ELECTRONIC SUPPLEMENTARY MATERIAL
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Evaluation of Cu-64 and Ga-68 Radiolabeled Glucagon-Like Peptide-1 Receptor Agonists as PET Tracers for Pancreatic βcell Imaging
Journal: Molecular Imaging and Biology
Nilantha Bandara,1 Alex Zheleznyak,1Kaavya Cherukuri,1 David A. Griffith,2 Chris Limberakis,3 David A. Tess,4 Chen Jianqing,5 Rikki Waterhouse,5 Suzanne E. Lapi1
1Department of Radiology, Washington University School of Medicine, St. Louis, MO63110, USA
2Worldwide Medicinal Chemistry,Pfizer Worldwide Research and Development,Cambridge, MA 02139, USA
3Worldwide Medicinal Chemistry,Pfizer Worldwide Research and Development,Groton, CT 06340, USA
4Pharmacokinetics, Dynamics and Metabolism, Pfizer Worldwide Research and Development,Cambridge, MA 02139, USA
5Clinical and Translational Imaging, Pfizer Worldwide Research and Development, Cambridge, MA 02139, USA
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Table S1. In vitro serum stability study results of [64Cu]DO3A-VS-Cys40-exendin-4, [64Cu]NODA-VS-Cys40-exendin-4, [68Ga]DO3A-VS-Cys40-exendin-4 and [68Ga]NODA-VS-Cys40-exendin-4 analogues. 64Cu was evaluated for 24 h and 68Ga for 3 h.
Time(h) / [64Cu]DO3A-VS-Cys40-exendin-4 / [64Cu]NODA-VS-Cys40
-exendin-4 / Time
(h) / [68Ga]DO3A-VS-Cys40
-exendin-4 / [68Ga]NODA-VS-Cys40
-exendin-4
0 / 100.0 / ± / 0.0 / 100.0 / ± / 0.0 / 0 / 100.0 / ± / 0.0 / 100.0 / ± / 0.0
0.5 / 100.0 / ± / 0.0 / 100.0 / ± / 0.0 / 0.5 / 99.2 / ± / 0.8 / 99.8 / ± / 0.3
1 / 100.0 / ± / 0.0 / 99.2 / ± / 1.1 / 1 / 97.8 / ± / 0.2 / 98.0 / ± / 0.1
2 / 100.0 / ± / 0.0 / 96.7 / ± / 0.2 / 2 / 95.5 / ± / 1.3 / 96.7 / ± / 0.6
4 / 98.8 / ± / 0.8 / 94.8 / ± / 1.6 / 3 / 91.9 / ± / 0.3 / 92.9 / ± / 1.0
24 / 83.1 / ± / 0.6 / 85.6 / ± / 1.4
Table S2.Biodistribution results of [64Cu]DO3A-VS-Cys40exendin-4 and [64Cu]NODA-VS-Cys40-exendin-4 analogues using normal SD rats 10, 60 and 240 min p.i.
