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8th December 2016 @ 23:56

deprotect.jpg

Compound

Mw (g/mol)

mmol

mL

g

equiv

Starting Material

526.6501

0.09494

 

50mg

1

HCl-Ether

38

0.0312

0.156 (2M HCl)

 

3.3

Product

Theoretical Values

339.17

0.09494

 

32.2mg

1

dioxane

88.11

 

2 mL (4M)

 

3.3

Product Actual Value

339.17

0.07371

 

25.0 mg

78% Yield

 

November 30, 2016

Procedure:

  1. Dissolve 50 mg of the starting material in 500 uL of anhydrous diethyl ether and place system under argon.

  2. Add 2M HCl-Ether (0.1 mL) dropwise to the solution while stirring.

  3. Age solution for 2 hours at room temperature and the reaction was monitored with TLC.

  4. Left in fridge overnight.

the nemesis deprot round 2.jpg

100% EtOAc solvent system, silica gel plate, UV visualization

S → Starting Material; C → Co-spot; P → Product.

This TLC is similar to the first deprotection we did, which we deemed unsuccessful in taking out that BOC group, though there was success in knocking out the THP. This can be determined by comparing the S and C to the P. The S and C show a dark spot higher up on the plate; P shows one lower with a faint smear above it. This suggests that the THP group was successfully knocked out and the BOC group only partially (this agrees with our NMR and GCMS results from the first deprotection). Another TLC was done to confirm this hypothesis and determine if the reaction should be hit harder to take out both the THP and BOC group.

  the nemesisdeproround2.jpg

100% EtOAc solvent system, silica gel plate, dipped in ninhydrin and treated with heat

S1→ Original Purified Starting Material; S2→ Starting Material from First Deprotection; C → Co-spot; P → Product.

Treating the TLC plate with ninhydrin and heat results in two dark spots on the base line of S2, and C and a faint brown spot on the base line of P. The difference in intensity can be attributed to concentration. The presence of these spots after being treated with heat suggest that the BOC group was successfully knocked off, and resulted in the formation of an amine, which is the desired product. The BOC was only successful knocked off with heat on the plate. To do this on a larger scale, these results suggests the deprotection reaction must be hit harder; the reaction must be done under heat with the addition of dioxane.

 

December 5, 2016

We were unsuccessful in removing the BOC with extra time . This suggests the reaction needs to be hit harder, specifically with the addition of dioxane and ran in an oil bath.

  1. Added 2 ml of 4M Dioxane and 0.1 mL of HCl  dropwise to the product while stirring and place in an oil bath at 50 degrees Celsius. System was placed under argon.  The reaction was monitored with TLC.

  2. After 2 hours at 50 degrees, TLC indicated that there was still starting material. The temperature was raised to 102 degrees to induce reflux. The reaction was then left to reflux for 45 minutes.

  3. After 45 minutes the reaction turned brown and a TLC was taken. The results of the TLC and a ninhydrin test suggest that the complete deprotection was successful due to the disappearance of the high Rf spot observed in previous attempts.

 

December 7, 2016

Mass Spec:

 
Deprotection.pdf
 

The peak at m/z 340 suggests that we successfully removed the Boc protecting group that we had trouble removing in the past. It is possible, however, some residual Boc protecting groups were removed in the mass spec instrument, so the product was also analyzed with proton and carbon NMR.

 
December 8, 2016 

NMR:

Still to be determined

 
Attached Files
19th November 2016 @ 19:12

deprotect.jpg
 
 
 

Compound

Mw (g/mol)

mmol

mL

g

equiv

Starting Material

526.6501

0.09494

 

50mg

1

HCl

38

0.0312

0.156 (2M HCl)

 

3.3

Product

Theoretical Values

339.17

0.09494

 

32.2

1

 

November 14, 2016

Procedure:

  1. Dissolve 50 mg of the starting material in 500 uL of anhydrous diethyl ether and place system under argon.

  2. Add 2M HCl-Ether (0.1 mL) dropwise to the solution while stirring.

  3. Age solution for 30 mins and the reaction was monitored with TLC.

IMAG0102.jpg

 

100% EtOAc solvent system, silica gel plate, UV visualization

S → Starting Material; C → Co-spot; P → Product

  1. The TLC of the reaction indicated that it was not complete, so another 500 uL of anhydrous ether and 50 uL of 2M HCl-ether was added to the reaction.

