Org Prep Daily

February 10, 2007

Ethanolamine-O-phosphate and N-palmitoylethanolamine-O-phosphate

Filed under: procedures — milkshake @ 7:21 am


In a 1L round flask, 95g of 85% H3PO4 (808 mmol) plus ice (100g) mixture was combined with ethanolamine 50g (819 mmol) with stirring (exothermic). The resulting solution was concentrated on rotavap from a hot water bath, the obtained oily residue was heated on oil bath at 185C under water-aspirator vacuum (20 Torr). After 3 hours – when the foaming ceased – the flask was cooled. 1 mL of water was added along the flask wall and the crystallization was induced by scratching the flask wall with a spatula. The mixture was left to crystallize at +5C in a fridge for 6 hours. The precipitated solid was collected by filtration using a large coarse-porosity fritted Buchner funnel. The obtained crude solid product was dissolved in water 150mL. Two small spoons of charcoal were added, the mix was gently refluxed for 10 min, cooled, filtered. The obtained pale yellow filtrates were diluted with hot ethanol to the point of cloudiness. The mixture was allowed to crystallize in a fridge overnight. The crystallized product was collected by filtration, dried by suction and on highvac. Y=57.6g (50.5%) of a pale-yellow crystalline solid, m.p. 230-234C(dec.)

A solution of ethanolamine-O-phosphate 10.0g (71mmol) and NaOH 10.0g (250mmol) in water 150mL was cooled to +5C. With vigorous stirring a solution of palmitoyl chloride 16.5g (60mmol) in anh. THF 20mL was added dropwise over 15 minutes. The stirring was continued at 5C  for additional 45 min, then at ambient temperature for 45min. The obtained heterogennous foamy reaction mixture was heated to 70C. After 10 minutes, a solution of BaCl2.2H2O 24.4g (100mmol) in hot water 150mL was added and the mixture was stirred for 15 min. The precipitated crude Ba salt was collected by filtration, washed with methanol, dried by suction and on highvac. Y=30.9g (100%) of a white solid. The material contained some Ba-palmitate impurity. (The crude Ba salt can be re-crystallized to analytical purity from hot AcOH  but the crude salt is suitable for the next step).

2.50g of the crude Ba-salt from the previous step was dissolved in neat acetic acid 25mL at 95C. The solution was placed at 70C hot bath and a solution of 70%  HClO4 1.45g in 5mL of AcOH was added dropwise over 5 min. The mixture was allowed to cool to ambient temperature, then inoculated mechanicaly and allowed to crystallize in refrigerator (+8C) overnight. The crystallized product (1.4g) was collected by filtration and re-crystallized from AcOH 10mL (at RT overnight). Y=1.1g (60%) of beautiful outcrops of white crystals, m.p. 111-115C.

1H(DMSO, 60C, 200MHz): 3.798(app quartet, 2H), 3.248(app t, 2H), 2.055(t, 2H), 1.463(app quintet, 2H), 1.32-1.18(br m, 24H), 0.843(t, 3H)
The spectra was acquired on a ho-hum instrument very long time ago – so don’t trust it too much. DMSO at 60C was used because of the poor product solubility.

Palmitoylethanolamine-O-phosphate is a detergent; it is quite impossible to extract it from an aqueous reaction mixture as a free acid (the reaction mixture should be stirred, not shaked). Hence the isolation through the insoluble Ba salt was used. The problem was then to find a solvent/acid combination that would allow decomposition of the isolated Ba salt.  

HClO4 in AcOH is explosive – it will produce a loud bang if grossly overheated. It is important to control the temperature. Large-scale  preparations with perchloric acid are not recommended. (TfOH would be probably a safer alternative but I did not have it in Prague, 20 years ago.)


  1. Aside from switching to triflic acid from perchloric acid, what other modifications would you recommend??

    Comment by Organic Chemistry — February 10, 2007 @ 11:39 am

  2. I don’t know. If you want to use this procedure as some kind of lab asignment, I would really recomend that you give your students something else – because this is not the most pleasant preparation, similar substances were used as detergents in textile industry. I just needed it as a building block for a phospholipid medchem project – and the next step, phosphate activation and coupling with a glycerol piece did not work at all. (I later found an alternative workaround, to get my N-palmitoylethanolamine-O-phoshoglycerol piece instaled into the molecule.)

