Synthesis and Characterization of CXCR4 Peptide Conjugates

Based on the crystal structures of human CXCR4 (accession codes 3ODU; 3OE0; 3OE6; 3OE8; 3OE9; 4RWS and previous SAR studies^{24 (link),25 (link)}, CXCR4 ectodomain peptides were selected. The crystal structures were imported into PyMOL Molecular Graphics System (Version 1.8.2.2 Schrödinger, LLC) and Jmol (http://www.jmol.org) for determining the C-to-N distance between residues 97–110 and 182–196^{29 (link),30 (link)}. Conjugates of 12-Ado with either 6-Ahx or O2Oc were visualized in three-dimensional space using Molview and Jmol. The estimated distances between the N- and C-terminal in both conjugates were similar to the ECL1-ECL2 distance. All CXCR4-derived peptides, including msR4M-L1(7xAla) and msR4M-L1(2xAla), were synthesized as C-terminal amides on Rink amide MBHA resin by SPPS using Fmoc chemistry^{28 (link)}. Couplings of Fmoc-6-Ahx-OH, Fmoc-12-Ado-OH and Fmoc-O2Oc-OH (Iris Biotech GmbH, Marktredwitz, Germany) were carried out with 3-fold molar excess of 2-(7-Aza-1H-benzotriazole-1-yl)- 1,1,3,3-tetramethyluronium hexafluorophosphate (HATU) and 4.5-fold molar excess of N,N-diisopropylethylamine (DIEA) in N,N-dimethylformamide (DMF). Fmoc-deprotection was in general carried out with 0.1 M hydroxybenzotriazole (HOBt) in 20% v/v piperidine in dimethylformamide (DMF) for 3 and 9 min to avoid aspartimide formation^{32 (link)}. To introduce N^α-fluorescein and TAMRA labels, 5(6)-carboxyfluorescein-N-hydroxysuccinimide ester (Sigma-Aldrich, Taufkirchen, Germany) and 5(6)-carboxytetramethylrhodamine (TAMRA, Novabiochem/Merck KGaA, Darmstadt, Germany) were coupled N-terminally to side chain-protected msR4M-L1 on solid phase, after Fmoc-deprotection^{32 (link)}. The N^α-biotinyl label was introduced as follows: after assembly of fully protected msR4M-L1 and N^α-Fmoc-cleavage, Fmoc-protected 6-Ahx was coupled followed by Fmoc-cleavage and coupling of biotin using the coupling protocols as above^{32 (link)}. Disulfide bridges in msR4M-L1ox and msR4M-L2ox were formed in 1 mg mL⁻¹ peptide solution in aqueous 3 M guanidinium hydrochloride (GdnHCl) and 0.1 M ammonium carbonate (NH₄HCO₃) solution, containing 40% dimethylsulfoxide (DMSO). msR4M-LS was produced similarly, using 0.3 mg mL⁻¹ ECL1 and 0.5 mg mL⁻¹ ECL2 and 20% DMSO. Reverse-phase high-performance liquid chromatography (RP-HPLC) was applied for the purification of crude and oxidized peptides by using Reprosil Gold 200 C18 (250×8 mm) or Reprospher 100 C18-DE (250 × 8 mm) columns with pre-column (30×8 mm) (Dr. Maisch-GmbH, Herrenberg, Germany). The mobile phase consisted of 0.058% (v/v) trifluoroacetic acid (TFA) in water (buffer A) and 0.05% (v/v) trifluoroacetic acid in 90% (v/v) acetonitrile and water (buffer B) (flow rate 2.0 mL/min). All peptides were purified with an elution program of 10% B for 1 min, followed by a gradient from 10 to 90% B over 30 min, except for msR4M-LS, which was eluted with 30% B for 7 min followed by an increase to 60% B over 30 min. Expected molecular weights were verified by matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS). Peptides were used as TFA salts. For in vivo experiments, the TFA anion was exchanged to chloride by four cycles of dissolution/lyophilization of pure msR4M-L1 in aqueous 5 mM HCl and one cycle of bidistilled water. MIF sequence-based peptides (Supplementary Table 2) were synthesized on Wang resin or purchased from Peptide Specialities GmbH (PSL, Heidelberg, Germany). MIF-derived peptides were N-terminally acetylated and their C-terminal end is a free carboxylate.