Supporting Information

Electronic Supplementary Material (ESI) for ChemComm. This journal is © The Royal Society of Chemistry 2018

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)GSSGSScKFRRRRE- S24 CONH2 and its conjugation to MB23

6.1 Synthesis of peptide H2N-C(Scm)GSSGSScKFRRRRE-CONH2 S24

6.2 Conjugation of MB23 to C(Scm)GSSGSS-cKFRRRRE 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

-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

-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. Coomassie stain (20 mL, InstantBlue Protein Stain, Expedeon) was added to the gel. It was allowed to develop for 1 hour on an orbital rocker then rinsed with water.

Western Blots were performed following SDS-PAGE on desired samples. SDS-PAGE were transferred onto a 0.2 mM nitrocellulose membrane using a Trans-Blot® Turbo™ transfer system (Bio-Rad). A solution of BSA (0.5 g, bovine serum albumin) in TBST (50 mL) was prepared. This was added to the membrane to block it, and placed on an orbital shaker at r.t. for 1 hour or at 4 °C overnight. A solution of BSA (0.5 g) and primary antibody (either anti- folic acid antibody, 1 in 1000, Sigma or anti-Alphabody antibody, 1 in 2500, produced in house) in TBST was added to the membrane and placed on an orbital shaker at r.t. for 45 minutes. The membrane was rinsed with TBST 3 times. A solution of BSA (0.5 g) and anti- mouse antibody-alkaline phosphatase conjugate (1 in 5000, Promega) in TBST was added to the membrane and placed on an orbital shaker at r.t. for 45 minutes. The membrane was rinsed with TBST 3 times. BCIP/NBT substrate (5 – 10 mL) was added and incubated with the membrane for a few minutes until staining was observed. The membrane was rinsed with water.

2. General procedures

2.1 Peptide Synthesis

S7 Automated peptide syntheses were performed on a fully-automated SYRO Multiple Peptide Synthesiser robot, equipped with a vortexing unit for the 24-reactor block (MultiSynTech GmbH), or an Intavis Multipep RSi, 72 column module synthesiser. Reactions were open to the atmosphere and executed at ambient temperature. Peptides were synthesised using the Fmoc/tBu strategy with HBTU/DIPEA-mediated couplings. Peptide synthesised using Rink amide resin (0.71 mmol/g)  ABA-C(Acm)GSSK (ABA = acetamidobenzoic acid)

Peptides synthesised using ChemMatrix H-Rink amide resin (0.54 mmol/g)  C(Acm)GSRGDS  C(Acm)RGDE(Alloc)  ABA-GVSSC(Acm)GSSK(Alloc)  C(Acm)GSSGSSK(Alloc)FRRRRE(Alloc)

2.2 Conversion of the Acm to the Scm group

Peptide on resin (100 mg, 54 mol, 0.54 mmol/g) was swollen in CH2Cl2 (3.5 mL) for 10 – 30 minutes at r.t. Methoxycarbonylsulfenyl chloride (5.8 L, 65 mol, 1.2 eq.) was added, and the reaction allowed to shake for 3 hours at r.t. Resin was then washed repeatedly with CH2Cl2,

DMF, MeOH and Et2O, and stored under Ar at – 20 °C. A small scale test cleavage was performed to check for conversion efficiency prior to larger scale peptide cleavage.

2.3 Manual Fmoc group removal

Peptide on resin (100 mg, 54 mol, 0.54 mmol/g) was swollen in DMF (3.5 mL) for 10 – 30 minutes at r.t. 40% piperidine in DMF (3 mL) was added and this was shaken for 5 mins at r.t

a total of 4 times. Resin was washed repeatedly with DMF, CH2Cl2, MeOH and Et2O, and stored under Ar at – 20 °C. A small scale test cleavage was performed to check for deprotection efficiency prior to larger scale peptide cleavage.

2.4 Small scale test cleavage

A few beads of washed resin were transferred to a small reaction vessel. Cleavage cocktail (200

L of 95% TFA, 2.5% TIPS and 2.5% H2O) was added, the reaction was left at r.t. for 2 – 4 hours. Longer incubation times were employed when an arginine with a Pbf protecting group was present. TFA was removed from the cleavage mixture under a flow of N2, and the resulting

S8 peptide dissolved in 50 – 100 L MeOH. The peptide was analysed by MALDI-TOF or LC/MS.

2.5 Large scale peptide cleavage

Cleavage cocktail (500 L – 1 mL of 95% TFA, 2.5% TIPS and 2.5% H2O) was added to peptide resin, and the reaction was shaken at r.t. for 2 – 4 hours. Longer incubation times were employed when an arginine with a Pbf protecting group was present. Cleavage cocktail containing peptide was precipitated into cold ether and centrifuged (10 mins, 10 kprm). Ether was poured off, and the pellet was resuspended in fresh cold ether and centrifuged (10 mins, 10 kprm). The resulting pellet peptide was either dried by lyophilisation or on an oil pump. The peptide was analysed by MALDI-TOF or LC/MS, and then purified by Prep-HPLC if purity was insufficient.

2.6 MB23 treatment with DTT

DTT (0.5 mg, 3.3 mol) was added to 100 L of MB23 (c = 4.6 mg/mL in 50 mM MES pH 6.0, 0.5 M NaCl) and shaken at r.t. for 15 minutes. After this time, the protein was separated from DTT and buffer exchanged into 10 mM Tris, pH 7.4 by means of a Micro BioSpin 6 column (Bio-Rad). Reduced protein was analysed by LC/MS, the associated ESI-MS is shown below (Fig S4). The absence of dimer was confirmed by SDS-PAGE as shown in Fig S5.

M kDa 20 15 10

2 S9 Figure S5. ESI-MS of reduced MB23 (above). Calculated mass 11471, observed mass 11469. SDS-PAGE of marker, non-reduced and reduced MB23 (below).

2.7 Verification of free thiol functionality by reaction of MB23 with Ellman’s reagent

OH O O OH O N S Cys59 2 S NO2 SH S S O 10 mM Tris-HCl, pH 7.4 r.t., 15 mins OH pre-reduced with NO2 10 eq. DTT Figure S6. Scheme for the reaction between MB23 and Ellman’s reagent

A solution of Ellman’s reagent was prepared (0.6 mg in 108 L PBS, pH 7.4). 10 L of this solution was added to 75 L of reduced MB23 (c = 0.2 mg/mL in 10 mM Tris-HCl, pH 7.4) and shaken at r.t. for 15 minutes. After this time, the protein was separated from excess Ellman’s reagent by means of a Micro BioSpin 6 column (Bio-Rad). Protein was analysed by LC/MS, the associated LC-MS is shown below.

