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Polyamides as Artificial Regulators of Gene Expression.

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The epoxidation of terminal olefins still requires prolonged
reaction times (usually longer than 92 h) under these conditions. A faster conversion of products can be achieved by
ligand-tuning using sterically undemanding, electron-deficient pyridines like 3-cyanopyridine.I"] The epoxidation is
usually complete in less than 30 h. Presumably, the electron
deficiency of these pyridines facilitates the binding of the
olefin substrate. For very acid-sensitive products, such as
styrene oxide, pyridine is required additionally to prevent
epoxide decomposition [Eq. (b)].
MTO (O.S%),
3-CN-CSHsN (12%),
aq. H202. 30 h, 25T, CH,C12 (1.3 M)
86 %
In summary the novel ligand-accelerated catalytic system[121
described by the Sharpless et al. offers a simple, safe,
economical, and easy to carry out epoxidation reaction that
will undoubtedly find numerous applications in the synthesis
of epoxides from olefins. With the recently devised efficient
synthesis and recycling procedure for MTO by Herrmann et al.,[13] the reaction seems to be especially wellsuited for large-scale applications and has the potential for
enantioselective catalysis by using chiral pyridine derivatives
as ligands.
German version: Angew. Chem. 1997,109.2701 -2702
Keywords: chemoselectivity
catalysis rhenium
- epoxidations - homogeneous
[l) A. S. Rao in Comprehensive Organic Synthesis, Vol. 7 (Eds.: B. M. Trost,
I. Fleming, G. Pattenden), Pergamon, Oxford, 1991, pp. 358-375.
[2] T. Yamada, T. Takai, 0. Rhode, T. Mukaiyama, BUN. Chem. Soc. Jpn. 1991,
2109 - 2117.
[3] S. Inoki, T. Takai, T. Yamada, T. Mukaiyama, Chem. Lett. 1991, 941 -944.
[4] a) W. Zhang, N. H. Lee, E. N. Jacobsen, J. Am. ChemSoc. 1994,116,425426; b) H. Sasaki, R. Irie, T. Katsuki, Synlett 1994, 356.
[5] W. A. Herrmann, R. W. Fischer, D. W. Marz, Angew Chem. 1991, 103,
1706-1709; Angew. Chem. fnt. Ed. Engl. 1991,30, 1157-1160.
[6] W. A. Herrmann, R. W. Fischer, W. Scherer, M. U. Rauch, Angew. Chem.
1993,105, 1209-1212; Angew. Chem. Int. Ed. Engl. 1991,30, 1638-1641.
[ 7 ] W. A. Herrmann, R. W. Fischer, M. U. Rauch, W. Scherer, J. Mol. Caral.
1994,86,243- 266.
[8] W. Adam, C. M. MitchelLAngew. Chem. 1996,108,578-581,Angew. Chem.
Int. Ed. Engt. 1996,35, 533-535.
[9] J. Rudolph, K. L. Reddy, J. P. Chiang, K. B. Sharpless, J. Am. Chem. Soc.
[lo] W. A. Herrmann. H. Ding, R. M. Kratzer, F. E. Kiihn, J. J. Haider, R. W.
Fischer, J. Organomet. Chem. 1997, in press.
[ l l ] C. Copbret, H. Adolfsson, K. B. Sharpless, J. Chem. SOC. Chem. Commun.
1997,1565- 1566.
[12] D. J. Berrisford, C. Bolm, K. B. Sharpless, Angew. Chem. 1995,lOj: 11591171, Angew. Chem. fnt. Ed. EngL 1995,34,1059-1070.
[13] W. A. Herrmann, R. M. Kratzer, R. W. Fischer, Angew. Chem. 1997, 109,
2767-2168, Angew. Chem. In!. Ed. Engl. lW,36,2652-2654.
Poiyamides as Artificial Regulators of Gene Expression
Klaus Weisz*
Numerous control mechanisms in the living cell are based
on the sequence-specific recognition of nucleic acids. Thus,
gene expression in organisms is controlled by proteins that
selectively recognize and bind to nucleic acid sequences. In
addition, a number of natural low molecular weight drugs are
able to interact with DNA thereby interfering with genetic
processes. The prospect of using DNA-specific ligands as
novel tools in molecular biology or as drugs for a directed
control of gene expression has greatly stimulated the rational
development of artificial DNA-binding compounds. The
major as well as minor groove of a DNA double helix may
serve as a specific binding site for ligands. Each of the four
base pairs has a characteristic pattern of available hydrogen
bond donor and acceptor sites, which allows the recognition
from both grooves under formation of specific hydrogen
bonds (Scheme 1). However, the nearly symmetric arrangement of the thymine 0 2 and adenine N3 acceptor sites
~ r K.. weisz
Institut fur Organische Chemie der Freien Universitat
Takustrasse 3, D-14195 Berlin (Germany)
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0 WILEY-VCH Verlag GmbH, D-69451 Weinheim, 1997
precludes a distinction of AT and TA base pairs in the minor
groove at least by means of hydrogen bond interactions.
