John Hanson's Research
My research interests focus on understanding the interactions between proteins and their
ligands or substrates, with particular emphasis on understanding the forces that lead to
ligand binding, how these forces are used to promote enzymatic catalysis, and how our
knowledge of protein-ligand interactions can be used to design more potent inhibitors.
My experimental approach involves synthesis of substrates and substrate analogs that can
be used in binding and enzymatic studies. My current research projects are described
below:
Phosphonate Analogs of DNA
Restriction endonucleases comprise a large class of enzymes that cleave double stranded
DNA at specific sites leaving 5' phosphates. Although these enzymes have played a
critical role in the development of modern molecular biology, relatively little is known
about the molecular mechanism by which they cleave DNA. We are synthesizing
phosphonate DNA analogs containing a non-hydrolyzable P-CH2 bond that can be used
in crystallographic studies with BamHI or other endonucleases.
O
O
G
O
Mg2+
O
P
O
O
O
BamHI
Endonuclease
O
G
O
O
O
G
G
OH
O
O
P
O
O
Hydrolysis of DNA by BamHI endonuclease.
O
O
G
CH2
O
P
O
O
G
O
O
O
Hydrolytically stable phosphonate
DNA analog.
The synthesis of phosphonate nucleotide analogs is quite challenging. We have
succeeded in preparing the phosphonate nucleoside analog 2 in several steps from the
commercially available sugar 1. Further functional group manipulation will produce the
protected nucleotide analog 3 suitable for incorporation into an oligonucleotide using an
automated DNA synthesizer.
O
O
HO
OH
9 steps
O
GP
O
GP
DMTrO
BzO
3 steps
O
O
O
EtO
1
CH3O
P
3
O
2
OEt
P
Phosphinate Analogs of DNA: Hydrolytically Stable
DNA
Recent studies show that short strands of synthetic DNA analogs are able to prevent the
expression of specific genes. There are a number of studies investigating the use of such
oligonucleotides as therapeutic agents. One requirement of these DNA analogs is that
they be hydrolytically stable, since foreign DNA is rapidly degraded when it enters a cell.
We are developing synthetic methods for preparing "phosphinate DNA" in which the two
P-O bonds in the phosphodiester linkage of DNA are replaced with hydrolytically stable
P-C bonds. Eventually we hope to develop a procedure for convenient production of short
oligonucleotides in which all the P-O bonds in the backbone are replaced by P-C bonds.
O
O
R
H2O2
P
O
EtOTf
H3PO2
P
H
O
G
H
R
R
EtO
HO
CH2
O
1. Base
R'
O
2. R'CH2X
radical
initiator
P
R'
O
O
CH2
O
P
R
G
P
R
EtO
Synthetic strategies for preparation of phosphinates.
CH2R'
O
EtO
Hydrolytically stable phosphinate
DNA analog.
T4 Lysozyme Substrates
From a structural standpoint, the lysozyme from T4 bacteriophage is one of the most
thoroughly studied enzymes. Brian Matthews and coworkers use this enzyme as a system
in which to study ideas about protein stability; numerous mutants have been made,
analyzed for their stability, and their crystal structures determined at high resolution.
Despite this wealth of structural data, many questions about the substrate specificity,
kinetics, and mechanism of T4 lysozyme remain unanswered, due in large part to the
complexity of the substrate and the lack of a convenient small molecule assay. We are
synthesizing small oligosaccharide fragments of the cell wall to probe the kinetics and
substrate specificity of T4 lysozyme. The ability to synthesize substrates of T4 lysozyme
will allow us to develop a convenient, continuous fluorescence assay that will facilitate
kinetic studies on various mutant enzymes. Eventually we hope to synthesize nonhydrolyzable substrate analogs to probe the mechanism of this enzyme.
HO
HO
O
O
OR
OH
O
H2N
H
N
CO2H
HN
O
O
N
H
CO2H
Proposed T4 Lysozyme Substrate