Supporting Information - Royal Society of Chemistry · Supporting Information ... General Experimental ... (200 mg, 0.917 mmol in 16.0 ml of CH 3 Cl) was added dropwise over three

0

Supporting Information

Photochemically re-bridging disulfide bonds and the discovery of

a maleimide mediated photodecarboxylation of C-terminal

cysteines

Daniel A Richards,a Sally A. Fletcher,a Muriel Nobles,b Lauren Tedaldi,a Hanno Kossen,a

Vijay Chudasama, a Andrew Tinker,b and James R. Bakera a Department of Chemistry, University College London, 20 Gordon St, London,

b

Barts and The London, Queen Mary's School of Medicine and Dentistry,

Charterhouse Square, London, UK.

Electronic Supplementary Material (ESI) for Organic & Biomolecular Chemistry.This journal is © The Royal Society of Chemistry 2015

1

Supporting Info General Experimental ............................................................................................................................. 2

Synthesis ............................................................................................................................................. 2

Buffers ................................................................................................................................................. 2

HPLC methods ..................................................................................................................................... 2

MALDI-TOF methods ........................................................................................................................... 2

LCMS methods .................................................................................................................................... 2

SDS-PAGE methods ............................................................................................................................. 3

Supplementary Schemes......................................................................................................................... 3

Synthesis of bromomaleimide-biotin 9b (SI. Scheme 1) .................................................................... 3

Synthesis of cysteine-maleimide 13 (SI. Scheme 2) ........................................................................... 3

Small Molecule Synthesis ........................................................................................................................ 4

Tert-butyl (3-aminopropyl)carbamate ................................................................................................ 4

Tert-butyl (D-Biotin) propylcarbamate ............................................................................................... 5

Monobromomaleimide-biotin 9b ....................................................................................................... 7

Fmoc-L-cysteine .................................................................................................................................. 8

Cysteine-maleimide 13 ..................................................................................................................... 10

(9H-fluoren-9-yl)methyl vinylcarbamate 16 ..................................................................................... 11

Octreotide bioconjugation .................................................................................................................... 13

Preparation of bioconjugate 5 ......................................................................................................... 13

Irradiation of bioconjugate 5 (SI Fig. 1-3) ......................................................................................... 14

Reaction Of Conjugate 5 with EDT (SI Fig. 4) ................................................................................... 17

Treatment Of Conjugate 6 with EDT (SI Fig. 5) ................................................................................. 18

Patch Clamping data ............................................................................................................................. 19

Cell culture ........................................................................................................................................ 19

Electrophysiology (SI Fig. 6) .............................................................................................................. 19

Herceptin bioconjugation ..................................................................................................................... 20

Preparation of Herceptin Fab 8 (SI Fig. 7) ........................................................................................ 20

Preparation of bioconjugate 10a (SI Fig. 8) ...................................................................................... 21

Irradiation of bioconjugate 10a (SI Fig. 9) ........................................................................................ 23

Preparation of bioconjuate 10b (SI Fig. 10) ...................................................................................... 24

Irradiation of bioconjugate 10b (SI Fig. 11) ...................................................................................... 25

Generation of bioconjuate 10c (SI Fig. 12) ....................................................................................... 27

Irradiation of bioconjugate 10c (SI Fig. 13) ....................................................................................... 28

Thiol stability of bioconjugate 10a (SI Fig. 14) .................................................................................. 30

ELISA .................................................................................................................................................. 30

References ............................................................................................................................................ 30

2

General Experimental

All reagents and starting materials were obtained from chemical suppliers, unless specifically stated

otherwise, and were used as received. Octreotide was purchased from LKT laboratories. Reactions were

monitored by thin layer chromatography using pre-coated SIL G/UV 254 plates purchased from VWR.

Flash chromatography was carried out using Kiesegel 60 M 0.04/0.063 mm silica gel. Petrol refers to

petroleum ether (40 – 60 °C). NMR spectra were recorded using a Bruker AC300, AC500 or AC600

spectrometer (300 MHz, 500 MHz and 600 MHz respectively). Chemical shifts (δ) are given in ppm

units relative to the solvent reference and coupling constants (J) are measured in Hertz. Proton (1H)

NMR multiplicities are shown as s (singlet), d (doublet), t (triplet), q (quartet), m (multiplet), dd (double

doublet), dt (double triplet), etc. Infrared spectra were recorded on a Perkin Elmer Spectrum 100 FTIR

spectrometer (ATR mode). High and low resolution mass spectrometry of organic molecules was

performed using a VG70 SE (EI or CI) or an LCT Premier mass spectrometer (ES). Irradiation was

performed using a commercial 5W LED torch purchased from Advanced NDT Ltd with an irradiance

measurement of approximately 20 mWcm-3 at 14 cm. Protein and peptide samples were placed in a 1

cm2 cuvette with the LED positioned 14 cm above, and the whole apparatus was encased in reflective

foil. For irradiation of compound 13 the UV torch was placed adjacent to the round bottomed flask

inside a reflective chamber at a distance of 14 cm and the beam directed through the solution. When a

mercury lamp is specified a 125 W lamp from Photochemical Reactors Ltd was used in combination

with a pyrex immersion well.