Netropsin and distamycin are two natural antibiotics that
preferentially bind at AIT sequences in the minor groove of a
DNA double helix (Scheme 2a, b).[l] The concave shape of
these oligopeptides with their structural complementarity to
the convex floor of the minor groove allows maximum
interactions with the DNA, which are further enforced by
the narrow groove of Am-rich DNA tracts. In addition to
electrostatic and van der Waals forces, hydrogen bonds
between the amide protons of the oligopeptides and the
adenine N3 as well as thymine 0 2 acceptor sites of the base
pairs contribute to the stability and A/T preference of these
drugs. The best-studied minor groove binding ligand is
netropsin and it has frequently been taken as the paradigm
for the rational design of synthetic drugs. The conversion of
one or both of the pyrrole rings in netropsin to imidazole was
a first attempt to extend the recognition code to G/C base
pairs.12]The resulting netropsin analogues were expected to
allow specific hydrogen bonds between the imidazole ring
nitrogen atom and the exocyclic amino group of the guanine
bases. Although G/C base pairs were increasingly tolerated in
the binding domain, there was an overall decrease in affinity
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Angew. Chem. Int. Ed. Engl. 1W,36, No. 23
major groove
minor groove
major groove
minor groove
Scheme 1. GC (top) and AT base pair (bottom). Free hydrogen bond donor and
acceptor sites in the major and minor groove of double-stranded DNA are
indicated by white and gray arrows, respectively.
and specificity of such ligands. Evidently, hydrogen bonds in
these complexes are of minor importance and the decrease of
other noncovalent interactions in the expanded minor groove
of GiC segments impairs effective ligand binding.
Unexpected NMR results from Pelton and Wemmer on
complexes of distamycin with double-helical DNA marked
the turning point in the quest for netropsin derivatives with
altered sequence specificity.13] At higher concentrations of
monocationic distamycin two molecules of the drug were
found to be bound simultaneously in the minor groove of the
central 5’-AAATT-3’segment of a DNA double helix. Within
these 2:l complexes the two ligands are stacked antiparallel,
side-by-side in the groove with each distamycin molecule
preferentially forming hydrogen bonds with nucleobases of its
adjacent DNA strand. Incorporation of a dimer results in the
expansion of the groove favoring such a 2:l motif in sequences
with a wide minor groove.
The breakthrough in the development of netropsin analogues with an expanded recognition code was finally
achieved by the groups of Dervan and Wemmer. 1-Methylimidazole-2-carboxamide-netropsin(Scheme 2 c), a synthetic
monocationic tripeptide with a N-terminal 1-methyl-2-carboxamide-imidazole residue and two N-methyl-2-carboxamide-pyrrole units recognized a 5’-TGACT-3’sequence with
Structural studies indicated the formation of
Angew Chem. In!. Ed. Engl 1997.36,No. 23
Scheme 2. Structure of DNA-binding natural and synthetic polyamides.
a) Netropsin, b) distamycin, c) l-methylimidazole-2-carboxamide-netropsin,
d) hairpin polyamide ImPyPy-y-PyPyPy-p-Dp (Im = N-methylimidazole, Py =
N-methylpyrrole. y = y-aminobutyric acid, 5,’ = p-alanine, Dp = N,N-dimethylaminopropylamide).
a 2:l complex with an antiparallel orientation of the two
ligands in the DNA minor groove, as was found earlier for
distamycin at high concentrations (Scheme 3 a) .is] The specific
recognition of GC and CG base pairs came as a surprise and
can be attributed to the expected interaction between the N3
imidazole ring nitrogen atom and the 2-amino group of
guanine protruding from the floor of the minor groove.
Evidently, a binary code consisting of two polyamide units was
required to distinguish GC, CG, and AT base pairs: an
imidazole ring paired with a pyrrole ring of the second ligand
in the dimeric motif recognizes a G C base pair; correspondingly, a pyrroleiimidazole combination targets a CG, and a
pyrroleipyrrole combination as well as N- and C-terminal
alkyl residues target AT or TA base pairs. The latter are
mostly degenerate. In contrast, the original netropsin ana-
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logues failed to bind in a 2:l motif because of their dicationic
structure, which resulted in repulsive electrostatic forces on
dimerization. The generality of this 2:l recognition code was
subsequently confirmed on various sequences.16] Shortly
thereafter further improvements in the design of pyrroleimidazole polyamides were introduced by Dervan et al. By
covalently connecting two polyamides with a y-aminobutyric
acid linker and by introducing a C-terminal P-alanine residue,
both DNA affinity and specificity were significantly increased
by the formation of a hairpin motif (Scheme 2 d and 3 b). Also,
synthesis of the polyamides was greatly facilitated by using
solid-phase method^.[^,^^']
Scheme 3. Model of the sequence-specific binding of a) l-methylimidazole-2carboxamide-netropsin and b) ImPyPy-y-PyPyPy-B-Dp in the minor groove of a
DNA double helix. Gray and black circles represent pyrrole and imidazole units
of the polyamide. respectively.
