A Novel Heterotrifunctional Peptide-Based Cross-Linking Reagent for Facile Access to Bioconjugates

Home , Sodium salts

A novel heterotrifunctional peptide-based cross-linking reagent for facile access to bioconjugates. Applications to peptide fluorescent labelling and immobilisation

Guillaume Clavé,a, b Hervé Boutal,c Antoine Hoang,d François Perraut,e Hervé Volland,c Pierre-Yves Renard*a, b, f and Anthony Romieu*a, b

a Equipe de Chimie Bio-Organique, COBRA - CNRS UMR 6014, rue Lucien Tesnière, 76131 Mont-Saint-Aignan, France. E-mail: [email protected] or [email protected]; Fax: +33 2-35-52-29-59 b Université de Rouen, Place Emile Blondel, 76821 Mont-Saint-Aignan, France c CEA, iBiTecS, Laboratoire d'Etudes et de Recherches en Immuno-analyse, Gif sur Yvette, F- 91191, France d Laboratoire de Fonctionnalisation Chimique de Microcomposants, Commissariat à l'Energie Atomique, 17 rue des Martyrs, F-38054 Grenoble, France e Laboratoire d'Imagerie et des Systèmes d'Acquisition, Commissariat à l'Energie Atomique, 17 rue des Martyrs, F-38054 Grenoble, France f Institut Universitaire de France

1 Supporting Information

Experimental : Detailed synthetic procedures for compounds 4, 7, 8, 9, 10, 11, 12, 13 and A. 3 1 H NMR spectrum of A recorded in CDCl3...... 10 13 C NMR spectrum of A recorded in CDCl3...... 11 ESI-MS spectrum of A recorded in the negative mode...... 11 ESI-MS spectrum of A recorded in the positive mode...... 12 1 H NMR spectrum of B recorded in CDCl3...... 13 13 C NMR spectrum of B recorded in CDCl3...... 14 ESI-MS spectrum of B recorded in the positive mode...... 14 1 H NMR spectrum of C recorded in D2O...... 15 13 C NMR spectrum of C recorded in D2O...... 16 ESI-MS spectrum of C recorded in the positive mode...... 16 1 H NMR spectrum of 5 recorded in CDCl3...... 17 13 C NMR spectrum of 5 recorded in CDCl3...... 18 ESI-MS spectrum of 5 recorded in the positive mode...... 18 RP-HPLC elution profile of 5 (system A)...... 19 ESI-MS spectrum of 19 recorded in the negative mode.a ...... 20 RP-HPLC elution profile of 19 (system B).a...... 21 UV-visible absorption of 19 in deionised water at 25°C (concentration = 4.2 µM)...... 21 ESI-MS spectrum of fluorescent substance P-tripod 23 recorded in the positive mode.a...... 22

2 Experimental : Detailed synthetic procedures for compounds 4, 7, 8, 9, 10, 11, 12, 13 and A

2-(2-(2-Azidoethoxy)ethoxy)ethanol (7)1. 2-(2-(2-Chloroethoxy)ethoxy)ethanol 6 (1.29 mL, 8.9 mmol) was added to a suspension of NaN3 (0.70 g, 10.7 mmol) and NaI (0.14 g, 0.93 mmol) in dry EtOH. The resulting yellow mixture was refluxed for 5 days under an argon atmosphere. The reaction was checked for completion by TLC (CH2Cl2-MeOH, 9 : 1, v/v). The mixture was filtered over Celite® 545 to remove sodium salts and evaporated to dryness. The resulting oily residue was dissolved in CH2Cl2 (ca. 10 mL) and stored at 4 °C for 1 h. After filtration over a cotton bowl and concentration, 1.8 g (10.7 mmol) of compound 7 was obtained as colorless oil (quantitative yield). Rf (CH2Cl2-MeOH, 9 : 1, v/v) 0.69; IR (neat): -1 1 νmax 935, 1118, 1287, 1346, 1453, 2110, 2874, 2915, 3390 (broad) cm ; H NMR (300 MHz, CDCl3): δ 2.63 (t, J = 6.0 Hz, 1H, OH), 3.43 (t, J = 4.9 Hz, 2H), 3.61-3.77 (m, 10H).

(2-(2-Azidoethoxy)ethoxy)acetic acid (8)2. 2-(2-(2-Azidoethoxy)ethoxy)ethanol 7 (1.56 g, 8.9 mmol) was dissolved in acetone (90 mL) and the resulting solution was cooled to 4 °C. Freshly prepared 3 M Jones' reagent (8.9 mL) was added dropwise (a green precipitate immediately formed) and the resulting reaction mixture was stirred at room temperature for 1 h. The reaction was checked for completion by TLC (CH2Cl2-MeOH, 9 : 1, v/v) and quenched by addition of propan-2-ol (ca. 4 mL). After 15 min, further amount of acetone (100 mL) was added and the green precipitate of Cr(III) salts was removed by filtration over Celite® 545. The filtrate was evaporated to dryness. The resulting oily residue was immediately purified by chromatography on a silica gel column with a step gradient of MeOH (0-5%) in CH2Cl2 as the mobile phase, giving 1.53 g (8.1 mmol) of carboxylic acid 8 as a yellow oil (yield 91%). Rf 1 (CH2Cl2-MeOH, 9 : 1, v/v) 0.23; H NMR (300 MHz, CDCl3): δ 3.43 (t, J = 4.9 Hz, 2H), 13 3.66-3.80 (m, 6H), 4.19 (s, 2H); C NMR (75.5 MHz, CDCl3): δ 50.7, 68.6, 70.2, 70.6, 71.4, 174.2.