DAD1 A, Sig=214,20 Ref=off (17-05-19\062-6101.D) mAU 5 4 7 . 2

6 2 4 . 1

400 0 6 9 . 0

0 5 9 6 6 . 7 2 5 1 0 1

4 . . 3 1 1 .

200 Product 6 3 0 . 5 0

-200

Figure S7. RP-HPLC trace of reaction mixture between MB23 and Ellman’s reagent.

-400

0 2 4 6 8 min S10 Figure S8. ESI-MS from LC-MS of MB23 reaction with Ellman’s reagent. Calculated mass 11668, observed mass 11673 (MW of unreacted MB23: 11471).

2.8 Verification of free thiol functionality by reaction of FasNb5 with Ellman’s reagent OH O O Cys113 OH O2N S SH S NO2 S S O 10 mM Tris-HCl, pH 7.4 r.t., 15 mins OH NO2 Figure S9. Scheme for the synthesis between FasNb5 with Ellman’s reagent

A solution of Ellman’s reagent was prepared (0.4 mg in 66.6 L PBS, pH 7.4). 2 L of this solution was added to 30 L of FasNb5 (c = 0.25 mg/mL in 10 mM Tris-HCl, pH 7.4) and shaken at r.t. for 15 minutes. After this time, the protein was separated from excess Ellman’s reagent by means of a Micro BioSpin 6 column (Bio-Rad). Protein was analysed by LC/MS, the associated ESI-MS is shown below indicating the availability of the cysteine-thiol functionality.

DAD1 A, Sig=214,20 Ref=off (17-01-26\013-1301.D) mAU

200 Product

-200 Unreacted

-400 FasNb5

-600

-800

-1000

0 2 4 6 8 min S11 Figure S10. RP-HPLC trace of reaction mixture between FasNb5 and Ellman’s reagent.

*MSD1 SPC, time=5.066:5.426 of D:\DATA\17-01-26\013-1301.D API-ES, Pos, Scan, Frag: 70

Max: 81555 6

5 + + M/10 + H M/9 + H 4 + M/11 + H 1410 1566 1282 3 + + M/12 + H M/8 + H 2 1175 1762

-1

1100 1200 1300 1400 1500 1600 1700 m/z

Figure S11. ESI-MS from LC-MS of FasNb5 reaction with Ellman’s reagent. Calculated mass 14092, observed multiply charged ions corresponding to 14092 (MW of unreacted FasNb5: 13895).

S12 3. Synthesis of peptide ABA-C(Scm)GSSK(folate)-CONH2 and its conjugation to MB23, BSA and FasNb5.

3.1 Synthesis of peptide ABA-C(Scm)GSSK(folate)-CONH2

0.2 eq. Pd(PPh ) Acm O 3 4 Acm O H 60 eq. PhSiH H S Gly Ser Ser N 3 S Gly Ser Ser N O CH2Cl2 O NH O 2 x 30 mins, r.t. NH O ABA ABA

HN O NH2

O O O O SCl O 5 eq. folic acid Acm H Scm H N N 5 eq. HBTU S Gly Ser Ser CH2Cl2 S Gly Ser Ser O 10 eq. DIPEA O 3 h, r.t. NH O NH O ABA ABA DMSO o/n, 37 °C HN Fol HN Fol 95% TFA O 2.5% TIPS O 2.5% H2O O OH 2 h, r.t. O O OH Scm H O N N H S Gly Ser Ser N O NH2 HN N O H NH O H2N N N ABA fol = folic acid HN Fol

Figure S12. Synthesis scheme for peptide ABA-C(Scm)GSSK(folate)-CONH2

Resin bound linear peptide was synthesised using automated SPPS on Rink amide resin (50 – 90 mesh, 0.71 mmol/g).

3.11 Alloc group removal

Peptide resin (100 mg, 71.0 mol, 0.71 mmol/g, 1 eq.) was swollen in CH2Cl2 for 30 minutes.

Pd(PPh3)4 (16.4 mg, 14.2 mol, 0.2 eq.) and phenylsilane (525.6 L, 4.26 mmol, 60 eq.) were added, and the reaction shaken for 30 mins at r.t., after which the resin was washed sequentially with DCM, DMF and DCM again. The deprotection was repeated and the resin then washed with DCM, DMF, MeOH and Et2O. A small scale test cleavage revealed full removal of the Alloc group (mass calcd. for [M+H]+ 712.3, obs. mass 712.4 [M+H]+, 734.4 [M+Na]+).

S13 3.12 Coupling of Folic Acid

ABA-C(Acm)GSSK-resin bound (15 mg, 10 mol, 0.71 mmol/g, 1 eq.) was swollen in DMSO for 30 minutes at r.t. In the meantime, a solution of folic acid (22 mg, 50 mol, 5 eq.), HBTU (19 mg, 50 mol, 5 eq.) and DIPEA (17.4 L, 0.1 mmol, 10 eq.) was premixed, and added to the peptide resin. The reaction was shaken at 37 °C overnight, and then washed sequentially with DMSO, DCM, DMF, MeOH and Et2O. A small-scale test cleavage and subsequent analysis of the crude reaction mixture by MALDI-TOF revealed full conversion to the folic acid-functionalised peptide (mass calcd. for [M+H]+ = 1135.4, obs. mass 1157.9 [M+Na]+, 1173.8 [M+K]+).

Voyager Spec #1[BP = 1157.9, 439]

1157.8773 100 439

60 1173.8538 y t i s n e

t 50 n I

30 550.8910

522.8582 20 641.6013 898.8188 684.7345 982.7782 1160.9102 10 507.6305 712.6491 1197.7732 1064.8521 1342.8131 673.2702 834.7147 516.2787 1008.0350 1238.0087 1385.9055 1794.5134 2206.2788 2700.8556 665.8457 843.7393 1543.6697 1998.0222 1785.8760 2465.0035 500.3892 837.0680 1016.2262 1164.1541 1306.8172 1599.1660 2000.4462 2174.4068 2676.3663 0 0 499.0 999.4 1499.8 2000.2 2500.6 3001.0 Mass (m/z) Figure S13. MALDI-TOF MS ABA-C(Acm)GSSK(folate).

3.13 Cys(Acm) to Cys(Scm) conversion Cys(Acm) was then converted to C(Scm) on solid support as described in section 2.2.

3.14 Cleavage and analyses

The peptide was cleaved from the solid support using the procedure described in section 2.5, and the peptide analysed by RP-HPLC and MALDI-TOF MS. Absorbance at 254 nm is indicative of the presence of folic acid. The observed peak splitting can be attributed to the non-entirely regioselective coupling of folic acid to the peptide which leads to two different regioisomeric products. Additionally, epimerization has presumably taken place during folic

S14 acid activation resulting in an additional two products being formed. This explains the presence of 4 different isomeric folic acid peptides as indicated by 4 peaks on the HPLC. The peptide was used without any further purification (mass calcd. for [M+H]+ = 1154.4, obs. mass 1062.6 - this may correspond to loss of the Scm group upon MALDI analysis).

