6. Experimental section
6.8 Phosphoserine modified zinc finger domains
Fmoc-O-benzyl-L-phosphoserine
PCl3 (0.70 mL, 8.00 mmol, 1.3 eq) was dissolved in THF (15.0 mL) and cooled to 0 °C.
BnOH (0.96 mL, 9.20 mmol, 1.5 eq) was added slowly to keep the temperature of the solution below 5 °C. The reaction mixture was allowed to stir for 60 min at 0 °C. 2,6-Lutidine (2.12 mL, 18.3 mmol, 3.00 eq) was slowly added to keep the internal temperature between -5 to 5 °C. During rigorous stirring, a thick slurry formed.
2,6-Lutidine (0.72 mL, 6.20 mmol, 1.0 eq) was added to a solution of Fmoc-L-serine-OH (2.00 g, 6.10 mmol, 1.0 eq) in THF (10.0 mL). The solution was subsequently added to the slurry while the temperature was kept at 0 °C. The reaction mixture was allowed to stirred at 5 °C for 12 h. The reaction was quenched by the addition of H2O (8.0 mL) at a rate that kept the temperature below 10 °C. A biphasic mixture was formed which was continuously stirred while NaBr (1.46 g, 14.2 mmol, 2.3 eq) was added at 0 °C. Subsequently, an aqueous solution of NaBrO3 (0.44 g, 3.0 mmol, 0.48 eq) was added to the solution at 0 °C. The reaction mixture was allowed to warm to room temperature and stirring was continued for 2 h. An aqueous solution of Na2S2O5 (10 wt% in H2O, 2.0 mL) was quickly added and stirring was continued for 1 h.
2-MeTHF (20 mL) was added to the biphasic mixture and the layers were separated. The organic layer was washed with brine (2 x 25 mL) and dried over MgSO4. The solvent was removed under reduced pressure to yield the crude product as a brown oil. The latter was diluted with 2-MeTHF (7 mL/g) and placed in a freezer at –22 °C over night. A white participate formed, which was filtrated and recrystallized from 2-MeTHF to give product 35 (2.52 g, 5.07 mmol, 83%) as a white solid.
1H-NMR (300 MHz, [D6]-DMSO): = 4.08 – 4.48 (m, 6H), 4.97 (d, JHH = 7.1 Hz, 2H), 7.27 – 7.46 (m, 8H), 7.73 (d, JHH = 7.4 Hz, 2H), 7.79 (d, JHH = 8.2 Hz, 1H), 7.87 (d, JHH = 7.5 Hz, 2H) ppm.
13C-NMR (300 MHz, [D6]-DMSO): = 46.61, 54.44 (d, J = 7.9 Hz), 65.33 (d, J = 7.6 Hz), 65.98, 67.56 (d, J = 7.6 Hz), 120.04, 125.27, 127.06, 127.56, 127.62, 127.99, 128.33, 136.76 (d, J = 7.6 Hz), 140.70, 143.75, 156.00, 170.73 ppm.
31P-NMR (122 MHz, [D6]-DMSO): = –1.48 ppm.
ESI-MS: 498.1 [M+H]+, 520.1 [M+Na]+.
ESI-HRMS: calculated for [C25H24NO8P]+ ([M+H]+) = 498.1312, found = 498.1311, calculated for [C25H24NO8PNa]+ ([M+Na]+) = 520.1132, found = 520.1127.
Zf3Pser70
Peptide 36 was synthesized on a pre-loaded Fmoc-L-Lys(Boc)-Wang resin (0.34 mmol/g) at a scale of 0.05 mmol using an automated peptide synthesizer (Liberty Blue, CEM). Standard amino acids were coupled according to protocol SPPS-A2 described in section 6.3.1.
