Spring Organic Chemistry Semester Project -- Part A
Multi-step Synthesis of3-alkoxy-4-methoxy-6-nitro-trans-cinnamic acids
Introduction
The semester project this term consists of two interconnected parts, A and B. In Part A of the project, you will be engaged in a multistep synthesis of a key synthetic intermediate. Once the synthesis is completed and the end product has been characterized, you will use this end product to synthesize (in Part B) a small molecule combinatorial library analogous to the example target structure shown below:
You should notice that within the context of this structure are three key points for potential diversification. As a first objective, we would like to incorporate structures that lead to diversification at position #2 (nitrocinnamic acids). This objective can be accomplished using commercially available 2-nitrocinnamic acids (the structures in the first row below) or through the synthesis of novel compounds (for example, like the other two compounds shown below). Herein lies the objective of Part A of your semester project. Our goal is to create molecules with enough molecular diversity to allow full investigation of the effects of substitution at position R3 of the aromatic ring on the binding of the target structures to other molecules of biological significance (for example key proteins and enzymes).
Your task in this part of the semester project will be to synthesize a structure similar to the one shown in the oval above. The synthetic scheme is shown below. The idea is to substitute various R groups in place of the isoamyl group that is present on the oxygen at position #5 of the ring.
Step one involves the protection of the aldehyde as the ethylene glycol acetal. Following the selective deprotection of the meta-methoxy ether under basic conditions, the free phenolic group is coupled with a primary or sterically accessible secondary alcohol under Mitsunobu conditions (alcohol, PPh3, and DEAD or DIAD). The acetal protecting group is removed under acidic conditions and then immediately reacted via Knoevenagel condensation to generate the desired nitrocinnamic acid target molecule. The chemistry for this series of steps has been derived in large part from J. Chem. Soc. Perkin I, 1978, 440-446.
Procedure
(1-24-05 or 1-25-05)
A mixture of 6-nitroveratraldehyde (47.4 mmol), ethylene glycol (1 equivalent), and a catalytic amount of p-toluenesulfonic acid (0.03 equivalent) in 60 mL of toluene was heated at reflux for 2.5 hours and the water was removed by Dean-Stark apparatus. Upon completion, the solvent was removed via rotary evaporation to yield a yellow solid. The product is recrystallized from EtOAc. The mass of the product is determined and a 30 mg sample is set aside for mp, IR, and LCMS analyses, as well as 1H-NMR and 13C- NMR.
(1-31-05 or 2-1-05)
You will set up a 48 hr reflux reaction for the selective deprotection of the aromatic methoxy group. The product from last week (16.11 mmol) is dissolved in 15 mL of 1,4-dioxane and 75 mL of 20% KOH(aq) is added to the mixture. The resulting solution is heated under reflux for 48 hrs. The resulting solution is cooled in an ice-bath and neutralized to slightly acidic (pH = 6.5) at which point the crude product precipitates from solution. BE CARFUL NOT TO OVER-ACIDIFY!!!!!!!!!!!!!!!!!!!!! It will ruin your reaction!!!!!!!!!!!!! The crude product is isolated by vacuum filtration, washed with water and recrystallized from EtOAc. The recrystallized product is isolated by vacuum filtration and dried on a vacuum pump overnight. The mass of product is determined and a 30 mg sample is set aside for mp, IR, and LCMS analyses, as well as 1H-NMR and 13C- NMR.
In addition, you'll have to work on a molecular modeling study to investigate the mechanism of this reaction.
You will need to search through the Aldrich catalog or our inventory to select a primary or secondary alcohol for the subsequent Mitsunobu reaction. This should be done in consultation with Dr. Timm.
(2-14-05 or 2-15-05)
To 2-neck 100 mL RBF, add the starting phenol (750 mg) and 1.3 equiv. of PPh3. Place a septum over the side outlet and a gas inlet adapter over the top outlet and introduce a strong stream of N2 gas to flush the flask. Under an N2 atmosphere, add 10 mL of dry THF by syringe followed by your chosen alcohol (1.5 eq.), by syringe and place the entire mixture in an ice bath for 15 minutes. At 0 ºC, add dropwise via syringe 1.6 eq. of DIAD that has been dissolved in 2 mL of dry THF in a small glass vial. After addition, the reaction is stirred at 0 ºC for 15 minutes followed by room temperature for 1 hour. An aliquot is removed from the reaction mixture and the progress of the reaction is checked by TLC in 3:1 hexane/EtOAc spotting against PPh3 and the starting phenol. If the reaction is incomplete an additional 0.3 eq. of PPh3 is added and the reaction is continued for another hour. The cycle of TLC monitoring and addition of PPh3 is continued until the reaction has gone to completion. Upon completion, the solvent is removed in vacuo to yield a yellow syrup.
(2-21-05 or 2-22-05)
The residue is redissolved in CH2Cl2 and then a small amount of alumina (neutral) is added to the flask. The contents are swirled and the solid is removed by filtration through a plugged filtration adapter. The solvent is again removed in vacuo. The product is purified by column chromatography as described below.
