Delta prep 4000

The protected AuIB and its variants were synthesized on Rink resin using the coupling agents HOBt/HBTU and DIEA, as described previously [12 (link)]. The peptide-resin (0.05 mmol) is cleaved in the cleavage solution (4.4 mL TFA, 0.25 mL H₂O, 0.25 g DTT, 0.1 mL TIPS) at room temperature for 3 h to remove the resin and side chain protecting groups. The released linear peptide was filtered, precipitated with cold anhydrous ether at 4 °C for 30 min, and folded in 0.1 M NH₄HCO₃ buffer (pH 8.0–8.2) at a concentration of 0.2 mg/mL for 24–48 h at room temperature. After the completion of folding, the folded solution was acidified with acetic acid to a pH of 4.0–5.0. The mixture was then filtered and directly loaded onto a 25 × 250 mm preparative C18 column using a preparative HPLC pump (Waters Delta Prep 4000). The column was washed with buffer A (0.1% TFA in water) at a flow rate of 2.5 mL/min and then with buffer B (0.1% TFA in acetonitrile) at a flow rate of 5 mL/min. The resulting peptide was further purified by semi-preparative RP-HPLC using a Kromasil 10.0 × 250 mm C18 column, and the target peaks were collected and analyzed for purity by HPLC. Confirmation of the correct molecular mass was ascertained by mass spectrometry on a ProFLEXTM-III MALDI-TOF spectrometer. The primary sequences of AuIB and its variants are listed in Table 1.

Freshly synthesized/purified CTP-amiodarone conjugate was HPLC characterized after lyophilization as the baseline measure. A small amount (1 mg/mL) was dissolved in the buffer used for injecting animals and placed in a water bath at 37 °C for up to 22 days. A small aliquot was taken at regular intervals, and an HPLC was run on the Waters Delta Prep 4000 chromatography system.

Cyclic Cy5.5 labeled lung targeting peptides (LTPs) were synthesized as above using 2-chlorotrityl resin as solid support. Cleavage of fully-protected peptide fragments were accomplished under mildly acidic conditions followed by head to tail cyclization using 3- (diethoxyphosphoryloxy)-1,2,3-benzotriazin-4(3H)-one (DEPBT) in Dimethylformamide/Dichloromethane (DMF/DCM) for 5 days at room temperature. Final cleavage and deprotection of the cyclized LTP peptides using Trifluoroacetic acid:Triisopropylsilane:H2O (TFA:TIPS:H2O - 90:25:25) was followed by precipitation in Diethyl Ether (EtO2) to isolate the crude peptide. The epsilon amino group of Lysine was then labelled with Cy5.5-NHS in 0.1M Triethylammonium bicarbonate (TEAbc)/acetonitrile at pH 8.5. The resulting Cy5.5 labelled cyclic LTP peptide was then directly purified by preparative C- 18 RP-HPLC on a Waters Delta Prep 4000 chromatography system using standard Acetonitrile/0.1%TFA gradient conditions followed by lyophilization. MALDI-TOF analysis of the purified conjugates on an Applied Biosystems Voyager workstation using α-Cyano-4-hydroxycinnamic acid (CHCA) matrix allowed for confirmation of the expected mass and identity of the final product.