(2-(2-Aminoethoxy)ethoxy)acetic acid (9)3. (2-(2-Azidoethoxy)ethoxy)acetic acid 8 (1.53 g, 8.2 mmol) was dissolved in EtOH (150 mL) and the solution was cooled to 4 °C. Pd-C (0.32 g, 10% Pd) was added and the resulting reaction mixture was stirred at room temperature for 12 h under an H2 atmosphere. The reaction was checked for completion by TLC (CH2Cl2- MeOH, 8 : 2, v/v) and the mixture was filtered over Celite® 545 to remove Pd-C. The filtrate was evaporated to dryness and the resulting oily residue was dried under vacuum to give 1.3 g (8.2 mmol) of amino acid 9 as a yellow oil (quantitative yield). Rf (CH2Cl2-MeOH, 8 : 2, v/v) 0.0; Spectroscopic data are identical to that reported in the literature.

(2-(2-(tert-Butyloxycarbonyl)aminoethoxy)ethoxy)acetic acid (10)4. (2-(2- Aminoethoxy)ethoxy)acetic acid 9 (0.7 g, 4.3 mmol) was dissolved in a mixture of THF-H2O (2 : 1, v/v, 15 mL). Freshly prepared 2 M aq. NaOH solution (6.5 mL) was added and the solution was cooled to 4 °C. Boc2O (1.4 g, 6.4 mmol) was added and the reaction mixture was stirred at room temperature for 1 h. The reaction was checked for completion by TLC (CH2Cl2-MeOH, 7 : 3, v/v) and acidified by adding 1 M aq. KHSO4 solution (ca. 3.5 mL). The mixture was evaporated close to dryness. H2O (30 mL) was added and the solution was extracted with CH2Cl2 (3 x 30 mL). The combined organic extracts were dried over Na2SO4, filtrated and evaporated to dryness. The resulting oily residue was purified by chromatography on a silica gel column with a step gradient of MeOH (0-3%) in CH2Cl2 as the mobile phase, giving 0.71 g (2.7 mmol, yield 63%) of protected amino acid 10 as a colorless 1 oil. Rf (CH2Cl2-MeOH, 7 : 3, v/v) 0.21; H NMR (300 MHz, CDCl3): δ 1.44 (s, 9H), 3.34 (bm, 2H), 3.50-3.77 (m, 6H), 4.17 (s, 2H), 4.97, (bs, 1H, NH).

3 (2-(2-Aminoethoxy)ethoxy)acetic acid methyl ester (11). (2-(2-Aminoethoxy)ethoxy)acetic acid 9 (0.70 g, 3.35 mmol) was suspended in 2,2-dimethoxypropane (30.9 mL) and 37% HCl (3.35 mL) was added. The resulting reaction mixture was stirred at room temperature for 1 h. The reaction was checked for completion by TLC (CH2Cl2-MeOH-TEA, 80 : 20 : 2, v/v/v) and the mixture was evaporated to dryness. Deionised water (10 mL) was added and the resulting aq. solution was lyophilised thrice to give 714 mg (3.35 mmol) of methyl ester 11 as a yellow oil (quantitative yield). This compound was used in the next step without further 1 purification. Rf (CH2Cl2-MeOH-TEA, 80 : 20 : 2, v/v/v) 0.46; H NMR (300 MHz, CD3CN + 5% D2O): δ 3.08 (t, J = 5.3 Hz, 2H), 3.56-3.76 (m, 11H), 4.13 (s, 2H); MS (ESI+): m/z 178.20 + [M + H] , calcd for C7H15NO4 177.20.

Boc-protected amino-PEG-acid spacer (A). A mixture of (2-(2-(tert- butyloxycarbonyl)aminoethoxy)ethoxy)acetic acid 10 (0.15 g, 0.83 mmol) and (2-(2- aminoethoxy)ethoxy)acetic acid methyl ester 11 (0.23 g, 0.87 mmol) was dissolved in dry CH3CN. DIEA (0.44 mL, 2.5 mmol) and BOP reagent (0.37 g, 0.83 mmol) were sequentially added and the resulting reaction mixture was stirred at room temperature overnight under an argon atmosphere. The reaction was checked for completion by TLC (CH2Cl2-MeOH, 8 : 2, v/v) and the mixture was evaporated to dryness. Thereafter, the resulting residue was taken up in ethyl acetate, washed with 10% aq. citric acid, sat. aq. NaHCO3, brine, dried over Na2SO4, filtrated and evaporated to dryness. The orange oily residue was dissolved in MeOH (5 mL) and the solution was cooled to 4 °C. 1 M aq. LiOH (0.83 mL) was added and the reaction mixture was stirred at room temperature for 30 min. The reaction was checked for completion by TLC (CH2Cl2-MeOH, 8 : 2, v/v) and acidified with 1 M aq. KHSO4 solution (ca. 1 mL). The solution was evaporated to dryness without warming and the resulting residue was purified by chromatography on a silica gel column with a step gradient of MeOH (0-50%) in CH2Cl2 as the mobile phase. 0.17 g (0.42 mmol, overall yield for the two steps 51%) of N- Boc pseudo-PEG linker A was obtained as a colorless oil. Rf (CH2Cl2-MeOH, 8 : 2, v/v) 0.54; IR (neat): νmax 559, 666, 771, 843, 1114 (broad), 1251, 1367, 1455, 1538, 1666 (broad), 2930, -1 1 3352 (broad) cm ; H NMR (300 MHz, CDCl3): δ 1.43 (s, 9H), 3.30 (bm, 2H), 3.47-3.66 (m, 14H), 4.01 (s, 2H), 4.04 (s, 2H), 5.16 (bs, 1H, NH), 7.34 (bs, 1H, NH); 13C NMR (75.5 MHz, CDCl3): δ = 28.5 (3C), 38.7, 40.3, 70.1 (2C), 70.4 (2C), 70.6 (2C), 70.9 (2C), 79.5, 156.3, 170.9 (2C); MS (ESI+): m/z 431.27 [M + Na]+, 453.27 [M + 2Na - H]+; MS (ESI-): m/z - 407.47 [M - H] , calcd for C17H32N2O9 408.45.