Synthesis Monobromomaleimide 41 and OEG-maleimide 9a2 were synthesised according to previously published

methods.

Buffers Assay buffer: 50:50 MeCN:phosphate buffer (50 mM NaH2PO4, pH = 6.2)

Digest buffer: 50 mM phosphate, 1 mM EDTA, 150 mM NaCl, pH 6.8

Borate buffered saline (BBS): 5 mM sodium borate, 25 mM NaCl, 0.5 mM EDTA, pH 8.0

HPLC methods

Octreotide HPLC was performed on a Kinetex 5u XN-C18 100A (260 x 4.60 mm) column at 40 C

using a Shimadzu LC-10AD liquid chromatograph equipped with a Shimadzu DGU-14A diode array.

Solvent A is H2O (0.1% formic acid), solvent B is MeCN (0.1% formic acid). Injection volume of 20

µl at 10 µM. Mobile phase: 80:20 A:B; gradient to 60:40 A:B over 24.5 minutes: gradient to 5:95 A:B

up to 26 minutes, followed by 80:20 A:B to completion of 30 minutes. For semi-preparative purification

of 5 an injection volume of 100 µl at 100 µM was employed.

MALDI-TOF methods MALDI-TOF was performed on a Shimadzu Biotech Axima CFR in reflectron mode, with laser power

set at 75.

LCMS methods LC–MS was performed on a Waters Acquity uPLC connected to Waters Acquity Single Quad Detector

and a photodiode array scanning at 254 nm. For Her-2 work a Hypersil Gold C4 (50 x 2.1 mm) column

at 50 C was used for separation. For Octreotide work an Acquity UPLC BEH C18 (50 x 2.1 mm)

column at 50 C was employed; Solvent A is H2O (0.1% formic acid), solvent B is MeCN (0.1% formic

acid). Mobile phase: 95:5 A:B; gradient over 4 min to 5:95 A:B. MS mode ES+ ; scan range: m/z ¼

3

250–2,000; scan time: 0.25 s. A capillary voltage of 3.5 kV and a cone voltage of 50 V was employed.

Deconvolution was performed using the MaxEnt 1 algorithm pre-installed on MassLynx software.

SDS-PAGE methods Non-reducing 12% acrylamide gels were made using standard procedures. A 4% stacking gel was

utilised. Samples (22 µM) were mixed 1:1 with a 1x SDS-loading buffer (1 g SDS, 3 ml glycerol, 6

ml 0.5 M Tris buffer pH = 6.8, 2 mg R-250 dye was diluted 1 in 6 with dd H2O), heated for 2 minutes

at 75 °C and loaded onto the gel with a total volume of 4 µl. Samples were run at constant current (30

mA) for 40 minutes in 1 x SDS running buffer and stained with Coomasie. Gel-Pro analyser version

3.1.00.00 from Media Cybernetics was used for densitometry analysis.

Supplementary Schemes

Synthesis of bromomaleimide-biotin 9b

Supplementary Information Scheme 1

The synthesis of monobromomaleimide-biotin 9b

Synthesis of cysteine-maleimide 13

Supplementary Information Scheme 2

The synthesis of cysteine-maleimide 13

4

Small Molecule Synthesis

Tert-butyl (3-aminopropyl)carbamate3

Di-tert-butyl dicarbonate (200 mg, 0.917 mmol in 16.0 ml of CH3Cl) was added dropwise over three

hours to a solution of 1,3-diaminopropane (765 µl, 9.17 mmol) in CHCl3 (50 ml) at 0 °C. After this time

the solution was returned to room temperature and allowed to stir for fifteen hours. The solution was

then diluted with CHCl3 (100 ml) and washed with saturated K2CO3 (2 x 50 ml) followed by H2O (2 x

50 ml). The organic layer was separated, dried over MgSO4, and concentrated in vacuo to produce the

title compound as an orange oil (155 mg, 0.890) in a 97% yield. Product was taken on without further

purification: 1H NMR (600 MHz, CDCl3) δ = 5.10 (1H, br. s), 3.10 (2H, q, J = 6.6), 2.68 (2H, t, J = 6.6),

1.54 (2H, q, J = 6.6), 1.45 (2H, br. s), 1.36 (9H, s); 13C NMR (125 MHz, CDCl3) δ = 156.3 (C), 79.5

(C), 39.7 (CH2) 38.4 (CH2), 33.3 (CH2) 28.5 (CH3); IR (film, cm-1) 3356, 2971, 2940, 2871, 1737, 1680,

1583; MS (EI) m/z [M, relative intensity] 175.2 [M+H, 100]. Spectra matched previously reported data.3

