Synthesis of Novel Peptide Linkers: Simultaneous Cyclization and Labeling

Transcription

1 UPPRTING INFRMATIN ynthesis of Novel Peptide Linkers: imultaneous Cyclization and Labeling Gajanan K. Dewkar, Pedro B. Carneiro, Matthew C. T. Hartman* Department of Chemistry and Massey Cancer Center, Virginia Commonwealth University, 401 College treet, Richmond, Virginia UA Table of Contents General Experimental -2 General procedure for synthesis of 3,5-bis(bromomethyl)benzyl ester -2 Characterization Data for Compounds 3, 3a-e and 5a-e -2 Table: 1. Mono-alkylation of 1,3,5-Tris(bromomethyl)benzene by Aliphatic and aromatic Carboxylic Acids -7 Characterization Data for Compounds 7a-g -8 General procedure for peptide cyclization (TRH-H Pro/ omatostatin with substituted 3,5-bis(bromomethyl)benzene 3, 3a-e, 5a-e and 7a-g -10 Larger scale cyclization of somatostatin cyclization with 5c -10 Table: 2. ubstituted 1,3-bis(bromomethyl)benzyl cyclization with TRH-H Pro and omatostatin H and 13 C NMR pectra of compound 3, 3a-e, 5a-e and 7a-g -12 MALDI-TF/M spectra of TRH-H Pro cyclized with compound 3, 3a-e, 5a-e and 7a-g MALDI-TF/M spectra of omatostatin cyclized with compound 3, 3a-e, 5b and 7a-g MALDI-TF/M spectrum and HPLC chromatogram of larger scale cyclization of somatostatin with 5c

2 General Experimental All reagents were used as received. All reactions were performed under argon atmosphere in oven-dried glassware. Flash chromatography was performed employing igma- Aldrich Mesh, 60Å. Thin-layer chromatography (TLC) was performed using silica gel coated aluminum plates with F-254 indicator (250 µm, 20x 20 cm, Whatman). Preparative thinlayer chromatography (PTLC) was performed using silica gel coated glass plates with F-254 indicator (250 µm, 20x 20 cm, Whatman). Visualization was accomplished by fluorescence quenching and/or Iodine. Nuclear magnetic resonance spectra ( 1 H and 13 C) were recorded on Varian 300 and 400 spectrometer using tetramethylsilane as an internal standard. Melting points were determined using a Thomas-Hoover melting point apparatus and are uncorrected. Mass spectra were recorded on a Waters/Micromass QTF-2 EI-M and/or Micromass MALDI-R MALDI-TF. MALDI-TF M spectra were calibrated externally using ACTH(18-39) [M + H] , Glu-fibrinopeptide [M + H] , and Angiotensin II [M + H] General procedure for synthesis of 3,5-bis(bromomethyl)benzyl esters: A solution of a carboxylic acid (1.05 mmol, 1.5 equiv), 1,3,5-tris(bromomethyl) benzene (1) (250 mg, 0.70 mmol, 1 equiv), and N,N-diisopropylethylamine (DIEA) (0.370 ml, 2.1 mmol, 3 equiv) in acetonitrile (5 ml) was stirred at rt for 24 h. After evaporation of the solvent under vacuum, CH 2 Cl 2 was added (25 ml). The solution was washed successively with water (3 x 25 ml) and brine (25 ml) and then dried over Mg 4. After filtration and evaporation of the solvent under reduced pressure, the crude product was purified by column chromatography (Hexanes/EtAc, 9:1) to give the desired product (45-75%). Characterization Data for Compounds 3 3,5-Bis(bromomethyl)benzyl propionate (3) white solid mp C; yield: 183 mg (75%); 1 H-NMR (CDCl 3, 300 MHz) δ: 1.17 (t, 3H, J = 7.6 Hz, CH 3 ), 2.40 (q, 2H, J = 7.6Hz, CH 2 C), 4.47 (s, 4H, CH 2 ), 5.09 (s, 2H, CH 2 ), 7.31 (d, 1H, J = 1.2 Hz, Ar-H), 7.38 (t, 2H, J = 1.7 Hz, 2

