supporting information for peptide-protein conjugation ... · s1 supporting information for...
TRANSCRIPT
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
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
pre-reduced with10 eq. DTT
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
SH10 mM Tris-HCl, pH 7.6
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-
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
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-
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.
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.
0.2 eq. Pd(PPh3)460 eq. PhSiH3
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
SH10 mM Tris-HCl, pH 7.4
37 °C, o/n
ABA-CGSS-K
S SO
O
pre-reduced with10 eq. DTT
S SCGSSKABA
CONH2
CONH2
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
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
SH10 mM Tris-HCl, pH 7.4
r.t., DMSO, o.n.
ABA-GVSSCGSS-K(FAM)-CONH2
pre-reduced with10 eq. DTT
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
SH10 mM Tris-HCl, pH 7.4
r.t., 10% DMSO, o.n.
pre-reduced with10 eq. DTT
S SCGSSGSS-cKFRRRRE
35 eq.C(Scm)GSSGSS-cKFRRRRE
Figure S36: Synthesis scheme for the conjugation of MB23 with peptide C(Scm)GSSGSS-
cKFRRRRE
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-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
Chemical Formula: C25H43N11O13S2Exact Mass: 769.25
Molecular Weight: 769.80
O
O
DMSO
S30
7.2 Conjugation of MB23 to C(Scm)GSRGDS-CONH2
SH10 mM Tris-HCl, pH 7.4
r.t., o.n.
NH2-CGSRGDS
pre-reduced with10 eq. DTT
S SCGSRGDS
20 eq.CONH2
CONH2
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.
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
0.2 eq. Pd(PPh3)60 eq. PhSiH3
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
Chemical Formula: C22H35N9O10S2Exact Mass: 649.19
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
SH10 mM Tris-HCl, pH 7.4
r.t., o.n.
pre-reduced with10 eq. DTT
S ScCRGDE
5 eq. cC(Scm)RDGE
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.
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
SH10 mM Tris-HCl, pH 7.4
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
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= 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
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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
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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
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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.
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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.