Amplitud de activación muscular con un 50% de carga máxima

All reagents were commercial grade and were used as received unless stated otherwise. MeOH and NMP were obtained from Biosolve. Toluene, EtOAc and PetEt (Riedel-de Haën) used for column chromatography were of technical grade and distilled before use. DCE, DCM, DMF, THF and dioxane (Biosolve) were of analytical grade and when used under anhydrous conditions stored over flame-dried 4 Å molecular sieves. Reactions were monitored by TLC-analysis using DC-alufolien (Merck, Kieselgel60, F254) with detection by UV-absorption (254/366 nm), spraying with a solution of (NH4)6Mo7O24∙4H2O (25 g/L) and (NH4)4Ce(SO4)4∙2H2O (10 g/L) in 10% aqueous sulfuric acid followed by charring at ~150 °C or spraying with an aqueous solution of KMnO4 (7%) and K2CO3 (2%). Column chromatography was performed on silica gel (Screening Devices BV, 0.040 - 0.063 mm, 60 Å). LC/MS analysis was performed on an LCQ Adventage Max (Thermo Finnigan) ion-trap spectrometer (ESI+) coupled to a Surveyor HPLC

51 system (Thermo Finnigan) equipped with a C18 column (Gemini, 4.6 mm x 50 mm, 5μm particle size, Phenomenex). The applied buffers were A: H2O, B: ACN and C: 1 % aqueous TFA. Reported gradients represent the percentage of buffer B in buffer A with 10% buffer C. HRMS analysis was performed on an LTQ Orbitrap (Thermo Finnigan) mass spectrometer equipped with an electronspray ion source in positive mode (source voltage 3.5 kV, sheath gas flow 10 mL min−1, capillary temperature 250 °C) with resolution R = 60000 at m/z 400 (mass range m/z = 150 - 2000) and dioctylphtalate (m/z = 391.28428) as a "lock mass". The high resolution mass spectrometer was calibrated prior to measurements with a calibration mixture (Thermo Finnigan). 1_{H- and} 13_{C-APT-NMR spectra were recorded on a} Jeol JNM-FX-200 (200/50) or Bruker AV-400 (400/100 MHz). Chemical shifts are given in ppm (δ) relative to the solvent peak or to tetramethylsilane as internal standard. Coupling constants (J) are given in Hz. All presented 13_C- APT spectra are proton decoupled. Peak assignments are based on 2D 1_{H-COSY and} 13_{C-HSQC NMR experiments.} Compounds 1a-d, 3 and 10 were described previously.38

(2S,3S)-diethyl oxirane-2,3-dicarboxylate (8)

D-(-)-diethyl tartrate (7) (29 g, 0.14 mol) was cooled to 0 °C, before a solution of 33% HBr in acetic acid (120 mL) was added dropwise over 45 min. After complete addition, the reaction mixture was stirred at 0 °C for 15 min and then at room temperature overnight. Next, the mixture was poored onto crushed ice/H2O (300 mL) and extracted with Et2O (3x). The combined organics were washed with H2O (3x), dried over anhydrous MgSO4, filtered and concentrated in vacuo. Remaining solvents were concentrated in the presence of toluene. The crude oil was dissolved in EtOH and acetyl chloride (5.1 mL, 70 mmol, 0.5 eq.) was added. The reaction mixture was stirred under reflux for 3.5 hrs, before being concentrated at a temperature of 30 °C. The remaining yellowish oil was dissolved in Et2O (175 mL), cooled to 0 °C and put under argon atmosphere. A solution of DBU (21 mL, 0.14 mol, 1.0 eq.) in Et2O (90 mL) was added dropwise over 100 min. The reaction mixture was then stirred at 0 °C for 1 hr, more DBU (2.1 mL, 14 mmol, 0.1 eq.) was added and the reaction mixture was stirred for an additional 2 hrs, before being quenched with H2O. The mixture was washed with 1M KHSO4 solution and H2O and the organic layer was dried over anhydrous MgSO4, filtered and concentrated in vacuo. Purification by column chromatography (PetEt → 15% EtOAc in PetEt) yielded title compound 8 (15 g, 79 mmol, 56% over 3 steps). 1_{H NMR (400 MHz, CDCl}

3): δ (ppm) 4.32-4.22 (m, 4H), 3.66 (s, 2H), 1.32 (t, J = 7.15, 7.15 Hz, 6H). 13_{C NMR (100 MHz, CDCl}

3): δ (ppm) 166.69, 62.14, 51.96, 13.96.