3 Ar-H); 13 C-NMR (CDCl 3, 75 MHz) δ: 9.2, 27.6, 32.6, 65.3, 128.7, 129.4, 137.6, 138.9, 174.3; EI-M [M+Na] + m/z calcd for C 12 H Na found a 3,5-Bis(bromomethyl)benzyl acetate (3a) white solid mp C (Lit C); yield: 176 mg (75%); 1 H-NMR (CDCl 3, 300 MHz) δ: 2.12 (s, 3H, CH 3 C), 4.47 (s, 4H, CH 2 ), 5.09 (s, 2H, CH 2 ), 7.31 (d, 1H, J = 1.5 Hz, Ar-H), 7.38 (t, 2H, J = 1.7 Hz, Ar-H); 13 C-NMR (CDCl 3, 75 MHz) δ: 21.0, 32.5, 65.4, 128.7, 129.4, 137.4, 138.8, 170.7; EI-M [M+Na] + m/z calcd for C 11 H Na found b 3,5-Bis(bromomethyl)benzyl isobutyrate (3b) white solid mp C; yield: 153 mg (60%); 1 H-NMR (CDCl 3, 300 MHz) δ: 1.20 (d, 6H, J = 7.0 Hz, CH 3 ), (septet, 1H, J = 7.0 Hz, CH), 4.47 (s, 4H, CH 2 ), 5.09 (s, 2H, CH 2 ), 7.31 (d, 1H, J = 1.7 Hz, Ar-H), 7.38 (t, 2H, J = 1.5, Ar-H); 13 C-NMR (CDCl 3, 75 MHz) δ: 19.1, 32.6, 34.0, 65.2, 128.5, 129.3, 137.8, 138.8, 176.9; EI-M [M+Na] + m/z calcd for C 13 H Na found c 3,5-Bis(bromomethyl)benzyl benzoate (3c) white solid mp C; yield: 153 mg (55%); - 1 H-NMR (CDCl 3, 300 MHz) δ: 4.46 (s, 4H, CH 2 ), 5.33 (s, 2H, CH 2 ), (m, 5H, Ar- H), (m, 1H, Ar-H), 8.07 (d, 2H, J = 8.2 Hz Ar-H); 13 C-NMR (CDCl 3, 75 MHz) δ: 32.5, 65.8, 128.5, 128.6, 129.5, 129.8, 129.9, 133.2, 137.5, 138.9, 166.3; EI-M [M+Na] + m/z calcd for C 16 H Na found

4 3d 3,5-Bis(bromomethyl)benzyl 2-(naphthalen-1-yl)acetate (3d) white solid mp C; yield: 170 mg (53%); 1 H-NMR (CDCl 3, 300 MHz) δ: 4.14 (s, 2H, CH 2 C), 4.32 (s, 4H, CH 2 ), 5.09 (s, 2H, CH 2 ), 7.04 (d, 2H, J = 1.5 Hz, Ar-H), 7.29 (t, 1H, J = 1.5, Ar-H), (m, 2H, Ar-H), (m, 2H, Ar-H), (m, 1H, Ar-H), (m, 1H, Ar-H), (m, 1H, Ar-H); 13 C-NMR (CDCl 3, 75 MHz) δ: 32.5, 39.2, 65.5, 123.8, 125.6, 125.9, 126.5, 128.0, 128.2, 128.3, 128.8, 129.2, 130.3, 132.0, 133.8, 137.3, 132.0, 171.1; EI-M [M+Na] + m/z calcd for C 21 H Na found Ph 3e 3,5-bis(bromomethyl)benzyl 2-benzylbenzoate (3e) gum. Yield: 205 mg (60%); 1 H-NMR (CDCl 3, 300 MHz) δ: 4.38 (s, 2H, Ar-CH 2 -Ar), 4.43 (s, 4H, CH 2 ), 5.23 (s, 2H, CH 2 ), 7.09 (d, 2H, J = 1.5 Hz, Ar-H), (m, 8H, Ar-H), 7.37 (t, 1H, J = 1.5 Hz, Ar-H), 7.47 (t, 1H, J = 7.6 Hz, Ar-H), 7.93 (d, 2H, J = 7.9 Hz, Ar-H); 13 C-NMR (CDCl 3, 75 MHz) δ: 32.5, 39.6, 65.8, , 128.4, 128.7, 128.9, 129.5, 129.7, 130.8, 131.8, 132.3, 137.3, 138.8, 140.8, 142.2, 167.2; EI-M [M+Na] + m/z calcd for C 23 H Na found a ( ) 12 3,5-Bis(bromomethyl)benzyl tetradecanoate (5a) white solid mp C; yield: 182 mg (52%); 1 H-NMR (CDCl 3, 300 MHz) δ: 0.87 (t, 3H, J = 7.0 Hz, CH 3 ), (m, 20H, CH 2 ), 1.64 (p, 2H, J = 7.3 Hz, CH 2 ), 2.36 (t, 2H, J = 7.6 Hz, CH 2 C), 4.46 (s, 4H, CH 2 ), 5.09 (s, 2H, CH 2 ), 7.30 (bs, 1H, Ar-H), 7.37 (bs, 2H, Ar-H); 13 C-NMR (CDCl 3, 75 MHz) δ: 14.2, 22.8, 4

