Supplementary Materials

Extraordinary Metabolic Stability of Peptides Containing -Aminoxy Acids

Fei Chen,1 Bin Ma,2Zong-Chang Yang,1Ge Lin,*2 and Dan Yang*1

1Department of Chemistry, The University of Hong Kong, Pokfulam Road,Hong Kong, P. R. China

Telephone: (852) 2859 2159

Fax: (852) 28592159

E-mail:

2School of Biomedical Sciences, The Chinese University of Hong Kong, Shatin, N. T., Hong Kong, P. R. China

Telephone: (852) 2609 6824

Fax: (852) 2603 5139

E-mail:

Preparation of peptide1: Peptide 1 was prepared according to standard protocols for peptide synthesis [1]. 1H NMR (400 MHz, CD3OD) δ 1.39 (t, J = 8.3 Hz, 6H), 2.93–3.02 (m, 2H), 3.18–3.31 (m, 2H), 4.06 (dd, J = 5.0, 8.1 Hz, 1H), 4.34–4.40 (m, 2H), 4.70 (dd, J = 5.4, 8.8 Hz, 1H), 7.20–7.36 (m, 10H); 13C NMR (75 MHz, CD3OD) δ 17.7, 18.3, 38.5, 38.8, 50.2, 55.4, 55.4, 56.1, 127.9, 128.9, 129.5, 130.1, 130.3, 130.5, 135.5, 138.2, 169.6, 172.6, 174.2, 175.8; HRMS (FAB) for C24H31N4O5 [M+H]+ calcd 455.2294, found 455.2269.

Scheme 1. Synthesis of Peptide 2

Preparation of peptide2: To a solution of compound A (0.47 g, 2 mmol) in THF (4 mL), MeOH (4 mL) and H2O (2 mL)was added lithium hydroxide monohydrate (0.252 g, 6 mmol). After stirring for 1 h, the reaction mixture was diluted with EtOAc (15 mL)and acidified with 1 N HCl(6 mL, 6 mmol). After extraction with EtOAc and wash with brine, the organic layer was dried over anhydrous MgSO4 and concentrated. The resulting acid (compoundB) was directly used for the next step reaction.

To a solution of compound C[2] (0.667 g, 2 mmol) in CH3OH (9 mL) and CH2Cl2 (3 mL) was added 35% hydrazine solution (0.55mL, 6 mmol). After stirring for 1 h at room temperature, the reaction mixture was diluted with Et2O and filtered. The filtrate was concentrated in vacuo. The residue was dissolved in CH2Cl2 and was washed with 5% NaHCO3 and then brine. The organic layer was dried over by anhydrous MgSO4 and concentrated to provide crude compoundD.

CompoundsB and Dweredissolved in freshly distilled CH2Cl2 (10 mL), and then HOBt (676 mg, 5 mmol)and EDCI (575mg, 3 mmol) were added. After stirring overnight, the reaction mixture was diluted with EtOAc. The organic layer was washed with 5% NaHCO3 and brine, then dried over anhydrous MgSO4 and concentrated. The crude oil was purified by flash column chromatography to afford compound E (0.39 g, 48%).

To a solution of compound E (0.5 mmol) in CH2Cl2 (3 mL) was carefully added equal volume of trifluoroacetic acid (3 mL) at 0 °C. After stirring for 1 h at room temperature, the reaction mixture was concentrated in vacuo, mixed with 1 M HCl solution in Et2O (0.5 mL),concentrated again in vacuo, and washed with EtOAc/Hexane to give Peptide 2. 1H NMR (400 MHz, CD3OD) δ 0.92 (d, J = 6.9 Hz, 3H), 0.94 (d, J = 6.6 Hz, 3H), 1.38 (t, J = 7.2 Hz, 6H), 1.43–1.48 (m, 1H), 1.51–1.60 (m, 1H), 1.76–1.88 (m, 1H), 3.18 (t, J = 11.8 Hz, 1H), 3.34–3.48 (m, 5H), 3.97 (dd, J = 3.9, 11.1 Hz, 1H), 4.33 (dd, J = 5.1, 8.0 Hz, 1H), 7.27–7.36 (m, 5H); 13C NMR (100 MHz, CD3OD) δ9.7, 22.4, 23.3, 25.6, 35.1, 40.8, 47.2, 64.5, 83.6, 128.8, 130.0, 130.7, 134.9, 164.2, 174.5; LRMS (FAB) m/z 351 [M+H]+ (100), 176 (35); HRMS (FAB) for C19H31N2O4 [M+H]+ calcd 351.2284, found 351.2289.

Scheme 2. Synthesis of Peptide 3

Preparation of peptide3: Peptide 3 was synthesized according to the procedure for the synthesis ofPeptide 2.1H NMR (400 MHz, CD3OD) δ1.36(t, J = 7.0Hz, 6H), 2.87–2.96(m, 2H), 3.03–3.19 (m, 2H), 3.36–3.43 (m, 4H), 3.96 (dd, J = 4.0, 11.2Hz, 1H), 4.42 (t, J = 6.1 Hz, 1H), 7.17–7.37 (m, 10H); 13C NMR (100 MHz, CD3OD) δ 9.5, 35.1, 37.7, 47.1, 64.6, 85.3, 127.8, 128.9, 129.3, 130.1, 130.5, 130.8, 135.0, 136.9, 164.0, 173.0; LRMS (FAB) m/z 385 [M+H]+ (100), 307(41), 154 (62); HRMS (FAB) for C22H29N2O4 [M+H]+ calcd 385.2127, found 385.2094.