DAD1 A, Sig=214,16 Ref=off (D:\DATA\17-01-09\WV000013.D) DAD1 B, Sig=254,16 Ref=off (D:\DATA\17-01-09\WV000013.D) mAU solvent peak 1400 DMSO

1200

1000

800

600

400

200

-200

0 5 10 15 20 25 min Figure S14. RP-HPLC of ABA-C(Scm)GSSK(folate). Peptide elutes between 11 and 12 mins. Red trace 254 nm, blue trace 214 nm.

Voyager Spec #1[BP = 604.2, 2305]

604.2278 100 2305.0 Peptide - Scm 90

666.2306 1062.6338 O H 80 N 1076.6351 HS Gly Ser Ser NH2 O 70 NH O N N NH2 H 60 1060.6232 N NH NH N

y H t i AcHN s O

n N e

t 50

n O I

% O HO O 40 1074.6458 Chemical Formula: C45H57N15O14S 653.4978 519.3113 Exact Mass: 1063.39 30 915.5279

887.5523 1031.8335 20 529.4791 1064.6022 628.2108 736.3390 511.3557 1078.6347 919.4939 573.5124 1042.6926 500.3386 1047.3067 578.1841 779.3183 917.4858 10 651.5445 1044.6630 537.3417 617.4685 755.3035 869.2305 994.3187 1070.6045 544.3100 1157.5259 657.4483 761.6947 881.7322 967.5514 1053.8190 1136.5872 1265.4966 1542.7060 1672.2573 1827.5400 773.5123 875.3536 1361.6249 1448.6746 1277.4787 1557.1225 1637.1780 1729.1190 1846.8628 0 0 499.0 799.4 1099.8 1400.2 1700.6 2001.0 Mass (m/z)

S15 Figure S15. MALDI-TOF MS spectra of ABA-C(Scm)GSSK(folate). Exact Mass Calculated

ABA-C(Scm)GSSLys(folate) H N OH O O O H H H O N N N N N NH2 H H O O O S OH O S

O O NH

Chemical Formula: C47H59N15O16S2 O Exact Mass: 1153.37 HO2C N O Molecular Weight: 1154.20 H N N NH H N N NH2 for C47H59N15O16S2 = 1153.27 (below), found Peptide – Scm = 1062.6338 (above)

3.2. Conjugation of MB23 to ABA-C(Scm)GSSK(folate)-CONH2 O O 11 eq. S S

ABA-CGSS-K(folate) CONH2 SH S S 10 mM Tris-HCl, pH 7.4 CGSSK(folate) CONH2 r.t., DMSO, o.n. ABA

pre-reduced with 10 eq. DTT Figure S16: Synthesis scheme for the conjugation of MB23 with peptide ABA-

C(Scm)GSSK(folate)-CONH2

MB23 was first reduced with DTT to remove any dimeric species formed during storage following the procedure described above. To 200 L of reduced MB23 (0.44 mg, 38.4 nmol, c

= 2.2 mg/mL in 10 mM Tris-HCl, pH 7.4) was added ABA-C(Scm)GSSK(folate)-CONH2 (175 L from 2.5 mM solution in DMSO, 0.43 mol, 11 eq.), and the reaction was allowed to shake at room temperature overnight. The reaction mixture was then centrifuged to remove peptide precipitate (10 mins, 13.2 krpm) and analysis by LC/MS showed conversion to the folic acid containing conjugate. Purification was carried out by RP-HPLC (Phenomenex Jupiter C18, 0 – 100% ACN over 15 mins, Figure S13). Solvent was removed by speed vac, and conjugated MB23 was resuspended in 10 mM Tris-HCl, pH 7.4 (300 L at 0.5 mg/mL).

S16 DAD1 A, Sig=214,16 Ref=off (D:\DATA\15-05-08\DVL000008.D) DAD1 B, Sig=254,16 Ref=off (D:\DATA\15-05-08\DVL000008.D) mAU

2000

11-12 mins - peptide

1500

1000 14.5 mins – MB23 conjugate DMSO 500

-500

0 5 10 15 20 25 min Figure S17. RP-HPLC trace of reaction. Luna C18 100 Å, 0-100% ACN over 15 mins. Red trace 254 nm, blue trace 214 nm.

Figure S18. ESI-MS of reaction of MB23-S-S-CGSSK(folate). Calculated mass 12532, observed mass 12529.

3.3 BSA conjugation to ABA-C(Scm)GSSK(folate)-CONH2 O O 10 eq. S S Cys34 ABA-CGSS-K(folate) CONH2 SH S S 10 mM Tris-HCl, pH 7.6 CGSSK(folate) CONH r.t., DMSO, o.n. 2 BSA ABA PDB 3v03 Figure S19: Synthesis scheme for the conjugation of BSA with peptide ABA-

C(Scm)GSSK(folate)-CONH2

To 1 mL of BSA (0.5 mg, 7.5 nmol, c = 0.5 mg/mL in 10 mM Tris-HCl, pH 7.6) was added

ABA-C(Scm)GSSK(folate)-CONH2 (30.4 L from 2.5 mM solution in DMSO, 75.2 nmol, 10

S17

Folic acid-containing conjugate

1 2 3 1 2 3 eq.), and the reaction mixture was shaken at r.t. overnight. Excess peptide was separated from protein with a PD MidiTrap G-25 (GE Healthcare). SDS-PAGE showed a slight shift in molecular weight and a Western blot staining with anti-folic acid antibody, as expected, allowed visualization of the conjugate and not BSA.

M kDa 75

Figure S20. SDS-PAGE (left) and Western blot (right) of BSA conjugation to ABA- C(Scm)GSSK(folate). Lane 1 – marker, lane 2 – BSA, lane 3 – BSA-S-S-GSSK(folate).

