Peptide Synthesis Procedure

Peptides were synthesized using standard solid phase peptide synthesis (SPPS) protocols using either manual synthesis or an automated peptide synthesizer (CEM Liberty Blue) using standard Fmoc-protected amino acids (Chemscene) on a Rink amide resin (Supra Sciences) unless otherwise noted. All amide couplings were done using O-(6- chlorobenzotriazol-1-yl)-N,N,N′,N′-tetramethyluronium hexafluorophosphate (HCTU) in DMF unless otherwise noted. For each coupling the amino acid, HCTU, and DIPEA were added in a 4:4:6 molar ratio to the peptide. During peptide synthesis a ninhydrin test was performed after every addition to test for the presence of free amines. Upon a positive test, the coupling was replicated until the test was negative. A capping step was then performed with acetic anhydride (Sigma-Aldrich) in a 10:5:100 acetic anhydride:DIPEA:DMF solution twice for 5 min, and then a ninhydrin test was performed to check for complete capping of the free amines. After successful coupling, the Fmoc group was removed, washing the resin with 20% piperidine in DMF twice for 5 min. A ninhydrin test was performed to check for a positive result.

For split-and-pool steps the resin was washed 3× with DMF and then the entire amount of resin was weighed on a scale. This number was divided by approximately 22 and this was split 19 ways into 19 separate tubes, with some excess to account for resin loss during transport and weighing. The reactions were performed in 15 mL tubes and upon successful coupling of all 19 amino acids all 19 fractions of resin were re-combined. Peptide libraries attached to the C-terminal end were completed in one vessel. Peptide libraries attached to the N-terminal end were washed with DMF 3X, weighed and split four ways followed by adding the desired chain-end chemistry. Once the N-terminal libraries were split four ways, they were not recombined again. Peptides with C-terminal carboxylic acids were synthesized using 2-chlorotrityl chloride resin. For the COOH library 2 mmol of resin was weight out and 52.6 μmol of each amino acid (1 mmol total synthesis scale) was added into a single 50 mL tube. 25 mL of DCM was added and then DMF was added dropwise until all of the amino acids went fully into solution. The 2-chlorotrityl chloride resin was washed with DCM for five minutes to swell the resin, at which time the DCM was drained and the amino acid solution was added with 5 equivalents of DIPEA. After 30 minutes another 1.5 equivalents of DIPEA was added. After another 30 minutes 5 mL of methanol was added to cap the resin, at which time the Fmoc groups were deprotected and the peptides were synthesized using standard Fmoc-peptide synthesis procedures.

Peptides libraries and all peptides containing tryptophan were cleaved using 92.5% trifluoroacetic acid (TFA), 2.5% H₂O, 2.5% triisopropylsilane (TIPS), and 2.5% dithiothreitol (DTT). Peptides which lacked a tryptophan were cleaved without DTT. Peptides were typically cleaved for 2–3 hours at room temperature using approximately 25 mL of cleavage solution per mM of peptide. However, peptides containing azides were cleaved for 30 minutes to prevent degradation of the azide group. Peptide mass was checked using electrospray ionization and if protecting groups remained the peptide was re-cleaved for 30 minutes. The masses of the peptide libraries were checked using liquid chromatography mass spectrometry (LCMS) and if protecting groups remained the peptides were re-cleaved for 30 minutes. At the end of the cleavage the peptides were precipitated in diethyl ether. These were then centrifuged for 5 minutes at 4,000 rpm, and the supernatant was discarded. The peptide pellet was washed with diethyl ether, and centrifuged, and this was repeated twice. The peptide pellet was allowed to dry, and then dissolved in water and neutralized with ammonium hydroxide prior to purification.

The cyclic RGD peptide was synthesized on a 2-chlorotrityl chloride resin. The first amino acid (0.3 mmol of amino acid per gram of resin) was dissolved in dichloromethane (DCM) and added to the resin in a shaker vessel. Five equivalents of DIPEA was then added, and after 5 min of shaking another 1.5 equivalents of DIPEA was added. After 1 h the unreacted 2-chlorotrityl chloride resin was capped with an excess of methanol for 30 min with another five equivalents of DIPEA. The rest of the amino acids were then coupled using standard solid phase Fmoc-synthesis protocols. After the last Fmoc group was deprotected the resin was washed 3× in DMF and 3× in DCM. The peptide was then cleaved under mild acidic conditions consisting of 5% trifluouroacetic acid and 2.5% triisopropylsilane in DCM. The mild cleavage solution was added to the resin for 5 minutes and then collected into a round bottom flask and this was repeated until the resin turned dark red or black. The collected liquid was then precipitated in ether. This cyclization was then dissolved in 50:50 acetonitrile:water, neutralized with 1M NH₄OH, and lyophilized. The protected linear NH₂-GRGDSK(N₃)-OH peptide was then cyclized. This was done by dissolving the peptide into DMF at 1 mg/mL and adding 3 equivalents of (1-[bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]- pyridinium 3-oxid hexafluorophosphate) (HATU), and three equivalents of DIPEA. After six hours the DMF was removed using rotary evaporation at 65°C.

The PanMMP crosslinking peptide was functionalized with 2-azido acetic acid on the N-terminus. 2-azido acetic acid was synthesized by mixing bromoacetic acid (70.168 g, 505 mmol) and sodium azide (32.504 g, 500 mmol) and water (250ml), the solution was stirred overnight at RT under ambient conditions. The next day the solution was acidified to pH ~ 1 using hydrochloric acid and extracted using ethyl acetate (5 × 100 mL). The organic layers were combined and dried in vacuo to afford the 2-azidoacetic acid as a colorless liquid. 2-azidoace+c acid was stored in a −20°C freezer until needed for synthesis.

All peptides were purified using HPLC using a Phenomenex Gemini 5 μm NX-C18 110 Å LC Column 150 × 21.2 mm. Gradients were run from 95% Mobile Phase A (water with 0.1% TFA) and 5% Mobile Phase B (acetonitrile with 0.1% TFA) to 100% Mobile Phase B. A typical HPLC run featured a two-minute equilibration step, followed by a 10 minute ramp from 95% Mobile Phase A to 100% Mobile Phase B, and then two minutes of equilibration at 100% Mobile Phase B, before ramping back down to the starting conditions. Notably, the split-and-pool libraries were ramped up to 100% Mobile Phase B over two minutes, since these libraries consisted of approximately 19 different peptides which weren’t intended to be separated from each other. The protected cyclic RGD peptides was purified using a gradient that ramped from 30% Mobile Phase B to 100% Mobile Phase B. After purification all peptides were lyophilized and were ready to use, except for the protected cyclic RGD peptide which was deprotected using 95% TFA, 2.5% TIPS, and 2.5% H₂O for one hour.

PEGylated RGEFV peptides were prepared using standard SPPS as described above. Immediately adjacent to the chain-end chemistry of interest, on the N or C terminus of the peptide, an Fmoc-Azidolysine-OH (AR001RXM, Aaron Chemicals) was added. Once seven variations of the azidolysine functionalized peptides were synthesized, HPLC purified, dissolved in water at 10mM, and verified via LCMS, and 20 mg of peptide was transferred to new Eppendorf tubes. 100 mg of m-dPEG₁₂-DBCO (QBD-10596, Vector Laboratories) was dissolved in water and evenly distributed 7 ways, amongst all azide-functionalized RGEFV peptides. The strained alkyne was allowed to click to the azide-functionalized peptides overnight. The next day, the pegylated peptides were HPLC purified, validated via LCMS, and lyophilized until needed.