1,2-diaminoethane (1) (20 g, 0.33 mol) was taken in dioxane:water (1:1, 500 ml) and cooled in an ice bath. Boc-azide (5 g, 35 mmol) in dioxane (50 ml) was slowly added with stirring and the pH was maintained at 10.0 by continuous addition of 4M NaOH. The mixture was stirred for 8 h and the resulting solution was concentrated to 100 ml. The N1, N2-di-Boc derivative not being soluble in water, precipitated, and it was removed by filtration. The corresponding N1-mono-Boc derivative was obtained by repeated extraction from the filtrate in ethyl acetate. Removal of solvents yielded the mono-N-Boc-diaminoethane 2 (3.45 g, 63% yield). 1H NMR (CDCl3) δ: 5.21 (br s, 1H, NH), 3.32 (t, 2H, J = 8 Hz), 2.54 (t, 2H, J = 8 Hz), 1.42 [s, 9H, C(CH3)3]. Mcalc (C7H16N2O2) = 160.21 and Mobs = 161.18 (M+1).
1,3-diaminopropane 13 (20 g, 0.27 mol) on reaction with Boc-anhydride (5.9 g, 27 mmol) under identical reaction conditions as above gave a mixture of N1,N3-di-Boc and the N1-mono-Boc derivative 14 from which the former compound was precipitated and removed by filtration. The product obtained by repeated extraction from ethyl acetate and removal of solvents yielded the mono-N-Boc-diaminopropane 14 (4 g, 60% yield). 1H NMR (CDCl3) δ: 4.78 (br s, 1H, NH), 3.32 (q, 2H), 2.74 (t, 2H), 1.59 (t, 2H), 1.42 [s, 9H, C(CH3)3]. Mcalc (C7H16N2O2) = 160.21 and Mobs = 174.18 (M+1).
Ethyl N–Boc-(aminoethyl)-α,α-dimethylglycinate (3)
The N1-(Boc)-1,2-diaminoethane 2 (3.2 g, 20 mmol) was treated with 2-bromoethylisobutyrate (3 ml, 20 mmol) in dry acetonitrile (50 ml) in the presence of anhydrous K2CO3 (5.5 g, 40 mmol) and the mixture was stirred at 70°C for 15 h. The reaction mixture was concentrated to remove the acetonitrile and work-up with ethylacetate-water and purification by silica gel column chromatography gave ethyl-N-Boc-(aminoethyl)-α,α-dimethylglycinate 3 as a yellowish oil (4.2 g, 76.6% yield). 1H NMR (CDCl3) δ: 5.29 (br s, 1H, NH), 4.22–4.11 (q, 2H), 3.26–3.21 (t, 2H), 2.7–2.65 (t, 2H), 1.43 [s, 9H, C (CH3)3], 1.34 [s, 6H, C(CH3)2], 1.30–1.23 (t, 3H); 13C NMR (CDCl3) δ: 176.8, 171.2, 156.6, 79.7, 62.5, 61.6, 43.9, 37.7, 28.1, 22.1, 21.1 and 13.8. Mcalc(C13H26N2O4) = 274.36 and Mobs = 275.21 (M+H+).
Ethyl N-Boc-(aminopropyl)-α,α-dimethylglycinate (9)
The N1-Boc-1,3-diaminopropane 14 (3.12 g, 18 mmol) was reacted with 2-bromoethylisobutyrate (2.65 ml, 18 mmol) under similar conditions, followed by usual work-up and chromatography gave ethyl N-Boc-(aminopropyl)-α,α-dimethylglycinate 15 as yellowish oil (4.5 g, 87% yield). 1H NMR (CDCl3) δ: 5.02 (br s, 1H, NH), 4.18–4.07 (q, 2H), 3.17–3.11 (t, 2H), 2.51–2.45 (t, 2H), 1.63–1.56 (t, 2H), 1.40 [s, 9H, C(CH3)3], 1.26 [s, 6H, C(CH3)2], 1.23–1.20 (t, 3H). 13C NMR (CDCl3) δ: 176.8, 155.9, 60.7, 58.8, 41.9, 38.9, 30.5, 28.3, 25.2, and 14.2. Mcalc (C14H28N2O4) = 286.38 and Mobs = 289.17 (M+3H+).
Ethyl N-(Boc-aminoethyl)-N-(chloroacetyl)-α,α-dimethylglycinate (4)
The ethyl N-Boc-(aminoethyl)-α,α-dimethylglycinate 3 (4.0 g, 14.6 mmol) was taken in 10% aqueous Na2CO3 (75 ml) and dioxane (60 ml). Chloroacetyl chloride (3.5 ml, 43.8 mmol) was added in two portions with vigorous stirring. The reaction was complete within an hour and was brought to pH 8.0 by addition of 10% aqueous Na2CO3 and concentrated to remove the dioxane. The product after work-up was purified by column chromatography to obtain the ethyl N-Boc-(aminoethyl)-N-(chloroacetyl)-α,α-dimethylglycinate 4 as a yellowish oil. (4.2 g, 82% yield). 1H NMR (CDCl3) δ: 5.13 (br s, 1H, NH), 4.27–4.17 (q, 2H), 4.13 (s, 2H), 3.57–3.5 (t, 2H), 3.36–3.30 (t, 2H), 1.49 [s, 6H, C(CH3)2], 1.43 [s, 9H, C(CH3)3], 1.27–1.20 (t, 3H). 13C NMR (CDCl3) δ: 173.9, 167.3, 156.0, 79.8, 72.6, 61.6, 61.2, 43.7, 41.8, 41.2, 28.2, 23.8 and 13.9. Mcalc (C15H27N2O5Cl) = 350.16 and Mobs = 352.01 (M+2).
Ethyl N-Boc-(aminopropyl)-N-(chloroacetyl)-α,α-dimethylglycinate (10)
The ethyl N-Boc-(aminopropyl)-α,α-dimethylglycinate 15 (3.6 g, 12.5 mmol) reacted with chloroacetyl chloride (5 ml, 62.5 mmol) under similar conditions, work up and chromatographic purification gave ethyl N-Boc-(aminopropyl)-N-(chloroacetyl)-α,α-dimethylglycinate 16 as a yellowish oil (3.8 g, 83.5% yield). 1H NMR (CDCl3) δ: 4.77 (br s, 1H, NH), 4.17–4.06 (q, 2H), 4.04 (s, 2H), 3.48–3.40 (t, 2H), 3.17–3.10 (t, 2H), 1.91–1.82 (t, 2H), 1.46 [s, 6H, C(CH3)2], 1.43 [s, 9H, C(CH3)3], 1.23–1.17 (t, 3H). 13C NMR (CDCl3) δ: 173.64, 166.22, 156.09, 61.66, 61.05, 42.19, 41.70, 32.33, 28.30, 23.75, and 13.97.Mcalc(C16H29N2O5Cl) = 364.16 and Mobs = 365.13 (M+1).
N-(Boc-aminoethyl-α,α-dimethylglycyl)-thymine ethyl ester (5)
Ethyl N-Boc-(aminoethyl)-N-(chloroacetyl)-α,α-dimethyl-glycinate 4 (4.0g, 11.4 mmol) was stirred with anhydrous K2CO3 (1.68 g, 12.2 mmol) and thymine (1.58 g, 12.5 mmol) in DMF. After completion of reaction (TLC), solvent DMF was removed under reduced pressure followed by aqueous work-up and column chromatographic purificationgave N-Boc-(aminoethyl-α,α-dimethyl glycyl)-thymine ethyl ester 5 (4.8 g, 96.5%). 1H NMR (CDCl3) δ: 6.94 (s, 1H), 5.43 (br s, 1H, NH), 4.57 (s, 2H), 4.17–4.06 (q, 2H), 3.57–3.50 (t, 2H), 3.41–3.34 (t, 2H), 1.87 (s, 3H, CH3), 1.48 [s, 6H, C(CH3)2], 1.42 [s, 9H, C(CH3)3], 1.23–1.16 (t, 3H). Mcalc (C20H32N4O7) = 440.49 and Mobs = 463 (M+Na+).
N-Boc-(aminopropyl-α,α-dimethylglycyl)-thymine ethyl ester (11)
Ethyl N-Boc-(aminopropyl)-N-(chloroacetyl)-α,α-imethyl-glycinate 16 (2.0 g, 5.5 mmol) was reacted with thymine (0.76 g, 6.1 mmol) under similar conditions in presence of K2CO3 in DMF followed by work-up and column chromatographic purification to give N-Boc-(aminopropyl)-α,α-dimethyl-glycyl-thymine ethyl ester 17 as white solid (2.2 g, 88% yield). 1H NMR (CDCl3) δ: 6.98 (s, 1H), 4.95 (br s, 1H, NH), 4.49 (s, 2H), 4.16–4.06 (q, 2H), 3.49–3.41 (t, 2H), 3.24–3.18 (t, 2H), 1.89 (s, 3H), 1.47 [s, 6H, C(CH3)2], 1.42 [s, 9H, C(CH3)3], 1.23–1.16 (t, 3H). 13C NMR (CDCl3) δ: 173.7, 166.4, 164.2, 156.4, 151.2, 110.6, 61.9, 61.19, 41.49, 31.92, 28.39, 23.98, 14.03, 12.32. Mcalc(C21H34N4O7) = 454.61 and Mobs = 477.18 (M+Na+).
Hydrolysis of the PNA ethyl esters 5 and 11
The ethyl ester 5 was hydrolyzed using aqueous NaOH (2N, 5 ml) in methanol (5 ml) and the resulting acid was neutralized with activated Dowex-H+ till the pH of the solution was 7.0. The resin was removed by filtration and the filtrate was concentrated to obtain the resulting Boc-protected acid 6 in excellent yield (85%), 1H NMR (CDCl3) δ: 10–11 (2H, thymine NH and –COOH), 7.18 (s, 1H), 5.43 (br s, 1H.NH), 4.2–4.16 (q, 2H), 3.5–3.56 (t, 2H), 3.39–3.32 (t, 2H), 1.87 (s, 3H, CH3), 1.48 [s, 6H, C(CH3)2], 1.42 [s, 9H, C(CH3)3]. Mcalc (C18H28N4O7) = 412.44 and Mobs = 435.57 (M+Na+).
Solid phase synthesis
The solid phase synthesis was performed on MBHA resin (Sigma, 100–200 mesh) having initial amine loading value of 0.85 mmol/g, which was lowered to 0.25 mmol/g by capping with acetic anhydride in dry DMF/DCM and pyridine as a base. The MBHA resin with free amine was functionalized by coupling with N (α)-Boc-N(ε)-Cl−Cbz- l-lysine using DCCI as coupling reagent. The lysine loaded resin was swollen in dry CH2Cl2 (30 min) and treated with 50% TFA in CH2Cl2 (1 ml × 2, 15 min each). After washing with CH2Cl2, neutralization of TFA salt with 5% DIPEA in CH2Cl2 (1 ml × 3, 2 min each) and washed further with CH2Cl2. The unmodified/modified PNA monomers were coupled to resin in presence of HBTU/HOBt/DIPEA in DMF/NMP and the synthesis continued using the same coupling agent to make PNA oligomers (). The efficiency of each coupling was monitored by Kaiser’s test. At the end of the assembly the PNA oligomers were cleaved from the resin with TFA/TFMSA in presence of scavenging agent thioanisole.
A typical cleavage reaction consisted of treating of resin-bound PNA oligomer (10 mg) in an ice bath with thioanisole (20 μl) and 1,2-ethanedithiol (8 μl) for 10 min and TFA (120 μl) followed by TFMSA (16 μl) and stirring continued for 2 h. After filtering the resin, the filtrate was evaporated under vacuum. The residue was dissolved in methanol (~0.1 ml) and precipitated by adding ether. The PNA oligomers were purified by RP-HPLC (C18 column) and characterized by MALDI-TOF mass spectrometry. The overall yields of the crude PNA oligomers were in the range 70–80%.
The concentration of PNA and DNA was calculated on the basis of absorbance using the molar extinction coefficients of nucleobases for A = 15.4, t = 8.8, C = 7.3 and G = 11.7 M−1cm−1. The complexes were prepared in sodium phosphate buffer (10 mM, pH 7.4) by annealing the samples at 85°C for 5 min followed by slow cooling to room temperature. Absorbance vs. temperature profiles were obtained by monitoring at 260 nm with a UV-VIS spectrophotometer scanning from 5 to 85°C at a ramp rate of 0.2°C per minute. Experiments were repeated at least thrice and the Tm values were obtained from the peaks in the first derivative plots. The reported Tm values are accurate to (±) 0.5°C.