  2. The solution was then aged for another 30 minutes.

  3. TLC of the reaction was taken after the additional 30 minutes. Starting material was present, indicating the  reaction incomplete (presence of a salt). 500 uL of anhydrous ether was added to the reaction to ensure it remained in solution.

100% EtOAc solvent system, silica gel plate, UV visualization


S → Starting Material; C → Co-spot; P → Product

  1. The reaction was then gravity filtered and washed with diethyl ether to isolate the product. The product was a fine yellow solid.

 

November 16, 2016

Procedure

  1. The fine pale yellow solid was then mixed with diethyl ether (~8 mL) in a small r.b. Flask.

  2. Because the product was found to be insoluble in diethyl ether, it was put in a sonicator bath to speed up dissolution/resuspension.

  3. The mixture was left to settle in the r.b. A pale yellow, very fine solid settled  at the bottom of the flask.

  4. Vacuum filtration was done to separate the solid product from the diethyl ether. It was then washed once with diethyl ether to yield  0.0125g of final product.

  5. In preparation for NMR, 1 mL of methanol was added to 5 mg of the final product. (The final product (5 mg) was found to be insoluble in (1 mL) D2O, even after being put in the sonicator; this resulted in resuspension rather than dissolution).

  6. NMR and MS were run to determine presence of a salt and the identity of the product.

    (11/28/16)

  7. LCMS was run on the product and the spectrum is shown below:

     

    NemesisDeprotonationMassSpec.JPG
  8. Carbon and Proton NMR were run.

 

Conclusions:

 

The 440 peak on the MS and the terbutyl groups seen in the NMR suggest that we were not successful in removing BOC protecting group from the compound. The MS peak at 340, however, suggests that some of the BOC was successfully removed to form the correct final product. It is possible that the deprotection must be run longer to successfully remove all of the BOC protecting groups. 

Attached Files
14th November 2016 @ 16:54

11/9/16

Purification:

 
  1. The 550 mg of MSLR 9-1 was dry loaded onto silica and the product was purified using the biotage using a solvent system of 100% ETOAc.

  2. Fractions containing product were checked using TLC. TLC shows that the product was successfully isolated. In 3 fractions, high Rf impurities might be present in low concentration, however it is unclear and this will be addressed if the NMR looks messy.

  3. The product was concentrated in vacuo. Yield: 0.145 grams

100% EtOAc solvent system, silica gel plate, UV visualizationTLC100ethylacetatemarcolincoln9-1.jpg

The numbers at the bottom of the TLC plates are the factions we identified our product to be in.  The curve seen between the factions on the TLC suggests there are diastereomers present in our final product.

 
 

11/10/2016

4. Used HNMR to determine if THP and BOC groups were present in the product.

The tall peak at approximately 1.5 ppm suggests the presence of a BOC group (the 9H on the quaternary carbon at the end). The presence of peaks between 3-4 pm, as well as others around 1 ppm suggest THP is present .

 
purificationCarbonNMR.pdf
 
Conclusions:
 
It appears that the protecting groups are present. A deprotection step will be done to see if the final product is successfully synthesized.
Attached Files
20th October 2016 @ 22:12

10/18

MRLS 4-2 (0.473 g, 1.4 mmol, 1 equiv) was added to toluene (10 mL) along with MRLS 8-1 (fraction 1, 0.2761 g, 1.5 mmol, 1 equiv), potassium hydroxide (0.3281 g, 5.8 mmol, 4.2 equiv) and 18-crown-6 (0.032 g, 0.12 mmol, 0.09 equiv).