[64Cu]DO3A-VS-Cys40-exendin-4 / [64Cu]NODA-VS-Cys40-exendin-4Organs / 10 min / 60 min / 240 min / 10 min / 60 min / 240 min
blood / 0.44 / ± / 0.05 / 0.15 / ± / 0.01 / 0.07 / ± / 0.00 / 0.40 / ± / 0.08 / 0.20 / ± / 0.02 / 0.12 / ± / 0.03
lung / 7.03 / ± / 2.12 / 15.46 / ± / 4.76 / 10.15 / ± / 1.54 / 13.06 / ± / 1.48 / 16.33 / ± / 0.93 / 9.97 / ± / 0.43
liver / 0.33 / ± / 0.03 / 0.485 / ± / 0.05 / 0.35 / ± / 0.03 / 0.17 / ± / 0.03 / 0.644 / ± / 0.09 / 0.51 / ± / 0.09
spleen / 0.19 / ± / 0.02 / 0.12 / ± / 0.01 / 0.11 / ± / 0.03 / 0.18 / ± / 0.04 / 0.39 / ± / 0.10 / 0.09 / ± / 0.02
kidney / 25.51 / ± / 3.63 / 53.11 / ± / 5.34 / 46.40 / ± / 2.96 / 24.16 / ± / 7.19 / 35.31 / ± / 2.96 / 25.74 / ± / 2.11
muscle / 0.10 / ± / 0.01 / 0.04 / ± / 0.00 / 0.01 / ± / 0.00 / 0.09 / ± / 0.04 / 0.04 / ± / 0.00 / 0.03 / ± / 0.00
fat / 0.08 / ± / 0.02 / 0.03 / ± / 0.00 / 0.01 / ± / 0.00 / 0.06 / ± / 0.01 / 0.03 / ± / 0.00 / 0.04 / ± / 0.03
heart / 0.18 / ± / 0.01 / 0.07 / ± / 0.01 / 0.04 / ± / 0.00 / 0.17 / ± / 0.04 / 0.13 / ± / 0.01 / 0.07 / ± / 0.01
brain / 0.03 / ± / 0.01 / 0.02 / ± / 0.00 / 0.01 / ± / 0.00 / 0.03 / ± / 0.00 / 0.03 / ± / 0.01 / 0.02 / ± / 0.01
bone / 0.08 / ± / 0.01 / 0.05 / ± / 0.00 / 0.05 / ± / 0.01 / 0.07 / ± / 0.02 / 0.06 / ± / 0.00 / 0.07 / ± / 0.01
adrenals / 0.24 / ± / 0.04 / 0.11 / ± / 0.03 / 0.07 / ± / 0.01 / 0.19 / ± / 0.03 / 0.15 / ± / 0.04 / 0.11 / ± / 0.03
pancreas / 0.22 / ± / 0.02 / 0.279 / ± / 0.050 / 0.20 / ± / 0.01 / 0.23 / ± / 0.03 / 0.286 / ± / 0.037 / 0.23 / ± / 0.02
stomach / 0.65 / ± / 0.04 / 0.54 / ± / 0.07 / 0.56 / ± / 0.18 / 0.70 / ± / 0.27 / 0.63 / ± / 0.16 / 0.49 / ± / 0.11
sm. int. / 0.26 / ± / 0.02 / 0.31 / ± / 0.04 / 0.22 / ± / 0.01 / 0.23 / ± / 0.07 / 0.36 / ± / 0.04 / 0.32 / ± / 0.05
u lg. int. / 0.16 / ± / 0.01 / 0.24 / ± / 0.02 / 0.24 / ± / 0.03 / 0.14 / ± / 0.03 / 0.26 / ± / 0.04 / 0.31 / ± / 0.05
l lg. int. / 0.08 / ± / 0.01 / 0.05 / ± / 0.00 / 0.18 / ± / 0.04 / 0.07 / ± / 0.03 / 0.07 / ± / 0.01 / 0.17 / ± / 0.02
Table S3.Biodistribution results of [64Cu]DO3A-VS-Cys40-exendin-4 analoguesusing normal SD rats after 1 -h p.i. with and without the presence of cold exendin-4 and exendin-(9–39)-amide injected 10 min prior to the radiolabeled compound injection.