N-Phthaloyl protected aminooxyacetic acid (12)5. (a) Synthesis of full-protected aminooxyacetic acid derivative: N-Hydroxyphthalimide (1.7 g, 10.3 mmol) was dissolved in dry NMP (24 mL). Anhydrous K2CO3 (2.1 g, 15.5 mmol) was then added and the resulting mixture was stirred at 40 °C under an argon atmosphere for 10 min. tert-Butyl bromoacetate (1.5 mL, 10.3 mmol) was then slowly added and the temperature increased to 50 °C for 3 h. The reaction was checked for completion by TLC (100% CH2Cl2). Precipitation of the desired product was achieved by adding cold deionised water. The solid was collected by filtration and washed with cold deionised water until the solid remains colorless. The resulting white solid was then dissolved in CH2Cl2, evaporated to dryness. Residual water was removed by lyophilisation to give tert-butyl phthalimidooxyacetate as a white powder (2.3 g, 8.3 mmol, yield 81%). Rf (100% CH2Cl2) 1 0.7; H NMR (300 MHz, CDCl3): δ 1.49 (s, 9H, tBu), 4.71 (s, 2H, CH2), 7.74-7.86 (m, 4H, 13 phthalimide); C NMR (75.5 MHz, CDCl3): δ 28.2, 73.6, 83.2, 123.8, 129.0, 134.8, 163.2, 166.5. (b) Removal of tert-butyl ester: tert-Butyl phthalimimidooxyacetate (2.3 g, 8.3 mmol) was dissolved in dry CH2Cl2 (20 mL) and the mixture was cooled to 0 °C. TFA (6.16 mL, 83

4 mmol) was then added dropwise and the reaction mixture was stirred at room temperature for 1 h under an argon atmosphere. The reaction was checked for completion by TLC (100% CH2Cl2). Further amount of TFA was added (1.05 mL, 16.6 mmol) and the mixture was stirred again for 20 min. Thereafter, the mixture was concentred under reduced pressure and the resulting residue was co-evaporated thrice with chloroform (3 x 10 mL). Finally, 10 mL of deionised water was added and the aq. solution was lyophilised to give phthalimimidooxyacetic acid 12 as a white powder (1.8 g, 8.3 mmol, quantitative yield). Rf 1 (100% CH2Cl2) 0.17; H NMR (300 MHz, CDCl3): δ 4.77 (s, 2H, CH2), 7.83 (s, 4H, 13 phthalimide); C NMR (75.5 MHz, CDCl3): δ 74.0, 124.3, 129.9, 135.9, 164.1, 168.3.

Synthesis of full-protected heterotrifunctional reagent (4).

The highly convergent synthetic strategy based on solution phase peptide couplings and developed for the preparation of N-Phthaloyl aminooxy heterotrifunctional cross-linking reagent 5 was used:

O O O O O N N O O H H HO N O H a, b O OH O N H S1 O building block S2 Scheme S1 Reagents and conditions: a) DCC (1.2 equiv), HOBt.H2O (1.2 equiv), CH3CN-DMF (1 : 1, v/v), rt, 2 ’ h; b) Boc-Lys-OH (1 equiv), DMF, rt, 2 h, 50%. DCC = N,N -dicyclohexylcarbodiimide, HOBt.H2O = hydroxybenzotriazole monohydrate.

(9-Fluorenylmethoxycarbonyl)aminooxyacetic acid (S1)6. Carboxymethoxylamine hemihydrochloride (0.5 g, 4.6 mmol) was dissolved in an aq. solution of Na2CO3 (1.2 g in 20 mL) and the resulting solution was cooled to 4 °C. Then 9-fluorenylmethyl chloroformate (1.31 g, 5.0 mmol) in 1,4-dioxane (10 mL) was added dropwise and the reaction mixture was stirred at room temperature overnight. The reaction mixture was partially evaporated, acidified to pH 4-5 with 5% aq. HCl (ca. 5 mL); the crude product quickly precipitated. It was collected by filtration and washed with deionised water and pentane. Residual water was removed by lyophilisation to give 1.05 g of crude product. Further purification by chromatography on a silica gel column was undertaken with a step gradient of MeOH (0- 50%) in CH2Cl2 as the mobile phase, giving 0.677 g (2.2 mmol, yield 47%) of Fmoc-Aoaa- 1 OH S1 as a white foam. H NMR (300 MHz, CD3OD): δ 4.22-4.27 (m, 3H), 4.44 (d, J = 6.8 Hz, 2H), 7.29-7.42 (m, 4H), 7.64 (d, J = 7.5 Hz, 2H), 7.80 (d, J = 7.5 Hz, 2H); HPLC (system B): tR = 17.8 min, purity 94%.