3.4 Conjugation of FasNb5 to ABA-C(Scm)GSSK(folate)-CONH2 O O 10 eq. S S

ABA-CGSS-K(folate) CONH2 SH S S 10 mM PBS, pH 8.0 CGSSK(folate) CONH2 r.t., DMSO, o.n. ABA Figure S21: Synthesis scheme for the conjugation of FasNb5 with peptide ABA-

C(Scm)GSSK(folate)-CONH2

To 40 L of FasNb5 (6 g, 0.43 nmol, c = 0.15 mg/mL in 10 mM PBS, pH 8.0) was added

ABA-C(Scm)GSSK(folate)-CONH2 (17.4 L from 0.25 mM solution in DMSO, 4.3 nmol, 10 eq.), and the reaction allowed to shake at room temperature overnight. Unreacted peptide was separated from protein using a MicroBio Spin 6 column. A slight shift in mass between FasNb5 and the reaction can be seen by SDS-PAGE. Analysis by Western blot, staining with anti-folic acid antibody revealed the presence of folic acid only in the reaction and not in the unreacted FasNb5.

S18 M kDa 15 Folic acid- FasNb5 containing Conj. conjugate

Figure S22. SDS-PAGE (left) and Western blot (right) of FasNb5 conjugation to ABA- C(Scm)GSSK(folate). Lane 1 – marker, lane 2 – FasNb5, lane 3 – FasNb5-S-S-GSSK(folate).

S19 4. Synthesis of peptide ABA-C(Scm)GSSK-CONH2 and its conjugation to MB23

4.1 Synthesis of peptide ABA-C(Scm)GSSK-CONH2

Peptide ABA-C(Scm)GSSK-CONH2 was synthesized using the protocols described in section 3.1 (Figure S19), with the exception that the folic acid coupling step was omitted.

0.2 eq. Pd(PPh ) Acm O 3 4 Acm O H 60 eq. PhSiH H S Gly Ser Ser N 3 S Gly Ser Ser N O CH2Cl2 O NH O 2 x 30 mins, r.t. NH O ABA ABA

HN O NH2 O O Cl O S

CH2Cl2 3 h, r.t. Scm O O H 95% TFA Scm H S Gly Ser Ser N 2.5% TIPS N NH2 S Gly Ser Ser O 2.5% H2O O NH O 2 h, r.t. NH O ABA ABA

NH2 NH2 Figure S23: Synthesis scheme for peptide ABA-C(Scm)GSSK-CONH2

4.2 Conjugation of MB23 to ABA-C(Scm)GSSK-CONH2

O O S S

ABA-CGSS-K CONH2 SH S S 10 mM Tris-HCl, pH 7.4 CGSSK CONH2 37 °C, o/n ABA

pre-reduced with 10 eq. DTT Figure S24. Synthesis scheme for the conjugation of MB23 to peptide ABA-C(Scm)GSSK-

CONH2

MB23 was first reduced with DTT to remove any dimeric species formed during storage following the procedure described above. To 15 L of reduced MB23 (69 g, 6.0 nmol, c =

4.6 mg/mL in 10 mM Tris-HCl, pH 7.4) was added ABA-C(Scm)GSSK-CONH2 (47 L from 0.6 mM solution in 10 mM Tris, pH 7.4, 30 nmol, 5 eq.), and the reaction allowed to shake at

S20 37 °C overnight. Full conversion to disulfide-modified MB23 was observed as no mass corresponding to the starting material was detected in the Total Ion Chromatogram after scanning the MS trace of the crude reaction mixture.

DAD1 A, Sig=214,20 Ref=off (17-08-16\033-2001.D) mAU 8 0 1 . 1

1250 8 8 . 4 MB23- 1000 Peptide region peptide 8 7 4 . 3

750 9 0 5

6 conjugate 4 . 7 3 .

500 3 3 . 3 2 8

6 3 3 7 3 6 2 7 6 . 4 . 0 4 3 5 3 . 0 3 7

5 .

4 . 9 6 1 . 3 5

1 4 3 6 .

0 2 3 2

1 . . 8

. 0 3

7 6 6 0 . 3 4 250 9

4 . 1 3

0 . 4

3 4 4 . 4

-250

-500

0 2 4 6 8 min Figure S25. RP-HPLC trace of reaction mixture between MB23 and peptide ABA-

C(Scm)GSSK(folate)-CONH2.

Figure S26. ESI-MS from LC-MS of crossed disulfide reaction via the Scm group with ABA-

C(Scm)GSSK-CONH2. Calculated mass 12205, observed mass 12206 (cfr MW of starting MB23: 11471).

S21 5. Synthesis of ABA-GVSSC(Scm)GSSK(FAM)-CONH2 and its conjugation to MB23

5.1 Synthesis of peptide ABA-GVSSC(Scm)GSSK(FAM)-CONH2

The peptides were synthesised on ChemMatrix Rink Amide resin via automated peptide synthesis on the MultiPep RSi (Intavis) (100 µmol scale). The N-terminal Fmoc was manually removed by 3' - 5' - 12' treatment with 40% piperidine/DMF. Folic acid or ABA was coupled manually at the N-terminus (5 eq. folic acid/ABA, 5 eq. HATU, 10 eq. DIPEA) for 3 h at room temperature. The coupling was repeated once more. The resin was swollen in CH2Cl2 and the Alloc protecting group on lysine was removed by 2 x 30 mins reaction at room temperature using 0.2 eq Pd(PPh3)4 and 60 eq. phenylsilane. The ninhydrin test was used to detect deprotection. 5(6)-carboxyfluorescein (FAM) was coupled overnight (10 eq. FAM, 10 eq. HATU, 20 eq. DIPEA) at room temperature in the dark. Coupling was tested using the ninhydrin colour test. The resin was washed extensively with 20% piperidine/DMF to remove

FAM dimers and then with DMF, MeOH and CH2Cl2. Cys(Acm) was converted to Cys(Scm) by swelling the resin in CH2Cl2, adding methoxycarbonylsulfenyl chloride (1.2 eq.) and shaking for 3 h at room temperature. After extensive washing of the resin with CH2Cl2, DMF,

MeOH and Et2O, the peptides were cleaved from the resin using 95% TFA-2.5% TIS-2.5%

H2O. The peptides were purified by prep-HPLC (0-100% ACN 15 mins).