5 25.0, 29.2, 29.3, 29.4, 29.5, 29.7, 29.7, 29.7, 32.0, 32.5, 34.3, 65.2, 128.6, 129.4, 137.7, 138.8, 173.6; EI-M [M+Na] + m/z calcd for C 23 H Na found b (5Z,8Z,11Z,14Z)-3,5-Bis(bromomethyl)benzyl icosa-5,8,11,14-tetraenoate (5b) The general procedure was followed except CH 2 Cl 2 was used as the solvent, and the reaction was scaled proportionally to 10 mg of 1,3,5-tris(bromomethyl)benzene. The crude product was purified by preparative TLC (Hexanes/EtAc, 9:1) giving a gum, 8.0 mg (50%). 1 H-NMR (CDCl 3, 300 MHz) δ: 0.88 (t, 3H, J = 7.0 Hz, CH 3 ), (m, 6H, CH 2 ), 1.73 (p, 2H, J = 7.3 Hz, CH 2 ), (m, 4H, CH 2 ), 2.38 (t, 2H J = 7.6 Hz, CH 2 ), 2.80 (p, 6H, J = 6.1 Hz, CH-CH 2 -CH), 4.46 (s, 4H, CH 2 ), 5.09 (s, 2H, CH 2 ), (m, 8H, CH), 7.30 (d, 1H, J = 1.5 Hz, Ar-H), 7.37 (t, 2H, J = 1.7 Hz, Ar-H); 13 C-NMR (CDCl 3, 75 MHz) δ: 14.2, 22.7, 24.8, 25.7, 26.6, 27.3, 29.4, 31.1, 31.6, 32.5, 33.7, 65.3, 127.6, 127.9, 128.2, 128.3, 128.7, 128.8, 128.9, 129.1, 129.5, 130.6, 137.6, 138.9, 173.4; MALDI-TF/M [M+Na] + calcd for C 29 H Na found H H H 5c 3,5-Bis(bromomethyl)benzyl cholate ester (5c) The general procedure was followed except CH 2 Cl 2 was used as the solvent. The crude product was purified by column chromatography (CHCl 3 /MeH, 9:1) giving a light yellow solid mp C; yield: 220 mg (46%); 1 H-NMR (CDCl 3, 300 MHz) δ: 0.66 (s, 3H, CH 3 ), 0.89 (s, 3H, CH 3 ), 0.98 (d, J = 6.0 Hz, 3H, CH 3 ), (m, 5H, CH 2 ), (m, 18H, CH 2 ), 3.13 (q, J = 7.4 Hz, 2H, CH 2 ), (m, 1H, CHH), 3.71 (p, J = 6.6 Hz, 2H, CH 2 ), 3.85 (bs, 1H, H), 3.97 (bs, 1H, H), 4.47 (s, 4H, CH 2 ), 5.09 (s, 2H, CH 2 ), 7.31 (d, 2H, J = 1.5 Hz, Ar-H), 7.38 (t, 1H, J = 1.7 Hz, Ar-H); 13 C- NMR (CDCl 3, 75 MHz) δ: 12.1, 12.6, 17.4, 22.6, 23.3, 26.7, 27.5, 28.4, 30.6, 30.9, 31.3, 32.6, 5