(2S,3S)-3-(ethoxycarbonyl)oxirane-2-carboxylic acid (9)

A solution of compound 8 (14 g, 76 mmol) in absolute EtOH (200 mL) was cooled to 0 °C and a solution of KOH (5.0 g, 76 mmol, 1.0 eq.) in absolute EtOH (100 mL) was added dropwise over 20 min. Next, the reaction mixture was stirred at 0 °C for 3 hrs and then at room temperature for 2 hrs, before being concentrated in vacuo. H2O (200 mL) was added to the residue and the basic aqueous mixture was washed with DCM (1x 30 mL). The aqueous layer was then acidified with concentrated HCl (7.0 mL), NaCl (60 g) was added and the mixture was extracted with EtOAc (4x 200 mL). The combined organics were dried over anhydrous MgSO4, filtered and concentrated in vacuo to give title compound 9 (11 g, 66 mmol, 86%). 1_{H NMR (400 MHz, CDCl}

3): δ (ppm) 6.24 (bs, 1H), 4.33-4.20 (m, 2H), 3.74-3.61 (m, 2H), 1.32 (t, J = 7.10, 7.10 Hz, 3H). 13_{C NMR (100 MHz, CDCl}

3): δ (ppm) 168.54, 166.92, 61.95, 51.49, 51.42, 13.38.

MBHA-Rink amide-Lys(Boc)-Ahx-Tyr(tBu)-Leu(Fmoc) (4)

4-methylbenzhydrylamine (MBHA) functionalizedRink amide resin (3.2 g, 0.64 mmol/g, 2.1 mmol) was washed with DCM and deprotected with 20% piperidine in NMP for 20 min. After washing with NMP (2x) and DCM (2x), the resin was coupled to Fmoc-Lys(Boc)-OH (2.4 g, 5.2 mmol, 2.5 eq.) in the presence of BOP (2.3 g, 5.2 mmol, 2.5 eq.) and DiPEA (1.1 mL, 6.2 mmol, 3.0 eq.) in NMP; the reaction mixture was shaken overnight, followed by washing with NMP (2x) and DCM (2x). The remaining free amines were capped with acetic anhydride (0.98 mL, 10 mmol, 5.0 eq.)

52

(2S,3S)-3-(ethoxycarbonyl)oxirane-2-carboxyl-Leu-Tyr-Ahx-Lys.TFA (5)

Resin-bound compound 4 (~0.90 mmol) was deprotected with 20% piperidine in NMP for 30 min. The resin was washed with NMP (2x) and DCM (3x) before being subjected to a condensation cycle with free acid 9 (0.36 g, 2.3 mmol, 2.5 eq.) in the presence of BOP (1.0 g, 2.3 mmol, 2.5 eq.) and DiPEA (0.45 mL, 2.7 mmol, 3.0 eq.) in NMP; the reaction mixture was shaken overnight, followed by washing with NMP (3x) and DCM (3x). The condensation cycle was repeated after which the Kaiser test indicated complete coupling. Cleavage from the resin was then accomplished by treatment with TFA/TIS/H2O (95/2.5/2.5, v/v/v) for 2 hrs at room temperature. After filtration and concentration in vacuo in the presence of toluene, the residue was recrystallized first from acetone/MeOH/EtOAc and then from MeOH/Et2O, yielding a 2:1 mixture of fully deprotected ester 5 and free acid 6 (total yield 0.73 g, 0.93 mmol, quant.) according to NMR analysis. LC/MS analysis: Rt 5.0 min (linear gradient 10 → 90% B in 15 min), m/z 677.4 [M+H]+_{, 1353.2 [2M+H]}+_. 1_{H NMR (400 MHz, MeOD): δ (ppm) 6.98 (d, J = 7.87 Hz, 2H), 6.66 (d, J = 7.76 Hz,} 2H), 4.44 (t, J = 7.23, 7.23 Hz, 1H), 4.37 (dd, J = 7.59, 5.82 Hz, 1H), 4.30 (dd, J = 7.96, 5.15 Hz, 1H), 4.26-4.16 (m, 1H), 3.79-3.61 (m, 1H), 3.61-3.46 (m, 1H), 3.17-3.07 (m, 2H), 3.06-2.98 (m, 2H), 2.98-2.91 (m, 2H), 2.91-2.73 (m, 2H), 2.20 (t, J = 6.87, 6.87 Hz, 2H), 1.66-1.63 (m, 2H), 1.59-1.31 (m, 9H), 1.26 (t, J = 6.99, 6.99 Hz, 3H), 1.23-1.13 (m, 2H), 0.88 (d, J = 5.77 Hz, 3H), 0.84 (d, J = 5.75 Hz, 3H). (2S,3S)-3-(ethoxycarbonyl)oxirane-2-carboxyl-Leu-Tyr-Ahx-((N)-(E)-hepta-4,6-dienoyl)Lys-H2N (2b)

