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Chapter 6
(HBTU),
N-[(dimethylamino)-1H-1,2,3-triazol[4,5-b]pyridine-1-ylmethylene]-Nmethylmethanaminium
hexafluorophosphate N-oxide (HATU), dichloromethane, N–
methylpyrrolidone (NMP) and N,N-dimethylformamide (DMF) were purchased from Applied
Biosystem (Foster City, CA, USA). Carboxymethyl-O-benzyl-guanine was purchased by
ASM (Hannover, Germany). Boc-Arg(Tos)-OH, Boc-Lys(2-Cl-Z)-OH, Val-OH, Boc-Pro-OH
and m-cresol were from Fluka (Buchs, Switzerland). (4-Methylbenhydryl)amine (MBHA) PS
resin was from Novabiochem (Laufelfingen, Switzerland). Carboxymethyl-thymine,
diisopropylethylamine (DIPEA), N,O-Bistrimethylsilyl acetamide (BSA), trifluoromethane
sulfonic acid (TFMSA), trifluoroacetic acid (TFA), 3-hydroxy-1, 2,3-benzotriazin-4-(3h)-one
(DhBtOH) and diisopropylcarbodiimide (DIC) were from Sigma-Aldrich (St. Louis, MO,
USA), Rhodamine. Oligonucleotides (guaranteed oligos grade) were purchased from
ThermoFisher Scientific (Ulm, Germany) and used without further purification.
6.4.2 Submonomer syntheses
General procedure for submonomer synthesis
L-amino acid methyl esters (Val, Lys, Arg) were prepared by dissolving the N-Boc and side
chain protected (Lys as 2-Cl-Z and Arg as Tosyl) amino acid derivatives, commercially
available, in methanol with magnetic stirring. The mixtures were cooled at 0 °C and added of
thionyl chloride dropwise up to a final concentration of 1M. The solutions were stirred
overnight at room temperature. Methanol was evaporated and the residues were added of
methanol and evaporated again (few times) to yield Boc-deprotected, side chain protected
chloridrated methyl esters as white foams in quantitative yields. In order to synthesize the
different substituted N-(2-aminoethyl)amino acid (PNA backbones), the corresponding
methyl esters were dissolved in the minimum amount of methanol at room temperature
together with diisopropylethylamine (DIPEA, 1 eq.) and the mixtures were stirred for 20
minutes. Boc-L-Lys(2-Cl-Z)-H aldehyde (1 eq.), synthesized as previously reported 31 , was
then added and the solutions were stirred for 30 min at room temperature, then cooled to 0°C
and NaBH 3 CN (3 eq) and CH 3 COOH (1.1 eq) were added. After 2h the methanol was
evaporated. The residues were dissolved in CH 2 Cl 2 and washed with saturated KHSO 4 (twice)
and NaHCO 3 (twice). The combined organic phases were dried over MgSO 4 and filtered, and
the solvent was evaporated under vacuum. The crude products were purified by flash
chromatography (eluent ethyl acetate : methanol = 95:5) and obtained as colourless foams.
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