Supporting Information for Peptide-Protein Conjugation ... · S1 Supporting Information for...

S1

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]

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

mailto:[email protected]

S2

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 reagent

S10

2.8 Verification of free thiol functionality by reaction of FasNb5 with Ellman’s reagent

S11

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

S13

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 conjugation to MB23

S19

4.1 Synthesis of peptide ABA-C(Scm)GSSK-CONH2 S19

S3

4.2 Conjugation of MB23 to ABA-C(Scm)GSSK-CONH2 S19

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

S21

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-CONH2 and its conjugation to MB23

S24

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 followed by purification

S28

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 conjugation to MB23 and FasNb5

S29

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

S4

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.

S5

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.

S6

min0 5 10 15 20 25

mAU

-300

-250

-200

-150

-100

-50

0

50

100

DAD1 A, Sig=214,16 Ref=off (D:\DATA\18-09-20C\AI000002.D)t = 9.135 min – solvent peak

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.

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.

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%

min0 2 4 6 8

mAU

-600

-400

-200

0

200

400

600

DAD1 A, Sig=214,20 Ref=off (18-09-12\079-0401.D) t = 2.738 min – solvent peak

S7

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

S8

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)FRRRRE(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

S9

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

201510

2

S10

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

SH

10 mM Tris-HCl, pH 7.4r.t., 15 mins

pre-reduced with10 eq. DTT

S S

SS

OOH

NO2

OHO

O2N

NO2

OH

O

Cys59

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.

min0 2 4 6 8

mAU

-400

-200

0

200

400

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

0.34

5

0.96

0 1.

079

1.12

6

1.42

6

1.65

0

2.74

5

5.03

6

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

Product

S11

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

10 mM Tris-HCl, pH 7.4r.t., 15 mins

S SS

S

OOH

NO2

OHO

O2N

NO2

OH

OSH

Cys113

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.

min0 2 4 6 8

mAU

-1000

-800

-600

-400

-200

0

200

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

Unreacted FasNb5

Product

S12

Figure S10. RP-HPLC trace of reaction mixture between FasNb5 and Ellman’s reagent.

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).

m/z1100 1200 1300 1400 1500 1600 1700

-1

0

1

2

3

4

5

6

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

Max: 81555

M/10 + H+

1410

M/9 + H+

1566

M/8 + H+

1762

M/11 + H+

1282M/12 + H

+

1175

S13

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(PPh3)460 eq. PhSiH3

CH2Cl22 x 30 mins, r.t.

5 eq. folic acid5 eq. HBTU

10 eq. DIPEA

DMSOo/n, 37 °C

Gly Ser Ser

NH O

S

ABA

O

HN

O

HN

O

O

Gly Ser Ser

NH O

S

ABA

O

HN

O

NH2

Gly Ser Ser

NH O

S

ABA

O

HN

O

HN

O

Fol

Acm Acm

Acm O SO

Cl

CH2Cl23 h, r.t.

95% TFA2.5% TIPS2.5% H2O

2 h, r.t.

Gly Ser Ser

NH O

S

ABA

O

HN

O

HN

O

Fol

Scm

O OH

NH

O

NH

NO

HN

H2N N N

O

OH

fol = folic acid

Gly Ser Ser

NH O

S

ABA

ONH2

HN

O

HN

O

Fol

Scm

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]+).

S14

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]+).

499.0 999.4 1499.8 2000.2 2500.6 3001.0Mass (m/z)

0

439

0

10

20

30

40

50

60

70

80

90

100

% In

tens

ity

Voyager Spec #1[BP = 1157.9, 439]

1157.8773

1173.8538

550.8910

522.8582

641.6013898.8188

684.7345 1160.9102982.7782712.6491507.6305 1197.77321064.8521 1342.8131834.7147673.2702 2700.85561794.5134516.2787 2206.27881385.90551008.0350 1238.0087 1998.0222843.7393 1543.6697665.8457 2465.00351785.8760 2174.40681016.2262 1306.81721164.1541 2676.3663500.3892 2000.4462837.0680 1599.1660

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

S15

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).

min0 5 10 15 20 25

mAU

-200

0

200

400

600

800

1000

1200

1400

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)

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.

