Nucleotide Chemistry and Biochemistry at SLU
Michael B. DoughtyAssociate Professor of Biochemistry
Department of Chem & PhysSoutheastern Louisiana University
1
Representative Nucleotides
-O
N
NN
N
NH2
OOP
O-
O
-O
N
NN
N
NH2
OOP
O-
O
OHOH OH
2'-deoxynucleotide 5'-phosphate (DNA form)
nucleotide 5'-phosphate (RNA form)
2'-deoxyribosesugar
base(purine)
phosphate ester
ribose sugar
5’
3’
2
DNA Synthesis by Polymerase
DNA template
DNA primer3’-OH
dNTP PPi
DNA template
DNA primerO-N-3’-OH
Inhibitors of DNA polymerase are traditionally used as anti-metabolites to treat cancer and DNA virus infections (e.g., Herpes simplex I and II; Cytomegalovirus; Hepatitis B virus, etc.)
3’ 5’DNA Pol
(repeat)
5’
3’ 5’
5’
3
N
NN
N SO
NH2
HO
N3
O
OPOPOPO-O
O-
O
O-
O
O-
template binding region
sugar binding region
triphosphate binding region
minor groove subsituent added to stabilize binary Pol-dNTP complex
Novel Template-Competitive DNA Polymerase Inhibitors
Doughty & Moore, 1996
4
DNA Synthesis by Reverse Transcriptase
Reverse transcriptase (RT) is a retroviral enzyme required for incorporation of a viral RNA genome into host DNA
RNA RNART
DNA
RT
RT
DNA
DNA
DNA Integration and viral particle production
RT inhibitors are or could be used to treat retroviral disease (HIV; Hepatitis A and C; Rabies virus; Mumps and Influenza viruses)
5
Template-Competitive RT Inhibitors
N
NN
O
HO
S
O
R
NH4O9P3O
triphosphate binding pocket
2'-deoxyribose binding pocket
minor groove subsituent added to stabilize binary RT-dNTP complex
lipophilictemplatebinding pocket
N
Required base modifications for RT inhibition
Doughty, Li, & Lin, 2001
6
Structural Comparison of RT and DNA Pol Inhibitors
Conformationally, the etheno group forces a population where the side chain is bent under the sugar
DNA Pol TC inhibitors RT inhibitors
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GTP Binding Proteins (G-Proteins)
8
GDP
activation
GTP GDP
GTP
activates cell processes:nerve conductionmetabolismsecretionreplication
+
H2O
Pi
GDP
Conformational Probes of G-Proteins
Seifert & Doughty, unpublished
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Techniques
1. Chemical synthesis of nucleotides.
2. Conformational analysis of nucleotides and enzyme/protein binding sites.
3. Kinetic analysis and other bioassays.
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