DMSO peak

Figure S27. RP-HPLC analysis of ABA-GVSSC(Scm)GSSK(FAM)-CONH2 (tr = 4.573 min)

S22 + Figure S28. ESI-MS of ABA-GVSSC(Scm)GSSK(FAM)-CONH2 (mass calcd. for [M+H] 1419.4, obs. mass 1419.3 [M+H]+). HO

O O NH O O HO

O H O N GSS O O GVSS N H NH2 N H S O S ABA-GVSSC(Scm)GSSK(FAM) O

Chemical Formula: C62H74N12O23S2 Exact Mass: 1418,44 Molecular Weight: 1419,46

Figure S29. Structure of peptide ABA-GVSSC(Scm)GSSK(FAM)-CONH2

S23 5.2 Conjugation of MB23 to ABA-GVSSC(Scm)GSSK(FAM)-CONH2 O

S O 11 eq. S

ABA-GVSSCGSS-K(FAM)-CONH2 SH S S 10 mM Tris-HCl, pH 7.4 CGSSK(FAM) CONH r.t., DMSO, o.n. 2 SSVG-ABA

pre-reduced with 10 eq. DTT Figure S30: Synthesis scheme for the conjugation of FasNb5 with peptide ABA-

C(Scm)GSSK(folic acid)-CONH2

For conjugation of the peptides to reduced MB23 (Valentine Alphabody), 38.4 nmol of MB23 was mixed with 0.43 µM of peptide (DMSO solution, 11 eq.) and the reaction allowed to shake at room temperature overnight. The reaction was then centrifuged to remove peptide precipitate (10 mins, 13,200 rpm) and purified by RP-HPLC (Phenomenex Jupiter C18, 0-100% ACN over 15 mins). Solvent was removed by SpeedVac, and conjugated MB23 was resuspended in 10 mM Tris HCl, pH 7.4.

DAD1 A, Sig=214,20 Ref=off (17-03-13\069-1501.D) solvent peak mAU 0 6 7 . 2

1000 Product 7 6 9 . 4 750 4 4

500 9 . 0

7 7 1 1 4 4 6 . 7 8 2 7 7 0 2 1 3 3 3 3 .

. 3 2 0 6 . 1 1 1 3 . . 9 .

. 2 1 1 7

1 0

2 . 3

250 1 4 6 . 4

-250

-500

0 1 2 3 4 5 6 7 8 9 min Figure S31. RP-HPLC trace of the RP-HPLC purified MB23- ABA-

GVSSC(Scm)GSSK(FAM)-CONH2 peptide conjugate

Figure S32. ESI-MS from LC-MS of MB23 conjugated to ABA-GVSSC(Scm)GSSL(FAM)-

CONH2. Calculated mass 12797, observed mass 12797.

S24 6. Synthesis and purification of H2N-C(Scm)GSSGSScKFRRRRE-CONH2 and its conjugation to MB23

6.1 Synthesis of peptide H2N-C(Scm)GSSGSScKFRRRRE-CONH2 The peptide C(Acm)GSSGSSK(Alloc)FRRRRE(Alloc) synthesised on ChemMatrix Rink Amide resin via automated peptide synthesis on the MultiPep RSi (Intavis) (50 µmol). Alloc

protecting group was removed by swelling the resin in CH2Cl2, followed by 2 x 30 mins

treatment with 0.2 eq. Pd(PPh3)4 and 60 eq. phenylsilane. The Kaiser colour test was used to detect deprotection. Cyclisation between the Lys and Glu residues was carried out using 5 eq. of PyBOP, 5 eq. of Oxyma and 10 eq. of DIPEA for 3 h at room temperature. The N-terminal Fmoc group was subsequently removed by 5 mins treatment with 40% piperidine/DMF and 15 mins treatment with 20% piperidine/DMF. Cys(Acm) was converted to Cys(Scm) by swelling

the resin in CH2Cl2, with subsequent addition of 1.2 eq. of methoxycarbonylsulfenyl chloride (3 h, r.t.). The peptide was cleaved using the standard procedure, and purified using prep-HPLC (0-60% ACN 15 mins). Qian exo

solvent peak

11.383

Figure S33. RP-HPLC trace of RP-HPLC purified peptide C(Scm)GSSGSS-cKFRRRRE

(tR = 11.4 min).

S25 Qian exo

Voyager Sp ec #1[BP = 1882.8, 10947]

18 82.75 1. 1E+4 100 1882.75

60 y t i s n e

t 50 n I

20 1707 .58 1909.84 1709.50 1810.61 1752 .64 19 89.85 10 18 95.81 1754.68 18 35.74 19 04 .85 19 51 .81

0 0 99 9. 0 1299.4 1599 .8 1900 .2 2200 .6 25 01 .0 Mas s (m/z) Figure S34. MALDI-TOF of C(Scm)GSSGSS-cKFRRRRE (mass calcd. for [M+H]+ 1880.9, obs. mass 1882.8 [M+H]+).

C(Scm)GSSGSScKFRRRE O O

N NH2 H HN R G S F R S S N NH R H S O O O R G S O O S HN H

Chemical Formula: C78H121N29O22S2 Exact Mass: 1879.87 Molecular Weight: 1881.13

Figure S35. Structure of peptide H2N-C(Scm)GSSGSScKFRRRRE-CONH2

S26 6.2 Conjugation of MB23 to C(Scm)GSSGSS-cKFRRRRE

35 eq. C(Scm)GSSGSS-cKFRRRRE SH S S 10 mM Tris-HCl, pH 7.4 CGSSGSS-cKFRRRRE r.t., 10% DMSO, o.n.

pre-reduced with 10 eq. DTT Figure S36: Synthesis scheme for the conjugation of MB23 with peptide C(Scm)GSSGSS- cKFRRRRE

MB23 was first reduced with DTT to remove any dimeric species formed during storage following the procedure described above. To 25 µL of reduced MB23 (12.5 µg, 1.1 nmol, c = 6.5 mg/mL in 10 mM Tris-HCl, pH 7.4) was added C(Scm)GSSGSS-cKFRRRRE (15 µL from 2.7 mM solution in 10 mM Tris-HCl pH 7.4, 40 nmol, 35 eq.), 2.5 µL DMSO and 5.6 µL 10 mM Tris-HCl pH 7.4 to give a final protein concentration of 0.5 mg/mL, and the reaction mixture was allowed to shake at room temperature overnight. Excess peptide was then separated from the protein using a Micro BioSpin 6 column (Bio-Rad). Analysis by LC/MS showed conversion to the desired conjugate, and SDS-PAGE revealed minimal dimer formation (and some remaining starting material).

S27 DAD1 A, Sig=214,20 Ref=off (17-06-07\017-0301.D) mAU 6 0 1 . 3

7 7 2 . 1 DMSO Peptide

1500

Dimer 1000 1 4 2 . 3

6 4 7 . 2

1 Product 9 8 9 6 . 3 8 8 2 .

3 5 0 2 0

4 8 . 500 . 9 1 . 4

6 8 1 2 4 . 3 . 3

2 5 4 5 9 8 . 6 9 3 .

7 . 4

6 0 5 . 5 3

7 6 . 4

-500

0 2 4 6 8 min

Figure S37. RP-HPLC trace of MB23 conjugated to C(Scm)GSSGSS-cKFRRRRE (tR = 4.984 min).

Figure S38. ESI-MS of MB23 conjugated to C(Scm)GSSGSS-cKFRRRRE. Calculated mass 13259, observed mass 13275. Probable oxidation product.

