Reactions were monitored by TLC on Silica Gel 60 F254 plates (Merck) using detection with ninhydrin. The melting points were determined on a Tottoli (Büchi) apparatus and were uncorrected. Column chromatography was performed on Silica Gel (Merck, 0.040–0.063). Chiral TLC was performed on chiral Silica Gel 60 F254 plates (Aldrich). NMR spectra were recorded on 600 and 300 MHz Bruker spectrometers, operating at 150. 92 or 75.47 MHz for 13C and 600.13 or 300.13 MHz for 1H nuclei. TMS was used as an internal standard. HRMS analysis was performed on MALDI-TOF/TOF mass spectrometer operating in reflectron mode. Mass spectra were acquired by accumulating three spectra after 400 laser shots per spectrum. Calibrant and analyte spectra were obtained in positive ion mode. Calibration type was internal with calibrants produced by matrix ionization (monomeric, dimeric and trimeric CHCA), with azithromycin and angiotensin II dissolved in a-cyano-4-hydroxycinnamic acid matrix in the mass range m/z 190.0499–749.5157 or 1046.5417. Accurately measured spectra were internally calibrated and elemental analysis was performed on Data Explorer v. 4.9 Software with mass accuracy better than 5 ppm. Samples were prepared by mixing 1 lμL of analyte methanol solution with 5 μL of saturated (10 mg/mL) solution of a-cyano 4-hydroxycinnamic acid (a-CHCA) and internal calibrants (0.1 mg/mL) dissolved in 50 % acetonitrile/0.1 % TFA. Microwave assisted reactions were performed on CEM Discovery System with infrared temperature control. The reaction mixtures were placed in a flask equipped with a magnetic stir bar and subjected to microwave irradiation.
N
α-benzyl-N
β-Boc amino acids 1 were prepared according to procedure described by Lelais and Seebach (2003). N-benzyl-α-amino acid (1 equiv.) was dissolved in dry MeOH and (CH3)4NOH (1 equiv.) was added at 0 °C. After 30 min solvent was evaporated and the residue dissolved in dry CH2Cl2. The solution was cooled to −78 °C and N-Boc-3-trichloromethyloxaziridine (1.3 equiv., prepared according to Vidal et al. (1998)) dissolved in CH2Cl2 was added dropwise. Reaction was stirred at room temperature overnight. Solution was washed three times with water, collected water layers were acidified with KHSO4 to pH 3 and product extracted with CH2Cl2. Solvent was evaporated and the product (yellow oil, 40 %) used without further purification. α-Hydrazino acids were prepared according the procedure described by Panda et al. (2013), with slight modifications. d-Amino acid was dissolved in 2.5 M H2SO4 (1.3 mL/mmol) and KBr (3.5 equiv.) was added. The solution was cooled down to 0 °C and then solution of NaNO2 (1.3 equiv.) was added dropwise. After 1 h at 0 °C, the reaction mixture was stirred at room temperature overnight. α-Bromo acid was extracted with EtOAc, washed with NaHCO3 and dried over MgSO4. After evaporation, product was obtained as yellow oil (85 % yield.). Obtained α-bromo acid was dissolved in EtOH (2 mL) and added dropwise to solution of hydrazine hydrate (3 equiv.) in EtOH (1 mL). The reaction mixture was irradiated under MW at 70 °C and 50 W for 1 h. A white suspension was obtained. Solvent was evaporated, and product recrystallized from EtOH/ether (40 % yield). Optical purity of prepared hydrazino acids has been checked by the chiral thin-layer chromatography and confirmed complete conversion.
General procedure for the synthesis of dipeptides 2
Dipeptides were prepared by acid mediated removal of the Boc group from the corresponding N-terminally protected dipeptides (TFA-water 9:1, v/v; 30 min.; r.t.; quant.). N-terminally protected dipeptides were obtained by the following procedure: Boc-Aaa-OH (Aaa = Leu, Val, Ala) (1 mmol) and HOSu (1.5 mmol) were dissolved in dry DMF (3 mL) and solution cooled down to 0 °C. DCC (1.5 mmol) dissolved in dry DMF (2 mL) was added dropwise. After 30 min reaction was stirred at room temperature and the consumption of starting dipeptide followed by TLC. The precipitate was filtered, and the filtrate added dropwise to the solution of H-Phe-R (R=OH, OEt or NH2) (1 mmol) and KHCO3 (2 mmol) in water (5 mL). Reaction mixture was stirred at room temperature overnight. Solvent was evaporated and the residue purified by the flash column chromatography. Mobile phase used for the chromatography was also used for the determination of R
f value and is given for each compound.
Boc-Leu-Phe-OH
Colourless oil (0.68 g, 82 %); R
f 0.40 (petrol ether-EtOAc-AcOH 7:5:0.5). 1H NMR (300 MHz, [D6]DMSO, 25 °C): δ = 12.6 (br s, 1H, OH), 7.88 (d, 3
J
NH,H = 7.9 Hz, 1H, NH Phe), 7.31–7.10 (m, 5H, δ, ε, ζ Phe), 6.83 (d, 3
J
NH,H = 8.6 Hz, 1H, NH Leu), 4.49–4.38 (m, 1H, α Phe), 4.00–3.88 (m, 1H, α Leu), 3.05; 2.90 (dd, 3
J
H,H = 5.1 Hz, 3
J
H,H = 8.6 Hz, 2
J
H,H = 13.9 Hz, 2H, β, β′ Phe), 1.55–1.46 (m, 3H, β, β′, γ Leu), 1.36 (s, 9H, CH3 Boc), 0.83; 0.80 (d, 3
J
H,H = 6.6 Hz, 6H, δ, δ′ Leu). 13C NMR (300 MHz, [D6]DMSO, 25 °C): δ = 172.8 (CO Phe), 172.3 (CO Leu), 155.1 (CO Boc), 137.3 (γ Phe), 129.0 (δ Phe), 128.0 (ε Phe), 126.4 (ζ Phe), 78.0 (C Boc), 53.1 (α Phe), 52.8 (α Leu), 41.0 (β Leu), 36.7 (β Phe), 28.1 (CH3 Boc), 24.1 (γ Leu), 22.8; 21.6 (δ, δ′ Leu).