Nα-(tert-Butyloxycarbonyl)-Nε-[(9-fluorenylmethoxycarbonyl)aminooxyacetyl]-L-lysine (S2). Fmoc-Aoaa-OH S1 (230 mg, 0.73 mmol) was dissolved in a mixture of dry CH3CN- DMF (1:1, v/v, 9 mL). Hydroxybenzotriazole monohydrate (119 mg, 0.88 mmol) and DCC (182 mg, 0.88 mmol) were sequentially added and the resulting reaction mixture was stirred at room temperature for 2 h under an argon atmosphere. Thereafter, a solution of Nα-Boc-L- lysine (180 mg, 0.73 mmol) in dry DMF (2 mL) was added and the resulting reaction mixture was stirred at room temperature. The reaction was checked for completion by TLC (CH2Cl2- MeOH 8 : 2, v/v). After 2 h, the mixture was evaporated to dryness. The resulting residue was taken up with ethyl acetate, washed by 10% aq. citric acid, deionised water, dried over Na2SO4, filtrated, concentrated by rotator evaporation, then purified by chromatography on a silica gel column with a step gradient of ethyl acetate (0-80%) in CH2Cl2 as the mobile phase,

5 giving 296 mg (0.36 mmol, yield 50 %) of lysine building block S2 as a white foam. Rf 0.59 1 (CH2Cl2-MeOH, 8 : 2, v/v); H NMR (300 MHz, CD3CN): δ 1.37-1.83 (m, 15H), 3.17-3.19 (d, J = 6.0 Hz, 2H), 4.00 (m, 1H), 4.04 (s, 2H), 4.26 (t, J = 6.8 Hz, 1H), 4.49 (d, J = 6.8 Hz, 2H), 5.61 (bd, J = 7.5 Hz, 1H, NH), 7.31-7.44 (m, 4H), 7.51 (bs, 1H, NH), 7.63 (d, J = 7.5 Hz, 13 2H), 7.83 (d, J = 7.5 Hz, 2H), 8.9 (bs, 1H, NH); C NMR (75.5 MHz, CD3CN): δ 23.5, 28.5 (3C), 29.5, 31.6, 38.9, 47.7, 54.2, 68.0, 76.3, 79.8, 120.9 (2C), 126.0 (2C), 128.1 (2C), 128.7 (2C), 142.1 (2C), 144.6 (2C), 156.7, 158.9, 169.3, 174.5; HPLC (system B): tR = 21.2 min, purity 95%; UV-visible (recorded during the HPLC analysis) λmax = 216, 262, 293 nm; MS (MALDI-TOF, positive mode): m/z 564.57 [M + Na]+, 580.55 [M + K]+, calcd for C28H35N3O8 541.61.

O O O

O H2N N N O NH2 H H + O S OH S O N H O building block C building block S2 (TFA salt)

a-b

O O O N N O H H O O H N O N NH2 H O S S

S3 (TFA salt)

O O O N N O H H O H N H2N NH2 O S S d building block A active OBt esters + building block S4 (TFA salt)

e-f

O O O N N O H H O O H H O N O N H2N O O N NH2 H O O S S S5 (TFA salt)

O O O N N O H H O O O O H H N O N O N O N O O N NH2 H H O O O S S Full-protected heterotrifunctional cross-linker 4 Scheme S2 Reagents and conditions: a) DCC (1.2 equiv), HOBt.H2O (1.2 equiv), CH3CN-DMF (2 : 1, v/v), rt, 2 h; b) DIEA (2 equiv), DCC (2 equiv), rt, overnight, 88% (a+b); c) 15% TFA, CH2Cl2, 4 °C to rt, 90 min, quant. yield; d) DCC (1.2 equiv), HOBt.H2O (1.2 equiv), CH3CN, rt, 2 h; e) DIEA (2.5 equiv), DCC (0.3 equiv), rt, overnight then acetic acid (1.9 equiv), 61% after RP-HPLC purification; f) 11% TFA, CH2Cl2, 4 °C to rt, 1 h, quant. yield; g) DSC (2.5 equiv), TEA (1 equiv), DMF, rt, 90 min, 74% after RP-HPLC purification. DSC = N,N'-disuccinimidyl carbonate, TEA = triethylamine.