Organ / Nonblock / Blocked with exendin-4 / Blocked with exendin-(9-39)-amideblood / 0.09 / ± / 0.02 / 0.11 / ± / 0.03 / 0.11 / ± / 0.02
lung / 8.77 / ± / 1.62 / 0.11 / ± / 0.01 / 0.24 / ± / 0.01
liver / 0.20 / ± / 0.02 / 0.21 / ± / 0.03 / 0.27 / ± / 0.02
spleen / 0.11 / ± / 0.01 / 0.06 / ± / 0.01 / 0.08 / ± / 0.01
kidney / 28.13 / ± / 2.53 / 25.16 / ± / 11.74 / 36.66 / ± / 6.10
muscle / 0.03 / ± / 0.01 / 0.03 / ± / 0.01 / 0.03 / ± / 0.00
fat / 0.02 / ± / 0.00 / 0.02 / ± / 0.00 / 0.02 / ± / 0.01
heart / 0.04 / ± / 0.01 / 0.05 / ± / 0.01 / 0.05 / ± / 0.01
brain / 0.01 / ± / 0.00 / 0.01 / ± / 0.00 / 0.01 / ± / 0.01
bone / 0.03 / ± / 0.00 / 0.03 / ± / 0.00 / 0.04 / ± / 0.00
adrenals / 0.05 / ± / 0.01 / 0.07 / ± / 0.02 / 0.07 / ± / 0.00
pancreas / 0.112 / ± / 0.018 / 0.051 / ± / 0.008 / 0.062 / ± / 0.006
stomach / 0.35 / ± / 0.10 / 0.05 / ± / 0.01 / 0.04 / ± / 0.00
sm. Int. / 0.17 / ± / 0.03 / 0.15 / ± / 0.04 / 0.12 / ± / 0.01
u. lg. int. / 0.12 / ± / 0.03 / 0.06 / ± / 0.01 / 0.15 / ± / 0.01
l. lg. int. / 0.03 / ± / 0.00 / 0.02 / ± / 0.00 / 0.03 / ± / 0.00
Table S4. Biodistribution results of [68Ga]NODA-VS-Cys40-exendin-4, [68Ga] DO3A-VS-Cys40-exendin-4 and [64Cu]NODA-VS-Cys40-exendin-4 analogues 1 -h p.i.with and without the presence of cold exendin-4 injected 10 min prior to the radiolabeled compound injection.
[68Ga]NODA-VS-Cys40-exenadin-4 / [68Ga]DO3A-VS-Cys40-exenadin-4 / [64Cu]NODA-VS-Cys40-exenadin-4Nonblock / Block / Nonblock / Block / Nonblock / Block
blood / 0.08 / ± / 0.009 / 0.08 / ± / 0.030 / 0.10 / ± / 0.003 / 0.10 / ± / 0.007 / 0.15 / ± / 0.031 / 0.09 / ± / 0.032
lung / 15.44 / ± / 2.579 / 0.25 / ± / 0.096 / 7.68 / ± / 0.300 / 0.11 / ± / 0.007 / 17.14 / ± / 3.791 / 0.11 / ± / 0.024
liver / 0.08 / ± / 0.014 / 0.07 / ± / 0.008 / 0.08 / ± / 0.007 / 0.08 / ± / 0.006 / 0.26 / ± / 0.019 / 0.16 / ± / 0.043
spleen / 0.09 / ± / 0.013 / 0.05 / ± / 0.008 / 0.08 / ± / 0.023 / 0.06 / ± / 0.009 / 0.16 / ± / 0.019 / 0.07 / ± / 0.022
kidney / 44.20 / ± / 0.583 / 32.97 / ± / 2.708 / 32.78 / ± / 1.761 / 26.19 / ± / 5.332 / 35.67 / ± / 2.905 / 29.94 / ± / 6.295
muscle / 0.02 / ± / 0.003 / 0.02 / ± / 0.007 / 0.02 / ± / 0.003 / 0.04 / ± / 0.019 / 0.03 / ± / 0.005 / 0.02 / ± / 0.009
fat / 0.01 / ± / 0.004 / 0.01 / ± / 0.010 / 0.02 / ± / 0.002 / 0.02 / ± / 0.012 / 0.03 / ± / 0.004 / 0.02 / ± / 0.011
heart / 0.04 / ± / 0.002 / 0.03 / ± / 0.019 / 0.04 / ± / 0.002 / 0.04 / ± / 0.003 / 0.07 / ± / 0.006 / 0.04 / ± / 0.011
brain / 0.01 / ± / 0.003 / 0.01 / ± / 0.003 / 0.01 / ± / 0.001 / 0.01 / ± / 0.001 / 0.01 / ± / 0.003 / 0.01 / ± / 0.003