    Comment by milkshake — February 10, 2007 @ 2:03 pm

  3. Purifying phosphates and sulfates as Ba salts is a nice trick.

    Comment by Jordan — February 10, 2007 @ 9:11 pm

  4. in practice it’s a lot less nice than it seems, especially on a small scale (imho)

    Comment by kiwi — February 10, 2007 @ 11:16 pm

  5. Getting the barium salt to form is not a problem. But getting the barium out… I was getting desperate – I had to get a crystalline material and stop the crude product from foaming. How do you think I got my nick name – during work on phospholipids. Aqueous workups are definitely not a favored technique with these compounds.

    Comment by milkshake — February 11, 2007 @ 4:20 am

  6. ever consider a more conventional approach to putting the phosphate on? say diphenyl chlorophosphate, followed by hydrogenation when you want the acid. no workup required, just filter and vac down. and bis(cyclohexylammonium) salts work well for phosphates

    Comment by kiwi — February 11, 2007 @ 5:38 pm

  7. Direct phosphorylation of N-palmitoylethanolamine produced oxazoline salt because of the carbonyl participation. The oxazoline is pretty unstable and opens up – depending on the workup – to produce O-palmitoyl ethanolamine hydrochloride or palmitoic acid N-(2-chloroethyl) amide…So, in order to use the phenyl-protected phosphate one would have to protect N in ethanolamine, then phosphorylate, deprotect N, put acyl on, then deprotect phosphate.

    This work was done in Prague, 20 years ago, and I did not have much experience or money to buy things or instrument access – so I had to keep things simple.

    Comment by milkshake — February 12, 2007 @ 7:15 am

  8. ah yes, fair enough

    Comment by kiwi — February 12, 2007 @ 4:51 pm

  9. Is this the correct synthesis of Ethanolamine-o-phosphate? What is the purpose of filteration using a large coarse-porosity fritted Buchner funnel after crystallize at +5C in a fridge for 6 hours??Please advise..

    Comment by Bryan — April 22, 2009 @ 4:24 am

  10. The procedure was taken from old German Houben-Weyl compendium, the volume about organic phosphates. It worked in my hands. The reason for using a coarse-porosity (as opposed to medium porosity) large glass Buchner funnel is that the mix is a thick slush and the supernatants are quite viscous, like pancake syrup, and if the filtration goes too slowly your mix will warm up on the buchner funnel and the precipitated product will start dissolving again.

    Comment by milkshake — April 22, 2009 @ 2:01 pm

  11. The filteration after crystallize in fridge 6 hours meaning to remove the un-react phosphoric acid? Only small 1mL of water difficult to soften the mixture in flask? please advice thanks

    Comment by Bryan — May 4, 2009 @ 4:13 am

  12. in my case the residue in the flask after evaporation was not hard but thick syrupy-like

    Comment by milkshake — May 4, 2009 @ 12:50 pm

  13. after vacuum 3 hours…cooled flask…mixture become harden..difficult to soften by small amount of water (1ml)…can I directly skip the filteration step and proceed to purification??

    Comment by Bryan — May 4, 2009 @ 8:53 pm

  14. I guess so. It has been a long time but I remember using a water-aspirator vacuum (that does not get below 20-25 Torr, depending on the tap water temperature). I did it just once, according to the Houben Weyl, and it went quite nicely. Maybe you have a better pump – I think you have dehydrated your sample a lot more. Please go ahead with the purification, but check the carbon and phosphorus spectra at the end to verify that your product is pure.

    Comment by milkshake — May 4, 2009 @ 9:34 pm

  15. I just proceed to purification, but the thick syrupy-like liquid unable to crystallize…now still keep in fridge.

    Comment by Bryan — May 5, 2009 @ 12:11 am

  16. My vacuum level just nice to be at 20~25 torr…

    Comment by Bryan — May 5, 2009 @ 2:50 am

  17. amino-ethyl phosphoric ester cannot be crystallize

    Comment by Bryan — May 5, 2009 @ 10:35 pm

  18. This procedure worked in my hands on the first try, without modification – taken as is from Houben Weyl monograph. I even reproduced the yield – and I was rather awful experimental chemist at the time. I think the problem is on your part.

    I believe what happened to you is that you dehydrated your reaction mix too much and got some polymeric stuff. Do it again. Watch it, the appearance of the foaming changes noticeably once the water is mostly gone. Stop it early before it turns into glass.

    Comment by milkshake — May 5, 2009 @ 11:05 pm

  19. Finally successful to recrystallize under fridge.. Thanks for Milkshake

    Comment by Bryan — May 7, 2009 @ 12:11 am

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