499.0 799.4 1099.8 1400.2 1700.6 2001.0Mass (m/z)

0

2305.0

0

10

20

30

40

50

60

70

80

90

100

% In

tens

ity

Voyager Spec #1[BP = 604.2, 2305]

604.2278

666.2306 1062.6338

1076.6351

1060.6232

1074.6458

653.4978519.3113

915.5279

887.5523 1031.83351064.6022529.4791 736.3390628.2108511.3557 1078.6347

919.4939573.5124 1042.69261047.3067500.3386 917.4858779.3183578.1841 651.5445 1044.6630537.3417 617.4685 869.2305755.3035 1070.6045994.3187544.3100 1157.5259657.4483 881.7322761.6947 1053.8190967.5514 1672.25731265.4966 1542.70601136.5872 1827.54001361.6249 1448.6746875.3536773.5123 1729.11901277.4787 1637.17801557.1225 1846.8628

Peptide - Scm

solvent peakDMSO

OHO

HN

O

HN

NO

NH

NH2NN

Gly Ser Ser

NH O

HSO

NH2

HN

O

NH

OAcHN

Chemical Formula: C45H57N15O14SExact Mass: 1063.39

S16

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

for C47H59N15O16S2 = 1153.27 (below), found Peptide – Scm = 1062.6338 (above)

3.2. Conjugation of MB23 to ABA-C(Scm)GSSK(folate)-CONH2

SH10 mM Tris-HCl, pH 7.4

r.t., DMSO, o.n.

ABA-CGSS-K(folate)

S SO

O


S SCGSSK(folate)ABA

11 eq.

CONH2

CONH2

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).

NH2

HN

NH

HN

NH

HN

SO

HN

S

O

OOH

O

O

NH

OO

OH

O

HO2C NH

O

NH

N

N

N

NH

O

NH2

Chemical Formula: C47H59N15O16S2Exact Mass: 1153.37Molecular Weight: 1154.20

O

O

ABA-C(Scm)GSSLys(folate)

S17

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


r.t., DMSO, o.n.

ABA-CGSS-K(folate)

S SO

O

S SCGSSK(folate)ABA

10 eq.

CONH2

CONH2

BSAPDB 3v03

Cys34

Figure S19: Synthesis scheme for the conjugation of BSA with peptide ABA-


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

1 2 3 1 2 3

min0 5 10 15 20 25

mAU

-500

0

500

1000

1500

2000

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)

14.5 mins – MB23 conjugate

11-12 mins - peptide

DMSO

Folic acid-containing conjugate

S18

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.

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

SH10 mM PBS, pH 8.0

r.t., DMSO, o.n.

ABA-CGSS-K(folate)

S SO

O

S SCGSSK(folate)ABA

10 eq.

CONH2

CONH2

Figure S21: Synthesis scheme for the conjugation of FasNb5 with peptide ABA-


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.

50

75

M kDa

S19

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).

FasNb515

M kDaFolic acid-containing conjugateConj.

S20

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.


CH2Cl22 x 30 mins, r.t.

Gly Ser Ser

NH O

S

ABA

O

HN

O

HN

O

O

Gly Ser Ser

NH O

S

ABA

O

HN

O

NH2

Acm Acm

O SO Cl

CH2Cl23 h, r.t.

95% TFA2.5% TIPS2.5% H2O

2 h, r.t.

Gly Ser Ser

NH O

S

ABA

O

HN

O

NH2

ScmGly Ser Ser

NH O

S

ABA

ONH2

HN

O

NH2

Scm

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

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


37 °C, o/n

ABA-CGSS-K

S SO

O


S SCGSSKABA

CONH2

CONH2

Figure S24. Synthesis scheme for the conjugation of MB23 to peptide ABA-C(Scm)GSSK-

CONH2


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

S21

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.

min0 2 4 6 8

mAU

-500

-250

0

250

500

750

1000

1250

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

0.344 0.9

56 1.047

1.108

1.423

1.553

2.745

3.266

3.334

3.478

3.609

3.662

3.703 3.7

38 3.8

23 3.9

36 4.0

14 4.067

4.443

4.888

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).

MB23-peptide

conjugatePeptide region

S22

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).

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

DMSO peak

S23

Figure S28. ESI-MS of ABA-GVSSC(Scm)GSSK(FAM)-CONH2 (mass calcd. for [M+H]+

1419.4, obs. mass 1419.3 [M+H]+).

GVSS GSSN

O

S

N

NH

S

O

O

OO

HO

O

HO

O

O

N

O

NH2

ABA-GVSSC(Scm)GSSK(FAM)

Chemical Formula: C62H74N12O23S2Exact Mass: 1418,44

Molecular Weight: 1419,46

O

H

H

H

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

S24

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


r.t., DMSO, o.n.

ABA-GVSSCGSS-K(FAM)-CONH2


S SCGSSK(FAM)SSVG-ABA

11 eq.

