Transcript PowerPoint-Präsentation - Universität Heidelberg

PRELIMINARY RESULTS ON THE SYNTHESIS OF METAL COMPLEX-PNA CONJUGATES
Felix Zelder, Andriy A. Mokhir, Roland Krämer, Igor Fritsky
Ruprecht-Karls-Universität Heidelberg, Anorganisch-Chemisches Institut Im Neuenheimer Feld 270, D-69120 Heidelberg. E-mail: [email protected]
RESULTS
ABSTRACT
Nonenzymatic hydrolysis of the phosphodiester
backbone of nucleic acids is an attractive research
aim in molecular biology, but the sequence specific
hydrolysis of ssDNA was achieved only in one case
with a conjugate consisting of a Ce(IV)-complex
attached to a DNA-oligonucleotide.[1] The
nonenzymatic sequence specific hydrolysis of
dsDNA is not reported. Since peptide nucleic acids
show interesting binding properties we are
focussing on the design of metal-complex-PNAs in
a way that allows the generation of combinatorial
libraries.
Their synthesis and characterization will be
discussed.
INTRODUCTION
Restriction
enzymes
that
hydrolyse the phosphodiester
backbone of DNA play an
important role in biological
processes and in biotechnology.
These enzymes recognize specific
sequences of 4-8 base pairs.
n+
M
Here we report on the synthesis
of some conjugates of PNA
(peptide nucleic acids) (blue)
with a varity of polydentate metal- binding fragments
(yellow) , which can hydrolyse
the phosphodiester bonds of
target oligonucleotides (black).
n+
M
Coupling of carboxylic acids onto 5’-terminus
of PNA was accomplished using an activation
mixture of HBTU, HOBT, and DIEA and
protected carboxylic acids. Yields were
consistently higher then 90 %.
After treatment with different transition metal
ions the chemical stability of the modified PNA
was determined by MALDI- TOF MS.
Metal-complex PNA conjugates were analysed
by MALDI- TOF MS.
POLYDENTATE LIGANDS ATTACHED TO 5´- TERMINUS OF PNA
Fig. 1: Mechanism of sequence specific cleavage
of polynucleotides with artificial nucleases.
n+
M
Although artificial restriction enzymes offer
unlimited sequence specifity and better chemical
stability, only one case is reported in which
sequence specific hydrolysis of ssDNA was
achieved by a conjugate consisting of a Ce(IV)complex attached to a DNA- oligonucleotide.[1]
NAE
n+
M
N
Mn+
n+
M
N
N
O
N
N
N
N
O
O
n+
M
O
O
O
N
Tetradentate ligands
Bidentate ligands
SOLID PHASE SYNTHESIS OF POTENTIAL SEQUENCE SPECIFIC NUCLEASES
R:
PNA (peptide nucleic acids)
T
T
O
NH
C
O O
N
N
H2N
N
O
N
N
NH
N
O
NH
O O
HN
N
O
N
O
NH
O O
HN
N
O
N
N
HN
T
T
C
T
T
C
T
T
T
C
NH2
R
O
T
TBhoc
C
T
T
CBhoc
T
T
T
CBhoc
T
TBhoc
C
T
T
CBhoc
T
T
T
CBhoc
N
HN
1
2
R
T
T
C
T
T
C
T
T
T
C
N
OH
O
HO
N
HO
O
Fig. 2: 1; RCOOH, HBTU, HOBT, DIEA, DMF. 2; TFA/ m-Cresol.
MALDI- TOF MS OF A CONJUGATE OF PNA AND METAL COMPLEX
ACKNOWLEDGEMENTS
This work is supported by Ruprecht-KarlsUniversität Heidelberg.
Authors thank Dr. Gross (Ruprecht-KarlsUniversität Heidelberg) for his kind
support
with
mass
spectrometric
measurements.
O
O
HO
N TTCTTCTTTC
H
N
HO
O
ZrIV
O
O
O
O
Fig. 3: MALDI- TOF MS spectra of crude NAE-TTCTTCTTTC [M+H]th+ = 2807,5.
and after treatment with Zr(IV) [M+Zr(IV)-3H th] = 2896,3.
Uncertainty of m/ z = 0. 1%.
O
Zr
N
H
N TTCTTCTTTC
2+
LITERATURE
[1]M.
Komiyama, J. Biochem. 1995, 118,
665- 670.