5

Tert-butyl (D-Biotin) propylcarbamate

Biotin (295 mg, 1.21 mmol), HBTU (398 mg, 1.05 mmol) and DIPEA (221 µl, 1.27 mmol) were

dissolved in DMF (5 ml) and stirred for twenty minutes at room temperature. This mixture was then

added dropwise over twenty minutes to a solution of tert-butyl (3-aminopropyl)carbamate (140 mg,

0.804 mmol) in DMF (3 ml) and stirred for two hours at room temperature. Toluene (7 ml) was

subsequently added and all solvent was removed in vacuo. The crude residue was purified by flash

chromatography (2% to 10% MeOH in CH2Cl2 followed by an additional separation in 70% EtOAc in

petrol), to yield the title compound as a crude yellow oil (225 mg, 0.512 mmol) in a 70% yield: 1H

NMR (600 MHz, CDCl3). δ = 6.91 (1H, s), 6.73 (1H, s), 5.80 (1H, s), 5.14 (1H, s), 4.50 (1H, m) ,

4.30 (1H, m), 3.10-3.15 (m, 3H), 2.89 (1H, dd, J = 12.6, J = 8.5), 2.72 (1H, dd, J = 12.6, J = 8.5),

2.32 (t, 2H, J = 7.5), 2.19 (2H, t, J = 7.8), 1.74-1.59 (6H, m), 1.37-1.47 (m, 11H); 13C NMR (125

MHz, CDCl3) δ = 173.9 (C) 164.3(C), 156.7 (C), 79.4 (C) 62.0 (CH), 60.3 (CH), 55.9 (CH), 40.7

(CH2), 37.4 (CH2) 36.6 (CH2), 36.1 (CH2), 30.0 (CH2), 29.8 (CH2), 28.5 (3 x CH3), 25.9 (CH2), 24.9

(CH2): IR (film, cm-1) 3450, 2960, 1744, 1608, 1550; MS (CI) m/z [M, relative intensity] 401.2

[M+H, 6] 301.2 [M-Boc+H, 100]; Exact mass calcd for [C18H33N4O4S] 401.22170, found 401.22256. Spectra matched previously reported data.4

6

7

Monobromomaleimide-biotin 9b

Tert-butyl (D-Biotin) propylcarbamate (100 mg, 0.250 mmol) was dissolved in trifluoroacetic acid (5

ml) and stirred for fifteen hours. After this time toluene (50 ml) was added and the solvent removed in

vacuo. The crude material was dissolved in acetic acid (10 ml) and bromomaleic anhydride (45.0 mg,

0.250 mmol) was added. The reaction was heated under reflux for three hours, then allowed to return

to room temperature. All solvents were removed in vacuo and the crude residue purified by flash

chromatography (2% to 10% MeOH in CH2Cl2) to afford title compound 9b as a white powder (75.0

mg, 0.164 mmol) in a 66% yield: 1H NMR (400 MHz, MeOD) δ = 7.96 (1H, br. s), 7.13 (1H, s), 4.48

- 4.53 (1H, m), 4.34 (1H, dd, J = 7.5, J = 4.4), 3.59 (2H, t, J = 7.2), 3.23 - 3.27 (1H, m), 3.20 (2H, q, J

= 9.2), 2.95 (1H, dd, J = 12.8, J = 5.0), 2.72 (1H, d, J = 12.8), 2.22 (2H, t, J = 11.6), 1.85 - 1.57 (6H,

m), 1.52 - 1.44 (2H, m); 13C NMR (151MHz, DMSO-d6) δ = 172.0 (C), 169.0 (C), 165.6 (C), 162.7

(C), 132.6 (CH), 130.4 (C), 61.0 (CH), 59.2 (CH), 55.4 (CH), 41.4 (CH2), 36.3 (CH2), 36.1 (CH2),

35.2 (CH2), 28.2 (CH2), 28.1 (2 x CH2) 25.3 (CH2); IR (solid, cm-1) 3287, 2932, 2861, 1779, 1700,

1645, 1590, 1548 cm-1; MS (ES+) m/z [M, relative intensity] 483.1 [M81Br + Na, 100], 481.1 [M79Br

+ Na, 98]; Exact mass calcd for [C17H2379BrN4O4SNa] 481.0521, found 481.0533.

8

Fmoc-L-cysteine

Triisopropylsilane (2.00 ml, 9.76 mmol) and trifluoroacetic acid (8.00 ml, 104 mmol) were added

sequentially to a suspension of Fmoc-L-Cys-(Trt)-OH (1.00 g, 1.71 mmol) in CH2Cl2 (68 ml). The

solution was stirred for ten minutes at room temperature, before all solvent was removed in vacuo.

