Supramolecular PEG-co-oligo(p-benzamide)s prepared on a peptide synthesizer

Transcription

1 Supporting Information Supramolecular PEG-co-oligo(p-benzamide)s prepared on a peptide synthesizer Hannah M. König, Tatiana Gorelik, Ute Kolb, Andreas F. M. Kilbinger* Experimental Chemicals All reactions were carried out in solvents of analytical quality (p.a.) purchased from Fischer Scientific. Poly(ethylene glycol) monomethyl ether (M W 2 g mol - ) was obtained from Fluka. All further chemicals were purchased from Acros Organics. Automated synthesis An Applied Biosystems Model 43A peptide synthesizer in standard Fmoc-mode was used for automated synthesis of oligomers. "Module a - Standard Fmoc: Activation" was modified for the use of pre-activated amino acids (see below). SEC SEC measurements (analytical) were performed with a set of Waters 77 plus autosampler, TSP Spectra Series P pump, and three PSS-SDV 5 µ columns (e4, e5, and e6 Å porosity). CHCl 3 was used as eluent (3 C, flow rate of ml min - ) by injection of 5 μl of polymer solution. RI spectra were detected at an Optilab DSP interferometric refractometer, UV spectra on a TSP Spectra Systems UV 2. Poly(styrene) standards were used for calibration. SEC purifications (preparative) were carried out in DMF and CHCl 3 using a Knauer HPLC Pump 64 and a Knauer Variable Wavelength Monitor (UV detector at 254 nm). Columns for DMF purifications were: MZ-GPC 25x3 mm SEC column, MZ-Gel SDplus, E3 Ǻ µm from MZ-Analysentechnik, Mainz, Germany. Columns for CHCl 3 purifications were: Mz- GPC 25x4 mm, MZ-Gel 5plus, E3 Ǻ µm from MZ-Analysentechnik. Mass spectrometry ESI mass spectra were recorded on a Micromass Q-ToF Ultima 3 from g/l solutions. NMR-spectra H-NMR spectra were recorded in d 7 -DMF (52 Scans) on a Bruker ARX 4 spectrometer, operating at 4 MHz. HPLC analysis RP-HPLC analysis was performed on a HP 9 Liquid Chromatograph of Hewlett Packard by using a PerfectSil column (MZ Analysentechnik, Mainz, Germany, 25 x 4. μm; 2 ODS-2 5 μm). The samples were eluted with an acetonitrile/water gradient that started from

2 % acetonitrile rising to 9 % over a period of 35 min. Both solvents were buffered with. % TFA. UV-detection was performed at 254 nm. TEM / STEM / cryo-tem A Philips EM 42 transmission electron microscope using a LaB 6 cathode at an acceleration voltage of 2 kv was used to obtain TEM-images. STEM images were detected on FEI Tecnai F3 at an acceleration voltage of 3 kv. Cryo-TEM images were recorded on FEI Tecnai F2 at an acceleration voltage of 2 kv. TEM grids (carbon film on copper, 3 mesh) were obtained from Electron Microscopy Sciences, Hatfield, PA, USA. For cryo-tem Quantifoil R.2/.3 copper grids were used. Amino acid activation When thionyl chloride is used for monomer activation, SO 2 is formed which is highly corrosive and therefore not suitable for automated synthesis in a peptide synthesizer. For this reason, alternatives for SOCl 2 activation of the monomer were investigated. C. Gilon et al.( J. Peptide Res. 999, 53, 57.) proposed 2-chloro-N-methyl--pyrrolinium chloride as the in situ activation intermediate when bis(trichloromethyl)carbonate (BTC) was added to NMP. According to H. Zollinger (Helvetica Chimica Acta 959, 75-76, 653.) we assumed the same intermediate to be responsible for activation using thionyl chloride and NMP. To gain more insights, we isolated the solid products formed in the reaction of thionyl chloride with NMP as well as BTC and NMP. The FT-IR spectra of both reaction products are shown in Figure SI-. Both spectra show the C=N valence band at around 63 cm -, the -CH 3, -CH 2 - valence bands at around 293 cm - as well as the C-Cl bands at 752 and 64 cm -. Furthermore, the isolated solids of both reactions could be used for the activation of N-PMB- N-Fmoc-p-aminobenzoic acid and subsequent coupling on solid support. (a) (b)