Coupling of the Fmoc-O-benzyl-L-phosphoserine (35) building block as well as of the subsequent amino acid was performed according to protocol SPPS-A3. Cleavage of the peptide from the resin was achieved by the addition of a cleavage cocktail containing TFA/H2O/EDT/TIS (94/2.5/2.5/1) and incubation for 2 h. The peptide was purified by RP-HPLC and subsequently lyophilized. Peptide 36 (103 mg, 33.3 mmol, 41.1%) was obtained as a white solid with a disulfide bond between the cysteine residues.
HPLC (RP-C18, semi-preparative, A (99.9% H2O, 0.10% TFA), B (79.9% ACN, 20.0% H2O, 0.10% TFA), gradient 10-60% B, 30 min, 3 mL/min): tR = 15.38 min.
ESI-MS m/z: 516.4 [M+6H]6+, 619.5 [M+5H]5+, 774.2 [M+4H]4+.
ESI-HRMS: calculated for [C126H215N46O39PS2]6+ ([M+6H]6+) = 516.2645, found = 516.2650, calculated for [C126H215N46O39PS2]5+ ([M+5H]5+) = 619.3159, found = 619.3163, calculated for [C126H215N46O39PS2]4+ ([M+4H]4+) = 773.8931, found = 773.8933.
Zf3Pser75
Peptide 37 was synthesized on a pre-loaded Fmoc-L-Lys(Boc)-Wang resin (0.34 mmol/g) at a scale of 0.05 mmol using an automated peptide synthesizer (Liberty Blue, CEM). Standard amino acids were coupled according to protocol SPPS-A2 described in section 6.3.1.
Coupling of the Fmoc-O-benzyl-L-phosphoserine (35) building block as well as of the subsequent amino acid was performed according to protocol SPPS-A3. Cleavage of the peptide from the resin was achieved by the addition of a cleavage cocktail containing TFA/H2O/EDT/TIS (94/2.5/2.5/1) and incubation for 2 h. The peptide was purified by RP-HPLC and subsequently lyophilized. Peptide 37 (98.0 mg, 31.6 mmol, 27.2%) was obtained as a white solid with a disulfide bond between the cysteine residues.
HPLC (RP-C18, semi-preparative, A (99.9% H2O, 0.10% TFA), B (79.9% ACN, 20.0% H2O, 0.10% TFA), gradient 10-60% B, 30 min, 3 mL/min): tR = 14.42 min.
ESI-MS m/z: 527.6 [M+6H]6+, 632.9 [M+5H]5+, 790.9 [M+4H]4+.
ESI-HRMS: calculated for [C129H220N49O38PS2]6+ ([M+6H]6+) = 527.4401, found = 527.4414,
calculated for [C129H220N49O38PS2]5+ ([M+5H]5+) = 632.7266, found = 632.7273, calculated for [C129H220N49O38PS2]4+ ([M+4H]4+) = 790.6565, found = 790.6570.
SerPEGFITC
Compound 38 was synthesized by the means of solid phase organic synthesis on a Rink amide MBHA resin (0.18 mmol/g) at a scale of 0.05 mmol. Coupling was performed manually with microwave support (CEM) according to the protocol SPPS-M1 described in section 6.3.1. Cleavage of the compound from the resin was achieved by the addition of a cleavage cocktail containing TFA/H2O/EDT/TIS (94/2.5/2.5/1) and incubation for 2 h. The peptide was purified by RP-HPLC and subsequently lyophilized. Compound 38 (21.0 mg, 0.32 mmol, 72%) was obtained as a white solid.
HPLC (RP-C18, semi-preparative, A (99.9% H2O, 0.10% TFA), B (79.9% ACN, 20.0% H2O, 0.10% TFA), gradient 10-70% B, 30 min, 3 mL/min): tR = 24.38 min.
ESI-MS m/z: 639.2 [M+H]+, 661.2 [M+Na]+.
ESI-HRMS: calculated for [C30H30N4O10S]+ ([M+H]+) = 639.1755, found = 639.1749, calculated for [C30H30N4O10SNa]+ ([M+Na]+) = 661.1575, found = 661.1565.