Excess trifluoroacetic acid was azeotropically removed by co-evaporation with CH2Cl2. The resulting

white solid was placed onto filter paper and washed with hexane (8 x 30 ml), before being dried under

vacuum to produce the title compound as a white solid (563 mg, 1.64 mmol) in a 96% yield: 1H NMR

(600 MHz, DMSO-d6) δ = 7.90 (2H, d, J = 7.4), 7.74 (2H, d, J = 7.4), 7.70 (1H, d, J = 8.0), 7.42 (2H,

t, J = 7.4), 7.33 (2H, t, J = 7.4), 4.31 (2H, d, J = 6.5), 4.24 (1H, t, J = 6.5), 4.12 (1H, dt, J = 8.0, J =

4.3), 2.93 - 2.85 (1H, m), 2.77 - 2.70 (1H, m), 1.23 (1H, s); 13C NMR (151 MHz, DMSO-d6) δ = 171.9

(C), 156.1 (C), 143.8 (2 x C), 140.8 (2 x C), 127.7 (2 x CH), 127.1 (2 x CH), 125.3 (2 x CH), 120.2 (2

x CH), 65.7 (CH2), 56.6 (CH), 46.6 (CH), 25.5 (CH2); IR (solid, cm-1) 3312, 1696, 1531, 1447, 1426;

MS (ES+) m/z [M, relative intensity] 366.1 [M+Na] 100], 179.1 [C14H11,7]; Exact Mass Calcd

for[C18H17NO4SNa] 366.0776, found 366.0748. Spectra matched previously reported data.5

9

10

Cysteine-maleimide 13

Fmoc-L-cysteine (115 mg, 0.335 mmol) and sodium acetate (60.0 mg, 0.737 mmol) were dissolved in

MeOH (7 ml) and stirred for five minutes under argon. Bromomaleimide 4 (65.0 mg, 0.369 mmol) was

added and the solution stirred for twenty minutes at room temperature. All solvent was removed in

vacuo, the crude material suspended in EtOAc (25 ml) and washed with 10% citric acid (10 ml). The

organic layer was dried over MgSO4 and all solvent removed in vacuo. Purification by flash

chromatography (2%-6% MeOH, 1% AcOH, in CH2Cl2) afforded the title compound 13 as a light

orange powder (129 mg, 0.294 mmol) in an 88% yield: 1H NMR (600 MHz, DMSO-d6) δ = 11.03 (1H,

s), 7.90 (1H, broad s), 7.89 (2H, d, J = 7.5), 7.70 (2H, dd, J = 7.5, J = 3.1), 7.42 (2H, t, J = 7.5), 7.32

(2H, t, J = 7.5), 6.49 (1H, s), 4.35 - 4.25 (3H, m), 4.23 (1H, t, J = 7.2), 3.43 (1H, dd, J = 13.5, J = 4.3),

3.25 (1H, dd, J = 13.5, J = 4.3); 13C NMR (151 MHz, DMSO-d6) δ = 171.4 (C), 170.9 (C), 169.3 (C),

156.0 (C-), 150.0 (C), 143.8 (2 x C), 140.7 (2 x C), 127.7 (2 x CH), 127.1 (2 x CH), 125.2 (2 x CH),

120.2 (2 x CH), 119.5 (CH), 65.9 (CH2), 52.5 (CH), 46.6 (CH), 32.7 (CH2); IR (solid, cm-1) 3316, 1735,

1705, 1685, 1529, 1448; MS (ES+) m/z [M, relative intensity] 461.1 [M+Na, 100], 366.1[M-

C4H2O2N+Na, 5], 179.1[C14H11,1]; Exact Mass Calcd for[C18H17NO4SNa] 461.0783, found 461.0760.

11

(9H-fluoren-9-yl)methyl vinylcarbamate 16

Cysteine-maleimide 12 (40.0 mg, 0.0912 mmol) and sodium acetate (8.22 mg, 0.100 mmol) were

dissolved in MeOH (240 ml) and the entire solution degassed with argon for thirty minutes. The

solution was irradiated for one hour using a 5W LED* at 365 nm. All solvent was removed in vacuo

at room temperature and the resulting crude material washed with Et2O (3 x 20 ml). The washings

were collected and solvent removed in vacuo to yied title enamide 16 as a white solid (22 mg, 0.0867

mmol) in a 95% yield. 5 mg (0.0188 mmol) of the product was dissolved in MeOD containing sodium

acetate** (5 mg, 0.0610 mmol) for stabilisation: 1H NMR (600MHz, MeOD) δ = 7.80 (2H, d, J = 7.5),

7.66 (2H, d, J = 7.5), 7.39 (2H, t, J = 7.5), 7.32 (2H, t, J = 7.5), 6.63 (1H, dd, J = 15.8, J = 9.0), 4.55

(1H, d, J = 15.8), 4.41 (2H, d, J = 6.8), 4.25 - 4.21 (2H, m); 13C NMR (151 MHz, MeOD) δ = 156.15

(C), 145.14 (2 x C), 142.61 (2 x C), 131.44 (CH), 128.83 (2 x CH), 128.17 (2 x CH), 126.11 (2 x CH),

120.95 (2 x CH) 93.84 (CH2) 67.95 (CH2), 48.28 (CH); IR (solid, cm-1) 3313, 2922, 2852, 1699, 1649;

MS (EI) m/z [M, relative intensity] 265.1 [M+H], 3], 178.1 [C14H11, 100]; Exact Mass Calcd

for[C17H15NO2Na] 288.1000 , found 288.0989.