6 34.7, 34.8, 35.2, 35.3, 39.6, 39.8, 41.5, 42.0, 42.4, 46.6, 47.1, 54.2, 65.3, 68.5, 72.0, 73.0, 128.8, 129.5, 137.6, 138.9, 174.0; MALDI-TF/M [M+Na] + calcd for C 33 H Na found HN H NH H ( ) 4 5d 3,5-Bis(bromomethyl)benzyl Biotin ester (5d) The general procedure was followed except DMF was used as the solvent. The crude product was purified by column chromatography (CHCl 3 /MeH, 9:1) giving a white solid mp C; yield: 182 mg (50%); 1 H-NMR (CDCl 3, 300 MHz) δ: (m, 2H, CH 2 ), (m, 4H, CH 2 ), 2.40 (t, 2H, J = 7.6 Hz, CH 2 C), 2.69 (d, 1H, J = 12.6 Hz, CH 2 ), 2.86 (dd, 1H, J = 4.7 Hz, J = 12.6 Hz, CH 2 ), 3.12 (m, 1H, CH), 4.26 (m, 2H, CH), 4.47 (s, 4H, CH 2 ), 5.08 (s, 2H, CH 2 ), 5.84 (bs, 1H, NH), 6.26 (bs, 1H, NH), 7.31 (bs, 2H, Ar-H), 7.37 (bs, 2H, Ar-H); 13 C-NMR (CDCl 3, 75 MHz) δ: 24.7, 28.2, 28.3, 32.6, 33.9, 40.5, 55.5, 60.1, 62.0, 65.3, 182.7, 129.4, 137.4, 138.8, 164.0, 173.4; MALDI-TF/M [M+Na] + calcd for C 19 H 24 2 N 2 3 +Na found CH 5e H 3,5-Bis(bromomethyl)benzyl 5-carboxyfluorescein ester (5e) The general procedure was followed except DMF was used as the solvent, and the reaction was scaled down proportionally to 50 mg of 1,3,5-tris(bromomethyl)benzene. The crude product was purified by column chromatography (CHCl 3 /MeH, 9:1) giving a orange solid: yield 40 mg (45%); mp 280 C dec.; 6

7 1 H-NMR (CDCl 3 +1 drop CD 3 D, 400 MHz) δ:4.41(s, 4H, CH 2 ), 5.31(s, 2H, CH 2 ), (m, 5H, Ar-H), 6.62 (d, 2H, J = 2.1 Hz, Ar-H), 7.18 (d, 1H, J = 8.0 Hz, Ar-H), 7.34 (s, 2H, Ar-H), 8.26 (d, 1H, J = 8.0 Hz, Ar-H), 8.58 (s, 1H, Ar-H); 13 C-NMR (CDCl 3 +1 drop CD 3 D, 75 MHz) δ:32.3, 66.6, 102.9, 109.4, 112.6, 124.6, 126.7, 127.4, 128.5, 128.9,129.3, 129.8, 131.7, 136.1, 136.7, 138.9, 139.0; MALDI-TF/M [M+Na] + calcd for C 30 H Na found Table: 1. Mono-alkylation of 1,3,5-Tris(bromomethyl)benzene by Aliphatic and aromatic Carboxylic Acids a + R-CH 6a-g DIEA CH 3 CN, rt, 24 h 7a-g R entry acid-6 product-7 (yield %) b 1 6a 2 6b 3 6c 4 6d 5 6e 6 6f 8 6g Me ( ) 2 ( ) 4 ( ) 6 ( ) 8 ( ) 10 ( ) 14 H H H H H H CH 7a (65) 7b (62) 7c (67) 7d (57) 7e (50) 7f (48) 7g (60) a Reaction conditions: bromo (1 equiv), acid (1.5 equiv), base (3 equiv), rt, 24 h. b Isolated yield after column chromatographic purification. 7