The recently published paper by Gottesfeld et al. adds one
more chapter to the successful story of pyrrole-imidazoIe
For the first time it was shown that polyamides
are capable of selectively interfering with gene expression as
artificial regulators in eukaryotic cells. An eight-ring polyamide targeted to the binding site of the transcription factor
TFIIIA in the minor groove specifically inhibited transcription of the 5s RNA gene in Xenopus kidney cells. These
experiments indicate that polyamides are capable of entering
the cell and the nucleus and displacing transcription factors
because of their higher affinity towards the target sequence.
However, despite these promising results the potential of
synthetic polyamides to act as specific gene regulators cannot
be definitely assessed yet. The majority of DNA-binding
proteins interact with nucleobases in the DNA major groove.
It remains to be seen whether and how minor groove binding
ligands are capable of effectively modulating protein-DNA
interactions and influencing the resulting regulatory mechanisms in a variety of cell types. Also, the question arises of how
selectively genes are recognized by synthetic polyamides.
Based on statistical arguments it can be shown that, depending on the base composition, approximately 17 base pairs must
be read by a ligand to recognize a unique sequence in the
human genome.[lO]However, due to the degeneracy of ATand
TA base pairs in the imidazole/pyrrole recognition code this
number must be set even higher. Unfortunately, there are still
some limitations with polyamide ligands. Polyamides having
0 WILEY-VCH Verlag GmbH, D-69451 Weinheim, 1997
more than five rings, that is with binding sites longer than 7
base pairs, show a continuous decrease in their affinity and
selectivity towards the target sequence.[”] This can be
attributed to the nonperfect adjustment of the polyamide
geometry to the double helix resulting in small irregularities,
which, in turn, are amplified with increasing length of the
binding site. By linking two three-ring polyamide subunits
with a spacer like P-alanine, the recognition sequence of such
an elongated ligand could be extended up to 13 base
However, in addition to the 2:l binding motif with fully
overlapping polyamides, “slipped” motifs with only partially
overlapped monomers are formed as well. Thus, it will be
important for the future to develop ligands that bind in a
definite and predetermined manner also to longer base
sequences with high affinity and specificity.
In contrast to polyamides, single-stranded “antigene”
oligonucleotides bind in the major groove of a double helix
forming a local triple helix. Recent studies on a covalently
linked polyamide-oligonucleotide conjugate reveal that binding occurs simultaneously to the major and minor groove even
at subnanomolar concentration^.['^] Since antigene oligonucleotides are modularly constructed from the same units as
the double helix itself and are thus optimally adjusted to the
target molecule, they have no limitations regarding the length
of their binding site. On the other hand, only ATand G C base
pairs in homopurine sequences are recognized, because an
effective recognition code for TA and CG base pairs is still
lacking. Combining both binding motifs in a chimeric ligand
not only enhances binding affinity and specificity, but may
also significantly expand the repertoire of possible target
sequences. In view of this development in the design of
artificial regulators of gene expression, DNA binding polyamides seem to be on their way to a promising future even
with some questions still unresolved.
German version: Angew. Chem. 1997,109,2702-2705
Keywords: DNA recognition
acids polyamides
- gene expression
[l] S. Neidle, Biopolymers 1997, 44, 105-121.
[2] J. w. Lown, K. Krowicki, U. G. Bhat, A. Skorobogaty, B. Ward. J. C.
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[3] J. G. Pelton, D. E. Wemmer. Proc. Natl. Acad. Sci USA 1989, 86. 57235727.
141 W. S. Wade, M. Mrksich, P. B. Dervan, J. Am. Chem. SOC. 1992,114,87838794.
[ 5 ] M. Mrksich, W. S. Wade, T. J. Dwyer, B. H. Geierstanger, D. E. Wemmer,
P. B. Dervan, Proc. Narl. Acad. Sci. USA 1992,89, 7586-7590.
[6] B. H. Geierstanger, M. Mrksich, P. B. Dervan, D. E. Wemmer, Science 1994,
[7] J. W. Trauger, E. E. Baird, P. B. Dervan, Nature 1996,382,559-561.
[8] a) E. E. Baird, P. B. Dervan, J. Am. Chem. SOC. 1996,118, 6141-6146; b)
M. E. Parks, E. E. Baird, l? B. Dervan, ibid. 1996,118,6147-6152; c) M. E.
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[9] J. M. Gottesfeld, L. Neely, J. W. Trauger, E. E. Baird, P. B. Dervan, Nature
I101 N. T. Thuong, C. Helene, Angew. Chem. 1993,105,697-723; Angew. Chem.
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[ l l ] J. J. Kelly, E. E. Baird, P. B. Dervan, Proc. Natl. Acad. Sci. USA 1996, 93,
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[12] J. W. Trauger, E. E. Baird, M. Mrksich, P. B. Dervan, J. Am. Chem. Soc.
[13] J. W. Szewczyk, E. E. Baird, P. B. Dervan, Angew. Chem. 1996, 108, 15961598; Angew. Chem. Int. Ed. Engl. 1996,35,1487- 1489.
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expressions, regulatory, polyamide, genes, artificial
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