M kDa 20 dimer 15 MB23 conjugate 10

Figure S39. SDS-PAGE of lane 1 – marker, lane 2 – reduced MB23 and, lane 3 – MB23-S-S-CGSSGSS-cKFRRRRE.

S28 7. Synthesis of H2N-C(Scm)GSRGDS-CONH2 and its conjugation to MB23 followed by purification

7.1 Synthesis of peptide H2N-C(Scm)GSRGDS-CONH2 Following peptide synthesis on an Intavis system, the N-terminal Fmoc group was removed and the Acm group converted to the Scm group using the procedures described above. The resulting peptide was cleaved from the resin and then purified on a Kinetex C18 column by RP-HPLC. DAD1 B, Sig=214,16 Ref=off (D:\DATA\16-01-27\MDV000021.D) DMSO mAU

1200

1000 13.2 mins

800

600

400

200

-200

-400

0 2.5 5 7.5 10 12.5 15 17.5 20 22.5 min

Figure S40. RP-HPLC trace of peptide H2N-C(Scm)GSRGDS-CONH2 (tR = 13.2 min). *MSD1 SPC, time=2.684:2.969 of D:\DATA\16-01-27\061-1101.D API-ES, Pos, Scan, Frag: 70 1 . 7

4 Max: 72574 1

100 HN NH2

H N-C(Scm)GSRGDS NH 2 O 80 O O O OH O H H H H2N N N N N N N NH2 H H H 60 O O O S OH OH O S 6 . 5

8 O 3

40 Chemical Formula: C25H43N11O13S2 Exact Mass: 769.25 Molecular Weight: 769.80 20 2 . 2 8 . 4 1 1 7

0 500 1000 1500 2000 m/z Figure S41. ESI-MS of purified CGSRGDS (mass calcd. for [M+H]+ = 770.25, obs. mass 771.2 [M+H]+).

S29 7.2 Conjugation of MB23 to C(Scm)GSRGDS-CONH2

20 eq.

NH2-CGSRGDS CONH2 SH S S 10 mM Tris-HCl, pH 7.4 CGSRGDS CONH2 r.t., o.n.

pre-reduced with 10 eq. DTT Figure S42: Synthesis scheme for the conjugation of MB23 with peptide C(Scm)GSRGDS-

CONH2

MB23 was first reduced with DTT to remove any dimeric species formed during storage following the procedure described above. To 11.5 L of reduced MB23 (25.2 g, 2.2 nmol, c

= 2.2 mg/mL in 10 mM Tris-HCl, pH 7.4) was added C(Scm)GSRGDS-CONH2 (21.2 L from

2.1 mM solution in MQ H2O, 44 nmol, 20 eq.), and the reaction made up to 40 L with 10 mM Tris, pH 7.4 and allowed to shake at room temperature overnight. LC/MS showed efficient conversion to the RGD-decorated MB23 and SDS-PAGE revealed minimal competing dimer formation as deduced from careful MS analysis.

Solvent peak DAD1 A, Sig=214,20 Ref=off (16-03-04\009-1001.D) 4.889 min mAU

600

400

200

-200

-400

-600

-800

0 2 4 6 8 min

Figure S43. RP-HPLC trace of MB23 conjugated to C(Scm)GSRGDS (tR = 4.889 min).

S30 20 kDa dimer

10 kDa conjugate

M MB23 conjugate

Figure S44. ESI-MS of MB23 conjugated to C(Scm)GSRGDS. Calculated mass 12148, observed mass 12146. SDS-PAGE – marker, MB23, MB23-CGSRGDS conjugate.

8. Synthesis and purification of cC(Scm)RGDE-CONH2 followed by conjugation to MB23 and FasNb5

8.1 Synthesis of peptide cC(Scm)RGDE-CONH2

HN NHPbf HN NHPbf NH O NH O O O O O 0.2 eq. Pd(PPh3) automated SPPS H H 60 eq. PhSiH H N H N N N 3 O O O O 2 2 N N N H H H H H H N N N O O 2 N N N S H H H O O HN O O S

O HN HO O

O HATU/HOBt/DIPEA, overnight

O O O O OH O O H O O H H O N O O N O O N O N N N H Methoxycarbonylsulfenyl H HN O HN O H HN O NH NH NH chloride H H H H TFA/TIS/H2O H H PbfHN N N N H N N N PbfHN N N N O H 2 O NH2 O H NH O NH O NH O S S S O S O S HN O O O

Chemical Formula: C22H35N9O10S2 Exact Mass: 649.19

Figure S45: Synthesis scheme for the peptide cC(Scm)RGDE-CONH2

S31 The linear peptide C(Acm)RGDE(Alloc) peptide was synthesised on a Syro II wave system. Following automated SPPS, Alloc group removal at the C-terminal Glu was carried out using the procedure described in section 10. Subsequently, head-to-tail cyclisation was performed by swelling the resin (100 mg, 0.54 mmol/g, 54 mol) in DMF for 30 mins at room temperature. In the meantime, a solution of HATU (100 mg, 2.7 mmol, 5 eq.), HOBt (41 mg, 2.7 mmol, 5 eq.) and DIPEA (47 L, 2.7 mmol, 5 eq.) in DMF (4 mL) was prepared and then added to the preswollen resin. Cyclisation was allowed to occur overnight with shaking at room temperature. The cysteine Acm group was converted to the Scm group using the procedure described in section 2.2. Peptide cleavage was carried out using TFA/TIS/H2O followed by precipitation with cold Et2O. The peptide was purified by RP-HPLC.

DAD1 A, Sig=214,20 Ref=off (18-03-12\086-0301.D) 2.920 min mAU 1250

1000

750

500

250

-250

-500

-750

-1000

0 2 4 6 8 min

Figure S46. RP-HPLC trace of the RP-HPLC purified peptide cC(Scm)RGDE-CONH2 (tR = 2.920) on a Phenomenex Kinetex C18 100 Å column. min)

*MSD1 SPC, time=2.948 of D:\DATA\18-03-12\086-0301.D API-ES, Pos, Scan, Frag: 70 7 . 5 2 3

Max: 8.3881e+006 1 100 . 0 5 6

1 . 1 5 6 80

60 1 . 2 5 6

40 6 . 2 3 3 20 1 . 6 3 . 5 0 6 7

6 2 .

