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Arg-PNA Microarrays<br />
complex mixtures simulating the real genotype profiles. Six different hybridization patterns<br />
related to six different genotypes in the ApoE gene were clearly obtained in microarray<br />
experiments, potentially allowing for an easy genetic assessment. Work is now in progress in<br />
order to apply this diagnostic system to human DNA samples amplified by PCR.<br />
4.4 Experimental Section<br />
4.4.1 Materials and reagents<br />
PNA N-Boc protected standard monomers, N-[1H-1,2,3-(benzotriazol-1-yl)-<br />
(dimethylamino)methylene]-N-methylmethanaminium hexafluorophosphate N-oxide<br />
(HBTU),<br />
N-[(dimethylamino)-1H-1,2,3-triazol[4,5-b]pyridine-1-ylmethylene]-Nmethylmethanaminium<br />
hexafluorophosphate N-oxide (HATU), dichloromethane, N–<br />
methylpyrrolidone (NMP) and N,N-dimethylformamide (DMF) were purchased from Applied<br />
Biosystem (Foster City, CA, USA). Carboxymethyl-O-benzyl-guanine was purchased by<br />
ASM (Hannover, Germany). Boc-Arg(Tos)-OH, Boc-D-Arg(Tos)-OH and m-cresol were<br />
from Fluka (Buchs, Switzerland). (4-Methylbenhydryl)amine (MBHA) PS resin was from<br />
Novabiochem (Laufelfingen, Switzerland). Diisopropylethylamine (DIPEA), N,O-<br />
Bistrimethylsilyl acetamide (BSA), trifluoromethane sulfonic acid (TFMSA), trifluoroacetic<br />
acid (TFA), 3-hydroxy-1, 2,3-benzotriazin-4-(3h)-one (DhBtOH) and<br />
diisopropylcarbodiimide (DIC) were from Sigma-Aldrich (St. Louis, MO, USA).<br />
Oligonucleotides (guaranteed oligos grade) were purchased from ThermoFisher Scientific<br />
(Ulm, Germany) and used without further purification.<br />
4.4.2 PNA synthesis<br />
The synthesis of chiral (2D,5L)-Arg-based PNAs was performed as described previously for<br />
similar PNAs 30 by using manual synthesis for the chiral residues and automated Boc-SPPS<br />
protocols on a ABI 433A synthesizer, following the procedures provided from the company,<br />
for the achiral residues. The chiral submonomers were introduced by using HATU/DIEA<br />
coupling protocol, Fmoc group was deprotected by piperidine/NMP (20%) and<br />
carboxymethyl-Z-adenine 31 or carboxymethyl-O-benzyl-guanine (commercialy available)<br />
85
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