from the corresponding ribonucleotides was accomplished with ribonucleotide reductase. Labeled inosine was prepared by enzymatic dephosphorylation and deamination of labeled AMP. These compounds have been used to measure the secondary kinetic isotope effects on the acid-catalyzed hydrolysis of the N-glycosidic bond of AMP, dAMP, and inosine and the corresponding primary kinetic isotope effects with AMP. Acid hydrolysis in 0.1 or 0.2 N HCl at 50°C gave 1H/3H secondary kinetic isotope effects of 1.23 ± 0.01, 1.26 ± 0.01, and 1.230 ± 0.003 for AMP, dAMP, and inosine, respectively. The primary kinetic isotope effect for 12C/14C was 1.049 ± 0.010 for AMP. The apparent rate constants for hydrolysis under these conditions were similar for inosine and AMP and were in the range 10-6-10-5 s-1. Acid hydrolysis of dAMP is approximately 1000-fold faster than AMP but gives a similar 1H/3H kinetic isotope effect. The results of secondary isotope effects indicate that the transition states for the acid-catalyzed hydrolysis of the N-glycosidic bonds of inosine, AMP, and dAMP have similar bonding to 1'-3H in the transition state and have considerable carboxonium character. Results with [1'-14C]AMP demonstrate that a significant primary isotope effect can be measured in the acid solvolysis of the N-glycosidic bond of AMP.

Original languageEnglish (US)
Pages (from-to)9411-9417
Number of pages7
JournalJournal of Biological Chemistry
Volume259
Issue number15
StatePublished - 1984
Externally publishedYes

Fingerprint

Inosine
Ribose
Adenosine Monophosphate
Isotopes
Hydrolysis
Nucleotides
Kinetics
Acids
Adenine
Pentoses
Ribonucleotides
Ribonucleotide Reductases
Salvaging
Deamination
Adenine Nucleotides
Structural analysis

ASJC Scopus subject areas

  • Biochemistry

Cite this

@article{b85dccdf21a948ec9a08b53e041ce5bf,
title = "Synthesis of nucleotides with specific radiolabels in ribose. Primary 14C and secondary 3H kinetic isotope effects on acid-catalyzed glycosidic bond hydrolysis of AMP, dAMP, and inosine",
abstract = "Adenosine 5'-phosphate was synthesized with 3H or 14C label specifically located as [1'-3H]AMP, [1'-14C] AMP, [5'-3H]AMP, and [5'-14C]AMP. The synthesis was accomplished from adenine and glucose or adenine and ribose using enzymes from the pentose pathway and/or from the purine salvage pathways. Structural analysis of the compounds confirmed the locations of the radiolabels. The methods provide a general scheme for the efficient synthesis of adenine nucleotides of high purity with 3H or 14C at any stable position on the ribose ring. Synthesis of [5'-14C]dAMP and [1'-3H]dAMP from the corresponding ribonucleotides was accomplished with ribonucleotide reductase. Labeled inosine was prepared by enzymatic dephosphorylation and deamination of labeled AMP. These compounds have been used to measure the secondary kinetic isotope effects on the acid-catalyzed hydrolysis of the N-glycosidic bond of AMP, dAMP, and inosine and the corresponding primary kinetic isotope effects with AMP. Acid hydrolysis in 0.1 or 0.2 N HCl at 50°C gave 1H/3H secondary kinetic isotope effects of 1.23 ± 0.01, 1.26 ± 0.01, and 1.230 ± 0.003 for AMP, dAMP, and inosine, respectively. The primary kinetic isotope effect for 12C/14C was 1.049 ± 0.010 for AMP. The apparent rate constants for hydrolysis under these conditions were similar for inosine and AMP and were in the range 10-6-10-5 s-1. Acid hydrolysis of dAMP is approximately 1000-fold faster than AMP but gives a similar 1H/3H kinetic isotope effect. The results of secondary isotope effects indicate that the transition states for the acid-catalyzed hydrolysis of the N-glycosidic bonds of inosine, AMP, and dAMP have similar bonding to 1'-3H in the transition state and have considerable carboxonium character. Results with [1'-14C]AMP demonstrate that a significant primary isotope effect can be measured in the acid solvolysis of the N-glycosidic bond of AMP.",
author = "Parkin, {D. W.} and Leung, {H. B.} and Schramm, {Vern L.}",
year = "1984",
language = "English (US)",
volume = "259",
pages = "9411--9417",
journal = "Journal of Biological Chemistry",
issn = "0021-9258",
publisher = "American Society for Biochemistry and Molecular Biology Inc.",
number = "15",

}

TY - JOUR

T1 - Synthesis of nucleotides with specific radiolabels in ribose. Primary 14C and secondary 3H kinetic isotope effects on acid-catalyzed glycosidic bond hydrolysis of AMP, dAMP, and inosine

AU - Parkin, D. W.

