Esquire ion trap mass spectrometer

Synthetic schemes in Figures S1A and S2A were followed to obtain PI-103 prodrug monomer and fluorescein monomer, respectively. PI-103 monomer was synthesized by conjugating the phenolic hydroxyl group of PI-103 to the carboxylic end of mono-2-(methacryloyloxy)ethyl succinate (SMA) using N-(3-dimethylaminopropyl)-N′-ethylcarbodimide hydrochloride (EDCI.HCl) as the coupling agent and N,N-dimethylpyridin-4-amine (DMAP) as the base. Fluorescein monomer was synthesized by a four-step synthetic pathway starting from 4-aminobutanoic acid using carbodiimide/DMAP coupling chemistry. The amine group was first protected with Boc group, and then coupled with 2-hydroxyethyl methacrylate (HEMA). Deprotection of the Boc group yielded amine terminated methacrylate derivative which upon conjugation with NHS activated 5-carboxyfluorescein afforded the fluorescein monomer. All the synthesized monomers and intermediates were purified by precipitation and/or silica gel column chromatography techniques. The successful synthesis and purity of the monomers were confirmed and characterized by ¹H NMR spectroscopy (Bruker Avance spectrometers 300 MHz) and Electrospray Ionization-Mass spectrometry (Bruker Esquire ion trap mass spectrometer) (Figure S1B, S1C, S2B, S2C).

All peptides were synthesized using standard Fmoc-solid phase peptide synthesis chemistry on a CEM Liberty Blue Microwave Synthesizer. 0.1 mmol resin was used and 5-fold excess amino acids were used for each coupling. N,N-diisopropylcarbodiimide (DIC) and oxyma were utilized as coupling reagents in 5-fold excess and 3 mL of 20% piperidine was used for deprotection. Peptides were cleaved in 9.5 mL trifluoroacetic acid, with 250 μL triisopropylsilane and 250 μL water used scavengers. Cleavage was allowed to occur over 90 minutes with mild shaking. Crude peptide was precipitated with cold diethyl ether.
Crude peptides were then purified using reverse phase HPLC. Peptides were dissolved in 5 to 7 ml of 50/50 water/acetonitrile and purified on a Varian Prostar 220 HPLC with an Agilent C18 column. A gradient of 10–60 % acetonitrile was used: eluent A was 1% TFA in water and eluent B was 0.085% TFA in acetonitrile. HPLC traces were monitored with UV detection at 220 nm and 280 nm wavelength. The collected fractions were lyophilized and stored as trifluoroacetate salts. Electrospray mass spectrometry (Bruker Esquire Ion Trap Mass Spectrometer) was used to analyze the purity of the peptide. A peptide purity at or above 97% was the criterion for further characterization.