6 1 1 . 3 0

4 5

0 200 400 600 800 1000 m/z

Figure S47. ESI-MS from LC-MS of the RP-HPLC purified peptide cC(Scm)RGDE-CONH2 + (tR = 2.920 min). E.M. calcd for C22H35N9O11S2 = 649.19, found M+H+ = 650.1, M/2 + H = 325.7

S32 8.2 Conjugation of MB23 to cC(Scm)RGDE-CONH2

5 eq. cC(Scm)RDGE SH S S 10 mM Tris-HCl, pH 7.4 cCRGDE r.t., o.n.

pre-reduced with 10 eq. DTT Figure S48: Synthesis scheme for the conjugation of MB23 with peptide cC(Scm)RGDE-

CONH2

MB23 was first reduced with DTT to remove any dimeric species formed during storage following the procedure described above. To 3.2 L of reduced MB23 (25.3 g, 2.2 nmol, c =

7.9 mg/mL in 10 mM Tris-HCl, pH 7.4) was added cC(Scm)RGDE-CONH2 (4.4 L from 2.5 mM solution in MQ H2O, 11 nmol, 5 eq.), and the reaction made up to 50 L with 10 mM Tris, pH 7.4 and allowed to shake at room temperature overnight. Analysis by LC/MS showed conversion to the cyclic RGD-decorated MB23, and SDS-PAGE showed minimal competing dimer formation. Solvent peak DAD1 A, Sig=214,20 Ref=off (18-03-16\019-2301.D) mAU

600 conjugate

400

200 Alphabody dimer

-200

-400

-600

0 1 2 3 4 5 6 7 8 9 min Figure S49. RP-HPLC trace of the crude reaction mixture between the MB23 and peptide cC(Scm)RGDE-CONH2. tR = 4.920 min for the conjugat on a Phenomenex Kinetex C18 100 Å column.

S33 Figure S50. ESI-MS from LC-MS of crossed-disulfide reaction of MB23 via Scm group of peptide cC(Scm)RGDE. Calculated mass 12028, observed mass 12035.63.

M kDa dimer 20 15 conjugate MB23 10 Figure S51. SDS-PAGE of lane 1 – marker, lane 2 – reduced MB23 and, lane 3 – MB23-S-S- cCRGDE.

S34 8.3 Conjugation of FasNb5 to cC(Scm)RGDE-CONH2

100 eq. cC(Scm)RDGE SH S S 10 mM Tris-HCl, pH 7.4 cCRGDE r.t., o.n. Figure S52. Synthesis scheme for the conjugation of FasNb5 with peptide cC(Scm)RGDE-

CONH2 To 25 L of FasNb5 (5.3 g, 0.39 nmol, c = 0.21 mg/mL in 10 mM Tris-HCl, pH 7.4) was

added cC(Scm)RGDE-CONH2 (15.6 L from 2.5 mM solution in MQ H2O, 39 nmol, 100 eq.), and the reaction was allowed to shake at room temperature overnight. Analysis by LC/MS showed conversion to the cyclic RGD-decorated FasNb5. Calculated mass 14452, observed mass 14447. Peptide region

DAD1 A, Sig=214,20 Ref=off (17-02-21\030-4801.D) mAU 4 8 9 . 2

800 4 9 4 4 2 . 7 . 3

2 conjugate 600 3 5 9 5 0 . 5 4 1 5 9

4 . . 3 0 4 1 0

. 3 4 6 1 5 3 3 0 2

. 6 1 2

400 1 . . 1

4 3 1 1 . .

3 0

2 9 3 1 6 . 8 . 3

4 4 2 200 . 4

-200

-400 7 3 0 . 9

-600

0 2 4 6 8 min

Figure S53. RP-HPLC trace of FasNb5-peptide cC(Scm)RGDE-CONH2 conjugate (tR = 4.244 min).

Figure S54. ESI-MS from LC-MS of crossed disulfide reaction of FasNb5 via Scm group of peptide cC(Scm)RGDE. Calculated mass 14452, observed mass 14447.

S35 8.4 Conjugation of FasNb5 with His and HA tags to cC(Scm)RGDE-CONH2 Solvent Peak

DAD1 A, Sig=214,20 Ref=off (18-05-17\050-0201.D) mAU 1 6 7 . 2 400

200 conjugate 4 2 9 . 0

8 1 9 7 0 1 3 6 4 7 5 9 9 4 2 2 1 0 0 7 9 0 6 9 5 9 7 0 7 1 9 6 1 6 0 5 1 0 3 4 6 2 0 2 4 2 0 6 8 8 7 5 5 . 0 1 1 3 4 4 5 6 7 8 8 3 4 7 1 2 2 3 5 5 5 6 9 0 8 1 ......

0 0 0 0 0 0 0 0 0 0 0 1 1 1 2 2 0 0 1 1 1 1 1 2 1

0 3 6 1 . 4

-200 3 7 0 . 5

-400

-600

-800 4 2 1 8 3 0 . . 9 9

-1000

0 2 4 6 8 min

Figure S55. RP-HPLC trace of FasNb5 nanobody with His and HA tags (tR = 4.163 min)

Figure S56. ESI-MS from LC-MS of FasNb5 nanobody with His and HA tags and deconvolution spectra. Calculated mass 16454, observed mass 16454.88 [M] and 16437.97 [M

+ – H2O + H ]

S36 Peptide region DAD1 A, Sig=214,20 Ref=off (18-05-15\022-2401.D) mAU 9 6 1 . 3 1500 2 2 2 . 3

6 6 0 5 1 3 . . 3 3

1000 9 3 8 7 9 . 4 2 .

3 Peptide-nanobody conjugate 1 1 3 8 1 7 . 0 1 0 4 3 4

500 1 2 2 . . . 8 2 . 2 2 2

0 region 3 3 9

6 9 2 . 1 2 3 . .

4 4

-500

2.5 3 3.5 4 4.5 5 5.5 min MSD1 TIC, MS File (D:\DATA\18-05-15\022-2401.D) API-ES, Pos, Scan, Frag: 70 8 0 2

3.5E8 . 3 Peptide

3E8

2.5E8 2 6 3 . 3

Peptide-nanobody conjugate 2E8

8 region 0 5 . 3 7 1.5E8 3 2 . 4

1 5 8 1E8 . 3

4 2 0 1 2 6 2 9 . 8 . 5 3 6 . 2 5

6 8 7 5

5 5 . 7

50000000 6 4 1 4 6 8 . 9

. 3 . 2 . 0 5 9 5 5 7 0 4 4 9 8

0 4 1 3 7 1 4 2 . 0 1 5

6 4 . . . . . 2 . 2

2 2 2 2 2

2.5 3 3.5 4 4.5 5 5.5 min

Figure S57. RP-HPLC trace (upper: UV trace, lower: TIC trace) of crude reaction mixture between FasNb5 nanobody with His and HA tags and cC(Scm)RGDE peptide.