Scheme 3. Synthesis of Peptide 4

Preparation of peptide4:Peptide 4 was synthesized according to the procedure for the synthesis ofPeptide 2. 1H NMR (400 MHz, CD3OD) δ 0.92–0.94 (m, 6H), 0.98 (d, J = 6.6 Hz, 3H), 1.03 (d, J = 6.6Hz, 3H), 1.59 (ddd, J = 4.4, 8.9, 14.2 Hz, 1H), 1.74 (ddd, J = 5.4, 8.9, 14.2 Hz, 1H), 1.88–2.09 (m, 2H), 3.12 (d, J = 7.7 Hz, 2H), 3.69 (d, J = 5.6 Hz, 1H), 3.90 (t, J = 7.7 Hz, 1H),4.42 (dd, J = 4.4, 8.9 Hz, 1H), 7.25–7.38 (m, 5H); 13C NMR (100 MHz, CD3OD) δ 17.6, 18.8, 22.4, 23.5, 25.8, 31.5, 38.5, 41.4, 53.7, 84.5, 91.0, 128.9, 130.2, 130.5, 135.6, 167.1, 168.6, 176.8;HRMS (FAB) for C20H32N3O6 [M+H]+ calcd 410.2291, found 410.2276.

Scheme 4. Synthesis of Peptide 5

Preparation of peptide5:Compound Q was synthesized according to the procedure for the synthesis of compound E.

A suspension of compound Q(0.31 g, 0.7 mmol) and 10% Pd/C (30mg) in CH3OH (5mL) was treated with H2 under 1 atm and stirred for 1.5 h at room temperature. The reaction mixture was filtered through celite and rinsed with CH3OH. The filtrate was concentrated in vacuo to give compoundR as the crude product.

To a solution of compound Q (0.7 mmol) in CH2Cl2 (5 mL) was carefully added equal volume of trifluoroacetic acid (5 mL) at 0 °C. After stirring for 1 h at room temperature, the reaction mixture was concentrated in vacuo, and azeotroped with toluene to get crude product S.

Compound T was synthesized according to the procedure for the synthesis of compound E.

A suspension of compound T(0.2 g, 0.3 mmol) and 10% Pd/C (20mg) in CH3OH (5mL) was treated with H2 under 1 atm and stirred for 1.5 h at room temperature. The reaction mixture was filtered through celite and rinsed with CH3OH. The filtrate was concentrated in vacuo.

And then peptide 5 was synthesized according to the procedure for the synthesis of peptide 2from compound E.1H NMR (300 MHz, D2O) δ 1.15 (d,J = 7.0 Hz, 3H), 1.38 (d, J = 7.1 Hz, 3H), 3.06–3.13 (m, 4H), 3.89 (q, J = 6.8 Hz, 1H), 4.11 (q, J = 7.3 Hz, 1H), 4.51–4.62 (m, 2H), 7.17–7.31 (m, 10H); 13C NMR (125 MHz, CD3OD) δ 17.5, 17.7, 38.2, 38.6, 50.2, 51.8, 85.9, 91.8, 127.6, 128.0, 129.3, 129.5, 130.5, 130.6, 136.7, 136.8, 167.9, 170.7, 171.5, 175.9; HRMS (FAB) for C24H31N4O7 [M+H]+ calcd 487.2193, found 487.2193.

Scheme 5. Synthesis of Peptides6 7

Preparation of peptide6:Peptide 6 was synthesized according to the procedure for the synthesis ofPeptide 2.1H NMR (300 MHz, D2O) δ 1.18 (d,J = 6.7 Hz 3H), 3.00–3.15 (m, 4H), 4.07–4.15 (m, 2H), 4.53 (t, J = 6.6 Hz, 1H), 7.13–7.30 (m, 10H); 13C NMR (75 MHz, D2O) δ 16.2, 36.2, 36.5, 46.8, 53.9, 84.0, 127.1, 127.2, 128.5, 128.9, 129.2, 129.3, 133.3, 135.1, 168.4, 170.3, 173.6; LRMS (FAB) m/z400 [M+H]+ (100), 154 (50); HRMS (FAB) for C21H26N3O5 [M+H]+ calcd 400.1872, found 400.1875.

Preparation of peptide7: Compound V was synthesized according to the procedure for the synthesis of compound E. Peptide 7 was synthesized from compound Vaccording to the procedure for the synthesis ofcompound B and compoundR. The crude product was purified according to the procedure for the purification of peptide 2. 1H NMR (300 MHz, D2O) δ 1.28 (d,J = 7.1 Hz 3H), 2.80–3.06 (m, 3H), 3.09 (dd, J = 5.0, 14.1 Hz, 1H), 3.80 (q, J = 7.3 Hz, 1H), 4.43–4.49 (m, 2H), 6.98–7.28 (m, 10H); 13C NMR (75 MHz, D2O) δ 16.2, 36.2, 36.6, 46.8, 53.6, 85.5, 127.1×2, 128.5, 128.6, 128.9, 129.2, 134.6, 136.1, 168.0, 171.4, 174.1; LRMS (FAB) m/z 400 [M+H]+ (13), 307 (85), 154 (100); HRMS (FAB) for C21H26N3O5 [M+H]+ calcd 400.1872, found 400.1882.

References:

[1]. Houben-Weyl, Methods of Organic Chemistry, Additional andSupplementary Volume E22 to the 4th Edition, Synthesis of Peptidesand Peptidomimetics (Eds.: M. Goodman, A. Felix, L.Moroder, C. Toniolo), Georg Thieme Verlag, Stuttgart, 2001.

[2]. Yang D, Li B, Ng FF, Yan YL, Qu J, Wu YD (2001)Synthesis and Characterization of Chiral N-O Turns Induced by -Aminoxy Acids. The Journal of Organic Chemistry 66 (22):73037312.

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