7, 18.9 Hz, 1H). Peptide modification–Labeled peptide (11a) To a two mM resolution of custom-synthesized peptide ten (1.82 mL, three.82 mol) in one hundred mM pH 7.0 NaH2PO4/Na2HPO4 buffer was 100 mM option of PTAD 9a (114 L, 11.5 mol) in MeCN was added (9.55 L 2 timies, interval 1 min.) at room temperature. The resulting solution was stirred at area temperature for 30 min. The crude reaction was analyzed straight by ESI-LC/MS at 254 nm UV absorption and corresponding MS. The reaction mixture was then diluted with MeCN (1.00 mL). The obtained crude material was purified by reversed phase HPLC (mobile phase; gradient of MeCN/0.1 TFA water, 30:70 to 50:50 more than 30 min, Rt; 14.five min, detection; UV 254 nm) to provide 11a (four.40 mg, 61 ) as white amorphous strong. HRMS: calcd for C73H93N21O17 (MH+) 1536.7131, located 1536.7125. Reversed phase HPLC purity 95.1 (mobile phase; gradient of MeCN/0.1 TFA water, 0:100 to 100:0 more than 30 min, Rt; 15.eight min, detection; UV 254 nm). Labeled peptide (11b). The compound 11b was prepared from custom-synthesized peptide ten (5 mg, 3.82 mol) and 9b (2.99 mg, 11.5 mol), and was obtained as white amorphous solid (four.40 mg, 60 ). Reverse phase HPLC circumstances for isolation: mobile phase – gradient of MeCN/0.Bleomycin site 1 TFA water, 30:70 to 50:50 over 30 min, Rt 15.6 min, detection at UV 254 nm). HRMS: calcd for C72H94N24O17 (MH+) 1567.7301, found 1567.7236. Reversed phase HPLC purity 91.three (mobile phase;: gradient of MeCN/ 0.1 TFA water, 0:100 to one hundred:0 more than 30 min, Rt 15.9 min, detection at UV 254 nm). Labeled peptide (11c). The compound 11c was ready from custom-synthesized peptide 10 (five mg, 3.82 mol) and 9c (two.84 mg, 11.5 mol), and was obtained as white amorphous solid (four.Phenylmethan-d2-ol In Vivo 60 mg, 63 ). Reverse phase HPLC conditions for isolation: mobile phase – gradient of MeCN/0.1 TFA water, 30:70 to 50:50 over 30 min, Rt 14.eight min, detection at UV 254 nm). HRMS: calcd for C73H95N21O18 (MH+) 1554.7236, located 1554.7220. Reversed phaseNIH-PA Author Manuscript NIH-PA Author Manuscript NIH-PA Author ManuscriptBioconjug Chem. Author manuscript; accessible in PMC 2014 April 17.Ban et al.PageHPLC purity 93.6 (mobile phase: gradient of MeCN/0.1 TFA water, 0:one hundred to 100:0 more than 30 min, Rt 15.2 min, detection at UV 254 nm).NIH-PA Author Manuscript NIH-PA Author Manuscript NIH-PA Author ManuscriptSequential bioconjugation of albumin working with 3 orthogonal chemistries (Step 1) Reaction of albumin with dansyl derivative: To a 1.PMID:23927631 five mL centrifuge tube have been added 300 L of filtered albumin solution in H2O (pH six.0) containing 1.five mg albumin followed by addition of ten L of 140 mM 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride in H2O (pH 6.0) and five L of 136 mM 11-(dansylamino) undecanoic acid in DMSO. Reaction tubes have been gently shaken at 250 rpm and incubated for 14.five hours at 37 . The reaction mixtures were applied to Micro Bio-Spin Chromatography Columns (Bio-Gel P-6 Gel, Bio Rad) and exchanged into nanopure water. The purified albumin construct was characterized by MALDI-TOF MS. The fluorescence signal on the modified albumins 12 and 13 were recorded by a Microplate spectrophotometer (Spectra Max Gemini; Molecular Devices) utilizing wavelength of excitation 320 nm and emission 555 nm. (Step two) Reaction of dansyl-albumins with PTAD derivative: The dansyl-albumin options 150 L have been applied to Micro Bio-Spin Chromatography Columns (Bio-Gel P-6 Gel, Bio Rad) to exchange the buffer to one hundred mM Na Phosphate buffer pH 7.four. The resulting solutions were diluted to 30 M.