*Irradiation with a medium pressure mercury lamp decreases reaction time to twenty minutes*.

**Under non-basic conditions degradation to the amide was observed which complicated NMR

spectra**

12

13

Octreotide bioconjugation

Preparation of bioconjugate 5

Tris(2-carboxyethyl)phosphine (TCEP) (30 mM, 1.5 equiv, 15 µl in assay buffer) was added to a

solution of Octreotide 3 (300 µM, in 1.00 ml assay buffer) and left to react at 37 °C for one hour. This

solution was added dropwise to a solution of bromomaleimide 4 (300 mM, 100 equiv, 100 µl in assay

buffer) over 5 minutes. After thirty minutes at room temperature the solution was placed into dialysis

cups (2 KDa MWCO) and dialysed with deionised water overnight. All solvent was subsequently

removed by lyophilisation, the peptide dissolved in deionised water and purified by HPLC. HPLC yield

of 70%. Analysis by MALDI-TOF confirmed successful conjugatation to produce 5. Expected mass

1211, observed mass 1211 (relative to pure Octreotide 1018) (SI. Fig. 2f).

14

Irradiation of bioconjugate 5

Purified conjugate 5 (30 µM, 60 µl in assay buffer) was placed in a 1 cm3 cuvette and irradiated for two

minutes using a 365 nm LED. The subsequent solution was analysed by HPLC (SI Fig. 1c). For

reference pure Octreotide 3 (15.24 minutes) and purified conjugate 5 (17.74 minutes) were also

analysed by HPLC (SI Fig. 1a, SI Fig. 1b). Extended irradiation times effected no change in the HPLC

trace, indicating the reaction had gone to completion: Peaks labelled A (16.14 minutes), B (16.49

minutes), C (17.26 minutes) and D (17.64 minutes) were analysed by MALDI-TOF (SI Fig. 2a-d).

Additionally, the eluent was collected between 15.0 and 20.0 minutes and analysed by MALDI-TOF

(SI Fig. 2e): Expected mass 1211, observed mass 1211.

Supplementary Information Fig. 1

(a) HPLC trace for pure Octreotide 3. (b) HPLC trace for purified conjugate 5. (c) HPLC trace for conjugate 5

after irradiation to produce 6.

a

b

c

15


MALDI-TOF spectrum for peak A (a), peak B (b), peak C (c), peak D (d), and the combined eluent from 15.0

to 20.0 minutes (e). (f) MALDI-TOF mass spectrum of pure conjugate 5.

b

a

c

d

e

Peak A

Peak B

Peak C

Peak D

15.00 - 20.0 minutes

Expected mass: 1211

Observed mass: 1211

Expected mass: 1211

Observed mass: 1211

Expected mass: 1211

Observed mass: 1211

Expected mass: 1211

Observed mass: 1211

Expected mass: 1211

Observed mass: 1211

f

Expected mass: 1211

Observed mass: 1211

+Na

+Na

+Na

+Na

+Na

+K

+K

+K

+K

+K

16


1H-NMR spectra for (A) Pure Octreotide 3. (B) Crude 1H-NMR of modified Octreotide 5 (zoomed in on

maleimide alkene protons). (C) Crude NMR of rebridged Octreotide 6 (zoomed in to show the

disappearance of alkene protons and appearance of cyclobutane protons). Spectra B and C contain

impurities at 2.20, 2.51, 3.65 and 3.75 ppm which are not related to the peptide and appear before

irradiation.

(A) Octreotide 3

(B) Conjugate 5

(C) Conjugate 6

17

Reaction Of Conjugate 5 with EDT

To a solution of conjugate 5 (100 µM, 30 µl in assay buffer) ethanedithiol (100 mM, 10 equiv, 0.3 µl)

was added. After thirty minutes LCMS confirmed successful reaction (SI Fig 3b): MS (ES+) m/z [M,

relative intensity] 1306 [M+H, 100], 1158 [M-Phe+H, 90]. Conjugate 5 was analysed under the same

conditions for comparison (SI Fig. 3a.): MS (ES+) m/z [M, relative intensity] 1211.5 [M+H, 100]

1064.3 [M-Phe+H, 30].


(a) LCMS of pure conjugate 5. (b) LCMS of conjugate 5 + ethanedithiol to generate conjugate 7.

a

b

Expected mass: 1211

Observed mass: 1211

Expected mass: 1306

Observed mass: 1306

18

Treatment Of Conjugate 6 with EDT

Purified conjugate 5 (100 µM, 30 µl in assay buffer) was placed in a 1 cm3 cuvette and irradiated for

two minutes using a 365 nm LED to produce conjugate 6. Ethanedithiol (100 mM, 10 equiv, 0.3 µl)

was added. After thirty minutes LCMS showed no change, indicating lack of reaction (SI Fig. 4b): MS

(ES+) m/z [M, relative intensity] 1211.6 [M+H, 100], 1064.5 [M-Phe+H, 90]. An LCMS of conjugate

6 was taken under the same conditions for comparison (SI Fig. 4a): MS (ES+) m/z [M, relative intensity]

1211.6 [M+H, 100] 1064.6 [M-Phe+H, 30].


