A Stable Evans Blue Derived Exendin-4 peptide for Type 2 Diabetes Treatment

Transcription

1 Supporting Information A Stable Evans Blue Derived Exendin-4 peptide for Type 2 Diabetes Treatment Yi Liu, Guohao Wang, Huimin Zhang, Ying Ma, Lixin Lang, Orit Jacobson, Dale O. Kiesewetter, Lei Zhu, Shi Gao, Qingjie Ma* and Xiaoyuan Chen* China-Japan Union Hospital of Jilin University, Changchun, Jilin, 1333, China Laboratory of Molecular Imaging and Nanomedicine (LOMIN), National Institute of Biomedical Imaging and Bioengineer-ing (NIBIB), National Institutes of Health (NIH), Bethesda, Maryland, 2892, United States Center for Molecular Imaging and Translational Medicine, State Key Laboratory of Molecular Vaccinology and Molecular Diagnostics, School of Public Health, Xiamen University, Xiamen, 36112, China S1

2 E X P E R I M E N T A L S E C T I O N S 3 Reagents a nd Inst ru mentatio n S3 A n i ma l M o d e l... S 3 Hypoglycemic efficacies of Abextide II in db/db mice.s4 C h e m i s t r y.. S 4 Scheme S1. The synthetic route of Abextide.. S4 MEB-Mal-COOH..S4 M EB -M a l S5 Abextide S5 MEB-C3-Mal S5 Abextide II S6 Figure S1. The 1 H NMR spectrum of MEB-C3-Mal in CD 3 OD S6 Figure S2. The MS of MEB-C3-Mal, (Cys 4 )exendin-4 and Abextide II S7 Figure S3. The 1 H NMR spectrum of MEB-Mal-COOH in CD 3 OD S7 Figure S4. The 1 H NMR spectrum of MEB-Mal in CD 3 OD S8 Figure S5. The changes of the powder of MEB-Mal in LC-MS at the room temperature S8 Figure S6. T he LC-MS of the solution of Abextide in saline at R.T S9 Figure S7. The MS spectra of the powder of Abextide II at R.T. S1 Figure S8. Hypoglycemic efficacies of exendin-4, Albiglutide and Abextide II S1 S2

3 EXPERIMENTAL SECTION Reagents and Instrumentation. Unless otherwise stated, all chemicals were obtained from commercial sources and used without further purification. 2-tolidine, 1-amino-naphthol-2,4-disulfonic acid monosodium salt 3-maleimidopropionic acid, N,N-Diisopropylethylamine (DIPEA), (Benzotriazol-1-yloxy)tripyrrolidinophosphonium hexafluorophosphate (PyBOP) were purchased from Sigma-Aldrich (USA). (Cys 4 )exendin-4 was prepared by solid-phase peptide synthesis (CS Bio, Menlo Park, CA, USA). The (Cys 4 )exendin-4 sequence is His-Gly-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Leu-Ser-Lys-Gly-Met-Glu-Glu-Glu-Ala-Val- Arg-Leu-Phe-Ile-Glu-Trp-Leu-Lys-Asn-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Pro-Pro-Ser- Cys-NH 2. All other chemicals were purchased from Fisher Scientific (USA). The water used in the experiments with (Cys 4 )exendin-4 (FW ) was deionized from Milli-Q Integral Water Purification System. Analytical thin layer chromatography (TLC) was performed on Merck pre-coated silica gel 6 F254 plates with visualization by ultraviolet (UV) irradiation at λ= 254 nm. Purifications were performed by silica gel chromatography. Waters 6 high-performance liquid chromatography (HPLC) system with a Waters 996 Photodiode Array Detector (PDA) and a semi-preparative C18 HPLC column (XTerra Prep RP18, 1 µm, 7.8 X 3 mm, Waters) was used for purification of products. A Perkin Elmer 2 series HPLC pump with a Waters 2487 UV detector and a Bioscan Flow-Count detector using an analytical C-18 HPLC column (XTerra 5 µm, 15 x 4.6 mm, Waters) was used for analyzing compounds. HPLC running a linear gradient starting from 5 A (.1 TFA in acetonitrile) and 95 B (.1 TFA in water) for 5 min and increasing to 65 A at 35 min with a flow rate of 5 ml/min for semi-prep HPLC and 1 ml/min for analytical HPLC. LC/MS analysis was conducted on a Waters LC-MS system (Waters, Milford, MA) that included an Acquity HPLC unit coupled to the Waters Q-Tof Premier high resolution mass spectrometer. An Acquity BEH Shield RP18 column ( mm) was employed for chromatography. Elution was achieved with isocratic 2 mm ammonium formate,.1 formic acid, and 5 CH 3 CN at.35 ml/min. The entire column elute was introduced into the Q-Tof mass spectrometer. Ion detection was achieved in ESI mode using a source capillary voltage of 3.5 kv, source temperature of 11 C, desolvation temperature of 2 C, cone gas flow of 5 L/Hr (N 2 ), and desolvation gas flow of 7 L/Hr (N 2 ). Animal Model. Type 2 diabetic C57BL/6 db/db mice (males, 6-8 weeks old) were obtained from the Nanjing BioMedical Research Institute of Nanjing University (Nanjing, China). Normal BALB/c mice (females, 4-5 weeks old), weighing 18-2 g, were purchased from Harlan Laboratories. All animal studies were conducted in accordance with the principles and procedures outlined in the Guide for the Care and Use of Laboratory Animals and were S3