Boc-Val-Phe-OH
Colourless oil (0.45 g, 54 %); R
f 0.38 (petrol ether-EtOAc-AcOH 7:5:0.5). 1H NMR (300 MHz, [D6]DMSO, 25 °C): δ = 8.04 (d, 3
J
NH,H = 7.8 Hz, 1H, NH Phe), 7.29–7.10 (m, 5 H, δ, ε, ζ Phe), 6.57 (d, 3
J
NH,H = 8.2 Hz, 1H, NH Val), 4.42 (m, 1H, α Phe), 3.76 (m, 1H, α Val), 3.05; 2.88 (dd, 3
J
H,H = 5.2 Hz, 3
J
H,H = 8.5 Hz, 2
J
H,H = 13.8 Hz, 2H, β, β′ Phe), 1.84 (m, 1H, β Val), 1.37 (s, 9H, CH3 Boc), 0.74; 0.77 (d, 3
J
H,H = 6.6 Hz, 6H, γ, γ′ Val). 13C NMR (300 MHz, [D6]DMSO, 25 °C): δ = 172.8 (CO Phe), 171.3 (CO Val), 155.2 (CO Boc), 137.0 (γ Phe), 129.1 (δ Phe), 128,1 (ε Phe), 126.4 (ζ Phe), 78.1 (C Boc), 59.7 (α Val), 53.2 (α Phe), 36.8 (β Phe), 30.5 (β Val), 28.2 (CH3 Boc), 19.1; 18.1 (γ, γ′ Val).
Boc-Ala-Phe-OH
Colourless oil (1.78 g, 36 %); R
f 0.30 (petrol ether-EtOAc-AcOH 7:5:0.5). 1H NMR (300 MHz, [D6]DMSO, 25 °C): δ = 7.83 (d, 3
J
NH,H = 7.7 Hz, 1H, NH Phe), 7.30–7.15 (m, 5H, δ, ε. ζ Phe), 6.81 (d, 3
J
NH,H = 6.5 Hz, 1H, NH Ala), 4.40 (m, 1H, α Phe), 3.95 (m, 1H, α Ala), 3.04; 2.91 (dd, 3
J
H,H = 5.1 Hz, 3
J
H,H = 8.4 Hz, 2
J
H,H = 13.9 Hz, 2H, β, β′ Phe), 1.36 (s, 9H, CH3 Boc), 1.12 (d, 3
J
H,H = 7.0 Hz, 3H, β Ala). 13C NMR (300 MHz, [D6]DMSO, 25 °C): δ = 172.6 (CO Phe), 172.5 (CO Ala), 154.8 (CO Boc), 137.3 (γ Phe), 129.1 (δ Phe), 128.0 (ε Phe), 126.3 (ζ Phe), 77.5 (C Boc), 53.1 (α Phe), 49.6 (α Ala), 18.1 (β Ala), 36.7 (β Phe), 28.1 (CH3 Boc).
Boc-Leu-Phe-NH2
White powder (0.31 g, 83 %); m.p. 140 °C; R
f 0.35 petrol ether:EtOAc:AcOH (10:5:0.5). 1H NMR (300 MHz, [D6]DMSO, 25 °C): δ = 7.70 (d, 3
J
NH,H = 7.8 Hz, 1H, NH Phe), 7.31–7.14 (m, 5H, δ, ε, ξ Phe), 7.36; 7.12 (br s, 2H, CONH2), 6.96 (d, 3
J
NH,H = 8.5 Hz, 1H, NH Leu), 4.45 (m, 1H, α Phe), 3.84 (m, 1H, α Leu), 3.00–2.82 (m, 2H, β, β′ Phe), 1.79–1.55 (m, 2H, β, β′ Leu), 1.55–1.44 (m, 1H, γ Leu), 1.37 (s, 9H, CH3 Boc), 0.84; 0.80 (d, 3
J
H,H = 6.6 Hz, 6H δ, δ′ Leu). 13C NMR (300 MHz, [D6]DMSO, 25 °C): δ = 172.5 (CO Leu), 155.7 (CO Boc), 137.2 (γ Phe), 129.1 (δ Phe), 128.0 (ε Phe), 126.3 (ζ Phe), 78.6 (C Boc), 53.9 (α Phe), 53.6 (α Leu), 48.0 (β Leu), 38.1 (β Phe), 28.1 (CH3 Boc), 24.6 (γ Leu), 23.3; 22.1 (δ, δ′ Leu).