(a) LCMS of pure conjugate 6. (b) LCMS of conjugate 6 + ethanedithiol.

b

a

Expected mass: 1211

Observed mass: 1211

Expected mass: 1211

Observed mass: 1211

19

Patch Clamping data

Cell culture Cell culture methods and the generation of stable cell lines were carried out as previously described.6

HEK293 cells (human embryonic kidney cell line) stably expressing Kir3.1 and Kir3.2A channels were

maintained in minimum essential medium supplemented with 10% foetal calf serum and 727 μg of

G418 (Invitrogen), at 37 °C in humidified atmosphere (95% O2, 5% CO2). Cells were transiently

transfected with SSTR2 DNA (Missouri S&T cDNA Resource Center) along with pEGFP-N1

(Clontech) for visualization of transfected cells using epifluorescence. Transfections were performed

with 5 μl of Fugene HD (Roche) and 800 ng SSTR2 DNA and 40 ng EGFP DNA .

Electrophysiology Whole cell patch-clamp current recordings were performed with an Axopatch

200B amplifier (Axon Instruments) using fire polished pipettes with a resistance of 4-5 M pulled

from filamented borosilicated glass capillaries (Harvard Apparatus, 1.5 mm OD x 1.17 mm ID). Data

was acquired and analysed via a Digidata 1322A interface (Axon Instruments) and pCLAMP software

(version 10.2, Axon Instruments). Cells were clamped at -50 mV. The extracellular solution was:

NaCl (135 mM), KCl (5.4 mM), CaCl2 (2 mM), MgCl2 (1 mM), NaH2PO4 (0.33 mM), H-HEPES (5

mM), Glucose (10 mM), buffered to pH 7.4 with NaOH. The intracellular solution was: K gluconate

(110 mM), KCl (20 mM), NaCl (10 mM), MgCl2 (1 mM), MgATP (2 mM), EGTA (2 mM), Na2GTP

(0.3 mM), buffered to pH 7.2 with KOH. Octreotide 3 and conjugates 5 and 6 were administrated by a

gravity driven system, at a concentration of 0.01, 0.1 or 1 μM until the current reaches a maximum

value. All experiments were done at room temperature. All peptides for testing were kept at -80 C

avoiding repetitive thawing/freezing. Currents were elicited by holding cells at -50 mV and stepping

to potentials between -120 and +60 mV in 10 mV increments for 360 ms. Current densities were

measured at -120 mV and all data are presented as means ± S.E.M. Data were analysed for statistical

significance using one way ANOVA with a post-hoc test.


(a) Current activation from native Octreotide 3 (0.01 µM). (b) Current activation from acyclic conjugate 5 (1

µM) (c) Current activation from bridged conjugate 6 (0.1 µM). (d) Current without agonist. The maximum

current values were: native Octreotide 3 (10 nM, -73.7 ± 5.9 pA/pF, n=10), conjugate 5 (1 µM, -2.1 ± 0.9

pA/pF, n=9, p<0.0001 compared to Octeotride 3) and conjugate 6 (100 nM, -16.1 ±4.7 pA/pF, n=10, p<0.0001

compared to Octeotride 3).

a b c

d

100 s

200 p

A

0.01 M native octeotride 3

100 s

200 p

A

1 M open chain conjugate 7

0.1 M photochemically bridged

compound 8

100 s

200 p

A

control

100 s

200 p

A

0.01 Octreotide 3 1 conjugate 5 0.1 conjugate 6

Control

20

Herceptin bioconjugation

Preparation of Herceptin Fab 87

Immobilized pepsin (Thermo scientific) (0.15 ml) was washed with acetate buffer (4 x 0.3 ml, 20 mM

sodium acetate trihydrate, pH 3.1). Herceptin (0.5 ml, 6.41 mg.ml-1 in digest buffer) was added and the

mixture incubated for five hours under constant agitation (1100 rpm) at 37 °C. The digest solution was

separated from the resin beads, which were washed with digest buffer (3 x 0.4 ml). The washes were

combined with the digest solution and the volume adjusted to 0.5 ml using spin filtration. Immobilised

papain (Thermo scientific) (0.5 ml, 0.25 mg.ml-1) was incubated in buffer (10 mM DTT in digest buffer)

at 37 °C with constant agitation (1100 rpm) for one hour. The papain resin was subsequently filtered

and washed with digest buffer (4 x 0.4 ml) and the pepsin digest solution prepared earlier was added.