6 Nα-(tert-Butyloxycarbonyl)-Nε-[(9-fluorenylmethoxycarbonyl)aminooxyacetyl]-L-lysinyl- S-(ethylthio)-L-cysteine carboxamide (S3). Lysine building block S2 (0.196 g, 0.36 mmol) was dissolved in a mixture of dry CH3CN-DMF (2 : 1, v/v, 3 mL). Hydroxybenzotriazole monohydrate (58.4 mg, 0.43 mmol) and DCC (89.1 mg, 0.43 mmol) were sequentially added and the resulting reaction mixture was stirred at room temprature for 2 h under an argon atmosphere. Thereafter, a solution of TFA salt of H-Cys(SEt)-OH C (106.0 mg, 0.36 mmol) in dry DMF (1 mL) was added and the resulting reaction mixture was stirred at room temperature under an argon atmosphere. The reaction was checked for completion by RP- HPLC (system B) and TLC (CH2Cl2-MeOH, 8 : 2, v/v). After 2, 4 and 6 h of stirring, DIEA (31 µL, 0.18 mmol) was added and a further amount of DCC (44.9 mg, 0.18 mmol) was added after 6 h. The round-bottom flask was stored at -20 °C overnight. Thereafter, further amounts of DIEA (31 µL, 0.18 mmol) and DCC (44.9 mg, 0.18 mmol,) were added. After 2 h, the reaction was checked to completion and the mixture was evaporated to dryness. The resulting residue was taken up with ethyl acetate, successively washed with 10% aq. citric acid, sat. NaHCO3, deionised water, dried over Na2SO4 and evaporated to dryness. The resulting residue was purified by chromatography on a silica gel column with a step gradient of MeOH (0-10%) in CH2Cl2 as the mobile phase, giving 222 mg (0.31 mmol, yield 88%) of full-protected dipeptide S3 as a white foam. Rf (CH2Cl2-MeOH, 8 : 2, v/v) 0.60; IR (KBr): νmax 586 (broad), 621, 741, 760, 861, 1045, 1118, 1167, 1254, 1367, 1392, 1451, 1516 -1 1 (broad), 1666 (broad), 2868, 2928, 3068, 3312 (broad) cm ; H NMR (300 MHz, CDCl3): δ 1.26-1.31 (t, 3H, J = 7.1 Hz, CH3(SEt) Cys), 1.42 (s, 9H, tBu) 1.51-1.92 (m, 6H, CH2 β, δ, γ Lys), 2.65-2.72 (q, 2H, J = 7.5 Hz, CH2(SEt) Cys), 3.08-3.10 (d, 2H, J = 6.0 Hz, CH2 β Cys), 3.26-3.33 (m, 2H, CH2 ε Lys), 4.04-4.06 (t, J = 6.0 Hz, 1H, CH α Lys), 4.20-4.25 (t, J = 7.2 Hz, 1H, CH Fmoc), 4.33 (s, 2H), 4.49-4.51 (d, 1H, J = 6.4 Hz, CH2 Fmoc), 4.71-4.78 (q, J = 13 6.4 Hz, 1H, CH α Cys), 7.26-7.78 (m, 8H, CH Fmoc); C NMR (75.5 MHz, CDCl3): δ 14.2, 22.3, 28.6, 30.1, 32.4, 38.0, 39.3, 46.7, 52.3, 52.2, 68.0, 76.1, 80.8, 120.3, 125.1, 127.4, 128.1, 141.4, 143.3, 156.4, 158.6, 169.0,172.6, 172.8. MS (MALDI-TOF, positive mode): m/z + + 726.77 [M + Na] , 742.74 [M + K] , calcd for C33H45N5O8S2 703.88.

Nε-[(9-Fluorenylmethoxycarbonyl)aminooxyacetyl]-L-lysinyl-S-(ethylthio)-L-cysteine carboxamide (S4). Dipeptide S3 (191 mg, 0.27 mmol) was dissolved in dry CH2Cl2 (7 mL) and the solution was cooled to 4 °C. TFA (1.2 mL, 16.6 mmol) was added dropwise and the resulting reaction mixture was stirred at room temperature for 90 min. The reaction was checked for completion by TLC (CH2Cl2-MeOH, 9 : 1, v/v) and the mixture was evaporated to dryness. The resulting oily residue was dissolved in deionised water and lyophilised to give the dipeptide building block S4 as a white amorphous powder (199 mg, 0.27 mmol, quantitative yield). This compound was used in the next coupling reaction step without further purification. Rf (CH2Cl2-MeOH, 9 : 1, v/v) 0.14; IR (KBr): νmax 740, 1117, 1257, 1456, 1506, 1538, 1558, 1652, 1689, 1717, 1732, 2103, 2351, 2928, 3288 cm-1; 1H NMR (300 MHz, CD3OD): δ 1.26-1.28 (t, 3H, J = 3.8 Hz, CH3(SEt) Cys), 1.40-1.56 (m, 4H, CH2 β, γ Lys), 1.83-1.90 (m, 2H, CH2 δ Lys), 2.67-2.74 (q, 2H, J = 7.2 Hz, CH2(SEt) Cys), 2.93-2.96 (d, 2H, J = 9.4 Hz, CH2 β Cys), 3.20-3.28 (m, 2H, CH2 ε Lys), 3.84-3.88 (t, J = 6.0 Hz, 1H, CH α Lys), 4.23 (s, 3H, CH Fmoc, CH2), 4.46-4.48 (d, 2H, J = 6.4 Hz, CH2 Fmoc), 4.63-4.68 (q, J = 4.9 Hz, 1H, CH α Cys), 7.26-7.78 (m, 8H, CH Fmoc), 8.32 (bs, 1H, NH); 13C NMR (75.5 MHz, CD3OD): δ 14.7, 22.3, 30.7, 32.2, 33.2, 39.5, 41.0, 48.2, 53.9, 54.2, 68.6, 76.5, 121.0, 126.0, 128.2, 129.0, 142.7, 144.8, 170.2, 171.2; MS (MALDI-TOF, positive mode): m/z + + + 604.70 [M + H] , 626.68 [M + Na] , 642.66 [M + K ], calcd for C28H37N5O6S2 603.76.