Boc-Val-Phe-NH2
White powder (0.33 g, 90 %); m.p. 148 °C; R
f 0.33 (petrol ether:EtOAc:AcOH 10:5:0.5). 1H NMR (300 MHz, [D6]DMSO, 25 °C): δ = 7.75 (d, 3
J
NH,H = 7.7 Hz, 1H, NH Phe), 7.30–7.14 (m, 5H, δ, ε, ξ Phe), 7.32; 7.02 (br s, 2H, CONH2), 6.64 (d, 3
J
NH,H = 8.0 Hz, 1H, NH Val), 4.60–4.35 (m, 1H, α Phe), 3.75–3.60 (m, 1H, α Val), 2.96; 2.83 (dd, 3
J
H,H = 5.0 Hz, 3
J
H,H = 8.5 Hz, 2
J
H,H = 13.8 Hz, 2H, β, β′ Phe), 1.75–1.68 (m, 1H, β Val), 1.37 (s, 9H, CH3 Boc), 0.72; 0.68 (d, 3
J
H,H = 6.6 Hz, 6H, γ, γ′ Val). 13C NMR (300 MHz, [D6]DMSO, 25 °C): δ = 173.2 (CO Val), 156.3 (CO Boc), 137.0 (γ Phe), 129.2 (δ Phe), 128.0 (ε Phe), 126.2 (ζ Phe), 78.6 (C Boc), 60.2 (α Val), 54.0 (α Phe), 37.6 (β Phe), 30.7 (β Val), 28.6 (CH3 Boc), 19.1; 18.1 (γ, γ′ Val).
Boc-Ala-Phe-NH2
White powder (0.24 g, 72 %); m.p. 157 °C; R
f 0.14 (petrol ether:EtOAc:AcOH 10:5:0.5). 1H NMR (300 MHz, [D6]DMSO, 25 °C): δ = 7.65 (d, 3
J
NH,H = 7.8 Hz, 1H, NH Phe), 7.25–7.15 (m, 5H, δ, ε. ζ Phe), 7.00 (d, 3
J
NH,H = 6.4 Hz, 1H, NH Ala), 7.45; 7.10 (br s, 2H, CONH2), 4.43 (m, 1H, α Phe), 3.87 (m, 1H, α Ala), 3.04–2.91 (m, 2H, β, β′ Phe), 1.36 (s, 9H, CH3 Boc), 1.07 (d, 3
J
H,H = 6.8 Hz, 3H, β Ala). 13C NMR (300 MHz, [D6]DMSO, 25 °C): δ = 172.6 (CO Phe), 172.3 (CO Ala), 156.6 (CO Boc), 137.1 (γ Phe), 129.0 (δ Phe), 128.1 (ε Phe), 126.3 (ζ Phe), 78.1 (C Boc), 53.2 (α Phe), 47.3 (α Ala), 36.7 (β Phe), 28.1 (CH3 Boc), 18.0 (β Ala).
General procedure for the synthesis of tripeptides 3a and 3b
N
α-benzyl-N
β-Boc amino acid 1 (0.5 mmol) was dissolved in dry DMF, NMM (0.5 mmol), BOP (0.55 mmol) and HOBt (0.55 mmol) were added. After 30 min solution of dipeptide 2 (0.5 mmol) and NMM (0.5 mmol) in dry DMF (1 mL) was added. Reaction was stirred at room temperature overnight. Solvent was evaporated and the residue purified by the flash column chromatography. Mobile phase used for the chromatography was also used for the determination of R
f value and is given for each compound.
N
α-benzyl-N
β-Boc-Leu-Leu-Phe-OH (3a)
Yellow oil (56 mg, 19 %); R
f 0.66 (petrol ether:EtOAc:AcOH 5:5:0.5). 1H NMR (600 MHz, [D6]DMSO, 25 °C): δ = 8.29 (d, 3
J
NH,H = 7.9 Hz, 1H, NH Phe), 7.88 (d, 3
J
NH,H = 8.8 Hz, 1H, NH), 7.78 (br s, 1H, NH), 7.65–7.55 (m, 5H, Bn), 7.26–7.16 (m, 5H δ, ε, ξ Phe), 4.53 (m, 1H, α Phe), 4.43 (m, 1H, α Leu), 4.34 (m, 1H, α hLeu), 3.79 (s, 2H, CH2 Bn), 3.00–2.89 (m, 2H β, β′ Phe), 1.53 (m, 3H, β, γ Leu), 1.42–1.34 (m, 3H, β, γ hLeu), 1.33–1.25 (m, 9H CH3 Boc), 0.84–0.74 (m, 12H, δ, δ′, Leu, hLeu). 13C NMR (151 MHz, [D6]DMSO, 25 °C): δ = 172.9, 171.6, 170.4 (CO Phe, Leu, hLeu), 154.9 (CO Boc), 137.6 (C Bn), 137.4 (γ Phe), 133.1, 132.4, 131.4 (CH Bn), 129.1, 128.1, 127.0 (δ, ε, ζ Phe), 78.2 (α hLeu), 60.1 (α Phe), 53.2 (CH2 Bn), 50.7 (α Leu), 37.0 (β Leu), 36.8 (β Phe), 36.6 (β hLeu), 28.0 (CH3 Boc), 24.2 (γ hLeu), 23.8 (γ Leu), 23.0 (δ hLeu), 22.8 (δ Leu), 21.7 (δ′ hLeu), 21.4 (δ′ Leu). HRMS (MALDI-TOF/TOF): calcd. for C33H48N4O6 [M + Na]+ 619.3466; found 619.3446.