bone / 0.02 / ± / 0.005 / 0.02 / ± / 0.005 / 0.03 / ± / 0.003 / 0.03 / ± / 0.003 / 0.05 / ± / 0.010 / 0.03 / ± / 0.010
adrenals / 0.04 / ± / 0.001 / 0.03 / ± / 0.021 / 0.05 / ± / 0.002 / 0.06 / ± / 0.009 / 0.10 / ± / 0.020 / 0.07 / ± / 0.028
pancreas / 0.26 / ± / 0.033 / 0.03 / ± / 0.009 / 0.11 / ± / 0.027 / 0.04 / ± / 0.008 / 0.14 / ± / 0.017 / 0.04 / ± / 0.013
stomach / 0.42 / ± / 0.032 / 0.01 / ± / 0.001 / 0.24 / ± / 0.063 / 0.01 / ± / 0.001 / 0.38 / ± / 0.111 / 0.02 / ± / 0.003
sm. Int. / 0.15 / ± / 0.025 / 0.05 / ± / 0.005 / 0.11 / ± / 0.014 / 0.06 / ± / 0.008 / 0.23 / ± / 0.024 / 0.13 / ± / 0.018
u lg. int. / 0.10 / ± / 0.024 / 0.02 / ± / 0.006 / 0.07 / ± / 0.013 / 0.03 / ± / 0.006 / 0.15 / ± / 0.033 / 0.04 / ± / 0.014
l. lg. int. / 0.03 / ± / 0.006 / 0.01 / ± / 0.001 / 0.02 / ± / 0.002 / 0.02 / ± / 0.004 / 0.04 / ± / 0.006 / 0.02 / ± / 0.005
Fig.S1. Blocking study with exendin-4 at 1 -h p.i..a [68Ga]NODA-VS-Cys40-exendin-4, b [68Ga]DO3A-VS-Cys40-exendin-4, and c [64Cu]NODA-VS-Cys40-exendin-4. Nonblock shown in the checkered bars and the uptake with the presence of a blocking dose is shown in dotted bars.
Fig.S2. Ex vivo Autoradiography imaging of pancreatic sections (20–40 µm) in normal SD rats 1 -h p.i. of i.v. injected [68Ga]NODA-VS-Cys40-exendin-4 and [68Ga]DO3A-VS-Cys40-exendin-4. Blocking dose of cold exendin-4 injected 10 min prior to the radiolabeled dose injection. Rat pancreatic section of [68Ga]NODA-VS-Cys40-exendin-4 without the exendin-4 block shown in a and with blocking displayed in b. [68Ga]DO3A-VS-Cys40-exendin-4 without and with blocking shown in c and d respectively.
Synthesis of DO3A-VS-Cys40-exendin-4 and NODA-VS-Cys40-exendin-4
Materials and Methods
Small Molecules
Commercial quality reagents and solvents were used without further purification. Proton NMR chemical shifts are given in parts per million downfield from tetramethylsilane and were recorded on a Bruker Avance III 400 MHz spectrometer. Multiplicities are given as s (singlet), br s (broad singlet), d (doublet), dd (doublet of doublets), and m (multiplet). Mass spectra were recorded using Agilent 1956A MSD and Agilent 6140 MSD spectrometers. Preparatory reversed phase HPLC purification and purity analysis were done using a Gilson GX-281 with a Shimadzu Prep LCMS 2010EV MS detectorand a Shimadzu SPD-20AV UV detector. ELSD data was collected using a Sedex 75 instrument.
Peptides
Commercial CTC resin and Fmoc-amino acids were used without further purification. Peptides were synthesized using standard solid phase peptide synthesis technique as described in this section.