4 approved by the Institutional Animal Care and Use Committee (IACUC) of the Clinical Center, National Institutes of Health and the Xiamen University IACUC. Animals were housed under a 12-h light/dark cycle (lights on at 6 a.m.), allowed food and water ad libitum, and acclimatized for 2 weeks. Hypoglycemic efficacies of Abextide in db/db mice. Hypoglycemic efficacies of exendin-4 analogues were investigated using an intraperitoneal glucose tolerance test (IPGTT) in male db/db mice (6-7 weeks old). Under nonfasting conditions with free access to food and water, animals received a single subcutaneous injection of saline, exendin-4, Albiglutide or Abextide II (25 nmol/kg body wt, n = 3/group). Blood glucose levels were then monitored using a convenient blood glucose meter (ACCU-CHEK Sensor, Roche Diagnostics Corp., USA). At predetermined times (, 1, 2, 4, 6, 8, 12, 24, 48, 72, 96,12 h), blood samples were collected from tail vein of each animal and blood glucose levels were measured as mentioned above. Hypoglycemic durations to a blood glucose level of <8.35 nm (15 mg/dl) were checked. Chemistry. Compounds MEB-NH 2, MEB-Mal and Abextide (Scheme S1) are known compounds and were prepared according to literature procedures. Their physical and spectroscopic data were in agreement with those previously reported. Scheme S1. The synthetic route of Abextide. S4

5 MEB-Mal-COOH: To a solution of MEB-NH 2 (.54 g, 1. mmol) in 1 ml DMF, DIPEA (646 mg, 5. mmol) and maleic anhydride (1.96 g, 2. mmol) were added and stirred at room temperature for overnight. The DMF was removed under reduced pressure, the mixture was purified by C-18 column to obtain the MEB-Mal-COOH in yield of 6. 1 H NMR (3 MHz, MeOD) δ 8.71 (s, 1H), 8.2 (dd, J = 9., 4.5 Hz, 2H), 7.81 (d, J = 8.5 Hz, 1H), 7.65 (d, J = 8.5 Hz, 1H), 7.61 (s, 1H), 7.56 (s, 1H), 7.5 (s, 1H), 7.22 (d, J = 9.8 Hz, 1H), 6.42 (d, J = 13.6 Hz, 1H), 6.6 (d, J = 13.2 Hz, 1H), 2.59 (s, 3H), 2.44 (s, 3H). MS (LC- MS): calcd. For C 28 H 24 N 4 O 1 S ; found [M-H] -. MEB-Mal: To a solution of MEB-Mal-COOH (64.1 mg,.1 mmol) in 1 ml DMF/(Ac) 2 O (v/v, 1:9), the NaOAc (4 mg,.5 mmol) was added and stirred at 9 C under N 2 atmosphere for 2 h. The solvents were removed under reduced pressure, and the residuum was purified by C-18 column to obtain the MEB-Mal in yield of 4. 1 H NMR (3 MHz, MeOD) δ 8.7 (s, 1H), 8. (d, J = 5.2 Hz, 1H), 7.97 (d, J = 6.7 Hz, 1H), (m, 3H), 7.56 (dd, J = 8.2, 1.9 Hz, 1H), 7.19 (d, J = 1.8 Hz, 1H), 7.16 (d, J = 3.6 Hz, 1H), 6.99 (d, J = 2.5 Hz, 2H), 2.55 (s, 3H), 2.17 (s, 3H). MS (LC-MS): calcd. For C 28 H 22 N 4 O 9 S ; found [M-H] -. Abextide : To a solution of MEB-Mal (9.3 mg,.15 mmol) in 2. ml PBS, the solution of (Cys 4 )exendin-4 (42.9 mg,.1 mmol) in 2. ml PBS was added drop wise. The mixture was stirred at room temperature for 2 h, and then purified by C-18 column to obatin the 3. mg Abextide II in the yield of 6. MS (LC-MS): calcd. For 4912; found 4911 [M-H] -. MEB-C3-Mal: 3-Maleimidopropionic acid (51. mg,.3 mmol), PyBOP (14. mg,.2 mmol) and DIPEA (13 mg, 1. mmol) were added to a solution of MEB-NH 2 (54.1 mg,.1 mmol) in dry DMF. The mixture was vigorously stirred at room temperature for 24 h and monitored by the HPLC. When HPLC showed complete conversion to desired compound, the solvent was evaporated under reduced pressure. The residue was purified by a Waters Xterra C-18 chromatography column running a linear gradient from 5 A (.1 TFA in acetonitrile) and 95 B (.1 TFA in water) for 2 min and increasing A to 65. The desired product was collected and lyophilized to afford compound MEB-C3- S5

