Memantine-Hcl Lab Report
An old well-known method to stabilize a peptide sequences or protein motifs is N-terminal and C-Terminal modification to resistance against exo-nucleases cleavage. Due to its feasibility, we decided to perform this type of modification in order to stabilize synthesized sequences based on previous protocols. Understanding the appropriate compound and choosing the way of performance, were sensitive and definitely time consuming. Finally, N-terminal modification with 5(6) Carboxyfluorescein (FAM) in sequences was used in uptake studies and Pyroglutamic acid (pGlu) in sequences was chosen in order to use in cytotoxicity and cell viability assays. On the other hand, C-terminal modification was done by applying Memantine (Mem) in sequences used in
…show more content…
Some deionized water was added and drug powder was dissolved in water. Then required amount of DCM was added. The pH adjustment was done by adding a few drops of TEA, afterwards two distinctive phases were formed. pH value of supernatant was measured quickly (pH-meter showed number 11-12). The reaction flask was heated and shaken for 30 minutes, followed by decantation to obtain base Memantine (without Hcl). The reaction flask was connected to rotary evaporator equipped with vacuum pump to remove excess solvent (i.e., …show more content…
= 17.70 µl) and HOBt (5 eq. = 36.48 mg) in the presence of TBTU (5 eq. = 86.70 mg) as a coupling reagent, DIPEA (5 eq. = 47 µl) as the base and DCM (10 ml) as solvent at room temperature. The mixture was stirred overnight.
3.3.3 Final Deprotection of protected peptide fragments
According to different yielding efficiency, there are several protocols correlated with peptide final cleavage. “Odorless” TIS Cleavage Cocktail was chosen due to its optimum yielding, as well as odorless contents in comparison with EDT (1,2-ethanedithiol) and thioanisole. TIPS (Triisopropylsylane) is used in Reagent B to scavenge cationic species. Reagent B is especially useful when the resin-product contains trityl-based protecting groups. The most important point in Reagent B utilization was immediate use after cocktail preparation. There were two process in our final deprotection and cleavage of peptides:
1) In the case of cleaving unmodified peptides or modified only at N-terminus like; peptide-MTX conjugates.
2) In the case of cleaving N-acetylated peptides which would be amidated at C-terminus in the next step.
Composition of Reagent
Some deionized water was added and drug powder was dissolved in water. Then required amount of DCM was added. The pH adjustment was done by adding a few drops of TEA, afterwards two distinctive phases were formed. pH value of supernatant was measured quickly (pH-meter showed number 11-12). The reaction flask was heated and shaken for 30 minutes, followed by decantation to obtain base Memantine (without Hcl). The reaction flask was connected to rotary evaporator equipped with vacuum pump to remove excess solvent (i.e., …show more content…
= 17.70 µl) and HOBt (5 eq. = 36.48 mg) in the presence of TBTU (5 eq. = 86.70 mg) as a coupling reagent, DIPEA (5 eq. = 47 µl) as the base and DCM (10 ml) as solvent at room temperature. The mixture was stirred overnight.
3.3.3 Final Deprotection of protected peptide fragments
According to different yielding efficiency, there are several protocols correlated with peptide final cleavage. “Odorless” TIS Cleavage Cocktail was chosen due to its optimum yielding, as well as odorless contents in comparison with EDT (1,2-ethanedithiol) and thioanisole. TIPS (Triisopropylsylane) is used in Reagent B to scavenge cationic species. Reagent B is especially useful when the resin-product contains trityl-based protecting groups. The most important point in Reagent B utilization was immediate use after cocktail preparation. There were two process in our final deprotection and cleavage of peptides:
1) In the case of cleaving unmodified peptides or modified only at N-terminus like; peptide-MTX conjugates.
2) In the case of cleaving N-acetylated peptides which would be amidated at C-terminus in the next step.
Composition of Reagent