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Supporting Information Electronic Supplementary Material (ESI) for ChemComm. This journal is © The Royal Society of Chemistry 2018 Supporting Information for Reviving Old Protecting Group Chemistry for Site-Selective Peptide-Protein Conjugation Smita B. Gunnoo,a Abhishek Iyer, a Willem Vannecke,a Klaas W. Decoene,a,b Tim Hebbrecht,b Jan Gettemans,b Mathias Laga,c Stefan Loverix,c Ignace Lastersc and Annemieke Madder*a a) Organic and Biomimetic Chemistry Research Group, Department of Organic and Macromolecular Chemistry, Krijgslaan 281 S4, Ghent University, Ghent, 9000 Belgium. b) Nanobody Lab, Department of Biochemistry, Faculty of Medicine and Health Sciences, Ghent University, Ghent, B-9000, Belgium c) Complix NV, BioVille, Agoralaan building A-bis, 3590, Diepenbeek, Belgium. *Corresponding author: [email protected] S1 Table of Contents Sr. No. Particulars Page # 1. Synthetic considerations S4 1.1 Proteins S4 1.2 Methods & Equipment S5 2. General procedures S7 2.1 Peptide synthesis S7 2.2 Conversion of the Acm to the Scm group S8 2.3 Manual Fmoc group removal S8 2.4 Small scale test cleavage S8 2.5 Large scale peptide cleavage S8 2.6 MB23 treatment with DTT S8 2.7 Verification of free thiol functionality by reaction of MB23 with Ellman’s S10 reagent 2.8 Verification of free thiol functionality by reaction of FasNb5 with Ellman’s S11 reagent 3. Synthesis of peptide peptide ABA-C(Scm)GSSK(folate)-CONH2 and its S13 conjugation to MB23, BSA and FasNb5 3.1 Synthesis of peptide ABA-C(Scm)GSSK(folate)-CONH2 S13 3.11 Alloc group removal S13 3.12 Coupling of Folic Acid S14 3.13 Cys(Acm) to Cys(Scm) conversion S14 3.14 Cleavage and analysis S14 3.2 Conjugation of MB23 to ABA-C(Scm)GSSK(folic acid)-CONH2 S16 3.3 BSA conjugation to ABA-C(Scm)GSSK(folic acid)-CONH2 S17 3.4 Conjugation of FasNb5 to ABA-C(Scm)GSSK(folic acid)-CONH2 S18 4. Synthesis of peptide peptide ABA-C(Scm)GSSK-CONH2 and its S19 conjugation to MB23 4.1 Synthesis of peptide ABA-C(Scm)GSSK-CONH2 S19 S2 4.2 Conjugation of MB23 to ABA-C(Scm)GSSK-CONH2 S19 5. Synthesis of ABA-GVSSC(Scm)GSSK(FAM)-CONH2 and its S21 conjugation to MB23 5.1 Synthesis of peptide ABA-GVSSC(Scm)GSSK(FAM)-CONH2 S21 5.2 Conjugation of MB23 to ABA-GVSSC(Scm)GSSK(FAM)-CONH2 S23 6. Synthesis and purification of H2N-C(Scm)GSSGSScKFRRRRE- S24 CONH2 and its conjugation to MB23 6.1 Synthesis of peptide H2N-C(Scm)GSSGSScKFRRRRE-CONH2 S24 6.2 Conjugation of MB23 to C(Scm)GSSGSS-cKFRRRRE S26 7. Synthesis of H2N-C(Scm)GSRGDS-CONH2 and its conjugation to MB23 S28 followed by purification 7.1 Synthesis of peptide H2N-C(Scm)GSRGDS-CONH2 S28 7.2 Conjugation of MB23 to C(Scm)GSRGDS-CONH2 S29 8. Synthesis and purification of cC(Scm)RGDE-CONH2 followed by S29 conjugation to MB23 and FasNb5 8.1 Synthesis of peptide cC(Scm)RGDE-CONH2 S30 8.2 Conjugation of MB23 to cC(Scm)RGDE-CONH2 S32 8.3 Conjugation of FasNb5 to cC(Scm)RGDE-CONH2 S34 8.4 Conjugation of FasNb5 with His tag to cC(Scm)RGDE-CONH2 S35 9. Circular Dichroism (CD) studies S37 10. ELISA Experiments S38 11. Serum stability S39 12. References S41 S3 1. Synthetic considerations All organic solvents were purchased from commercial suppliers and used without further purification or drying. DMF and NMP (peptide synthesis grade) were purchased from Biosolve. Acetonitrile, methanol, diethyl ether, DIPEA (supplied as extra dry, redistilled, 99.5 % pure) and triisopropylsilane (TIPS) were purchased from Sigma Aldrich. Milli-Q grade water was obtained in-house either from a Millipore ROs 5 purification system or a Sartorius Arium 611 DI. H-Rink amide ChemMatrix (35 – 100 mesh, manufacturer’s loading: 0.4-0.6 mmol/g) was obtained from Sigma Aldrich. All reagents were acquired from commercial sources and used without prior purification. HBTU, HATU, HOBt, TFA (peptide synthesis grade) and Nα-Fmoc protected amino acids used for peptide synthesis were obtained from Iris Biotech GmbH. All chiral α-amino acids used in this paper possessed the L configuration. Throughout this work, residues with standard acid-sensitive side-chain PGs were used: Cys(Trt) [C], Asp(OtBu) [D], Arg(Pbf) [R], Lys(Boc) [K], Ser(tBu) [S], as well as those with alternative sensitivities: Cys(Acm) [C], Glu(Alloc) [E] and Lys(Alloc) [K], used as described below for modification purposes. Some peptides were N-terminally capped with acetamidobenzoic acid (ABA, Sigma Aldrich). DL-Dithiothreitol, methoxycarbonylsulfenyl chloride, folic acid and tetrakis(triphenylphosphine)palladium(0) were purchased from Sigma Aldrich. Bovine serum albumin (BSA) was purchased from Sigma. 