Purification of final peptides: Final peptides were purified using a Waters 4000 system connected to a Waters Delta-PakTM C18, 15 micron, 100 Å reversed-phase HPLC column (25 × 200mm) eluting with a solvent gradient of 63:37 (A:B) to 40:60 (A:B) or 58:42 (A:B) to 48:52 (A:B) over 60 min at a flow rate of 20.0 mL/min (solvent A: 0.1% TFA in water; solvent B: 0.1% TFA in 80% acetonitrile and 20% water).
Purity check of peptides. Final peptides were analyzed using a HP1090 system with a 4.6 × 150 mm Phenomenex C18 (1), 5 micron 100 Å column eluting with a solvent gradient of 58:42 to 48:52 (A:C) over 20 min at a flow rate of 1.0 mL/min. solvent A:H2O (0.1% TFA), solvent C:acetonitrile:water (4:1, 0.09% TFA).The specific retention times and UV purity (220 nm) are listed with the final peptides.
Mass spectrometeryof the peptides. A Voyager DE system was used to collect mass data via the matrix-assisted laser desorption/ionization (MALDI) technique.
Abbreviations: CTC, 2-chlorotrityl; DCM, dichloromethane; DMF, dimethylformamide; DIPEA, diisopropylethylamine; DMSO, dimethylsulfoxide; EDT, 1,2-ethanedithiol; ELSD: evaporative light scattering detector; ESI-MS, electrospray ionization mass spectrometry; Fmoc, Fluorenylmethyloxycarbonyl;HBTU, N,N,N′,N′-Tetramethyl-O-(1H-benzotriazol-1-yl)uroniumhexafluorophosphate;; 1H NMR, proton nuclear magnetic resonance; HPLC, high performance liquid chromatography; MALDI, matrix-assisted laser desorption/ionization;MeOH, methanol; NMM, N-methylmorpholine; TFA, trifluoroacetic acid; UV, ultra-violet.
Tri-tert-butyl 2,2',2''-(10-(2-(vinylsulfonyl)ethyl)-1,4,7,10-tetraazacyclododecane-1,4,7-triyl)triacetate(DO3A-VSderivative). To a solution of tri-tert-butyl 2,2',2''-(1,4,7,10-tetraazacyclododecane-1,4,7-triyl)triacetate1(1.0 g, 1.9 mmol) in THF (10 mL) was added divinylsulfone (1.15 g, 9.7mmol) andDIPEA (0.74 g, 5.7 mmol). The mixture was stirred at room temperature for 30 min. The mixture was diluted in ethyl acetate, washed with water, dried over sodium sulfate and filtered. The filtrate was then concentrated under reduced pressure to afford the crude product. The crude product was then waspurified by reversed phase prep-HPLC (column: DIKMA Diamonsil (1) C18 200×20×5 mm; gradient: 80:20 to 60:40 [water (0.1%TFA):acetonitrile (0.1% TFA)] to give 800 mg (65%) ofthe desired vinylsulfone as a white solid.1H NMR (400 MHz, CDCl3) 6.75 (dd, J=10.0, 16.6 Hz, 1 H), 6.44 (d, J=16.6 Hz, 1 H), 6.22 (d, J=10.0 Hz, 1 H), 3.77– 3.63 (m, 6 H), 3.60–3.03 (m, 20 H) 1.53 (s, 1.5 H), 1.49 (s, 10 H), 1.48 (s, 14 H), 1.41 (s, 1.5 H); MS (ESI) m/z 633.3 (M+1); HPLC purity (220 nm) = 99.6%.
