I am synthesising N-(Benzyloxycarbonyl)-L-serine benzyl ester starting from N-(benzyloxycarbonyl)-L-serine by using benzyl bromide in aq. NaOH and Acetonitrile under reflux condition (by following a research article “Optimized synthesis of phosphatidylserine” By Guanti, Giuseppe et. al., From Amino Acids, 39(2), 367-373; 2010)
My questions
1) why no racemization was observed in such conditions?
2) If I run the reaction for whole night, is it possible to get racemization under such condition?
Exact procedure is N-(benzyloxycarbonyl)-L-serine (10.63 g, 44.4 mmol) was suspended in CH3CN (50 ml), and treated with 4 M aqueous NaOH (11.175 ml, 44.7 mmol). The resulting solution was treated with n-Bu4NHSO4 (1.52 g, 4.48 mmol) and BnBr (6.40 ml, 53.9 mmol) and refluxed for 4 h. After cooling to r.t. (a white solid separates), 5% aqueous NaHCO3 (55 ml) was added and the suspension stirred for 20 min. After removal of the solid through filtration, the two phases were separated and the aqueous one extracted three times with AcOEt (40 ml each). The reunited organic phases were washed with brine, well dried with Na2SO4 and evaporated to dryness to give an oil. It was taken up in n-hexane (40 ml) and stirred until a well solid separates. It was filtered on a Buchner filter and washed with n-hexane. Finally, the solid was crystallized from Et2O/PE to give pure N-(Benzyloxycarbonyl)-L-serine benzyl ester as white solid, yield (11.03 g, 75%). m.p. 81.9-82.6°C. Rf = 0.34 (PE/Et2O 3:7).
Racemisation is possible, but the Cbz (urethane protection) helps to reduce racemisation potential. If you go overnight, more likely to cause racemisation, as now you have lots of fully protected Ser exposed to base (the ester is more prone to racemise as the deprotonated acid group helps prevent the racemisation reaction). The final crystallisation step also helps to remove any D-isomer generated, so the final product is likely enantiomerically purer than the crude product. You need to use a derivatisation method to accurately determine if racemisation is occurring and your starting material is not necessarily pure. We showed that commercial samples of Ser actually are not pure - around 96-98% ee (see J. Am. Chem. Soc. 1993 115, 5021, paragraph before the conclusions).
I agree with Mark. Besides, NaOH is not strong enough to give substantial enolization. Check the corresponding pKa and see by yourself.
How are you monitoring if you have any of the d isomer.
I agree with Mark that your crystallization will remove most of the racemer
I would like to ask the same question as Paul did. How did you monitor the racemization? Of course, NMR, TLC, IR, MS analyses give you no information, because both enantiomers (D- and L-) give exactly the same results. You should go into the analyses by optical rotation and chiral HPLC etc. For chiral HPLC, you need a racemate to determine the separation conditions and the retention times for both enantiomers.
Chiral shift reagents also allow to use NMR to measure enantiomeric excesses.
Thanks Marcelo, for your additional information. It is totally true, but be careful to choose a suitable shift reagent, Sameer. If there is no shift to the signals, it is useless, because the shift reagent does not recognize your compound. Good luck!
Paul Jones sir, I didn't check it. I just thought the possibility because of presence of strong alkaline condition.
I agree with Kohei Torikai sir. Thanks for your valuable suggestions.
If you have access to a HPLC and a high performance C18 column running a gradient you will often see the racemer . The d isomer often elutes after the product.
Dear Paul, I am afraid I should disagree with you. Enantiomers cannot be separated by polarity-based chromatography. A Chiral column should be used, and it is also dangerous to believe D-isomer comes later than L.
I have used 5 u C18 only for analytical purposes . You are right, both are generalizations, that I have observed.
I would probably prepare this ester via the imidazolide. The reported conditions seem to be a bit harsh which may be the cause your concerns. Do the authors give an optical rotation (OR) for this compound? If so, does the optical rotation of your sample match with the reported value? You can have a tight melting point , a good TLC and CHN, but the optical purity could be low (
Hi. Sorry but the first thing I would do was to register the optical purity of your sample, the optical rotation. After that you and we may start to discuss or theorize concerning conditions, base, time of reaction and eventual implications and ways to tackle the EVENTUAL problem.
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