PserPEGFITC
Compound 39 was synthesized by the means of solid phase organic synthesis on a Rink amide MBHA resin (0.18 mmol/g) at a scale of 0.05 mmol. Coupling was performed manually with microwave support (CEM) according to the protocol SPPS-M1 described in section 6.3.1. Cleavage of the compound from the resin was achieved by the addition of a cleavage cocktail containing TFA/H2O/EDT/TIS (94/2.5/2.5/1) and incubation for 2 h. The peptide was purified by RP-HPLC and subsequently lyophilized. Compound 39 (18.3 mg, 0.25 mmol, 64%) was obtained as a white solid.
HPLC (RP-C18, semi-preparative, A (99.9% H2O, 0.10% TFA), B (79.9% ACN, 20.0% H2O, 0.10% TFA), gradient 10-70% B, 30 min, 3 mL/min): tR = 25.31 min.
ESI-MS m/z: 717.1 [M-H]-, 358.1 [M-2H]-2-.
ESI-HRMS: calculated for [C30H31N4O13PS]- ([M-H]–) = 717.1273, found = 717.1272, calculated for [C30H30N4O10S]-2 ([M-2H]-2) = 358.0600, found = 358.0604.
Zf3FITC wildtype
Peptide 40 was synthesized on a pre-loaded Fmoc-L-Lys(Boc)-Wang resin (0.34 mmol/g) at a scale of 0.05 mmol using an automated peptide synthesizer (Liberty Blue, CEM). Standard amino acids were coupled according to protocol SPPS-A2 described in section 6.3.1.
Coupling of the N-terminal PEG spacer (8-(Fmoc-amino)-3,6-dioxaoctanoic acid) and of the FITC fluorophore was performed manually according to protocol SPPS-M1. Cleavage of the peptide from the resin was achieved by the addition of a cleavage cocktail containing TFA/H2O/EDT/TIS (94/2.5/2.5/1) and incubation for 2 h. The peptide was purified by RP-HPLC and subsequently lyophilized. Peptide 40 (42.0 mg, 11.1 mmol, 22.2%) was obtained as a white solid with a disulfide bond between the cysteine residues.
HPLC (RP-C18, semi-preparative, A (99.9% H2O, 0.10% TFA), B (79.9% ACN, 20.0% H2O, 0.10% TFA), gradient 10-60% B, 30 min, 3 mL/min): tR = 18.45 min.
ESI-MS m/z: 517.7 [M+7H]7+, 603.8 [M+6H]6+, 724.4 [M+5H]5+.
ESI-HRMS: calculated for [C156H243N51O43S3]7+ ([M+7H]7+) = 517.4010, found = 517.4006, calculated for [C156H243N51O43S3]6+ ([M+6H]6+) = 603.4666, found = 603.4661, calculated for [C156H243N51O43S3]5+ ([M+5H]5+) = 723.9584, found = 723.9582.
Zf3Pser75NBD
Peptide 41 was synthesized on a pre-loaded Fmoc-L-Lys(Boc)-Wang resin (0.34 mmol/g) at a scale of 0.05 mmol using an automated peptide synthesizer (Liberty Blue, CEM). Standard amino acids were coupled according to protocol SPPS-A2 described in section 6.3.1.
Coupling of the Fmoc-O-benzyl-L-phosphoserine (35) building block as well as of the subsequent amino acid was performed according to protocol SPPS-A3. Coupling of the N-terminal NBD fluorophore was achieved manually. Cleavage of the peptide from the resin was achieved by the addition of a cleavage cocktail containing TFA/H2O/EDT/TIS (94/2.5/2.5/1) and incubation for 2 h. The peptide was purified by RP-HPLC and subsequently lyophilized. Peptide 41 (12 mg, 0.04 mmol, 8.2%) was obtained as a white solid with a disulfide bond between the cysteine residues.
HPLC (RP-C18, semi-preparative, A (99.9% H2O, 0.10% TFA), B (79.9% ACN, 20.0% H2O, 0.10% TFA), gradient 10-60% B, 30 min, 3 mL/min): tR = 21.45 min.