3 Figure SI-: FT IR spectrum of (a) the solid precipitated from the reaction of NMP with BTC and (b) the one of the reaction of NMP with thionyl chloride. Both products appear to be identical and are assumed to be 2-chloro-N-methyl--pyrrolinium chloride. 2-chloro-N-methyl--pyrrolinium chloride: using BTC Freshly distilled NMP ( g, mmol) was dissolved in abs. diethyl ether ( ml). The solution was stirred under a N 2 -atmosphere while cooling with an ice bath. BTC (.97 g, 3.3 mmol) was dissolved in abs. diethyl ether ( ml) and added dropwise to the mixture. After a reaction time of 2 min. a colorless solid precipitated. The reaction was allowed to proceed for further 3 min. The colorless precipitate was recovered by filtration and washed with abs. diethyl ether. The ether was removed under vacuum and the solid stored over phosphorus pentoxide to give the highly hygroscopic 2-chloro-N-methyl--pyrrolinium chloride. (9 %,.4 g, 9 mmol). using SOCl 2 Following the same reaction conditions the conversion of NMP with thionyl chloride was performed. The colorless precipitate was collected after min. of reaction time and analyzed by FT-IR (see Figure SI- b). If the reaction was allowed to proceed for reaction times longer than min. the precipitate started to change its color from colorless to black. As yields were low after min. of reaction time, the BTC activation described above was favored. Solid Phase Synthesis: First residue attachment on Wang resin: Wang resin (3.6 g,.9 mmol) was swollen in the minimum amount of NMP for 3 min. Amino acid (9 g, 9 mmol) was dissolved in NMP (2 ml) and thionyl chloride (.3 ml, 9 mmol) was added. After a reaction time of h, this solution was added to Wang resin to react for another 24 h. After the resin was drained and washed with NMP (5x), it was capped with 4-nitrobenzoyl chloride (3 g, 8 mmol) in NMP ( ml). Analysis by RP-HPLC gave a degree of functionalization of 99 % (comparing the signal intensities for 4-nitrobenzoic acid and amino acid ). N-(p-Methoxy benzyl) protected hepta(p-benzamide) (2g): Functionalized Wang resin (476 mg,.25 mmol) was placed in the reaction vessel of the peptide synthesizer. A mixture of amino acid (2.9 g, 6 mmol) and bis(trichloromethyl)carbonate (.7 g, 2 mmol) was dissolved in NMP (2 ml). After dissolution of the reagents, the solution was filtered through a 4 micron syringe filter and filled into 6 amino acid cartridges of the peptide synthesizer (each containing mmol of activated in 2 ml of NMP). Following each cartridge containing monomer, a cartridge filled with a solution of 4- nitrobenzoyl chloride (85 mg, mmol) in NMP (2 ml) was placed in the amino acid sequence of the synthesizer as a capping reagent. The automated synthesis started with the removal of the Fmoc-group of the functionalized Wang resin followed by six monomer and capping reagent coupling cycles. The success of the complete reaction sequence was analyzed by RP-HPLC. Pentynoic acid chloride (3):