Di-tert-butyl 2,2'-(7-(2-(vinylsulfonyl)ethyl)-1,4,7-triazonane-1,4-diyl)diacetate(NODA-VSderivative). To a solution of di-tert-butyl 2,2'-(1,4,7-triazonane-1,4-diyl)diacetate2(1.0 g, 2.8 mmol) in THF (20 mL) was added divinylsulfone (1.65 g, 14 mmol) and DIPEA (1.1 g, 8.5mmol). The mixture was stirred at ambient temperature for 30 min. The mixture was diluted in ethyl acetate, washed with water, dried over sodium sulfate and filtered. The filtrate was then concentrated under reduced pressure to afford the crude product. The crude product was then was purified by reverse phase prep-HPLC [column: DIKMA Diamonsil (1) C18, 200×20×5 mm; gradient: 85:15 to 65:35 [water (0.1% formic acid):acetonitrile (0.1% formic acid)] to give 560 mg (42%) of the desired vinylsulfone as a white solid. 1H NMR (DMSO-d6, T = 80 °C) 7.03 (dd, J = 10.1, 16.6 Hz, 1 H), 6.23 (d, J = 16.6 Hz, 1 H), 6.16 (d, J = 10.1 Hz, 1 H), 4.27 (br s, 2 H), 3.99 (br s, 2 H), 3.66 (br s, 2 H), 3.50 (br s, 2 H), 3.26–3.21 (m, 2 H), 3.09 (br s, 2 H), 2.80–2.69 (m, 8 H), 1.46 (s, 8 H), 1.44 (s, 10 H); MS (ESI) m/z476.3 (M+1); HPLC purity (ELSD) = 99.4%.
Preparation of the Fmoc-Cys(Trt)-CTC resin and synthesis of the template peptide (Cys40-exendin-4)
CTC resin (950 g, 0.988 mol) was suspended in DCM (8 L) in a peptide synthesis vessel equipped with a mechanical stirrer. To the suspension was added Fmoc-Cys(Trt)-OH (579 g, 0.988 mmol) and DIPEA (860 mL, 4.94mol). The mixture was stirred gently at room temperature for 3 h. To the mixture was then added MeOH (0.95 L) and resulting mixture was stirred at room temperature for 30 min. Reaction mixture was filtered. Solid was washed with DCM (3×15 L) and then MeOH (2 ×10 L). The resulting solid was dried under vacuum overnight to furnish 1.25kg of Fmoc-Cys(Trt)-CTC resin.Loading level of the product thus obtained was established to be 0.303 mmol/g via standard UV absorption method upon cleavage of small aliquots of the product resin.
Fmoc-Cys(Trt)-CTC resin (10 g, loading level: 0.303 mmol/g, 3.03 mmol) was suspended in DMF (200 mL) at room temperature overnight in a peptide synthesis vessel equipped with a mechanical stirrer. DMF was then removed via filtration. To the solid was added 20% piperidine in DMF (1000 mL). The resulting mixture was stirred at 18 °C for 30 min and filtered. The H-Cys(Trt)-CTC resin was washed with DMF (6×200 mL). Fmoc-Ser(tBu)-OH (3.45 g, 9.00mmol) and HBTU (3.25 g, 8.57mmol) were dissolved in DMF (20mL) at room temperature. To this solution was added NMM(1.98 mL, 18 mmol) at 0 oC in one portion, and the solution was kept at 0 °C for 15 min. Thissolution was then added to H-Cys(Trt)-CTC resin, and mixture was stirred at 18 °C for 1 h, at which point the Kaiser ninhydrin test indicated reaction completion. The mixture was filtered, and the resulting solid was washed with DMF (5× 200 mL), and the Fmoc-Ser(tBu)-Cys(Trt)-CTC resin product was used in subsequent step without further treatment. The remaining amino acids were coupled to the peptidyl resin sequentially to give