Figure S58. ESI-MS from LC-MS of crude reaction mixture between FasNb5 nanobody with His and HA tags and cC(Scm)RGDE peptide and deconvolution spectra. Calculated masses of

+ + conjugate M = 17013, M+2xTris (121.14) +H2O+H = 17274.28, M+Tris+2xACN (41.05) +H

S37 = 17217.24, M+Tris (121.14) +HCl (36.5) +H+ = 17170.6, observed deconvoluted masses 17279.84, 17220.96 and 17177.70 respectively.

The reaction mixture was purified by dialysis as follows: 200 µL of the nanobody conjugate was pipetted into a dialysis membrane (1 kDa MW cutoff). After sealing the membrane it was put in a beaker of 1 L filled with 20 mM phosphate buffer (pH 7). The dialysis was carried out for 18 hours and the buffer was changed 3 times.

Solvent Peak

conjugate

Figure S59. RP-HPLC trace of the FasNb5 nanobody with His and HA tags conjugated to the cCRGDE peptide after using a Micro Biospin 6 column and dialysis of the crude reaction mixture.

Figure S60 ESI-MS from LC-MS of reaction mixture between FasNb5 nanobody with His and HA tags and cC(Scm)RGDE peptide and deconvolution spectra after dialysis. Calculated

+ masses of conjugate M = 17013, M+2xTris (121.14) +H2O+H = 17274.28, M+2xTris (121.14) +2xACN (41.05) + H+ = 17338.38 observed deconvoluted masses 17279.84 and 17339.87 respectively.

S38 9. Circular Dichroism (CD) studies

The secondary structure of the nanobody, alphabody and their conjugates were analysed via CD measurements. The proteins were dialysed into a 20mM phosphate buffer (pH 7) and diluted to a concentration of 0.2 mg/mL. The spectra were recorded on a Jasco (J-710) using a scanning speed of 100 nm/min and 2 s response time. The results were expressed as molar ellipticity (deg.cm2/dmol).

Figure S61. A. Graph showing molar ellipticity (deg.cm2/dmol) vs wavelength for the nanobody and nanobody conjugate. B. Graph showing molar ellipticity (deg.cm2/dmol) vs wavelength for the alphabody conjugate and the alphabody.

10. ELISA

The antigen of the nanobody (fascin) (100 ng/well) was immobilized into the wells of a Nunc maxisorb plate by addition of 100 µL of a solution of the antigen in carbonate buffer (1 µg/mL, pH 9.6) overnight at 4°C. The wells for the blank measurement were left empty. Cortactin was immobilized in the same way as a negative control. The plate was washed 3 times with 0.5% Tween in PBS. The complete plate was incubated with blocking buffer (1% BSA in PBS) for 1.5 hours at 25 °C. The plate was washed 3 times with 0.5% Tween in PBS. Nanobody and conjugate tenfold dilution series (from 10 µg/mL to 10-6 µg/mL) in PBS were prepared and 100 µL of these dilutions were added to the wells, each row on the plate representing a different dilution. The plate was incubated for 1.5 hours at 25 °C. The plate was washed 3 times with 0.5% Tween in PBS. 100 µL of Rabbit anti-HA antibody (Zymed 71-5500) (0.1 µg/mL) in blocking buffer was added. The plate was incubated for 1.5 hours at 25 °C. The plate was washed 3 times with 0.5% Tween in PBS. Following this each well was incubated with 100 µL of the secondary antibody: Goat anti-Rabbit HRP F(ab’)2 (GE Healthcare NA9340) (0.1

S39 µg/mL) in blocking buffer for 1.5 hours at 25 °C. Subsequently the plate was washed 6 times with 0.5% Tween in PBS. The detection was done using the Pierce TMB substrate kit, equal volumes of the TMB solution and peroxide solution were mixed directly before adding 100 µL of the mixture to each well. A blue colour develops when the TMB is oxidized and the reaction

was stopped by adding 50 µL of 1N H2SO4. The absorbance at 450 nm was measured using a plate reader (Versamax tunable microplate reader).

Figure S62. Graph showing absorbance at 450 nm (A.U.) vs concentration (µg/mL) for the negative control, nanobody and the nanobody-peptide conjugate.

11. Serum stability experiments

10 µg of the HA/His tagged nanobody conjugate (20 µL of a 0.5 mg/mL solution) was added to 80 µL of human serum. Samples were incubated at 37 °C while shaking for 30 min, 1 hour, and 2 hours. As a positive control the HA/His tagged nanobody conjugate was incubated for 2 hours with PBS, as a negative control serum was used without adding nanobody conjugate. After this, 50 µL of a 1/1 suspension of Talon beads (Clontech) was added, followed by 900 µL of Tris buffer (pH 7.4). The beads were incubated for 1 hour at 37 °C while rotating.

Afterwards the beads were washed three times with 1 mLwash buffer (50 mM NaH2PO4, 500 mM NaCl, 20 mM imidazole, pH 8). After removing the wash buffer for the last time, 30 µL of elution buffer (50 mM NaH2PO4, 500 mM NaCl and 500 mM imidazole) was added and the

S40 beads were left shaking overnight. After centrifuging the beads, the samples were desalted using a Bio-rad micro spin column. Samples were analysed via LC/MS. Due to the high dilution of the sample and overlap of the retention time between the nanobody-peptide conjugate and proteins in the serum sample, the conjugate was not clearly visible on the UV spectrum. In some instances, intact conjugate could be detected via MS.

Figure S63. ESI-MS from LC-MS of the nanobody-peptide conjugate after 30 min treatment

+ in human serum. Calculated masses of conjugate M = 17013, M+2xTris (121.14) +H2O+H = 17274.28, observed 17278.47.

Figure S64. ESI-MS from LC-MS of the nanobody-peptide conjugate after 2-hour min treatment in human serum. Calculated masses of conjugate M = 17013, M+2xTris (121.14)

+ + +H2O+H = 17274.28, M+Tris+2xACN (41.05) +H = 17217.24 observed 17280.49 and 17221.40.

S41 12. References

1 J. Desmet, K. Verstraete, Y. Bloch, E. Lorent, Y. Wen, B. Devreese, K. Vandenbroucke, S. Loverix, T. Hettmann, S. Deroo, K. Somers, P. Henderikx, I. Lasters and S. N. Savvides, Nat. Commun., 2014, 5, 5237. 2 I. Van Audenhove, C. Boucherie, L. Pieters, O. Zwaenepoel, B. Vanloo, E. Martens, C. Verbrugge, G. Hassanzadeh-Ghassabeh, J. Vandekerckhove, M. Cornelissen, A. De Ganck and J. Gettemans, FASEB J., 2014, 28, 1805–1818.

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