7 PEG-peptide (S5). (a) Coupling reaction: Boc-protected amino-PEG-acid spacer A (78 mg, 0.19 mmol) was dissolved in dry CH3CN (2 mL). Hydroxybenzotriazole monohydrate (31 mg, 0.23 mmol) and DCC (47.3 mg, 0.23 mmol) were sequentially added and the resulting reaction mixture was stirred at room temperature for 2 h under an argon atmosphere. Thereafter, TFA salt of dipeptide S4 (136 mg, 0.19 mmol) was added and the resulting reaction mixture was stirred at room temperature for 2 h. The round-bottom flask was stored at -20 °C overnight. Thereafter, DIEA (32 µL, 0.19 mmol) was added. The reaction was checked for competion by RP-HPLC (system B). After 3, 5 and 7 h of stirring, DIEA (16 µL, 95 µmol) was added a further amount of DCC (12 mg, 58 µmol) was added after 3 h. Finally, reaction was quenched by the addition of acetic acid (21 µL, 360 µmol) and the round-bottom flask was stored at -20 °C overnight. After dilution in a mixture of CH3CN-H2O (2 : 1, v/v, 2 mL), the crude coupling product was purified by RP-HPLC (system D, 3 injections, tR = 35.8-27.2 min). The product-containing fractions were lyophilised to give the coupling product as a white amorphous powder (119 1 mg, 0.12 mmol, yield 61%). H NMR (300 MHz, CDCl3): δ 1.25-1.30 (t, 3H, J = 7.1 Hz, CH3(SEt) Cys), 1.43 (s, 9H, tBu), 1.52-1.90 (m, 4H, CH2 γ, δ Lys), 2.32 (bs, 2H, CH2 β Lys), 2.64-2.69 (q, 2H, J = 7.2 Hz, CH2(SEt) Cys), 2.97-3.16 (m, 2H, CH2 β Cys), 3.30 (s, 2H, CH2 linker), 3.45-3.65 (m, 11H, CH2 ε Lys + 4 x CH2 linker), 4.0 (s, 3H, CH2 linker + CH Fmoc), 4.21-4.25 (q, J = 6.8 Hz, 1H, CH α Lys), 4.34 (s, 2H, CH2), 4.44-4.50 (d, 2H, J = 10.0 Hz, CH2 Fmoc), 4.70-4.72 (q, 1H, J = 2.3 Hz CH α Cys), 5.23 (bs, 1H, NH), 6.08 (bs, 1H, NH), 13 6.9 (bs, 1H, NH), 7.26-7.77 (m, 8H, CH Fmoc); C NMR (75.5 MHz, CDCl3): δ 14.4, 22.6, 28.5, 28.7, 31.0, 31.6, 32.5, 38.7, 38.8, 39.5, 40.5, 52.5, 53.3, 53.6, 70.0, 70.2, 70.5, 70.5, 70.6, 71.0, 71.1, 77.8, 79.5, 124.1, 128.6, 135.2, 156.1, 163.8, 167.1, 170.4, 170.7, 171.9; HPLC (system B): tR = 20.8 min, purity 86%; MS (MALDI-TOF, positive mode): m/z + 1016.66 [M + Na] , calcd for C45H67N7O14S2 994.20. (b) Removal of Boc group: Full-protected pseudo-peptide (62 mg, 62 µmol) was dissolved in dry CH2Cl2 (3 mL). The resulting solution was cooled to 4 °C and TFA (368 µL, 4.96 mmol) was added dropwise. The resulting reaction mixture was stirred at room temperature for 1 h. The reaction was checked for completion by RP-HPLC (system B) and the mixture was evaporated to dryness. Deionised water was added and the resulting solution was lyophilised to give the PEG-peptide S5 as a white foam (83 mg, 82 µmol, yield 86%). This compound was used in the next step without further purification. HPLC (system B): tR = 16.5 min, purity 88%. MS (MALDI-TOF, positive mode): m/z 894.64 [M + H]+, calcd for C40H59N7O12S2 894.08.

Full-protected heterotrifunctional cross-linker (4). TFA salt of PEG-peptide S5 (36 mg, 36.7 µmol) was dissolved in dry DMF (500 µL). TEA (5 µL, 36.7 µmol) and 50 µL of a solution of DSC reagent in dry DMF (24 mg, 91.8 µmol) were sequentially added and the resulting reaction mixture was stirred at room temperature for 90 min. The reaction was checked for completion by RP-HPLC (system B). Finally, the reaction mixture was quenched by dilution with aq. TFA 0.1% (pH 2, 4 mL) and purified by RP-HPLC (system E, 2 injections, tR = 31.3-32.7 min). The product-containing fractions were lyophilised to give 4 as 1 a white amorphous powder (25 mg, 24.2 µmol, yield 74%). H NMR (300 MHz, CDCl3): δ 1.24-1.27 (t, 3H, J = 7.1 Hz, CH3(SEt) Cys), 1.32-1.90 (m, 6H, CH2 β, γ, δ Lys), 2.63-2.69 (q, 2H, J = 7.1 Hz, CH2(SEt) Cys), 2.81 (s, 4H, 2 x CH2 succinimide), 2.24-3.28 (m, 2H, CH2 β Cys), 3.24-3.69 (m, 11H, 5 x CH2 linker + CH2 ε Lys), 4.22-4.27 (t, 1H, CH Fmoc), 4.32 (s, 2H, CH2), 4.51-4.53 (d, 2H, J = 6.4 Hz, CH2 Fmoc), 4.58-4.66 (q, J = 7.5 Hz, 1H, CH α Lys), 4.70-4.76 (q, 1H, J = 6.0 Hz CH α Cys), 6.17 (bs, 1H, NH), 6.83 (s, 1H, NH), 7.27-7.83 (m, 8H, CH Fmoc), 9.2 (bs, 1H, NH); HPLC (system B): tR = 18.5 min, purity 97%; MS

8 (MALDI-TOF, positive mode): m/z 1035.69 [M + H]+, 1057.71 [M + Na]+, 1073.68 [M + + K] , calcd for C45H62N8O16S2 1035.17.