6 Mal as a purple solid (7). 1 H NMR (3 MHz, MeOD) δ 8.71 (s, 1H), 7.99 (d, J = 9.9 Hz, 1H), 7.93 (d, J = 8.5 Hz, 1H), 7.59 (d, J = 8.7 Hz, 1H), 7.54 (s, 1H), 7.49 (s, 1H), (m, 2H), 7.34 (d, J = 8.2 Hz, 1H), 7.15 (d, J = 9.9 Hz, 1H), 6.87 (s, 1H), 3.93 (t, J = 6.8 Hz, 2H), 2.75 (t, J = 6.9 Hz, 2H), 2.52 (s, 3H), 2.26 (s, 3H). MS (LC-MS): calcd. For C 31 H 27 N 5 O 1 S ; found [M-H] -. O H 2 N Exendin-4(Cys 4 ) S H HO N N N O N O MEB-C3-(Cys 4 )exendin-4 Abextide II SO 3 H SO 3 H Abextide II: To a solution of MEB-C3-Mal (13.9 mg, 2. µmol) in 2. ml PBS, the solution of (Cys 4 )exendin-4 (64.4 mg, 15. µmol) in 2. ml PBS was added drop wise. The mixture was stirred at room temperature for 4 h and monitored with HPLC and LC- MS. After the reaction was completed, the mixture was purified with semi-prep HPLC in 3 injections. The fractions containing the product were collected and lyophilized to give 35. mg of desired product Abextide II in the yield of MS (LC-MS): calcd. For 4983; found 4982 [M-H] -. Figure S1. The 1 H NMR spectrum of MEB-C3-Mal in CD 3 OD. S6

7 Figure S2. The mass spectra of MEB-C3-Mal (a), (Cys 4 )exendin-4 (b) and Abextide II (c). Figure S3. The 1 H NMR spectrum of MEB-Mal-COOH in CD 3 OD. S7

8 Figure S4. The 1 H NMR spectrum of MEB-Mal in CD 3 OD. NEB-Mal 18-May :54:11 Ying : TOF MS ES- 1 TIC 2.21e4 NEB-Mal Ying (4.519) 1 1 Time May :54:11 1: TOF MS ES m/z NEB-Mal 18-May :54:11 Ying (4.966) 1: TOF MS ES Ying (4.892) : TOF MS ES m/z Figure S5. The changes of the powder of MEB-Mal in LC-MS at the room temperature for 1 day (the mass peaks were belong to the LC peak at 4.52, 4.89 and 4.97 min). S8

9 Figure S6. The LC-MS spectra of the solution of Abextide in saline at room temperature for h and 24 h. The top is LC spectrum and the bottom is mass spectrum of the main peaks. Ying : TOF MS ES _ Da 2.66e Time e mass Figure S7. The MS spectra of the powder of Abextide II at room temperature for 3 days. S9

10 Figure S8. (a, c) Hypoglycemic efficacies of free exendin-4 (25 nmol/kg), Albiglutide (14, 123 nmol/kg, ) and Abextide II (25 nmol/kg) for 12 h. Data represent three rats and are presented as mean ± SDs. (b, d) The focused profile of a, c respectively, (-32 h, 3-16 mmol/l). Arrows depict hypoglycemic duration rebound to 8.35 mmol/l (normal blood glucose level). S1