CONH2

SS

O

O

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.

min0 1 2 3 4 5 6 7 8 9

mAU

-500

-250

0

250

500

750

1000

DAD1 A, Sig=214,20 Ref=off (17-03-13\069-1501.D)

0.337

0.944

1.038

1.137 1.2

61 1.3

71 1.477

1.632

2.204

2.760

3.279

4.641

4.967

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.

Product

solvent peak

S25

6. Synthesis and purification of H2N-C(Scm)GSSGSScKFRRRRE-CONH2 and its

conjugation to MB23

6.1 Synthesis of peptide H2N-C(Scm)GSSGSScKFRRRRE-CONH2

The peptide C(Acm)GSSGSSK(Alloc)FRRRRE(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).

Figure S33. RP-HPLC trace of RP-HPLC purified peptide C(Scm)GSSGSS-cKFRRRRE

(tR = 11.4 min).

Qian exo

11.383

solvent peak

S26

Figure S34. MALDI-TOF of C(Scm)GSSGSS-cKFRRRRE (mass calcd. for [M+H]+

1880.9, obs. mass 1882.8 [M+H]+).

C(Scm)GSSGSScKFRRRE

GS

SG

SS F

R

RR

R

NH

O

NH2

HN

OO

HHN

O SS

O

NH

O

NH

O

Chemical Formula: C78H121N29O22S2Exact Mass: 1879.87

Molecular Weight: 1881.13

Figure S35. Structure of peptide H2N-C(Scm)GSSGSScKFRRRRE-CONH2

Qian exo

99 9. 0 1299 .4 1599 .8 1900 .2 2200 .6 25 01 .0Mas s (m/z)

0

1. 1E+4

0

10

20

30

40

50

60

70

80

90

100

% In

tens

ity

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

18 82 .75

1707 .581909 .84

1810 .611709 .5019 89 .851752 .64 18 95 .81

18 35 .741754 .68 19 04 .8519 51 .81

1882.75

S27

6.2 Conjugation of MB23 to C(Scm)GSSGSS-cKFRRRRE


r.t., 10% DMSO, o.n.


S SCGSSGSS-cKFRRRRE

35 eq.C(Scm)GSSGSS-cKFRRRRE

Figure S36: Synthesis scheme for the conjugation of MB23 with peptide C(Scm)GSSGSS-

cKFRRRRE


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-cKFRRRRE (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).

S28

min0 2 4 6 8

mAU

-500

0

500

1000

1500

DAD1 A, Sig=214,20 Ref=off (17-06-07\017-0301.D)

0.328

0.945 1.0

33

1.277

2.746

2.868 2.9

91 3.1

06 3.2

41

3.416

3.552

3.769

4.673

4.808

4.984

5.506

Figure S37. RP-HPLC trace of MB23 conjugated to C(Scm)GSSGSS-cKFRRRRE (tR =

4.984 min).

Figure S38. ESI-MS of MB23 conjugated to C(Scm)GSSGSS-cKFRRRRE. Calculated

mass 13259, observed mass 13275. Probable oxidation product.

Figure S39. SDS-PAGE of lane 1 – marker, lane 2 – reduced MB23 and, lane 3 –

MB23-S-S-CGSSGSS-cKFRRRRE.

101520

M kDa

MB23 conjugate

dimer

Product

Peptide

Dimer

DMSO

S29

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.

min0 2.5 5 7.5 10 12.5 15 17.5 20 22.5

mAU

-400

-200

0

200

400

600

800

1000

1200

DAD1 B, Sig=214,16 Ref=off (D:\DATA\16-01-27\MDV000021.D)

Figure S40. RP-HPLC trace of peptide H2N-C(Scm)GSRGDS-CONH2 (tR = 13.2 min).

m/z500 1000 1500 20000

20

40

60

80

100

*MSD1 SPC, time=2.684:2.969 of D:\DATA\16-01-27\061-1101.D API-ES, Pos, Scan, Frag: 70

Max: 72574

771

.2

148

.2

385

.6

147

.1

Figure S41. ESI-MS of purified CGSRGDS (mass calcd. for [M+H]+ = 770.25, obs. mass

771.2 [M+H]+).

13.2 mins

NH2H2N

O

S

NH O

HN

O

OH

NH O

NH2HN

NH

HN

O

NH O

OH

O

HN

O

OHS

H2N-C(Scm)GSRGDS


Molecular Weight: 769.80

O

O

DMSO

S30

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


r.t., o.n.

