Fluorescein-Conjugated Lysine Monomers for Solid Phase Synthesis of Fluorescent Peptides and PNA Oligomers

Abstract

Fluorescein ethyl ester, I, was used to prepare the fluorescent mixed ester/ether 6-O-(carboxymethyl)-fluorescein ethyl ester, III. Conjugation of III to the epsilon-amino group of alpha-N-Boc-L-lysine, via the N-hydroxysuccinimde ester, IV, gave the Boc-protected fluorescein-conjugated lysine monomer V. Removal of the Boc group, followed by reaction with Fmoc chloride, gave the Fmoc-protected monomer, VI (Figure 1). These Boc- and Fmoc-protected fluorescein-conjugated lysines were readily incorporated into peptides and PNA oligomers during solid phase synthesis to give fluorescent products. Mass spectroscopy and UV studies showed that the fluorophore remains unchanged during solid phase synthesis. In contrast to fluorescein, the photophysical properties of these derivatives are pH independent from pH 3 to 8, with a molar absorption coefficient, epsilon max 456, of 2.9 x 10(4) M-1 cm-1 and fluorescence quantum yield, phi f, of 0.18.

An Efficient Biodelivery System for Antisense Polyamide Nucleic Acid (PNA)

“…The PNA sequences 8-10 were synthesized on solid support (MBHA resin) by well-established protocols (see Materials and Methods). For the preparation of conjugate 3, a N-Boc N-ε uorescein-labeled lysine residue was introduced on the resin before PNA elongation (Lohse et al, 1997).…”

“…TBTP 5 was synthesized as described elsewhere (Mehiri et al, 2007). 6-O-(carboxymethyl) uorescein ethyl ester (Flu-OH) and N-α-Boc-Lys(N-ε-Flu)-OH were prepared as described by Lohse et al (1997). PNA monomers Boc(T) OH, Boc(C Z )OH, Boc(G Z )OH, and Boc(A Z )OH were synthesized as described elsewhere (Meltzer et al 1995;Coull et al, 1996;Aldrian-Herrada et al, 1998).…”

PNA-mediated modulation and redirection of Her-2 pre-mRNA splicing: Specific skipping of erbB-2 exon 19 coding for the ATP catalytic domain

“…N-(9-aminoaciridinyl)-6-aminohexanoic acid (Acr) was linked to PNA on solid support at the N-terminal of the PNA through the eg1 (8-amino-3,6-dioxaoctanoyl) linker (Acr-PNA). Fluorescein (Fl)-labeled PNA oligomers were prepared using a previously described 'Fl-lysine' monomer (44).…”

Efficiency of Cellular Delivery of Antisense Peptide Nucleic Acid by Electroporation Depends on Charge and Electroporation Geometry

“…The PNAs were dissolved in sterile water and the concentration was determined spectrophotometrically using the extinction coefficient 177,200 L/ (mol cm) at 260 nm. PNAs with different charges (at neutral pH) were included in the experiments, namely PNA 3325: H-(Lys(bisP-4)) 6 (Lohse et al, 1997)]. …”