References

1 S. S. Iyer, A. S. Anderson, S. Reed, B. Swanson and J. G. Schmidt, Tetrahedron Lett., 2004, 45, 4285. 2 H. Kato, C. Boettcher and A. Hirsch, Eur. J. Org. Chem., 2007, 2659. 3 C. Visintin, A. E. Aliev, D. Riddall, D. Baker, M. Okuyama, P. M. Hoi, R. Hiley and D. L. Selwood, Org. Lett., 2005, 7, 1699. 4 Y. Zhang, M.-K. So, A. M. Loening, H. Yao, S. S. Gambhir and J. Rao, Angew. Chem. Int. Ed., 2006, 45, 4936. 5 U.S. Pat., 2006016752, 2006. 6 L. Cipolla, M. Rescigno, A. Leone, F. Peri, B. La Ferla and F. Nicotra, Bioorg. Med. Chem., 2002, 10, 1639.

9 Boc-protected amino-PEG-acid spacer A

O O H O N O O N O O OH H O

1 H NMR spectrum of A recorded in CDCl3.

8.1153 7.4045 7.3990 7.2600 5.2489 4.0980 4.0748 4.0011 3.6725 3.6439 3.6243 3.5926 3.5731 3.5555 3.5481 3.5299 3.5128 3.4982 3.4805 3.2933 3.2799 2.6130 2.5813 1.4048 Integral 1.4167 0.8897 0.5861 1.8472 2.0421 13.248 1.9544 9.0000 9.5 9.0 8.5 8.0 7.5 7.0 6.5 6.0 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5 (ppm)

10 13 C NMR spectrum of A recorded in CDCl3.

170.8654 156.3267 79.4874 77.5818 77.3637 77.1600 76.7382 70.8689 70.3961 70.1052 40.3295 38.7222 36.8312 29.7473 28.4672

190 180 170 160 150 140 130 120 110 100 90 80 70 60 50 40 30 20 10 (ppm)

ESI-MS spectrum of A recorded in the negative mode.

407.47 100 [M-H]- 90

40 Relative Abundance 30

20 262,40 10 837,47 451,40 692,27 944,27 1220,53 1503,67 1786,13 1964,4 0 200 400 600 800 1000 1200 1400 1600 1800 2000 m/z

11 ESI-MS spectrum of A recorded in the positive mode.

431.27 100 [M+Na]+ 90

70 453.27453,27 60 [M+2Na-H]+ 50

40 Relative Abundance Relative 30

20 347,27

10 469,20 286,20 732,20 576,20 883,13 1211,53 1326,20 1504,00 1830,33 0 200 400 600 800 1000 1200 1400 1600 1800 2000 m/z

12 Aminooxy-containing lysine building block B

O O O N N H O O OH O N H O

1 H NMR spectrum of B recorded in CDCl3.

7.9889 7.9706 7.9522 7.8379 7.8264 7.8204 7.8102 7.7957 7.7795 7.7663 7.7552 7.7492 7.7377 7.7258 7.7002 7.3956 7.3824 7.3671 7.2613 6.0747 6.0589 5.3548 5.3283 5.2626 5.2601 4.6843 4.2573 4.2390 4.2160 3.3365 3.3152 3.2943 3.2725 1.8300 1.8121 1.7285 1.7042 1.6791 1.6560 1.6155 1.5929 1.5716 1.5490 1.3767 Integral 4.8291 0.4905 0.5592 1.5965 0.5978 1.6088 0.8321 2.5583 9.0000 9.5 9.0 8.5 8.0 7.5 7.0 6.5 6.0 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5 (ppm)

13 13 C NMR spectrum of B recorded in CDCl3.

175.5513 167.7531 163.7702 155.7740 135.2048 128.4194 124.1396 79.9777 77.5865 77.1600 76.7412 76.6117 53.5370 53.2628 39.1057 31.9396 28.6574 28.3451 22.5041

190 180 170 160 150 140 130 120 110 100 90 80 70 60 50 40 30 20 10 (ppm)

ESI-MS spectrum of B recorded in the positive mode.

449.27 100 [M+H]+ 90

40 102,07 225,20 Relative Abundance 30 774,87

20 377,00 326,13 627,27 471,87 791,67 10 213,00 641,60 922,80

0 100 200 300 400 500 600 700 800 900 1000 m/z

14 Cysteine building block C (TFA salt)

H2N NH2 S S

1 H NMR spectrum of C recorded in D2O.

4.7900 4.3469 4.3306 4.3205 4.3029 3.4242 3.4091 3.3740 3.3577 3.3125 3.2962 3.2635 3.2472 3.2133 3.1857 3.1405 3.1141 3.0915 3.0639 2.7965 2.7727 2.7488 2.7237 1.3077 1.2839 1.2588 Integral 1.0000 2.2417 1.7854 3.0577 9.5 9.0 8.5 8.0 7.5 7.0 6.5 6.0 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5 (ppm)

15 13 C NMR spectrum of C recorded in D2O.

170.8143 52.2269 38.2983 31.9394 13.8375

190 180 170 160 150 140 130 120 110 100 90 80 70 60 50 40 30 20 10 (ppm)

ESI-MS spectrum of C recorded in the positive mode.

