2.6.1. General consideration
All reagents were purchased from commercial sources and were used without any further purification unless mentioned otherwise. The o-nitrobenzenesulfonyl chloride and DIPEA purchased from Sigma-Aldrich and DCM purchased from Merck. DCM is distilled and dried using standard procedure. Melting points were uncorrected. Thin layer chromatograms were run on glass plates coated with silica gel G for TLC, using solvent systems EtOAc/Hexane. Compounds were purified by column chromatography using Silica Gel (60-120 mesh) with EtOAc/Hexane (of specific proportion as required) as an eluent. 1H NMR (400 MHz and 600 MHz) and 13C NMR (100 MHz and 150 MHz) were recorded using CDCl3 as solvent. Chemical shifts (δ) were reported in parts per million (ppm), internal reference (0.05% to 1%) tetramethylsilane. Coupling constants (J) are reported in Hz singlet (s), doublet (d), triplet (t), doublet of doublet (dd), multiplet (m), or broad (br). High resolution mass spectrometry (HRMS) was recorded on a Q-TOF ESI- MS instrument and a Q-TOF LC/MS system and GCMS were recorded on a gas chromatograph. HPLC analysis was carried out with either a C8 (5 µm, 3.5×150 mm) or a C18 (5 µm, 4.6×250 mm) reverse phase column and a chiral column (5 µm, 2.1×150 mm) attached with an UV detector. HPLC grade solvents were used for HPLC analysis. IR spectra were recorded on an IR spectrometer; X-ray data were collected on a diffractometer equipped with a CCD area detector using Mo. Optical rotation was recorded on Rudolph research analytical autopol II automatic polarimeter using one ml glass cell and chloroform as solvent. Data for previously reported compounds (cited) matched well with our observed data.
2.6.2. Procedure for the synthesis of the coupling reagent ethyl 2-cyano-2-(2- nitrobenzenesulfonyloxyimino)acetate (o-NosylOXY, I)
DIPEA (1 equiv) was added to a solution of Oxyma (1 equiv) in 2 mL of DCM under nitrogen. The reaction mixture was cooled up to 0 °C and o-nitrobenzenesulfonyl chloride (1 equiv) was added drop wise during 30 min. The reaction mixture was then stirred for another 2 h at room temperature. After completion of the reaction, the reaction mixture was diluted with 10 mL of DCM and washed with 5% of HCl (3×5 mL). Finally organic layer was dried by anhydrous CaCl2 and recrystallized with hexane.
2.6.3. General procedure for the synthesis of amide
o-NosylOXY (1 equiv) was added to a solution of carboxylic acid (1 equiv) and DIPEA (1 equiv) in 2 mL of DCM. The reaction mixture was stirred for 3-5 min for pre- activation, followed by the addition of amine (1 equiv). The reaction mixture was stirred at room temperature for 2-3 h. After completion, the reaction mixture was diluted with 50 mL of ethyl acetate; the organic phase was washed with 5% citric acid (3×20 mL), 5%
NaHCO3 (3×20 mL) and dried by anhydrous Na2SO4. Finally, Na2SO4 was filtered off and the solvent was evaporated to obtain the product which was purified by column chromatography.
2.6.4. General procedure for the synthesis of dipeptides
To a solution of N-protected amino acid (1 equiv) and o-NosylOXY (1 equiv) in 2 mL of DCM, DIPEA (1.1 equiv) was added. The reaction mixture was stirred for 5 min for preactivation followed by the addition of methyl ester of the second amino acid (1.1 equiv) and DIPEA (1.1 equiv) in 1 mL of DCM. The reaction mixture was stirred at room
Chapter 2 Ethyl 2-Cyano-2-(2-nitrobenzenesulfonyloxyimino)acetate (o-NosylOXY): A New Coupling Reagent for Racemization-Free Synthesis of Amide and Peptide
temperature for more 2-3 h. After completion, the reaction mixture was diluted with 50 mL of ethyl acetate, the organic phase was washed with 5% citric acid (3×20 mL), 5%
NaHCO3 (3×20 mL), brine and dried over anhydrous Na2SO4. Finally, Na2SO4 was filtered and the solvent was evaporated. The product was purified by silica gel column chromatography.
2.6.5. Synthesis of Boc-VVIA-OMe in solution phase
Boc-isoleucine (1 equiv) and o-NosylOXY (1 equiv) was taken in a 25 mL RB, after that DIPEA (1.5 equiv) was added and kept 5 min for pre-activation. In another oven dried 50 mL RB, methyl ester of alanine (1.5 equiv) was taken in DCM and DIPEA was added to it till basic pH was reached. Finally, that solution was added in first RB and stirring was continued until completion of the reaction. Then, the reaction mixture was diluted by 50 mL of EtOAc, washed by 5% NaHCO3 solution (2×5 mL) and washed by 5% citric acid solution (2×5 mL). Finally, combined organic layer was dried using anhydrous Na2SO4. Solid product (Boc-IA-OMe) was obtained after evaporation of EtOAc by rotary vacuum evaporator.
In an oven dried 50 mL RB, Boc-IA-OMe was taken and 70% TFA solution in DCM was added to it. Stirring continued up to 2.5 h. After that, TFA was evaporated by rotary evaporator and washed 3 times with diethyl ether and finally white solid (IA-OMe) was obtained.
After Boc deprotection, resulted IA-OMe was coupled with Boc-V-OH following the above mentioned procedure to obtain Boc-VIA-OMe. Another cycle of Boc-deprotection and coupling with Boc-V-OH, resulted white solid Boc-VVIA-OMe, which was
characterized by reverse phase HPLC, retention time was 12.8 min on linear gradient of 20 to 100% CH3CN/0.09% TFA in H2O/0.09% TFA over 18 min, symmetry C8 analytical column and LRMS (ESI): m/z [M+H]+ calculated for C25H47N4O7 is 537.3264 found 537.3421. Yield was 78% with respect to starting material Boc-isoleucine.
2.6.6. Solid phase synthesis of NFGAILG-NH2 and VQAAIDYING-NH2
Specific amino acids were manually assembled on Fmoc-Rink Amide MBHA resin (loading 1.1 mmol/g) following Fmoc-tBut orthogonal protection strategy. Fmoc-amino acids (3.2 equiv), o-NosylOXY (3 equiv) and DIPEA (5 equiv) was kept for pre- activation for 5 min. Amino acid coupling was performed for 2 h. Fmoc deprotection was carried out with 25% piperidine/DMF (3×7 min). The peptide was cleaved from the resin using TFA/DCM/H2O (9/0.5/0.5) mixture for 2 h 30 min. Purification of the peptide was performed using semi-preparative HPLC using a linear gradient of 20 min (5 to 100%
CH3CN in water with 0.09% TFA) and characterized using ESI-MS.
For NFGAILG-NH2, we took 300 mg of Fmoc-Rink Amide resin (loading 1.1 mmol/g), after final coupling, peptide was cleaved from resin and purified by semi-preparative HPLC. The purified yield of peptide was 90 mg, 40%, HPLC profile and ESI-MS spectrum (Figure S25-S26). And for VQAAIDYING-NH2, we took 300 mg of Fmoc- Rink Amide resin (loading 1.1 mmol/g), after final coupling, this peptide was also cleaved from resin and purified by semi-preparative HPLC. Yield of the purified peptide was 175 mg, 50%. HPLC profile and ESI-MS spectrum are provided in Figure S29-S30.
Chapter 2 Ethyl 2-Cyano-2-(2-nitrobenzenesulfonyloxyimino)acetate (o-NosylOXY): A New Coupling Reagent for Racemization-Free Synthesis of Amide and Peptide