AU - Leung, H. B.

AU - Schramm, Vern L.

PY - 1984

Y1 - 1984

N2 - Adenosine 5'-phosphate was synthesized with 3H or 14C label specifically located as [1'-3H]AMP, [1'-14C] AMP, [5'-3H]AMP, and [5'-14C]AMP. The synthesis was accomplished from adenine and glucose or adenine and ribose using enzymes from the pentose pathway and/or from the purine salvage pathways. Structural analysis of the compounds confirmed the locations of the radiolabels. The methods provide a general scheme for the efficient synthesis of adenine nucleotides of high purity with 3H or 14C at any stable position on the ribose ring. Synthesis of [5'-14C]dAMP and [1'-3H]dAMP from the corresponding ribonucleotides was accomplished with ribonucleotide reductase. Labeled inosine was prepared by enzymatic dephosphorylation and deamination of labeled AMP. These compounds have been used to measure the secondary kinetic isotope effects on the acid-catalyzed hydrolysis of the N-glycosidic bond of AMP, dAMP, and inosine and the corresponding primary kinetic isotope effects with AMP. Acid hydrolysis in 0.1 or 0.2 N HCl at 50°C gave 1H/3H secondary kinetic isotope effects of 1.23 ± 0.01, 1.26 ± 0.01, and 1.230 ± 0.003 for AMP, dAMP, and inosine, respectively. The primary kinetic isotope effect for 12C/14C was 1.049 ± 0.010 for AMP. The apparent rate constants for hydrolysis under these conditions were similar for inosine and AMP and were in the range 10-6-10-5 s-1. Acid hydrolysis of dAMP is approximately 1000-fold faster than AMP but gives a similar 1H/3H kinetic isotope effect. The results of secondary isotope effects indicate that the transition states for the acid-catalyzed hydrolysis of the N-glycosidic bonds of inosine, AMP, and dAMP have similar bonding to 1'-3H in the transition state and have considerable carboxonium character. Results with [1'-14C]AMP demonstrate that a significant primary isotope effect can be measured in the acid solvolysis of the N-glycosidic bond of AMP.

AB - Adenosine 5'-phosphate was synthesized with 3H or 14C label specifically located as [1'-3H]AMP, [1'-14C] AMP, [5'-3H]AMP, and [5'-14C]AMP. The synthesis was accomplished from adenine and glucose or adenine and ribose using enzymes from the pentose pathway and/or from the purine salvage pathways. Structural analysis of the compounds confirmed the locations of the radiolabels. The methods provide a general scheme for the efficient synthesis of adenine nucleotides of high purity with 3H or 14C at any stable position on the ribose ring. Synthesis of [5'-14C]dAMP and [1'-3H]dAMP from the corresponding ribonucleotides was accomplished with ribonucleotide reductase. Labeled inosine was prepared by enzymatic dephosphorylation and deamination of labeled AMP. These compounds have been used to measure the secondary kinetic isotope effects on the acid-catalyzed hydrolysis of the N-glycosidic bond of AMP, dAMP, and inosine and the corresponding primary kinetic isotope effects with AMP. Acid hydrolysis in 0.1 or 0.2 N HCl at 50°C gave 1H/3H secondary kinetic isotope effects of 1.23 ± 0.01, 1.26 ± 0.01, and 1.230 ± 0.003 for AMP, dAMP, and inosine, respectively. The primary kinetic isotope effect for 12C/14C was 1.049 ± 0.010 for AMP. The apparent rate constants for hydrolysis under these conditions were similar for inosine and AMP and were in the range 10-6-10-5 s-1. Acid hydrolysis of dAMP is approximately 1000-fold faster than AMP but gives a similar 1H/3H kinetic isotope effect. The results of secondary isotope effects indicate that the transition states for the acid-catalyzed hydrolysis of the N-glycosidic bonds of inosine, AMP, and dAMP have similar bonding to 1'-3H in the transition state and have considerable carboxonium character. Results with [1'-14C]AMP demonstrate that a significant primary isotope effect can be measured in the acid solvolysis of the N-glycosidic bond of AMP.

UR - http://www.scopus.com/inward/record.url?scp=0021229835&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=0021229835&partnerID=8YFLogxK

M3 - Article

VL - 259

SP - 9411

EP - 9417

JO - Journal of Biological Chemistry

JF - Journal of Biological Chemistry

SN - 0021-9258

IS - 15

ER -