N
α-benzyl-N
β-Boc-Val-Val-Phe-OH (3b)
Yellow oil (66 mg, 12 %); R
f 0.50 (petrol ether:EtOAc:AcOH 10:5:0.5). 1H NMR (600 MHz, [D6]DMSO, 25 °C): δ = 7.62–7.56 (m, 5H, Bn), 7.45–7.24 (m, 5H, δ, ε, ξ Phe), 4.43 (m, 1H, α Phe), 4.33–4.12 (m, 2H, α Val, hVal), 3.84–3.66 (m, 2H, CH2 Bn), 3.05–2.95 (m, 2H, β Phe), 2.02–1.85 (m, 2H, β Val, hVal), 1.29–1.17 (m, 9H, CH3 Boc), 0.93–0.70 (m, 12H, γ, γ′ Val, hVal). 13C NMR (151 MHz, [D6]DMSO, 25 °C): δ = 172.7, 170.6, 163.8 (CO Phe, Val, hVal), 154.3 (CO Boc), 137.5 (C Bn), 137.4 (γ Phe), 133.1, 132.4, 131.4 (CH, Bn), 128.7, 128.0, 127.8 (δ, ε, ζ Phe), 78.6 (C Boc), 78.1 (α hVal), 60.8 (α Phe), 57.3 (CH2 Bn), 53.1 (α Val), 36.6 (β Phe), 34.7 (β hVal), 30.4 (β Val), 28.0 (CH3 Boc), 19.3, 18.8 (γ, γ′ hVal), 18.3, 17.9 (γ, γ′ Val). HRMS (MALDI-TOF/TOF): calcd. for C31H44N4O6 [M + K]+ 607.2892; found 607.291.
General Procedure for the synthesis of tripeptides 3c–3g
N
α-benzyl-N
β-Boc amino acid 1 (0.5 mmol) and dipeptide 2 (0.5 mmol) were dissolved in dry DMF; NMM (1 mmol), BOP (0.55 mmol) and HOBt (0.55 mmol) were added. Reaction was stirred at room temperature overnight. Solvent was evaporated and the residue purified by the flash column chromatography. Mobile phase used for the chromatography was also used for the determination of R
f value and is given for each compound.
N
α-benzyl-N
β-Boc-Leu-Leu-Phe-OEt (3c)
Yellow oil (32 mg, 10 %); R
f 0.75 (petrol ether:EtOAc:AcOH 10:5:0.5). 1H NMR (600 MHz, [D6]DMSO, 25 °C): δ = 8.57 (d, 3
J
NH,H = 7.4 Hz, 1H, NH), 8.32 (d, 3
J
NH,H = 7.2 Hz, 1H, NH), 7.79 (d, 3
J
NH,H = 7.5 Hz, 1H, NH), 7.42–7.08 (m, 10H, Bn, δ, ε, ξ Phe), 4.50–4.33 (m, 3H, α Phe, CH2 OEt), 4.07–3.95 (m, 2H, CH2 Bn), 3.80 (m, 1H, α Leu), 3.48–3.32 (m, 1H, α hLeu), 3.07–2.90 (m, 2H, β, β′ Phe), 1.66–1.40 (m, 6H, β, β′, γ Leu, hLeu), 1.40–1.17 (m, 9H, CH3 Boc), 1.12–1.06 (m, 3H, CH3 OEt), 0.93–0.75 (m, 12H, δ, δ′ Leu, hLeu). 13C NMR (151 MHz, [D6]DMSO, 25 °C): δ = 171.8, 171.2, 170.6 (CO Phe, Leu, hLeu), 156.0 (CO Boc), 137.5 (C Bn), 137.1 (γ Phe), 129.0, 128.8, 128.1 (CH Bn), 127.8, 127.0, 126.5 (δ, ε, ζ Phe), 78.2 (α hLeu), 76.3 (C Boc), 60.4 (CH2 OEt), 60.1 (CH2 Bn), 53.7 (α Leu), 53.5 (α Phe), 51.4 (β Phe), 40.1 (β hLeu), 36.4 (β Leu), 28.0 (CH3 Boc), 24.2 (γ hLeu), 24.0 (γ Leu), 22.9; 22.8 δ, δ′ hLeu), 21.5; 21.2 (δ, δ′ Leu), 13.8 (CH3 OEt). HRMS (MALDI-TOF/TOF): calcd. for C35H52N4O6 [M + Na]+ 647.3779; found 647.3766.
N
α-benzyl-N
β-Boc-Val-Val-Phe-OEt (3d)
Yellow oil (82 mg, 27 %); R
f 0.71 (petrol ether:EtOAc:AcOH 10:5:0.5). 1H NMR (600 MHz, [D6]DMSO, 25 °C): δ = 8.65 (d, 3
J
NH,H = 7.2 Hz, 1H, NH), 8.27 (d, 3
J
NH,H = 7.3 Hz, 1H, NH), 7.65 (d, 3
J
NH,H = 7.4 Hz, 1H, NH),7.66–7.53 (m, 5H, Bn), 7.31–7.16 (m, 5H, δ, ε, ξ Phe), 4.45 (m, 2H, CH2 OEt), 4.31–4.24 (m, 1H, α Phe), 4.23–4.17 (m, 1H, α Val), 4.17–4.10 (m, 1H, α hVal), 4.01 (m, 2H, CH2 Bn), 3.01–2.94 (m, 2H, β, β′ Phe), 1.93 (m, 1H, β hVal), 1.72 (m, 1H, β Val), 1.65–1.25 (m, 9H, CH3 Boc), 1.16–1.00 (m, 6H, γ, γ′ Val), 0.94–0.86 (m, 3H, CH3 OEt), 0.85–0.75 (m, 6H, γ, γ′ hVal). 13C NMR (151 MHz, [D6]DMSO, 25 °C): δ = 171.2, 170.7, 160.7 (CO Phe, Val, hVal), 156.6 (CO Boc), 133.1 (Bn), 132.4 (γ Phe), 132.1, 131.5, 131.4 (CH Bn), 129.0, 128.7, 128.1 (δ, ε, ζ Phe), 60.4 (α hVal), 57.2 (α Val), 55.5 (α Phe), 53.5 (CH2 OEt), 47.5 (β Bn), 36.5 (β Phe), 33.3 (β hVal), 30.7 (β Val), 28.0 (CH3 Boc), 25.3, 24.4 (γ, γ′ hVal), 19.1, 17.9 (γ, γ′ Val), 13.9 (CH3 OEt). HRMS (MALDI-TOF/TOF): calcd. for C33H48N4O6 [M + Na]+ 619.3466; found 619.3478.