8 7a ( ) 2 3,5-Bis(bromomethyl)benzyl butyrate (7a) white solid mp C; yield: 165 mg (65%); 1 H-NMR (CDCl 3, 300 MHz) δ: 0.95 (t, 3H, J = 7.3 Hz, CH 3 ), (m, 2H, CH 2 ), 2.35 (t, 2H, J = 7.3 Hz, CH 2 C), 4.45 (s, 4H, CH 2 ), 5.08 (s, 2H, CH 2 ), 7.30 (d, 1H, J = 1.4 Hz, Ar- H), 7.36 (t, 2H, J = 1.7 Hz, Ar-H); 13 C-NMR (CDCl 3 ) δ: 13.7, 18.4, 32.5, 36.1, 65.1, 128.5, 129.3, 137.6, 138.7, 173.3; EI-M [M+Na] + m/z calcd for C 13 H Na found b ( ) 4 3,5-Bis(bromomethyl)benzyl hexanoate (7b) white solid mp C; yield: 170 mg (62%); 1 H-NMR (CDCl 3, 300 MHz) δ: 0.88 (t, 3H, J = 7.0 Hz, CH 3 ), (m, 4H, CH 2 ), 1.65 (p, 2H, J = 7.3 Hz, CH 2 ), 2.36 (t, 2H, J = 7.6 Hz, CH 2 C), 4.46 (s, 4H, CH 2 ), 5.08 (s, 2H, CH 2 ), 7.30 (d, 1H, J = 1.5 Hz, Ar-H), 7.37 (t, 2H, J = 1.7 Hz, Ar-H); 13 C-NMR (CDCl 3, 75 MHz) δ: 14.0, 22.0, 24.7, 31.3, 32.5, 34.3, 65.2, 128.6, 129.4, 137.6, 138.8, 173.6; EI-M [M+Na] + m/z calcd for C 15 H Na found c ( ) 6 3,5-Bis(bromomethyl)benzyl octanoate (7c) white solid mp C; yield: 197 mg (67%); 1 H-NMR (CDCl 3, 300 MHz) δ: 0.87 (t, 3H, J = 7.0 Hz, CH 3 ), (m, 8H, CH 2 ), 1.65 (p, 2H, J = 7.2 Hz, CH 2 ), 2.37 (t, 2H, J = 7.6 Hz, CH 2 C), 4.46 (s, 4H, CH 2 ), 5.09 (s, 2H, CH 2 ), 7.30 (bs, 1H, Ar-H), 7.37 (bs, 2H, Ar-H); 13 C-NMR (CDCl 3, 75 MHz) δ: 14.1, 22.6, 24.9, 28.9, 29.1, 31.7, 32.5, 34.3, 65.1, 128.6, 129.3, 137.6, 138.8, 173.5; EI-M [M+Na] + m/z calcd for C 17 H Na found

