1-pyrenehexanoic acid was used as the 5-carbon linker and fluorescent component of the target molecule "PYRET-5". This molecule was purchased pure along with the sugar Trehalose, the molecule to which the pyrene was to be attached. Other reactants and solvents include HATU, HBTU, N, N-Diisopropylethylamine (DIPEA), sodium azide, Dimethylformamide (DMF), and Diphenyl phosphoryl chloride and were bought pure or used as received. All reactions were run at room temperature (298.15 K) and standard pressure unless otherwise noted.
The routine lab equipment included a rotary evaporator (evaporating solvents off by lowering boiling point through pressure and temperature changes), analytical balances measuring to one tenth of a milligram, and a lyophilizer (freeze-dryer).
Synthesizing "PYRET-5" molecules
"PYRET-5" (refer to Scheme 1 for structure) was the target molecule to be synthesized by replacing one of the –OH groups with 1-pyrenehexanoic which would be connected by either an ester or an amide group. Both compounds, the ester and the amide, were synthesized with the pyrene as the limiting reagent, which was measured out to be about 50 milligrams (~0.16 millimoles) for each reaction. The first reaction to synthesize the ester was run in a round bottom flask starting with 1-pyrenehexanoic acid (refer to Table 1. for all exact weights and moles). One equivalent of HBTU (O-benzotriazole- N,N,N′,N′ -tetramethyluroniumphosphate) was added as a coupling agent along with 1.2 equivalents DIPEA which serves as a non-nucleophilic base and tertiary amine for the coupling step (1). The reagents were all dissolved over DMF and allowed to stir for five minutes before adding three equivalents of trehalose (dehydrated). A color change to a dull gold hue was observed upon the addition of trehalose. This reaction was left to stir for approximately 72 hours before confirming the reaction’s progress with (TLC) thin-layer chromatography. The second reaction to synthesize the ester was also run in a round bottom flask with 1-pyrenehexanoic acid (refer to Table 1. For all exact weights and moles), however, 1 equivalent of HATU (O-(7-Azabenzotriazol-1-yl)-N,N,N′,N′-tetramethyluronium hexafluorophosphate) was used as the coupling agent along with 2 equivalents of DIPEA. HATU, when compared to HBTU as a coupling agent, is more efficient at producing the active ester intermediate, but mostly used in the formation of amides (2). All reagents were dissolved over DMF, allowed to stir for five minutes before adding trehalose, and left to stir for two days before performing TLC. The first amide reaction involved first making the -OH group on trehalose a good leaving group by attaching diphenyl phosphochloridate. Trehalose was first dissolved over pyridine because the reaction would produce a proton, which the lone pair on nitrogen in pyridine bonds to. One equivalent of diphenyl phosphochloridate was added and the mixture was left to stir for 48 hours. Following confirmation of producing the phosphorylated trehalose molecule by TLC and mass spectrometry, the mixture was extracted and 0.9 equivalents of sodium azide were added. This mixture was dissolved over DMF and left to stir in a round bottom flask that was heated, however, not to boiling. A snorkel was used to collect potentially hazardous fumes produced during the reaction (3) which was determined to be finished after 48 hours by TLC. The second amide reaction used one equivalent of P-TsCl (4-Toluenesulfonyl chloride) to make the -OH group on trehalose a good leaving group. Trehalose and P-TsCl were dissolved over pyridine and one milliliter of the nucleophile ammonia (NH3) hydroxide was mixed with 10 …show more content…
Following TLC, the sample was evaporated with the Rotavap® and then extracted using 4:1 1-butanol and ethyl acetate as the organic layer and water. The sample failed to dissolve, so the organic layer was decanted off, and dichloromethane was used instead. The bottom-layer (dichloromethane and sample) was run through the Rotavap® and the sample residue that was left was reacted with 1-pyrenehexanoic acid. The second step of forming the amide also required TLC, and was done using the same reagents, ethyl acetate and water. The spots, however, on the plate could not be differentiated visually, and thus the reaction may not have run to completion. Mass spectrometry was used instead, and it was determined that the reaction had failed by producing a
The routine lab equipment included a rotary evaporator (evaporating solvents off by lowering boiling point through pressure and temperature changes), analytical balances measuring to one tenth of a milligram, and a lyophilizer (freeze-dryer).
Synthesizing "PYRET-5" molecules
"PYRET-5" (refer to Scheme 1 for structure) was the target molecule to be synthesized by replacing one of the –OH groups with 1-pyrenehexanoic which would be connected by either an ester or an amide group. Both compounds, the ester and the amide, were synthesized with the pyrene as the limiting reagent, which was measured out to be about 50 milligrams (~0.16 millimoles) for each reaction. The first reaction to synthesize the ester was run in a round bottom flask starting with 1-pyrenehexanoic acid (refer to Table 1. for all exact weights and moles). One equivalent of HBTU (O-benzotriazole- N,N,N′,N′ -tetramethyluroniumphosphate) was added as a coupling agent along with 1.2 equivalents DIPEA which serves as a non-nucleophilic base and tertiary amine for the coupling step (1). The reagents were all dissolved over DMF and allowed to stir for five minutes before adding three equivalents of trehalose (dehydrated). A color change to a dull gold hue was observed upon the addition of trehalose. This reaction was left to stir for approximately 72 hours before confirming the reaction’s progress with (TLC) thin-layer chromatography. The second reaction to synthesize the ester was also run in a round bottom flask with 1-pyrenehexanoic acid (refer to Table 1. For all exact weights and moles), however, 1 equivalent of HATU (O-(7-Azabenzotriazol-1-yl)-N,N,N′,N′-tetramethyluronium hexafluorophosphate) was used as the coupling agent along with 2 equivalents of DIPEA. HATU, when compared to HBTU as a coupling agent, is more efficient at producing the active ester intermediate, but mostly used in the formation of amides (2). All reagents were dissolved over DMF, allowed to stir for five minutes before adding trehalose, and left to stir for two days before performing TLC. The first amide reaction involved first making the -OH group on trehalose a good leaving group by attaching diphenyl phosphochloridate. Trehalose was first dissolved over pyridine because the reaction would produce a proton, which the lone pair on nitrogen in pyridine bonds to. One equivalent of diphenyl phosphochloridate was added and the mixture was left to stir for 48 hours. Following confirmation of producing the phosphorylated trehalose molecule by TLC and mass spectrometry, the mixture was extracted and 0.9 equivalents of sodium azide were added. This mixture was dissolved over DMF and left to stir in a round bottom flask that was heated, however, not to boiling. A snorkel was used to collect potentially hazardous fumes produced during the reaction (3) which was determined to be finished after 48 hours by TLC. The second amide reaction used one equivalent of P-TsCl (4-Toluenesulfonyl chloride) to make the -OH group on trehalose a good leaving group. Trehalose and P-TsCl were dissolved over pyridine and one milliliter of the nucleophile ammonia (NH3) hydroxide was mixed with 10 …show more content…
Following TLC, the sample was evaporated with the Rotavap® and then extracted using 4:1 1-butanol and ethyl acetate as the organic layer and water. The sample failed to dissolve, so the organic layer was decanted off, and dichloromethane was used instead. The bottom-layer (dichloromethane and sample) was run through the Rotavap® and the sample residue that was left was reacted with 1-pyrenehexanoic acid. The second step of forming the amide also required TLC, and was done using the same reagents, ethyl acetate and water. The spots, however, on the plate could not be differentiated visually, and thus the reaction may not have run to completion. Mass spectrometry was used instead, and it was determined that the reaction had failed by producing a