Fmoc-D-Lys(Dde)-OH

Pyrimidine nucleobases bearing 5-phenylethynyl substitution represent compact and intrinsically fluorescent nucleobases. Such nucleobases are capable of selective recognition of a complementary base and may fluorimetrically report on hybridization events. Our past work has demonstrated that the fluorescence of 5-phenylethynyluracils is sensitive to substitution on the phenyl ring, however these are relatively weak fluorophores. We currently are pursuing the functionalization of the phenyl group of these modified nucleobases in order to further improve their fluorescence response, increase their aqueous solubility and stabilize hybrids formed with complementary nucleic acids. As an example of this work, we have synthesized the 5-((9H-fluoren-2-yl)ethynyl)uracil PNA monomer that will be incorporated into oligomers using Fmoc-based chemistry. Initial evaluation of the fluorescence of the 5-((9H-fluoren-2-yl)ethynyl)uracil derivative shows that the fluorescence intensity is approximately 50 times greater than a similar 5-phenylethynyluracil derivative when under identical conditions.

Fluorescent labeling of biological RNA is complicated by the narrow range of nucleoside triphosphates that can be used for biological synthesis (i.e., transcription) as well as the inability to site-specifically incorporate them into long RNA transcripts. Noncovalent strategies for labeling RNA rely on attaching fluorescent dyes to hybridization probes which deliver the dye to a specific region of the RNA through Watson-Crick base pairing. This report demonstrates the use of high-affinity peptide nucleic acid (PNA) probes in labeling mRNA transcripts with thiazole orange donor and Alexa-594 acceptor fluorophores. The PNA probes were targeted to sequences flanking splice sites in a pre-mRNA such that before splicing the PNAs were separated by >300 nucleotides (nts) whereas after splicing the separation decreased to

3. 8-Vinylguanine nucleo amino acid: a fluorescent PNA building block

Stefan Müllar, Julian Strohmeier, Ulf Diederichsen Org Lett. 2012 Mar 16;14(6):1382-5. doi: 10.1021/ol3000603. Epub 2012 Feb 27.

Attachment of a vinyl group at guanine position 8 provides fluorescent properties of the nucleobase. Therefore, 8-vinylguanine was introduced as a 2-aminoethylglycine peptide nucleic acid (PNA) building block. Incorporation of the guanine analog in short PNA sequences by Fmoc solid phase peptide synthesis allowed the differentiation between hybridization states of specific double strands with DNA, RNA, and PNA as well as quadruplex forming RNA/PNA oligomers based on fluorescence intensity.