N
α-benzyl-N
β-Boc-Leu-Leu-Phe-NH2 (3e)
Yellow oil (212 mg, 71 %); R
f 0.48 (petrol ether:EtOAc:AcOH 7:5:0.5). 1H NMR (600 MHz, CD3OD, 25 °C): δ = 7.42–7.04 (m, 12H, δ, ε, ξ Phe, Bn, CONH2), 4.65; 4.58 (m, 1H, α Phe), 4.40; 4.19 (m, 1H, α Leu), 3.46; 3.32 (m, 1H, α hLeu), 3.14–2.77 (m, 2H, β, β′ Phe), 1.87–1.45 (m, 6H, β, β′, γ Leu, hLeu), 1.29 (br s, 9H, Boc), 0.94–0.80 (m, 12H, δ, δ′ Leu, hLeu). 13C NMR (151 MHz, CD3OD, 25 °C): δ = 175.8, 174.4, 172.6 (CO Phe, Leu, hLeu), 158.7 (CO Boc), 137.9 (γ Phe), 137.0, 129.6, 129.5, 129.4 (Bn), 129.1 (ε Phe), 128.5 (δ Phe), 127.8 (ε Phe), 80.8 (C Boc), 61.8 (CH2 Bn), 55.6, 55.5 (α Phe), 53.8, 53.0 (α Leu), 49.7 (α hLeu), 41.4, 41.1 (β Leu), 40.2, 40.1 (β Phe), 38.9, 38.3 (β hLeu), 28.6 (CH3 Boc), 25.8, 25.4 (γ Leu, hLeu), 23.4, 23.0, 22.5, 21. 8 (δ, δ′ Leu, hLeu). HRMS (MALDI-TOF/TOF): calcd. for C33H49N5O5 [M + Na]+ 618.3625; found 618.3618.
N
α-benzyl-N
β-Boc-Val-Val-Phe-NH2 (3f)
Yellow oil (195 mg, 69 %); R
f 0.46 (petrol ether:EtOAc:AcOH 5:5:0.5). 1H NMR (600 MHz, [D6]DMSO, 25 °C): δ = 8.11, 7.96, 7.75 (br d, 3H, NH Phe, Val, hVal), 7.42-7.19 (m, 10H, Bn, Phe), 7.16 (br s, 2H, NH2), 4.49 (m, 1H, α Phe), 4.22 (m, 1H, α Val), 3.76 (m, 2H, CH2 Bn,), 3.08-2.91 (m, 2H, β, β′ Phe), 2.81 (m, 1H, α hVal), 1.96 (m, 1H, β Val), 1.86 (m, 1H, β hVal), 1.32-1.13 (br s, 9H CH3 Boc), 1.07-0.65 (m, 12H, γ, γ′ Val, hVal). 13C NMR (151 MHz, [D6]DMSO, 25 °C): δ = 172.6 (CO Phe), 170.3, 170.1 (CO Val, hVal), 163.7 (CO Boc), 137.7 (γ Phe),), 137.6 (C Bn), 132.0, 131.5, 128.7 (CH Bn), 128.0, 127.8, 126.9, (δ, ε, ζ Phe), 78.1 (C Boc), 60.3 (CH2 Bn), 57.6 (α Val), 53.5 (α Phe), 37.6 (β Phe), 30.2 (β Val), 28.0 (β hVal), 19.3 (CH3 Boc), 18.9, 18.3 (γ, γ′ Val, hVal). HRMS (MALDI-TOF/TOF): calcd. for C31H45N5O5 [M + Na]+ 590.3313; found 590.3334.
N
α-benzyl-N
β-Boc-Ala-Ala-Phe-NH2 (3g)
Yellow oil: (176 mg, 69 %); R
f 0.31 (EtOAc:EtOH:AcOH:H2O 70:10:2:2). 1H NMR (600 MHz, [D6]DMSO, 25 °C): δ = 7.40–7.35 (br s, 3H. NH Ala, hAla, Phe), 7.30–7.18 (m, 10H, Bn, Phe), 7.15 (s, 2H, NH2), 4.41 (m, 1H, α Phe), 4.22 (m, 1H, α Ala), 3.82 (s, 2H, CH2 Bn), 3.57 (m, 1H, α hAla), 3.04–2.81 (m, 2 H, β, β′ Phe), 1.40 (m, 6H, β Ala, hAla), 1.22 (s, 9H, CH3 Boc). 13C NMR (151 MHz, [D6]DMSO, 25 °C): δ = 172.6 (CO Phe), 171.9 (CO, Ala), 171.6 (CO, hAla), 137.8 (Bn), 136.9 (γ Phe), 129.2, 128.2, 127.9 (CH Bn), 127.8, 127.0, 126.8, (δ, ε, ζ Phe), 78.5 (C Boc), 62.2 (α hAla), 53.6 (α Phe), 48.0 (α Ala), 37.4 (β Phe), 28.2 (CH3 Boc), 21.0 (β Ala), 17.8 (β hAla). HRMS (MALDI-TOF/TOF): calcd. for C27H37N5O5 [M + Na]+ 534.2686; found 534.2699.