The mixture was incubated at 37 °C with constant agitation (1100 rpm) for sixteen hours. The resin was

separated from the digest via spin filtration, washed with borate buffer (3 x 0.4 ml, 25 mM sodium

borate, 25 mM NaCl, 0.5 mM EDTA). The digest and the washes were combined and buffer swapped

for BBS using spin filtration columns (10000 MWCO), and the volume was adjusted to 0.5 ml. Yield

of Her-Fab 8 was determined by UV/Vis spectroscopy (280 = 68590 M-1. cm-1, 2.55 mg.ml-1, 61%).

Her-Fab 8 was confirmed using SDS-PAGE and LCMS (SI Fig. 6a-c). Expected mass 47742

a

21


(a) Ion trace for Her-Fab 8. (b) Deconvoluted mass spectrum for Her-Fab 8. (c) SDS-PAGE characterisation for

Her-Fab 8 (lane 1).

Preparation of bioconjugate 10a

Fab-Her 8 (22 μM, 1 mg⋅ml-1, 200 μl in BBS) was reduced with TCEP (22 mM in BBS, 1 μl, 5 equiv.)

at 37 °C for ninety minutes. A sample (10 μl) was taken and reacted with maleimide (22 mM in MeCN,

0.2 μl, 20 equiv.) to provide a reduced control. Bromomaleimide 4 (22 mM in MeCN, 19 μl, 100 equiv.)

was added to the remainder and the reaction was left at room temperature for thirty minutes. The

solution was subsequently purified by desalting column (G-25, GE Healthcare), buffer swapped into

PBS pH = 6 using spin filtration (5 KDa MWCO), and the concentration adjusted to 22 μM (1 mg.ml-

1) (280 = 68590 M-1.cm-1). Analysis by SDS-PAGE and LCMS confirmed conjugate 10a (SI. Fig. 7a-

b

c

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1

22

c). Expected mass of light chain 23545, observed mass 23545. Expected mass of heavy chain 24307,

observed mass 24306 (expected masses relative to reduced Her2 Fab 8).


(a) Ion trace for conjugate 10a. (b) Deconvoluted mass spectrum for conjugate 10a. (c) SDS-PAGE

characterisation for conjugate 10a (lane 5).

a

b

c

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40

Expected mass: 23545

Observed mass: 23545



23

Irradiation of bioconjugate 10a

Bioconjugate 10a (22 μM, 1 mg⋅ml-1, 120 μl in PBS pH = 6) was added to a cuvette and irradiated using

a 365 nm LED for two minutes. Analysis by SDS-PAGE and LCMS confirmed successful

photocycloaddition to produce bioconjugate 11a (SI Fig. 8a-c). Expected mass 47851, observed mass

47851 (Expected mass relative to unbridged conjugate 10a). Side product 12a was also observed;

Expected mass of light chain 23372, observed 23370. Densitometry gave a bridging yield to 11a of

85%.

a

b







24


(a) Ion trace for conjugate 11a + 12a. (b) Deconvoluted mass spectrum for conjugate 11a + 12a. (c) SDS-PAGE

characterisation for conjugate 11a + 12a (lane 6).

Preparation of bioconjugate 10b

Procedure was followed as with bioconjugate 10a (Page 20), though DMF was used in place of

MeCN to dissolve maleimide 9a. Analysis by SDS-PAGE and LCMS confirmed conjugate 10b (SI

Fig. 9a-c). Expected mass of light chain 23692, observed mass 23690. Expected mass of heavy chain

24454, observed mass 24452 (expected masses relative to reduced Her-Fab).

c

a

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1 2 3 4 5 6

25


(a) Ion trace for conjugate 10b. (b) Deconvoluted mass spectrum for conjugate 10b. (c) SDS-PAGE

characterisation for conjugate 10b (lane 5).

Irradiation of bioconjugate 10b

Irradiation was performed as in the irradiation of bioconjugate 10a (Page 22). Reaction was analysed

by SDS-PAGE and LCMS (SI Fig. 10a-c). Expected mass of 11b 48146, observed mass 48155

b

c

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1 2 3 4 5





26

(Expected mass relative to unbridged conjugate 10b). Side product 12b was also observed; Expected

mass of light chain 23372, observed 23367. Densitometry gave a bridging yield to 11b of 80%.

a

b

c

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1 2 3 4 5 6





27


(a) Ion trace for conjugate 11b+12b. (b) Deconvoluted mass spectrum for conjugate 11b+12b. (c) SDS-PAGE

characterisation for conjugate 11b+12b (lane 6).

Generation of bioconjuate 10c

Procedure was followed as with bioconjugate 10a (Page 20), though DMF was used in place of

MeCN to dissolve maleimide 9b. Analysis by SDS-PAGE and LCMS confirmed conjugate 10c (SI

Fig. 11a-c). Expected mass of light chain 23829, observed mass 23828. Expected mass of heavy chain

24591, observed mass 24592 (expected masses relative to reduced Her-Fab).

a

b


Observed mass: 23828 Expected mass: 24591


28


(a) Ion trace for conjugate 10c. (b) Deconvoluted mass spectrum for conjugate 10c. (c) SDS-PAGE

characterisation for conjugate 10c (lane 4).