His(Trt)-Gly-Glu(OtBu)-Gly-Thr(tBu)-Phe-Thr(tBu)-Ser(tBu)-Asp(OtBu)-Leu-Ser(tBu)-Lys(Boc)-Gln(Trt)-Met-Glu(OtBu)-Glu(OtBu)-Glu(OtBu)-Ala-Val-Arg(Pbf)-Leu-Phe-Ile-Glu(OtBu)-Trp(Boc)-Leu-Lys(Boc)-Asn(Trt)-Gly-Gly-Pro-Ser(tBu)-Ser(tBu)-Gly-Ala-Pro-Pro-Pro-Ser(tBu)-Cys-CTC resin (16.2 g) using the standard HBTU/NMM coupling conditions.The His(Trt)-Gly-Glu(OtBu)-Gly-Thr(tBu)-Phe-Thr(tBu)-Ser(tBu)-Asp(OtBu)-Leu-Ser(tBu)-Lys(Boc)-Gln(Trt)-Met-Glu(OtBu)-Glu(OtBu)-Glu(OtBu)-Ala-Val-Arg(Pbf)-Leu-Phe-Ile-Glu(OtBu)-Trp(Boc)-Leu-Lys(Boc)-Asn(Trt)-Gly-Gly-Pro-Ser(tBu)-Ser(tBu)-Gly-Ala-Pro-Pro-Pro-Ser(tBu)-Cys-CTC resin (16.2 g) was placed in a 1 L round-bottom flask, and a solution ofTFA: thioanisole: EDT: phenol: H2O (87.5:5:2.5:2.5:2.5, 200 mL) was added. Then, the reaction mixture was stirred at room temperature for 2.5 h. Cold ether (1.5L) was added which afforded a white solid. The mixture was centrifuged to give the crude template peptide His-Gly-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Leu-Ser-Lys-Gln-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(4g, 30% pure by HPLC), which was purified by reversed phase prep HPLC to give 300 mg (90% by HPLC) of the desiredpeptide.
Synthesis of DO3A-VS-Cys40-exendin-4
His-Gly-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Leu-Ser-Lys-Gln-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(43mg, 10μmol) and tert-butyl 2,2',2''-(10-(2-(vinylsulfonyl)ethyl)-1,4,7,10-tetraazacyclododecane-1,4,7-triyl)triacetate(6.4mg, 10μmol) were added to a mixture of saturated aqueous NaHCO3 /MeOH (0.2 mL). The reaction mixture was stirred for 2 h at room temperature and after workup furnished 49.4 mg ofthe conjugate addition adduct. To this peptidewas added a mixture of TFA/triethylsilane/H2O (95:2.5:2.5, 1 mL) at 0 °C.The mixture was stirred at room temperature for 2 h. TFA was removed under reduced pressure to give deliverthe crude peptide which was purified by reversed phase HPLCLike fractions were combined and lyophilized to give the final peptide (TFA salt) as a white solid (14 mg, 29% over two steps). HPLC purity = 96.6% (at 220 nm, retention time = 9.04 min); MS(MALDI)m/z 4755.31.
Synthesis of NODA-VS-Cys40-exendin-4
His-Gly-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Leu-Ser-Lys-Gln-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(54mg, 12.6mol) and tert-butyl 2,2'-(7-(2-(vinylsulfonyl)ethyl)-1,4,7-triazonane-1,4-diyl)diacetate(6.0mg, 12.6mol) were added to a mixture of saturated aqueous NaHCO3 /MeOH(0.2 mL). The mixture was stirred for 2 h at room temperature and after workup 60 mg of the conjugate addition peptide was delivered. To this peptide was added a mixture of TFA:triethylsilane:H2O (95:2.5:2.5, 1 mL) at 0 oC.The mixture was stirred at room temperature for 2 h. The TFA was removed under reduced pressure to give the crude peptide. The residue was then purified by reversed phase preparatory HPLC. Like fractions were combined and lyophilized to give the final peptide (TFA salt) as a white solid (18 mg, 31% over two steps). HPLC purity = 97.7% (at 220 nm, retention time = 9.86 min); MS (MALDI) m/z 4653.54.
References
1. Shetty D, Choi SY, Jeong JM et al(2011) ChemCommun47: 9732-9734.