181.00 100 [M+H]+ 95

45 Relative Abundance Relative

20 261.13

15 239.13

10 136.00 82.80 681.00 221.87 119.07 655.20 839.40 914.93 522.93 579.47 696.93 790.53 986.47 5 465.80 283.73 389.47 437.40

0 100 200 300 400 500 600 700 800 900 1000 m/z

16 Heterotrifunctional cross-linking reagent 5

O O O N N H O O O O O H H N O N O N O N O O N NH2 H H O O O S S

1 H NMR spectrum of 5 recorded in CDCl3.

7.8042 7.7954 7.7847 7.6284 7.6096 7.5437 7.5192 7.4973 7.4715 7.2600 7.0027 6.3361 5.8258 4.7161 4.6979 4.6797 4.6722 4.6527 4.4864 4.4619 4.4412 4.4173 4.0489 4.0332 3.6823 3.6666 3.6440 3.6258 3.6183 3.6014 3.5838 3.5505 3.5317 3.5166 3.4444 3.4269 3.4112 3.3949 3.3741 3.3522 3.3315 3.3095 3.1720 3.1538 3.1256 3.1080 3.0471 3.0189 3.0001 2.9724 2.8130 2.7201 2.6956 2.6718 2.6473 1.9895 1.9663 1.9443 1.9186 1.8991 1.8740 1.8019 1.7780 1.7523 1.7322 1.7058 1.6556 1.6324 1.6092 1.5859 1.4880 1.4623 1.4372 1.4140 1.3079 1.2834 1.2589 Integral 5.0539 2.9250 1.6746 0.8318 3.1021 2.9431 0.9618 3.9269 17.508 2.0835 3.7684 2.0574 0.9824 0.9965 1.9647 2.0186 3.0000 9.5 9.0 8.5 8.0 7.5 7.0 6.5 6.0 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5 (ppm)

17 13 C NMR spectrum of 5 recorded in CDCl3.

173.3710 172.0255 171.2328 171.1891 170.3309 167.5381 163.7634 151.8502 135.2023 128.4675 124.1255 77.4692 77.2510 77.0474 76.6255 71.0253 70.8435 70.2107 70.1307 70.0507 69.9271 69.3962 53.2355 52.5155 41.7515 39.1041 38.8932 38.7404 38.6095 32.3693 31.4093 28.6601 25.5254 22.6671 14.2886

190 180 170 160 150 140 130 120 110 100 90 80 70 60 50 40 30 20 10 (ppm)

ESI-MS spectrum of 5 recorded in the positive mode.

943.27 100 [M+H]+ 95

45 Relative Abundance Relative

965.20 10 981.20 763.20 1885.00 472.60 927.20 5 287.13 1433.80 434.27 735.07 1096.33 1797.80 1905.87 571.20 1173.93 1540.13 1709.27 0 200 400 600 800 1000 1200 1400 1600 1800 2000 m/z

18 RP-HPLC elution profile of 5 (system A).

1100 PDA-280nm 1100 Retention Time Name

1000 25.177 1000

900 900

800 800

700 700

600 600

500 500 mAU mAU

400 400

300 300

200 200 25.035 100 100 22.625 22.827 0 0

-100 -100

0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 32 34 36 38 40 Minutes

19 R6G-WS labeled aminooxy reagent 19

O O N NH2 H O O O H H O N O N O N O O N NH2 H H O O S H N H O N N H O =

N O N H H

- O SO3H SO3 H H N N O O O O

R6G-WS

ESI-MS spectrum of 19 recorded in the negative mode.a

1670.47 100

- 95 [M-H]

45 Relative Abundance

835.53835.53 35 [M-2H]2- 30

20 1008.20

15 853.27 1626.47 1831.87 725.13 1165.47 1890.40 877.80 1340.33 1742.73 10 1524.40 1961.67 277.33 913.60 1039.47 1189.80 502.87 5 219.93430.13 677.33

0 200 400 600 800 1000 1200 1400 1600 1800 2000 m/z aUnder our ionisation conditions, the aminooxy group of 19 was converted into the corresponding alcohol (∆M of -15.0 Da). Such degradation reaction has been already reported by Bruné et al. (Rapid. Commun. Mass Spectrom. 2000, 14, 2158) for the mass analysis of (aminooxy)acetyl peptides involved in oxime ligation reactions.

20 RP-HPLC elution profile of 19 (system B).a

PDA-530nm Retention Time Name 450 450 27.585

400 400

350 350

300 300

250 250 mAU

mAU 200 200

150 150

100 100 23.943

50 50 25.718 36.437 28.143 27.987 35.095 25.963 33.903 0 0

-50 -50 0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 32 34 36 38 40 42 44 Minutes aPartial cleavage of Boc group was occurred during the HPLC analysis due to the acidity of aq. mobile phase.

UV-visible absorption of 19 in deionised water at 25°C (concentration = 4.2 µM).

21 ESI-MS spectrum of fluorescent substance P-tripod 23 recorded in the positive mode.a

1347.47 100

2+ 95 [2M+2H2O+2H]

45 Relative Abundance Relative

20 1325.27 1796.47 15 1602.20 898.67 1093.60 1978.8 10 1170.07 1284.93 1442.40 1774.60 1838.27 855.80 1016.67 1692.47 5 510.67 582.07 726.40 803.87

0 500 600 700 800 900 1000 1100 1200 1300 1400 1500 1600 1700 1800 1900 2000 m/z a 2+ Only the doubly charged hydrated ion of 23 ([M + 2H2O + 2H] : m/z calcd mass 1347.10, found 1347.47) was observed under our ionisation conditions. Production of such solvated ions through ESI ionisation has been already reported especially for peptidyl biopolymers (see: Rodriguez-Cruz et al. J. Am. Soc. Mass Spectrom., 1999, 10, 958 and Wyttenbach et al. Int. J. Mass Spectrom., 2005, 240, 221).