(using column chromatography to purify a reaction mixture): Our crude reaction mixture from Week #3 (above) can be efficiently purified using standard column chromatography. The theory underlying the separation of components is the SAME as that for TLC. In this technique, we will pack a glass column with silica gel (the same material that is on the rough surface of your TLC plate) that will serve as the stationary phase. In order to pack the column, we must saturate completely with our mobile phase before we develop it. During the development part of the experiment, we will collect fractions of the solution that elutes from the column. These fractions can be analyzed using the mobile phase/TLC system we developed last semester. We combine like fractions containing our spot of interest and remove the solvent using rotary evaporation. The remaining residue in the round-bottom flask should be our PURE product.
Here's the procedure in a little more detail:
We begin by introducing a small (1-2 cm) layer of washed sea sand, followed by 10-15 cm of silica gel (70-200 mesh) -- see the figure below. We then select a relatively nonpolar mobile phase to pack the column (90% hexane/10% EtOAc should work well). The solvent is carefully introduced into the column and then is forced through with the help of an air or compressed gas line (this is performed behind a blast shield). Additional solvent is introduced so that the column does not dry out. Once the column is saturated (we determine by visual inspection), the solvent level is lowered to just even with the top of the silica gel. At this point, we prepare a concentrated solution of our crude material. This solution is CAREFULLY pipetted onto the top of the column being careful to not disrupt the surface. The solvent level is returned to even with the top of the solid material and capped with a 3 cm layer of washed sea sand. Our mobile phase of choice is then reintroduced and fractions are collected (usually in medium size test tubes or 50 mL Erlenmeyer flasks). Be careful to never let the column dry out and to drain solvent to the top of the column when changing to a different mobile phase. Fractions are TLC'd and combined as appropriate for concentration.
Step-by-step summary of the procedure:
(1) clean the column and dry before packing.
(2) mount the column using the clamps (provided)
(3) fill the bottom of the column with approximately 1/2 inch of sea sand
(4) fill the next 10 inches of the column with 60 x 200 micron silica gel
(5) introduce the initial mobile phase (1:9 EtOAc:Hexane) and pack with nitrogen gas
(6) once you've generated a homogeneous mixture, introduce your sample by dissolving in the minimum amount of dichloromethane
(7) drain off solvent until the liquid level matches the top of the solid silica gel column
(8) cap your column with 1/2 inch of sea sand
(9) fill the column with the initial mobile phase
From here you will run solvent through until the level of yellow bands is approximately 1 inch above the frit. At this point, begin collecting fractions in 15 x 125 mm test tubes. Periodically, check your fractions by TLC using 1:3 EtOAc:Hexane as the mobile phase in the chamber. If it does not appear that your sample has come off the column by fraction #10, consult with Dr. Timm to see if you might switch mobile phases (to something more polar) --- NOTE: you'll probably have to do this. Once you've run all of your desired sample off of the column, TLC fractions to determine which ones can be combined. Combine like fractions in a 500 mL RBF and remove the solvent on the rotary evaporator, followed by further drying under a stream of N2 gas to a constant mass. Be sure to preweigh this flask! Be sure to record the mass of product recovered.
Any questions and you should consult Lehman Operation #18 (pages 93-104).
A portion (30 mg) of the DRY acetal protected product isolated from column chromatography is removed for confirmation of the structure by 1H- and 13C NMR, IR, mp, and LCMS analyses.
The remaining product (~650 mg) is dissolved in 8 mL of acetone and 4 mL of 10% HCl (aq) is added in a 50 mL RBF. The flask is fitted with a water-cooled condenser and warmed to 60 ºC in a water bath while stirring for 35 minutes. Upon completion, the acetone is removed from the reaction mixture in vacuo. The remaining solution is left to sit at room temperature until the yellow solid completely forms in the bottom of the flask. The solid is collected by vacuum filtration, washed with water, and dried under vacuum. The mass is determined and a 30 mg sample is set aside for mp, IR, and LCMS analyses, as well as 1H-NMR and 13C- NMR.
(3-7-05 or 3-8-05)
If you have not collected 1H- and 13C NMR data for the product from last week (after the HCl deprotection of the acetal), you'll need to start doing this first.
The product from last week (1.95 mmol) is dissolved in 1 mL of pyridine at room temperature in a 25 mL RBF. To this solution was added 2 equiv. of malonic acid along with a catalytic amount of piperidine (0.2 equiv.). The reaction is heated at reflux for an additional 1.5 hours at 115 C. At the end of the reflux period, the reaction is cooled to room temperature and poured into 8 mL of cold water. The mixture is carefully acidified with conc. HCl to strongly acidic. The resulting sludge is mixed vigorously with a spatula to generate a light brown solid material. The solid is collected by vacuum filtration and washed thoroughly with water. The residue is dissolved in 15 mL of 0.67 M NaOH and filtered by gravity. The solid is washed with an additional 10 mL of water and the filtrate is acidified with 6 M HCl with stirring. The resulting solid is isolated by vacuum filtration and washed thoroughly with water. The solid is dried until later in the week and analyzed by 1H- and 13C NMR, IR, mp, and LCMS. Don't forget to get the mass!!