NH2-CGSRGDS


S SCGSRGDS

20 eq.CONH2

CONH2

Figure S42: Synthesis scheme for the conjugation of MB23 with peptide C(Scm)GSRGDS-

CONH2


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.

min0 2 4 6 8

mAU

-800

-600

-400

-200

0

200

400

600

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

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

4.889 minSolvent peak

S31

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

NH

HNO

S

NH

O

PbfHN

NH

HN

HN

O

NH

OO

O

HN O

O

HN

H2NNH

H2NO

S

NH O

NHPbfHN

NH

HN

O

NH O

O

O

HN

O

O OHN

O

automated SPPS

Methoxycarbonylsulfenylchloride

NH

H2NO

S

NH O

NHPbfHN

NH

HN

O

NH O

O

O

HN

O

HO OHN

OHATU/HOBt/DIPEA,overnight

O


NH

HNO

S

NH

O

PbfHN

NH

HN

HN

O

NH

OO

O

HN O

O

S

O

NH2

HN

O

S

NH

O

H2N

NH

HN

HN

O

NH

OOH

O

HN O

O

S

O


O O

TFA/TIS/H2O

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

M MB23 conjugate

20 kDa

10 kDa

dimer

conjugate

S32

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.

min0 2 4 6 8

mAU

-1000

-750

-500

-250

0

250

500

750

1000

1250

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

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)

m/z200 400 600 800 10000

20

40

60

80

100

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

Max: 8.3881e+006

540.1 316.6 270.6 653.1

332.6

652.1 651.1

650.1 325.7

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

2.920 min

S33

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


r.t., o.n.


S ScCRGDE

5 eq. cC(Scm)RDGE

Figure S48: Synthesis scheme for the conjugation of MB23 with peptide cC(Scm)RGDE-

CONH2


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.

min0 1 2 3 4 5 6 7 8 9

mAU

-600

-400

-200

0

200

400

600

DAD1 A, Sig=214,20 Ref=off (18-03-16\019-2301.D)

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.

conjugate

Alphabody dimer

Solvent peak

S34

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.

Figure S51. SDS-PAGE of lane 1 – marker, lane 2 – reduced MB23 and, lane 3 – MB23-S-S-

cCRGDE.

MB23

dimerM kDa

2015

10conjugate

S35

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


r.t., o.n.

S ScCRGDE

100 eq. cC(Scm)RDGE

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.

min0 2 4 6 8

mAU

-600

-400

-200

0

200

400

600

800

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

0.312

0.959

1.035

1.134 1.364 1.4

40 1.553

1.653

2.749

2.984

3.244

3.420

3.632

3.819

4.244

9.037

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.

Peptide region

conjugate

S36

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

min0 2 4 6 8

mAU

-1000

-800

-600

-400

-200

0

200

400

DAD1 A, Sig=214,20 Ref=off (18-05-17\050-0201.D)

0.079

0.117

0.190

0.240

0.327

0.361

0.413

0.466

0.504

0.657

0.715

0.809

0.839 0.9

24 1.0

18

1.344

1.462

1.509

1.560

1.586

1.689

1.722

1.857

1.975

2.059

2.101

2.220

2.761

4.163

5.073

9.082

9.314

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+]

Solvent Peak

conjugate

S37

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+

min2.5 3 3.5 4 4.5 5 5.5

mAU

-500

0

500

1000

1500

DAD1 A, Sig=214,20 Ref=off (18-05-15\022-2401.D)

2.11

8

2.20

1 2.

247

2.73

9 3.10

6 3.

169

3.22

2

3.31

1 3.

356

3.49

8

3.60

2 3.84

0

4.19

3 4.

229

min2.5 3 3.5 4 4.5 5 5.5

0

50000000

1E8

1.5E8

2E8

2.5E8

3E8

3.5E8

MSD1 TIC, MS File (D:\DATA\18-05-15\022-2401.D) API-ES, Pos, Scan, Frag: 70

2.00

9

2.14

5

2.26

0

2.41

8

2.51

7

2.63

4

2.79

2

3.20

8

3.36

2

3.50

8

3.60

4

3.85

1

4.03

4

4.23

7

4.56

8

4.84

8

4.96

7

5.15

2 5.52

1

5.67

6 5.92

2

Peptide region

Peptide-nanobody conjugate region

Peptide-nanobody conjugate region

Peptide

S38

= 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.

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.

conjugate

Solvent Peak

S39

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

S40

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

S41

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.

S42

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.

Supporting Information for Peptide-Protein Conjugation ... · S1 Supporting Information for...

Documents

Transcript of Supporting Information for Peptide-Protein Conjugation ... · S1 Supporting Information for...