9 7d ( ) 8 3,5-Bis(bromomethyl)benzyl decanoate (7d) white solid mp C; yield: 178 mg (57%); 1 H-NMR (CDCl 3, 300 MHz) δ: 0.87 (t, 3H, J = 6.7 Hz, CH 3 ), (m, 12H, CH 2 ), 1.64 (p, 2H, J = 7.0 Hz, CH 2 ), 2.36 (t, 2H, J = 7.3 Hz, CH 2 C), 4.45 (s, 4H, CH 2 ), 5.08 (s, 2H, CH 2 ), 7.30 (d, 1H, J = 1.5 Hz, Ar-H), 7.36 (t, 2H, J = 1.7 Hz, Ar-H); 13 C-NMR (CDCl 3, 75 MHz) δ: 14.1, 22.7, 24.9, 29.1, 29.3, 29.4, 31.9, 32.5, 34.3, 65.1, 128.6, 129.3, 137.6, 138.8, 173.5; EI-M [M+Na] + m/z calcd for C 19 H Na found e ( ) 10 3,5-Bis(bromomethyl)benzyl dodecanoate (7e) white solid mp C; yield: 165 mg (50%); 1 H-NMR (CDCl 3, 400 MHz) δ: 0.87 (t, 3H, J = 7.3 Hz, CH 3 ), (m, 16H, CH 2 ), 1.65 (p, 2H, J = 7.3 Hz, CH 2 ), 2.36 (t, 2H, J = 7.6 Hz, CH 2 C), 4.46 (s, 4H, CH 2 ), 5.09 (s, 2H, CH 2 ), 7.30 (d, 1H, J = 1.5 Hz, Ar-H), 7.37 (t, 2H, J = 1.6 Hz, Ar-H); 13 C-NMR (CDCl 3, 300 MHz) δ: 14.2, 22.7, 25.0, 29.2, 29.3, 29.4, 29.5, 29.7, 32.0, 32.5, 34.3, 65.2, 128.6, 129.4, 137.6, 138.8, 173.6; EI-M [M+Na] + m/z calcd for C 21 H Na found f ( ) 14 3,5-Bis(bromomethyl)benzyl palmitate (7f) The general procedure was followed except the crude product was purified by preparative TLC (Hexanes/EtAc, 9:1) giving a white solid. mp C; yield 180 mg (48%); 1 H-NMR (CDCl 3, 300 MHz) δ: 0.87 (t, 3H, J = 7.0 Hz, CH 3 ), (m, 24H, CH 2 ), 1.64 (p, 2H, J = 7.3 Hz, CH 2 ), 2.36 (t, 2H, J = 7.6 Hz, CH 2 C), 4.46 (s, 4H, CH 2 ), 5.09 (s, 2H, CH 2 ), 7.30 (bs, 1H, Ar-H), 7.37 (bs, 2H, Ar-H); 13 C-NMR (CDCl 3, 75 MHz) δ: 14.2, 22.8, 25.0, 29.2, 29.3, 29.4, 29.5, 29.7, 29.7, 29.7, 32.0, 32.5, 34.3, 65.2, 128.6, 9

10 129.4, 137.7, 138.8, 173.6; EI-M [M+Na] + m/z calcd for C 25 H Na found Me 7g 3,5-Bis(bromomethyl)benzyl 4-methoxybenzoate (7g) white solid mp C; yield: 180 mg (60%); 1 H-NMR (CDCl 3, 300 MHz) δ: 3.85 (s, 3H, Me), 4.47 (s, 4H, CH 2 ), 5.31 (s, 2H, CH 2 ), 6.93 (d, 2H, J = 8.8 Hz, Ar-H), 7.39 (s, 3H, Ar-H), 8.03 (d, 2H, J = 8.8 Hz, Ar-H); 13 C-NMR (CDCl 3, 75 MHz) δ: 32.6, 55.5, 65.6, 113.8, 122.2, 128.6, 129.4, 131.9, 137.8, 138.9, 163.6, 166.1; EI-M [M+Na] + m/z calcd for C 17 H Na found General procedure for peptide cyclization (TRH-H Pro or omatostatin with substituted 3,5-bis(bromomethyl)benzenes 3, 3a-e, 5a-e and 7a-g: Peptide cyclization reactions (10 μl) contained ammonium carbonate (20 mm, ph ~ 9.5) or ammonium bicarbonate (20 mm, ph ~ 7.0), tris-carboxyethylphosphine (200 μm), peptide (TRH-H Pro, 10 μm, or omatostatin, 100 μm), and linkers 3-7g (1.1 mm) in an aqueous solution containing 30-70% acetonitrile to maximize solubility of the linkers. The reactions were incubated at rt for 1-2 h and the reaction products was analyzed by adding 2 μl to 8 μl of MALDI matrix (10 mg/ml α-cyano-p-hydroxycinnamic acid in 1:1 MeCN:0.1% aq. trifluoroacetic acid). 1 μl was spotted onto a MALDI plate and analyzed. cale-up of omatostatin cyclization with 3,5-Bis(bromomethyl)benzyl cholate ester (5c): A 100 ml oven dried flask was charged with acetonitrile (24.4 ml) and water (18.4 ml) and was degassed with argon for 5 min. Then 200 mm ammonium bicarbonate (6.1 ml, ph 7.9), TCEP (3.5 mg, 12.1 μmol) in 6.10 ml of water, and somatostatin (10 mg, 6.1 μmol) were added and reaction was kept under argon. After 20 min 3,5-bis(bromomethyl)benzyl cholate ester (5c) (45.54 mg, 66.5 μmol) was added as a solution in 6.10 ml of acetonitrile. The reaction was incubated at rt, and the progress was monitored by MALDI-TF. After 6 h the reaction was frozen and lyophilized. The resulting white powder was washed with acetone (3 x 25 ml) giving 13 mg of a white solid (98%). A small amount of the product (0.5 mg) was dissolved in 10