A mixture of 5 and 6 (0.31 g, 0.39 mmol, non-hydrolyzed/hydrolyzed 2/1) was dissolved in DCE/DMF under argon atmosphere and made basic (pH 8.5) using DiPEA (0.13 mL, 0.78 mmol, 2.0 eq.), before a solution of OSu-ester 10

(0.23 g, 1.0 mmol, 2.6 eq.) in DCE/DMF was added. After stirring overnight at room temperature under argon atmosphere, the reaction mixture was concentrated in vacuo. The residue was taken up in MeOH/acetone and the soluble fraction was purified by column chromatography (CHCl3 → 10% MeOH in CHCl3), yielding diene-modified title compound 2b (0.16 g, 0.20 mmol, 76% from non-hydrolyzed starting material 7). 1_{H NMR (400 MHz, dmso-d6):} δ (ppm) 8.54 (d, J = 8.19 Hz, 1H), 8.11 (d, J = 8.24 Hz, 1H), 7.82-7.74 (m, 2H), 7.30 (s, 1H), 6.96 (d, J = 8.40 Hz, 2H), 6.61 (d, J = 8.32 Hz, 2H), 6.28 (td, J = 17.03, 10.26, 10.26 Hz, 1H), 6.04 (dd, J = 15.16, 10.53 Hz, 1H), 5.74-5.64 (m, 1H), 5.02 (dd, J = 50.10, 13.54 Hz, 2H), 4.37-4.28 (m, 2H), 4.23-4.08 (m, 3H), 3.73-3.69 (m, 1H), 3.62-3.57 (m, 1H), 3.07-2.88 (m, 4H), 2.82 (dd, J = 13.69, 5.57 Hz, 1H), 2.67 (dd, J = 13.64, 8.85 Hz, 1H), 2.26 (dd, J = 14.23, 7.06 Hz, 2H), 2.17-2.05 (m, 4H), 1.59 (td, J = 9.47, 6.89, 6.89 Hz, 1H), 1.54-1.26 (m, 12H), 1.23 (t, J = 7.10, 7.10 Hz, 3H), 1.20-1.12 (m, 2H), 0.83 (dd, J = 14.79, 6.46 Hz, 6H).

Diels-Alder reaction of maleimide-tag 3 with diene-functionalized probe 1c

Fluorescent maleimide 3 (5 mM) was reacted with diene-derivatized probe 1c (5 mM) in a 1/1 (v/v) mixture of H2O and DMSO at room temperature. The reactions were monitored by LC/MS analysis at various time points (t = 0, 24, 48, 72 hrs). LC/MS analysis (linear gradient 10 → 90% B in 15 min): Bodipy-maleimide 3: Rt 8.6 min, m/z 589.3 [M-

53 F]+_{, 631.4 [M+Na]}+_{, 1217.0 [2M+H]}+_{; Probe 1c: Rt 9.3 min (linear gradient 10 → 90% B in 15 min), m/z 637.2 [M+H]}+_, 1295.1 [2M+Na]+_{; Diels-Alder adduct [3 + 1c]: Rt10.2 min (linear gradient 10 → 90% B in 15 min), m/z 1245.3} [M+H]+_.

B. Biochemistry