General procedure for the synthesis of N
α-benzyl-N
β-Boc-Aaa-Gly-OH 4a and 4b
N
α-benzyl-N
β-Boc-Aaa-OH (Aaa = Leu, Val) (0.45 mmol) and HOSu (0.5 mmol) were dissolved in 3 mL dry DMF and solution cooled down to 0 °C. DCC (0.5 mmol) dissolved in 2 mL dry DMF was added dropwise. After 30 min reaction was stirred at room temperature and the consumption of starting dipeptide followed by TLC. The precipitate was filtered, and the filtrate added dropwise to the solution of glycine (0.45 mmol) and KHCO3 (0.45 mmol) in 5 mL of water. Reaction mixture was stirred at room temperature overnight. Solvent was evaporated and the residue purified by the flash column chromatography. Mobile phase used for the chromatography was also used for the determination of R
f value and is given for each compound.
N
α-benzyl-N
β-Boc-Leu-Gly-OH (4a)
Yellow oil: (117 mg, 52 %); R
f 0.57 (EtOAc:petrol ether:AcOH 10:5:0.5). 1H NMR (600 MHz, CDCl3, 25 °C): δ = 7.57–7.03 (m, 5H, Bn), 5.52 (s, 2H, CH2 Bn), 4.12 (br s, 2H, α Gly), 3.46–3.36 (m, 1H, α Leu), 1.95–1.88 (m, 2H, β Leu), 1.59 (m, 1H, γ Leu), 1.54–1.21 (m, 9H, CH3 Boc), 1.15–0.75 (m, 6H, δ, δ′ Leu). 13C NMR (151 MHz, CDCl3, 25 °C): δ = 175.8 (CO Gly), 163.1 (CO Leu), 157.9 (CO Boc), 129.7, 128.9, 128.0 (CH Bn), 64.0 (α Leu), 61.3 (CH2 Bn), 49.7 (α Gly), 36.9 (β Leu), 28.5 (CH3 Boc), 25.2 (γ Leu), 21.1, 21.0 (δ, δ′ Leu). HRMS (MALDI-TOF/TOF): calcd. for C20H31N3O5 [M + Na]+ 416.2156; found 416.2166.
N
α-benzyl-N
β-Boc-Val-Gly-OH (4b)
Yellow oil (82 mg, 46 %); R
f 0.70 (petrol ether:EtOAc:AcOH 7:5:0.5). 1H NMR (600 MHz, [D6]DMSO, 25 °C): δ = 8.51–7.09 (m, 5H, Bn), 5.09 (s, 2H, CH2 Bn), 3.96–3.68 (m, 1H, α Val), 3.60–2.96 (m, 2H α Gly), 1.39 (m, 1H, β Val), 1.32–1.18 (m, 9H, CH3 Boc), 1.08–0.99 (m, 6H, γ, γ′ Val). 13C NMR (151 MHz, [D6]DMSO, 25 °C): δ = 173.5 (CO Gly), 171.0 (CO Val), 156.6 (CO Boc), 128.9, 127.8, 126.8 (CH Bn), 78.6 (C Boc), 76.8 (α Val), 65.5 (CH2 Bn), 47.5 (α Gly), 28.0 (CH3 Boc), 25.0 (β Val), 19.3, 18.2 (γ, γ′ Val). HRMS (MALDI-TOF/TOF): calcd. for C19H29N3O5 [M + Na]+ 402.1999; found 402.2005.
Synthesis of N
α-benzyl-N
β-Boc-Leu-OMe (5a)
N
α-benzyl-N
β-Boc-Leu-OH (100 mg, 0.3 mmol) was dissolved in dry DMF (10 mL), KHCO3 (60 mg, 0.59 mmol) was added and then CH3I (30 μL, 0.48 mmol) dropwise. Reaction mixture was stirred at room temperature overnight. Water was added to the reaction and product extracted with EtOAc. Yellow oil: (102 mg, 95 %); R
f 0.64 (petrol ether:EtOAc:AcOH 15:5:0.5). ESI–MS: m/z 373 [M + Na]+.
Synthesis of N
α-benzyl-N
β-Boc-Ala-OMe (5b)
N
α-benzyl-N
β-Boc-Ala-OH (100 mg, 0.3 mmol) was dissolved in dry DMF (10 mL), KHCO3 (60 mg, 0.59 mmol) was added and then CH3I (30 μL, 0.48 mmol) dropwise. Reaction mixture was stirred at room temperature overnight. Water was added to the reaction and product extracted with EtOAc. Yellow oil (109 mg, 100 %); R
f 0.83 (petrol ether:EtOAc:AcOH 7:5:0.5). 1H NMR (600 MHz, CD2Cl2, 25 °C): δ = 7.32 (br d, 1H, NH hAla), 7.40–7.24 (m, 5H, Bn), 4.02–3.91 (m, 2H, CH2 Bn), 3.71 (s, 3H, OCH3), 3.63 (m, 1H, α hAla), 1.36 (d, 3
J
H,H = 7.3 Hz, 3H, β hAla), 1.34 (s, 9H, CH3 Boc). 13C NMR (151 MHz, CD2Cl2, 25 °C): δ = 164.6 (CO Ala), 137.8 (C Bn), 132.5, 128.7, 127.9 (CH Bn), 79.8 (C Boc), 76.5 (α hAla), 61.8 (CH2 Bn), 52.1 (OCH3), 28.5 (CH3 Boc), 16.6 (β hAla). ESI–MS: m/z 331 [M + Na]+, m/z 209 [M-Boc]+.
Synthesis of N
α-benzyl-hLeu-OMe (6a)
Crude product 5a was dissolved in TFA-water 9:1, v/v) and the reaction was stirred at room temperature 60 min. Solvent was evaporated and the residue dried in vacuum.
Synthesis of N
α-benzyl-N
β-Boc-Leu-N
α-benzyl-Leu-OMe (7)
N
α-benzyl-N
β-Boc-Leu-OH (100 mg; 0.3 mmol) was dissolved in dry DMF, NMM (55 μL, 0.50 mmol) and HATU (125 mg, 0.33 mmol) were added. After 15 min solution of N
α-benzyl-hydrazino leucine (100 mg; 0.3 mmol) and NMM (55 μL, 0.50 mmol) in 1 mL dry DMF was added. Reaction was stirred at room temperature overnight. Solvent was evaporated and the residue purified by flash column chromatography in petrol ether:EtOAc:AcOH 10:5:0.5. Yellow oil: (80 mg, 47 %); R
f 0.53 (petrol ether:EtOAc:AcOH 15:5:0.5). ESI–MS: [M + H]+
m/z 569.3; [M + Na]+
m/z 591.3. 1H NMR (600 MHz, CD2Cl2, 25 °C): δ = 7.49–7.14 (m, 10H, Bn), 6.86 (br s, 1H, NH), 5.83 (d, 3
J
NH,H = 10.5 Hz, 1H, NH), 3.99–3.94 (m, 2H, α hLeu), 3.78–3.62 (m, 7 H, CH2, Bn, CH3 O–CH3), 1.63–1.56 (m, 4H, β, β′ hLeu), 1.38–1.31 (m, 9H, CH3 Boc), 0.99–0.87 (m, 14H, γ, δ, δ′ hLeu). 13C NMR (151 MHz, CD2Cl2, 25 °C): δ = 172.8, 172.7 (CO hLeu), 137.1, 137.0 (C Bn), 128.9, 128.1, 127.7, 127.6, 127.0, 126.0 (CH Bn), 80.9 (C Boc), 66.0 (CH2 Bn), 66.0, 62.2 (α hLeu), 60.6 (CH2 Bn), 50.9 (OCH3), 38.7, 38.5 (β hLeu), 27.4 (CH3 Boc), 24.2, 23.7 (γ Leu), 22.1, 20.9 (δ, δ′, hLeu). HRMS (MALDI-TOF/TOF): calcd. for C32H48N46O5 [M + K]+ 607.3256; found 607.3256.
Synthesis of N
β-Boc-Leu-OMe (8a)
N
α-benzyl-N
β-Boc-Leu-OMe (240 mg, 0.71 mmol) was dissolved dry MeOH (45 mL), acetic acid (3 mL) and 10 % Pd/C (90 mg) were added. Reaction was performed under 15 atm H2 at room temperature for 3 days. Catalyst was filtered off, solvent evaporated and the residue purified by flash chromatography. Yellow oil: (88 mg; 48 %); R
f = 0.58 (petrol ether:EtOAc:AcOH 15:5:0.5). ESI–MS: [M-Boc]+
m/z 161.2; [M + H]+
m/z 261.2.
Synthesis of N
β-Boc-Phe-OMe (8b)
N
α-benzyl-N
β-Boc-Phe-OMe (100 mg, 0.26 mmol) was dissolved in dry MeOH (40 mL), acetic acid (3 mL) and 10 % Pd/C (26 mg) were added Reaction was performed under 15 atm H2 at room temperature for 3 days. Catalyst was filtered off, solvent evaporated and the residue purified by the flash column chromatography. Yellow oil: (57 mg; 74 %); R
f = 0.44 (petrol ether:EtOAc:AcOH 15:5:0.5). 1H NMR (600 MHz, CD2Cl2, 25 °C): δ = 7.33–7.22 (m, 6H, NH, δ, ε, ζ Phe), 6.16 (br. s, 1H, NH Phe), 3.93 (m, 1H, α hPhe), 3.69 (s, 3H, O–CH3), 3.05, 2.91 (m, 2H, β, β′ hPhe), 1.40 (s, 9H, CH3 Boc). 13C NMR (151 MHz, CD2Cl2, 25 °C): δ = 172.4 (CO hPhe), 155.6 (CO Boc), 136.5 (γ hPhe), 128.7, 127.0, 126.3 (δ, ε, ζ hPhe), 79.8 (C Boc), 63.6 (α hPhe), 51.3 (OCH3), 36.4 (β hPhe), 27.4 (CH3 Boc). ESI–MS: [M-Boc]+
m/z 195.1; [M + H]+
m/z 295.1; [M + Na]+
m/z 317.1; [2 M + H]+
m/z 589.3; [2 M + Na]+
m/z 611.3
Synthesis of Boc-Lys(Boc)-hLeu-OH (9)
Boc-Lys(Boc)-OH (236 mg, 0.68 mmol) and HOSu (117 mg, 1.02 mmol) were dissolved in dry DMF (3 mL) and solution cooled down to 0 °C. DCC (210 mg, 1.02 mmol) dissolved in dry DMF (2 mL) was added dropwise. After 30 min reaction was stirred at room temperature and the consumption of starting dipeptide followed by TLC. The precipitate was filtered, and the filtrate added dropwise to the solution of hLeu (100 mg, 0.68 mmol) and KHCO3 (136 mg, 1.36 mmol) in water (5 mL). Reaction mixture was stirred at room temperature overnight. Solvent was evaporated and the residue purified by the flash column chromatography (mobile phase: EtOAc:AcOH 70:2). Yellow oil: (156 mg, 48 %); R
f 0.38 (EtOAc:AcOH 70:2). ESI–MS: [M + H]+
m/z 475, [M-Boc]+
m/z 375, [M-2Boc]+
m/z 275. 1H NMR (600 MHz, CDCl3, 25 °C): δ = 4.83–4.62 (m, 1H, α Lys), 3.83–3.60 (m, 1H, α hLeu), 3.16–3.07 (m, 2H, ε Lys), 1.90–1.50 (m, 8H, β, δ Lys, β hLeu, γhLeu,), 1.42 (m, 18H, CH3 Boc), 1.33 (m, 2H, γ Lys), 0.99, 0.96 (d, 3
J
H,H = 6.6 Hz, 6H, δ, δ′ hLeu). 13C NMR (151 MHz, CDCl3, 25 °C): δ = 175.4 (CO hLeu), 52.9 (α Lys), 47.7 (α hLeu), 40.3 (β hLeu), 39.6 (ε Lys), 32.3 (β Lys), 29.8 (δ Lys), 28.4 (CH3 Boc), 25.1 (γ hLeu), 23.1 (γ Lys), 22.7, 22.1 (δ, δ′ hLeu). HRMS (MALDI-TOF/TOF): calcd. for C22H42N4O7 [M + Na]+ 497.2945; found 497.2937.
Synthesis of Boc-Lys(Boc)-hLeu-Leu-OH (10)
Compound 9 (100 mg, 0.21 mmol) and HOSu (37 mg, 0.32 mmol) were dissolved in dry DMF (3 mL) and solution cooled down to 0 °C. DCC (65 mg, 0.32 mmol) dissolved in dry DMF (2 mL) was added dropwise. After 30 min reaction was stirred at room temperature and the consumption of starting dipeptide followed by TLC. The precipitate was filtered, and the filtrate added dropwise to the solution of leucine (28 mg, 0.21 mmol) and KHCO3 (42 mg, 0.42 mmol) in water (5 mL). Reaction mixture was stirred at room temperature overnight. Solvent was evaporated and the residue purified by the flash column chromatography (mobile phase: EtOAc:AcOH 70:2). Yellow oil: (61 mg, 49 %); R
f 0.73 (EtOAc:AcOH 70:2). ESI–MS: [M + H]+
m/z 588.7, [2 M + H]+
m/z 1176.1. 1H NMR (600 MHz, CDCl3, 25 °C): δ = 5.27 (m, 1H, α Leu), 3.88–3.59 (m, 1H, α Lys), 3.46 (m, 1H, α hLeu), 3.12 (m, 2H, ε Lys), 1.75–1.68 (m, 2H, β Lys), 1.67–1.58 (m, 2H, δ Lys), 1.58–1.51 (m, 2H, β Leu, hLeu), 1.51–1.41 (m, 18H, CH3 Boc), 1.38–1.32 (m, 2H, γ Lys), 1.22–0.82 (m, 12H, δ, δ′ Leu, hLeu). 13C NMR (151 MHz, CDCl3, 25 °C): δ = 176.2 (CO Leu), 175.3 (CO hLeu), 171.7 (CO Lys), 157.2 (CO Boc), 76.5 (C Boc), 63.9 (α hLeu), 61.5 (α Lys), 55.5 (α Leu), 41.1 (ε Lys), 40.4 (β Leu), 33.8 (β hLeu), 31.5 (β Lys), 29.7 (δ Lys), 28.4, 28.3 (CH3 Boc), 25.6 (γ hLeu), 24.9 (γ Leu), 23.5 (γ Lys), 23.1, 23.0 (δ, δ′ hLeu), 22.7, 22.5 (δ, δ′ Leu). HRMS (MALDI-TOF/TOF): calcd. for C28H53N5O8 [M + K]+ 626.3524; found 626.3542.
Synthesis of Boc-Lys(Boc)-hLeu-hLeu-OH (11)
Compound 9 (100 mg, 0.21 mmol) was dissolved in dry DMF; NMM (23 μL, 0.21 mmol) and HATU (88 mg, 0.23 mmol) were added. After 15 min solution of hLeu (31 mg, 0.21 mmol) and NMM (23 μL, 0.21 mmol) in dry DMF (1 mL) was added. Reaction was stirred at room temperature overnight. Solvent was evaporated and the residue purified by the flash column chromatography (mobile phase: EtOAc:AcOH 70:2). Yellow oil: (68 mg, 53 %); R
f 0.35 (EtOAc:AcOH 70:2). ESI–MS: [M + H]+
m/z 603.6, [M-Boc]+
m/z 503.6. 1H NMR (600 MHz, MeOD, 25 °C): δ = 3.96 (m, 1H, α Lys), 3.49 (m, 2H, α hLeu), 3.06 (m, 2H, ε Lys), 1.89 (m, 2H, β Lys), 1.77–1.73 (m, 2H, δ Lys), 1.66–1.55 (m, 6H, β, β′, γ hLeu), 1.47 (m, 18H, CH3 Boc), 1.41–1.37 (m, 2H, γ Lys,), 1.02–0.98 (m, 12H, δ, δ′, hLeu). 13C NMR (151 MHz, CDCl3, 25 °C): δ = 178.1 (CO hLeu), 172.1 (CO hLeu), 170.9 (CO Lys), 157.2 (CO Boc), 82.1 (C Boc), 79.1 (C Boc), 66.3 (α hLeu), 63.9 (α Lys), 57.8 (α hLeu), 41.3 (β hLeu), 40.1 (ε Lys), 38.6 (α hLeu), 33.8 (β Lys), 29.6 (δ Lys), 28.4, 28.3 (CH3 Boc), 25.6 (γ hLeu), 24.8 (γ hLeu), 22.8 (γ Lys), 22.1, 22.0, 21.9 (δ,δ′ hLeu). HRMS (MALDI-TOF/TOF): calcd. for C28H54N6O8 [M + Na]+ 625.3895; found 625.3914.