11 100 ul of 1:1 acetonitrile:dm (100 ul) and was analyzed for purity by HPLC (Vydac 218 TP x 100 mm column) under the following conditions: 10 min at 10% acetonitrile in water, 30 min of a gradient from % acetonitrile, followed by 5 minutes at 100% acetonitrile. The product was injected at 5 minutes into the program. Both the aqueous and acetonitrile mobile phases contained 0.1% TFA. The solvent was passed through the column at 1 ml/min and peaks were detected at 280 nm. Table: 2. ubstituted 1,3-bis(bromomethyl)benzyl cyclization with TRH-H Pro and omatostatin entry ubstrate TRH-Pro omatostatin calc/obs calc/obs / / a / / b / / c / / d / / e / / a / / b / / c / / d / / e / / a / / b / / c / / d / / e / / f / / g / /

12 1 H and 13 C NMR pectra of compound 3, 3a-e, 5a-e and 7a-g (-12-47) 3 12

13 3 13

14 3a 14

15 3a 15

16 3b 16

17 3b 17

18 3c 18

19 3c 19

20 3d CH 2 Cl 2 20

21 3d 21

22 Ph 3e 22

23 Ph 3e 23

24 5a ( ) 12 24

25 5a ( ) 12 25

26 5b 26

27 5b 27

28 H H H 5c 28

29 H H H 5c 29

30 HN H NH H ( ) 4 5d 30

31 HN H NH H ( ) 4 5d 31

32 32 5e

33 33 5e

34 7a ( ) 2 34

35 7a ( ) 2 35

36 7b ( ) 4 36

37 7b ( ) 4 37

38 7c ( ) 6 38

39 7c ( ) 6 39

40 7d ( ) 8 40

41 7d ( ) 8 41

42 7e ( ) 10 42

43 7e ( ) 10 43

44 7f ( ) 14 44

45 7f ( ) 14 45

46 Me 7g 46

47 Me 7g 47

48 MALDI-TF/M spectra of TRH-H Pro cyclized with compound 3, 3a-e, 5a-e and 7a-g bs: Calc:

49 3a bs: Calc:

50 3b bs: Calc:

51 3c bs: Calc:

52 3d bs: Calc:

53 3e bs: Calc:

54 ( ) 12 5a bs: Calc:

55 bs: Calc: b 55

56 H 5c H H bs: Cal:

57 bs: d Cal:

58 CH H 5e bs: Calc:

59 7a bs: Calc:

60 ( ) 4 7b bs: Calc:

61 ( ) 6 7c bs: Calc:

62 ( ) 8 7d bs: Calc:

63 7e ( ) 10 bs: Calc:

64 7f ( ) 14 bs: Calc:

65 7g bs: Calc: Me 65

66 MALDI-TF/M spectra of omatostatin cyclized with compound 3, 3a-e, 5b and 7a-g bs: Calc:

67 3a bs: Calc:

68 3b bs: Calc:

69 3c bs: Calc: [M + Na] + 69

70 3d bs: Calc:

71 3e bs: Calc: [M+Na] + 71

72 bs: Calc: b 72

73 7a bs: Calc:

74 ( ) 4 7b bs: Calc:

75 7c ( ) 6 bs: Calc: [M+Na] + 75

76 ( ) 8 7d bs: Calc:

77 7e ( ) 10 bs: Calc:

78 7f ( ) 14 bs: Calc:

79 7g bs: Calc: Me 79

80 MALDI-TF M spectrum of the product of larger scale somatostatin cyclization with 5c before HPLC purification H 5c H H Calc: bs: bs: Cal:

81 HPLC chromatogram of the product of larger scale cyclization of 5c and somatostatin H 5c H H bs: Cal:

82 MALDI-TF M spectrum of the peak with retention time min H 5c H H Calc: bs: bs: Cal: