The nuclease stability and melting temperatures (Tm) were compared for fully modified oligoribonucleotide sequences containing 2'-fluoro, 2'-O-methyl, 2'-O-propyl and 2'-O-pentyl nucleotides. Duplexes formed between 2' modified oligoribonucleotides and RNA have typical A-form geometry as observed by circular dichroism spectroscopy. Modifications, with the exception of 2'-O-pentyl, were observed to increase the Tm of duplexes formed with complementary RNA. Modified homoduplexes showed significantly higher Tms, with the following Tm order: 2'-fluoro:2'fluoro > 2'-O-propyl:2'-O-propyl > 2'-O-methyl:2'-O- methyl > RNA:RNA > DNA:DNA. The nuclease stability of 2'-modified oligoribonucleotides was examined using snake venom phosphodiesterase (SVPD) and nuclease S1. The stability imparted by 2' modifications was observed to correlate with the size of the modification. An additional level of nuclease stability was present in oligoribonucleotides having the potential for forming secondary structure, but only for 2' modified oligoribonucleotides and not for 2'-deoxy oligoribonucleotides.
A technique for lead discovery vs RNA targets utilizing mass spectrometry (MS) screening methods is described. The structure-activity relationships (SAR) derived from assaying weak binding motifs allows the pharmacophores discovered to be elaborated via "SAR by MS" to higher affinity ligands. Application of this strategy to a subdomain of the 23S rRNA afforded a new class of compounds with functional activity.
High-throughput screening of in-house compound libraries led to the discovery of a novel antibacterial agent, compound 1 (MIC: 12-25 microM against S. pyogenes). In an effort to improve the activity of this active compound, a series of 2-substituted quinazolines was synthesized and evaluated in several antibacterial assays. One such compound (22) displayed improved broad-spectrum antibacterial activity against a variety of bacterial strains. This molecule also inhibited transcription/translation of bacterial RNA, suggesting a mechanism for its antibiotic effects. Structure-activity relationship studies of 22 led to the synthesis of another 24 compounds. Although some of these molecules were found to be active in bacterial growth assays, none were as potent as 22. Compound 22 was tested for its ability to cure a systemic K. pneumonia infection in the mouse and displayed moderate effects compared with a control antibiotic, gentamycin.
Chemical modification of pre-formed asymmetric polyazaphane
scaffolds by simultaneous addition of
functionality (letters) in solution has been developed for the
preparation of tertiary nitrogen-based combinatorial
chemistry libraries. This approach has some significant advantages
over the more commonly employed solid phase
bead splitting/reaction/mixing procedures for the preparation of
libraries. Three novel, asymmetric polyazaphanes
32, 33, and 37 have been synthesized
in high yields by an efficient cyclization of
2,6-bis(bromomethyl)pyridine (31)
with new orthogonally protected triamines 29, 30,
and 35, respectively. Selective deprotection of
32, 33, and 37
provided mono-t-Boc-protected scaffolds
1−3 suitable for solution phase, simultaneous
addition of functionalities.
Model studies of small libraries of scaffold 2 using
CZE analyses indicated that simultaneous addition of 10
benzylic
bromide alkylating functionalities would result in libraries containing
approximately equimolar amounts of all possible
compounds. Sixteen purified tertiary amine libraries
4−19 (total complexity of 1600 compounds) were
generated
by this procedure from scaffold 2. A “fix-last”
combinatorial method was devised to minimize chemical
reactions.
Several first-round sublibraries of scaffold 2,
containing a mixture of 100 compounds, exhibited potent
antimicrobial
activities. Twenty single compounds 63−82
with uniform functionalities at the combinatorialized sites
were
synthesized. Some of these pure compounds were more active, while
others were less active, compared with the
parent mixtures 5 and 10.
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