The reaction was stirred at room temp for 30 minutes and then heated to 40°C (bath temperature) for 4 h.

Planned on leaving stirring at room temperature for only 10 minutes, but problems with hot plate meant lukewarm water for 15 minutes until I got one that worked properly.

Came back later that night (11:30) to monitor reaction via TLC. Also noticed that hot plate had stopped heating. TLC showed almost no starting material—I decided to replace the hot plate and keep it stirring over night (~10 more hours) to make sure all starting material had completely reacted.

10/19

TLC showed no SM and baseline spots (tested w/ 60% EtOAc/Hexanes, KMnO4 stain) (shown below).

The sample was cooled to room temperature and diluted with 8 mL of water. The mixture was extracted with EtOAc (3 x 20 mL). The combined organic layer was washed with water (2 x 8 mL) until the aqueous layer became neutral. The organic layer was then washed with brine (7 mL) and dried over Na2SO4. The brown/red solution was gravity filtered and dried via rotovap to yield a red brown oil. It was placed under high vac over night.

10/20

Yield: 0.55 g/.6811

80.75% yield

Took NMR.

Unfortunately, ran out of time so could not run a column on the crude product to verify a successful synthesis. Hopefully, another group will pick up this project!

 

 

Attached Files
13th October 2016 @ 20:02

Started 10/4

 

 Based on AEW 221-2.

Procedure:

Dried 3-necked round bottom flask+stir bar on high-vacuum w/ heat gun. Immediately placed under Argon. (attempting to conduct reaction under anhydrous conditions)

Transferred LiAlH4 (about 0.5 g) to the round bottom flask. Added 12 mL Et2O (anhydrous) via syringe. Stirred in ice bath (~0 C).

Dissolved MRLS 6-1 (4.57 g, 18.26 mmol) in anhydrous Et2O (8 mL). Transferred this mixture to the LiAlH4 suspension via syringe. Added dropwise over ~5 minutes. Stirred in ice bath for about 1 hour. Removed the round bottom from the ice bath and stirred at room temperature for 3 hours. Then, refluxed the mixture for about 16 hours. 

10/5

Quenched the LAH by stirring with rochelle salts and Et2O overnight.

10/6

Filtered the reaction mixture through celite. Washed round bottom with EtOAc. Performed extraction (3x30 mL EtOAc) of the mixture, and combined the organic layers. Dried the layers with sodium sulfate, followed by gravity filtration and rotovap. Yield: 3.39 g, 83.5%. Took NMR of the crude.

10/11

Attempted to purify the crude with the Biotage Isolera. 

Used KP-Sil 100 g column. Loaded sample by dissolving in DCM, adding silica gel, and then rotovap (dry loading instead of using a samplet). Unfortunately, had issue with attaching solvent lines to the column, resulting in a spill (possibly affecting sample?). Collected 6 fractions.

10/13

Rotovapped fractions. Although I had expected the product to be in the second fraction, there was almost no product after rotovapping. The other fractions, however, did have some product (test tubes 20-29 and 30-40).

 

10/14

Took proton NMR of fractions 20-29 and 30-40. Ethyl acetate solvent impurities near 1.25 ppm (t), 2.03 ppm (s), 4.11 ppm (q). Will attempt to run NMR again after removing solvents.

Redid NMR (removed EtOAc/Hexanes by rotovapping CDCl3 mixture, then adding CDCl3, rotovapping once more, and then dissolving that sample in CDCl3). Attached below. The NMRs seem to be very similar, but unsure why there are differences. I will try asking for assistance on our GitHub post (issue #430).

10/17

It seems like the fractions are diastereomers of one another (thanks to Chase Smith (@MedChemProf) for his help here!) .

stereoisomers

I've also added GC-MS results (TIC and MS) for both fractions. The mass peak matches that of the structure above with a cleaved CH2OH (M+ of ~191).

 

 

Attached Files