4 Pentynoic acid (2 mg, 2 mmol) was dissolved in dry NMP (2 ml). Then, SOCl 2 (3.5 μl,.8 mmol) was added to the mixture and stirred at rt for min. The NMP-solution of activated 3 was used on the solid support without further purification. Alkyne functionalized N-(p-Methoxy benzyl) protected hepta(p-benzamide) (4): The freshly prepared NMP solution of 3 was given to solid supported 2g and reacted for 2 h to give 4. Solid supported 4 was washed with NMP (5x) and used directly in further reaction steps. An analytical amount of 4 was cleaved off the solid support by applying TFA/DCM (5 %) for 3 min and was analyzed by ESI MS. ESI MS [ H] + Azide functionalized poly(ethylene glycol) monomethyl ether (5): Poly(ethylene glycol) monomethyl ether (4 g,.2 mol) was dissolved in pyridine (2 ml) and 4-toluenesulfonyl chloride (38.2 g,.2 mol) was added. The reaction mixture was stirred for 2 h at room temperature while its color changed from slightly pink to yellow. The solution was poured into ice cold water (2 ml) and was then extracted three times with DCM. The organic layer was washed twice with cold hydrochloric acid (6 M) and three times with cold distilled water. Then it was dried with magnesium sulfate and the solvent was removed in vacuum. *) The resulting white precipitate was dissolved in DMF (2 ml) and sodium azide (3 g,.2 mol) was added. The resulting yellow solution was stirred for 24 h at 2 C until its color turned red. The solvent was removed in vacuum and the resulting yellow solid was dissolved in DCM (5 ml) to get precipitated in ice cold ether. After precipitation the yellow product was obtained by filtration and was dried in the vaccum oven at 4 C (34 g,.7 mol, 85 %). FT-IR (cm - ): 2874 (-CH 2 CH 2 -), 24 (-N 3 ); ESI-MS: [ Na] + *) According to J. A. Opsteen, J. C. M. Hest, van, Chem. Comm. 25, 57. Investigation of coupling conditions by RP-HPLC: (a) (a) (b) (b) (c) (c) elution time elution time Figure SI-2: RP-HPLC elugrams: (a) N-protected hepta-p-benzamide 2g; (b) block copolymer 7 via CuI catalyzed click reaction (c) block copolymer 7 via CuSO 4 x 5 H 2 O / sodium ascorbate catalyzed click reaction. Left: Full elugrams, right: magnification of the elugrams on the left. Elugram (b) shows the presence of residual OPBA. The Huisgen [2+3]-cycloaddition was performed with CuI as well as CuSO 4 x 5 H 2 O / sodium ascorbate. The success of both reactions was established by analysis of the crude reaction

5 mixture by RP-HPLC. The elugram of the reaction catalyzed by CuI is shown in Figure SI-2 (b), the one catalyzed by CuSO 4 x 5 H 2 O / sodium ascorbate in Figure SI-2 (c). Even though both click reactions showed a clear disappearance of the N-PMB protected hepta-p-benzamide signals (see Figure SI-2 (a)), the reaction catalyzed with CuSO 4 x 5 H 2 O / sodium ascorbate turned out to operate slightly better than the one catalyzed with CuI. Huisgen [2+3]-cycloaddition catalyzed by CuSO 4 x 5 H 2 O / sodium ascorbate (6): Solid supported 4 ( mg,.2 mmol) was swollen in the minimum amount of NMP and 5 (72 mg,.36 mmol) dissolved in NMP (3 ml) was added. Aqueous sodium ascorbate solution ( M,.6 ml) and aqueous CuSO 4 x 5 H 2 O ( M,.4 ml) was added and the suspension was reacted while shaking for 24 h at room temperature. 6 was obtained by draining the resin and washing with NMP (3x) and DCM (5x). Huisgen [2+3]-cycloaddition catalyzed by CuI / DBU (6): Solid supported 4 ( mg,.2 mmol) was swollen in the minimum amount of DMF and 5 (72 mg,.36 mmol) dissolved in DMF (3 ml) was added. DBU ( mg,.6 mmol) and CuI (93 mg, 6 mmol) was added and the suspension reacted while shaking for 24 h at 5 C. 6 was obtained by draining the resin and washing with DMF (x 3) and DCM (x 5). N-(p-Methoxy benzyl) protected hepta(p-benzamide)-b-poly(ethylene glycol) monomethyl ether (7): The block copolymer was cleaved from Wang resin (initial loading.25 mmol) by treatment with a TFA / DCM solution (5 %, 3 ml). After a reaction time of 3 min., the solid support was removed by filtration followed by washing with DCM several times. Solvent and cleaving reagent was removed under vacuum to give crude 7 (8 mg,.2 mmol, 83 %), which was purified by preparative GPC in chloroform (26 mg,.6 mmol, 3 %). ESI MS: [ K Na + ] +++ H-NMR (DMF-d 7 ): δ (ppm) 7.82 (m, 3 H), (m, 53 H), (m, 4 H), 4.83 (s, H), 4.53 (t, 2 H, 5.3 Hz), 3.87 (t, 2 H, 5.3 Hz), 3.74 (m, 2 H), 3.58 (s, 76 H), 3.29 (s, 3 H)

