nucleotide chemistry and biochemistry at slu

Nucleotide Chemistry and Biochemistry at SLU

Michael B. DoughtyAssociate Professor of Biochemistry

Department of Chem & PhysSoutheastern Louisiana University

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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

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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)

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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Seifert & Doughty, unpublished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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