ESI-MS m/z: 554.8 [M+6H]6+, 665.5 [M+5H]5+, 831.6 [M+4H]4+.
ESI-HRMS: calculated for [C135H221N52O41PS2]6+ ([M+6H]6+) = 554.6070, found = 554.6070, calculated for [C135H221N52O41PS2]5+ ([M+5H]5+) = 665.3270, found = 665.3274, calculated for [C135H221N52O41PS2]4+ ([M+4H]4+) = 831.4069, found = 831.4073.
Zf3Pser70FITC
Peptide 42 was synthesized on a pre-loaded Fmoc-L-Lys(Boc)-Wang resin (0.34 mmol/g) at a scale of 0.05 mmol using an automated peptide synthesizer (Liberty Blue, CEM). Standard amino acids were coupled according to protocol SPPS-A2 described in section 6.3.1. The building block Fmoc-O-benzyl-L-phosphoserine (35) as well as the subsequent amino acid were coupled according to protocol SPPS-A3. Coupling of the N-terminal PEG spacer (8-(Fmoc-amino)-3,6-dioxaoctanoic acid) and of the FITC fluorophore was performed manually according to protocol SPPS-M1. Cleavage of the peptide from the resin was achieved by the addition of a cleavage cocktail containing TFA/H2O/EDT/TIS (94/2.5/2.5/1) and incubation for 2 h. The peptide was purified by RP-HPLC and subsequently lyophilized. Peptide 42 (23.0 mg, 0.63 mmol, 15.2%) was obtained as a white solid with a disulfide bond between the cysteine residues.
HPLC (RP-C18, semi-preparative, A (99.9% H2O, 0.10% TFA), B (79.9% ACN, 20.0% H2O, 0.10% TFA), gradient 10-60% B, 30 min, 3 mL/min): tR = 22.35 min.
ESI-MS m/z: 605.6 [M+6H]6+, 726.5 [M+5H]5+, 907.9 [M+4H]4+.
ESI-HRMS: calculated for [C153H237N48O47PS3]6+ ([M+6H]6+) = 605.2828, found = 605.2826, calculated for [C153H237N48O47PS3]5+ ([M+5H]5+) = 726.1379, found = 726.1384, calculated for [C153H237N48O47PS3]4+ ([M+4H]4+) = 907.4205, found = 907.4214.
Zf13Pser70
Peptide 43 was prepared by NCL between Zf3Pser70 (36) and the expressed Zf12-thioester domain 27 according to the method described in section 6.2.
Yield: 1.18 mg (0.17 mol, 22%)
HPLC (RP-C18, analytical, A (99.9% H2O, 0.10% TFA), B (79.9% ACN, 20.0% H2O, 0.10%
TFA), gradient 20-50% B, 30 min, 1 mL/min): tR = 25.35 min.
ESI-MS m/z: 717.5 [M+15H]15+, 768.4 [M+14H]14+, 827.4 [M+13H]13+.
ESI-HRMS: calculated for [C452H729N156O134PS7]15+ ([M+15H]15+) = 717.0927, found = 717.0936, [C452H729N156O134PS7]14+ ([M+14H]14+) = 768.2416, found = 768.2522.
Zf13Pser75
Peptide 44 was prepared by NCL between Zf3Pser75 (37) and the expressed Zf12-thioester domain 27 according to the method described in section 6.2.
Yield: 0.94 mg (0.09 mol, 12%)
HPLC (RP-C18, analytical, A (99.9% H2O, 0.10% TFA), B (79.9% ACN, 20.0% H2O, 0.10%
TFA), gradient 20-50% B, 30 min, 1 mL/min): tR = 25.12 min.
ESI-MS m/z: 721.8 [M+15H]15+, 773.2 [M+14H]14+, 832.6 [M+13H]13+.
ESI-HRMS: calculated for [C455H736N159O133PS7]15+ ([M+15H]15+) = 721.6973, found = 721.6981, [C455H736N159O133PS7]14+ ([M+14H]14+) = 773.1751, found = 773.1756.