6 7, triply charged 7, quadruply charged m/z C 3 H 4 N O 7 (C 2 H 4 O) 45 (Na + ) 2 K + C 3 H 4 N O 7 (C 2 H 4 O) 46 (Na + ) 2 K + C 3 H 4 N O 7 (C 2 H 4 O) 46 (Na + ) 3 C 3 H 4 N O 7 (C 2 H 4 O) 45 Na + (K + ) m/z Figure SI-3: ESI-mass spectrum of 7. Top: full spectrum; bottom: section of the triply charged mass distribution. Hepta(p-benzamide)-b-poly(ethylene glycol) monomethyl ether (8): Purified 7 ( mg,.25 mmol) was dissolved in a mixture of TFA (.8 ml) and EDT (.2 ml, as scavenger for the PMB cations). The cleavage of the PMB protection groups was allowed to take place for 48 h at room temperature. During the reaction, a colorless precipitation occurred. For this reason, the reaction mixture was kept in a sonicator for an additional.5 h to ensure de-aggregation and hence complete cleavage of the PMB-protective groups. After evaporation of the volatile reagents, the block copolymer was purified by preparative GPC in DMF to give pure 8 (3 mg,.9 mmol, 4 % ). H-NMR (DMF-d 7 ): δ (ppm).67 (s, H),.57 (s, 6 H), 8.6 (s, 2H), (m, 2 H), 7.38 (d, 2 H), 7.9 (d, 2 H), 6.88 (d, 2 H), 4.95 (s, H), 4.55 (t, 2 H, 5 Hz), 3.89 (t, 2 H, 5.4 Hz), 3.57 (s, 76 H), 3.28 (s, 3 H)

7 PMB-protective groups deprotected elution volume / ml Figure SI-4: Removal of cleaved PMB-protective groups by prep GPC in DMF. toluene internal standard elution volume / ml Figure SI-5: GPC elugram (UV detection at 254 nm) of polymers 7 (solid line) and 8 (dashed line) in chloroform. The aggregate of 8 elutes close to the exclusion limit (= high molecular weight limit) of the GPC setup.

8 Figure SI-6: Cryo-TEM image of 8 in chloroform (c = g L-). For vitrification a FEI Vitrobot was used. The temperature in the chamber was kept constant at C. Preparation of the TEM and STEM grids: 8 (.5 mg) was dissolved in CHCl3 p.a. ( ml) and was allowed to equilibrate at 22 C for 48 h. The TEM grid was positioned on a filter paper and the prepared solution of 8 drop cast onto the grid. To remove solvent residues, the grid was dried for min in vacuum. Protocol of Synthesis using the Applied Biosystems Model 43A Peptide Synthesizer: 43A Chemistry Fmoc (Standard) [.25 mmol]: middle cycle with capping deprotection and NMP wash NMP wash only rinse cycle GgbdefffGgeff gbd gd E Only module G and E do not correspond to standard modules of the peptide synthesizer and were modified: Module: G > Activated-Monomer Solution Needle Down Interrupts function Drain amino acid cartridge Vent amino acid cartridge Needle Up Eject Cartridge Advance Cartridge Needle Down Mix Activator Drain Activator #9 thru top valve block #9 to top of ACT with drain Drain Activator # thru bottom valve block

9 5 Gas thru bottom valve block Mix amino acid cartridge Cartridge to Activator BEGIN LOOP Cartridge to Activator 5 2 Wait END LOOP # to amino acid cartridge 2 23 Gas thru bottom valve block Mix Activator 25 4 # thru bottom valve block 26 Gas thru bottom valve block # thru bottom valve block 28 Wait 5 29 Gas thru bottom valve block Mix Activator 3 2 #9 thru bottom valve block 32 Wait 5 33 Gas thru bottom valve block # thru bottom valve block 35 9 Gas thru top valve block 36 Gas thru bottom valve block 37 6 Vent amino acid cartridge Drain amino acid cartridge 39 6 Vent amino acid cartridge 4 62 Drain amino acid cartridge Module: E -> Rinse Cycle 5 Needle Down 2 36 # ACT thru bottom valve bloc 3 2 Mix Activator Drain Activator #9 to Activator 6 2 Mix Activator Drain Activator Drain amino acid cartridge # to amino acid cartridge 8 6 Mix amino acid cartridge 62 Drain amino acid cartridge #9 to amino acid cartridge Mix amino acid cartridge 4 62 Drain amino acid cartridge # to amino acid cartridge Mix amino acid cartridge 7 22 Drain Activator Cartridge to Activator Drain Activator #9 to amino acid cartridge Cartridge to Activator Drain Activator #9 to top of ACT with drain Drain Activator Drain amino acid cartridge Vent amino acid cartridge Drain amino acid cartridge Vent amino acid cartridge Drain amino acid cartridge 6