Luna 5u c8

Acetone/lithium perchlorate was used to precipitate 100 nmol of ODN Ip3-am, which was then dissolved in 100 μl of buffer containing 0.1 M NaHCO₃ at pH 8.3 and 80% DMSO. Next, 50 μl of 100 mM DBCO-PEG₄-NHS (5 μmol) was added, and the reaction mixture was incubated at room temperature for 1 h. The oligonucleotide was precipitated using acetone/lithium perchlorate and purified by gel-filtration on an EMP CentriPure N2 while simultaneously transferring it into a PBS solution. An aliquot of the reaction products was analyzed using RP HPLC (Phenomenex Luna 5u C8, linear gradient of acetonitrile in 50 mM sodium acetate; results are not provided). The reaction products were evaporated using a vacuum centrifuge and dissolved in 50 μl of PBS. The reaction progress was controlled by mixing an aliquot of modified oligonucleotide with a 10–20× molar excess of azido derivative of sulfo-Cy5 (sCy5-N3) (Lumiprobe, Russian Federation), followed by purification using RP HPLC.

Dopamine isotopic enrichment was determined as native dopamine and as the n-propyl derivative [27] (link) (diester, amide) by LC-MS/MS (Shimazu UFLCXR, QTRAP 6500) by negative electrospray ionization. Three parent-daughter ion pairs were monitored to confirm peak identification as dopamine (154/137, 154/119, and 154/91), or as its n-propyl derivative (322/137, 322/210, and 322/266). A gradient elution Solvent A: aqueous ammonium acetate 12 mM, Solvent B: acetonitrile) using a C8 reverse phase HPLC column (on a Phenomenex Luna 5u C8, 100A, 150 × 4.6 mm, 5 microns) was used for dopamine (gradient: 5–20% solvent B) and the dopamine n-propyl derivative (gradient: 50–70% solvent B). Isotopic enrichments were determined by monitoring parent-daughter ion pairs of dopamine (and the n-propyl derivative) formed from the labeled precursor: dopamine m+3 from [2, 5, 6-D3] L-Dopa, dopamine m+6 from ring [13C3]tyrosine, and dopamine m+9 from [13C9, 15N]tyrosine. Tyrosine isotopic enrichment was determined following derivatization as the n-trifluoracetyl-n-butyl ester [28] by Chemical ionization GC-MS (Agilent: HP-1 column, G1530A GC, and 5975C MS) using isobutane as the reagent gas, He gas at a flow rate of 1 ml/min was the carrier, and a temperature program ramped from 100 to 200 °C.