Irradiation of bioconjugate 10c

Irradiation was performed as in the irradiation of bioconjugate 10a (Page 22). Reaction was analysed

by SDS-PAGE and LCMS (SI Fig. 12a-c). Expected mass of 11c 48420, observed mass 48428

(Expected mass relative to unbridged conjugate 10c). Side product 12c was also observed; Expected

mass of light chain 23372, observed 23369. Densitometry gave a bridging yield to 11c of 80%.

c

a

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25

1 2 3 4

29


(a) Ion trace for conjugate 11c+12c. (b) Deconvoluted mass spectrum for conjugate 11c+12c. (c) SDS-PAGE

characterisation for conjugate 11c+12c (lane 5).

b

c

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40

1 2 3 4 5


Observed mass: 48428 Expected mass: 23372


30

Thiol stability of bioconjugate 11a.

Bioconjugate 11a (SI Fig. 14., lane 6) was separated into 3 x 30 μl aliquots (22 μM, 1 mg⋅ml-1, PBS pH

= 7.4) and each aliquot reacted with a specific thiol (dithiothreitol, glutathione, β-mercaptoethanol in

PBS pH = 6.4, 220 mM, 1.5 μl, 500 equiv, total thiol concentration 11 mM). The solutions were

incubated at 37 °C for 4 hours. Analysis by SDS-PAGE demonstrated the stability of the bridge (SI Fig.

13, lanes 7-9).


(a) SDS-PAGE characterisation demonstrating thiol stability of conjugate 11a

ELISA7

A 96-well plate was coated overnight at 4 °C with HER2 (100 μl of a 0.25 μg⋅ml-1 solution in PBS). As

a negative control one row was coated with only PBS. The solutions were removed and each well

washed (2 x PBS). The wells were subsequently coated with a 1% BSA solution in PBS for one hour at

room temperature. After this the wells were emptied and washed (3 x PBS). Solutions of Fab-Her2 8,

bioconjugate 10a and bioconjugate 11a in PBS pH = 7.4 were prepared in the following dilutions: 22

nM, 7.33 nM, 2.44 nM, 0.814 nM, 0.272 nM and 0.0905 nM. The dilutions were placed into the wells,

each dilution repeated 3 times, and incubated for two hours at room temperature. As negative controls

a PBS only and Fab-Her 8 at 22 nM in the absence of HER2 were also subjected to the same protocol.

The solutions were removed and the wells washed (2 x 0.1% Tween 20 in PBS, 3 x PBS). Detection

antibody (100 μl of anti-human IgG, Fab-specific-HRP solution, 4 μl of a 1:5000 solution diluted further

in 20 ml of PBS) was added and left for one hour at room temperature. The solutions were removed and

the wells washed (2 x 0.1% Tween 20 in PBS, 3 x PBS). Finally, an OPD solution (100 μl of 0.5 mg⋅ml-

1 OPD in phosphate-citrate buffer with sodium perborate) was added to each well. After 2 minutes the

reaction was stopped through addition of 4 M HCl (50 μl). Absorbance was measured at 490 nm and

corrected by subtracting the average of negative controls (SI Fig. 14)


ELISA against HER2 for Native Her Fab 8, open conjugate 10a and bridged conjugate 11a.

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31

References

1 C. Marculescu, H. Kossen, R. E. Morgan, P. Mayer, S. A. Fletcher, B. Tolner, K. A. Chester, L. H. Jones and J. R. Baker, Chem. Commun. (Camb)., 2014, 50, 7139–42.

2 L. Castañeda, Z. V. F. Wright, C. Marculescu, T. M. Tran, V. Chudasama, A. Maruani, E. A. Hull, J. P. M. Nunes, R. J. Fitzmaurice, M. E. B. Smith, L. H. Jones, S. Caddick and J. R. Baker, Tetrahedron Lett., 2013, 54, 3493–3495.

3 D. Muller, I. Zeltser, G. Bitan and C. Gilon, J. Org. Chem., 1997, 62, 411–416.

4 U. Boudi, J. Fiet and H. Galons, Synth. Commun., 1999, 29, 3137–3141.

5 I. Ramos-Tomillero, H. Rodríguez and F. Albericio, Org. Lett., 2015, 17, 1680–1683.

6 J. L. Leaney, G. Milligan and A. Tinker, J. Biol. Chem., 2000, 275, 921–929.

7 A. Maruani, M. E. B. Smith, E. Miranda, K. A. Chester, V. Chudasama and S. Caddick, Nat. Commun., 2015, 6, 6645.

Documents

Supporting Information - Royal Society of Chemistry · Supporting Information ... General Experimental ... (200 mg, 0.917 mmol in 16.0 ml of CH 3 Cl) was added dropwise over three