1.1 Proteins MB23 was expressed and purified as described elsewhere.1 PDB entries of related structures: 5MJ3 and 5MJ4. Figure S1. ESI-MS of MB23. Calculated mass 11471, observed mass 11469. S4 Figure S2. ESI-MS of BSA. Calculated mass 66463, observed mass 66464. The FasNb5 nanobody and a related FasNb5 nanobody with His and HA tags (vide infra) were expressed and purified as described elsewhere.2 After purification, fractions containing FasNb5 were pooled and were in 20 mM Tris-HCl, 500 mM NaCl, 1 mM EDTA + trace amounts of DTT (pH 8). FasNb5 samples were buffer exchanged using a Micro BioSpin 6 column (Bio-Rad) into 10 mM Tris-HCl, pH 7.4 prior to conjugation attempts. Figure S3. ESI-MS of FasNb5. Calculated mass 13895, observed mass 13892. 1.2 Methods & Equipment Reversed-Phase HPLC analysis and purification was performed on an Agilent 1100 Series instrument with diode array detector (set to 214, 254, 280, 310, 360 nm), equipped with a Phenomenex Luna C18(2) 100 Å column (250 x 4.6 mm, 5 μm, at 35 °C) for peptides and a Phenomenex Jupiter C4 300 Å column (250 x 4.6 mm, 5 μm, at 35 °C) for proteins and protein conjugations. Linear gradient elution was performed by flushing 2 min with A followed by 0 to 100% buffer B in 15 minutes and finally by a 5 min flushing with B using a binary solvent system composed of buffer A: 0.1% TFA in H2O and B: MeCN with a flow of 1.0 mL/min at 35°C. MALDI-TOF-MS data was acquired on an Applied Biosystems Voyager-DE STR Biospectrometry Workstation, equipped with a high performance nitrogen laser (337 nm). All spectra were recorded in the positive and reflector mode, with delayed extraction. S5 LC-TIC-MS data (reversed phase) were recorded on an Agilent 1100 Series instrument with diode array detector (set to 214, 254, 280, 310, 360 nm), equipped with a Phenomenex Kinetex C18 100 Å (150 x 4.6 mm, 5 µm, at 35 °C), hyphenated to an Agilent ESI-single quadrupole MS detector type VL. Mass detection operated in the positive mode. Linear gradient elutions were performed by flushing 0.5 min with A followed by 0 to 100% buffer B in 6 minutes and finally by a 2 min flushing with B using a binary solvent system composed of buffer A: 0.1% HCOOH in H2O and B: MeCN with a flow of 1.5 mL/min at 35 °C. A solution of 4-5 mg α- cyano-4-hydroxycinnamic acid in 500μL MeCN, 490μL mQ, 10μL 1 M ammonium citrate, 1μL TFA was used as a matrix for MALDI-TOF-MS. DAD1 A, Sig=214,20 Ref=off (18-09-12\079-0401.D) t = 2.738 min – solvent mAU peak 600 400 200 0 -200 -400 -600 0 2 4 6 8 min Figures S4. A. HPLC trace of pure water at wavelength 214 nm. Gradient: 0.5 min 100% 0.1% HCOOH in H2O, 0-100% ACN in 6 min, 2 min 100% ACN, 100-0% ACN in 0.25 min, 2 min 100% 0.1% HCOOH in H2Owith a flow of 1.5 mL/min at 35 °C using a Kinetix C18 column. Peak observed at 2.7 min is due to the gradient change and buffer system. DAD1 A, Sig=214,16 Ref=off (D:\DATA\18-09-20C\tA I0=000 09.1352.D) min – solvent mAU 100 peak 50 0 -50 -100 -150 -200 -250 -300 0 5 10 15 20 25 min Figure S4. B. HPLC trace of pure water at wavelength 214 nm. Gradient: 5 min 100% 0.1% TFA in H2O, 0-100% ACN in 15 min, 5 min 100% ACN, 100-0% ACN in 1 min, 5 min 100% S6 0.1% TFA in H2O with a flow of 1.5 mL/min at 35 °C using a Kinetix C18 column. Peak observed at 2.7 min is due to the gradient change and buffer system. Semi-preparative purification was performed on an Agilent prepstar system using a Phenomenex Luna 5µm C18(2) 100A, Axia packed column. The analyses were executed with a flow rate of 5 mL/min with the following solvent systems: H2O containing 0.1% TFA (A) and CH3CN (B). For SDS-PAGE, Novex Bis-Tris gels (Life Technologies) were used (4 – 12 %). Gels were placed in the gel tank, and the gel tank filled with MES running buffer (800 mL, prepared from 10x concentrate). Samples for the gel were prepared by adding sample (8 L) to loading dye (2 L, NuPage® LDS sample buffer, Novex), and then loading into the gel. Please note, non- reducing conditions were required in order to see disulfide bond formation. The gel was run at 180V for 38 minutes or the time taken for the dye to reach the bottom of the well.
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