[pigsknow]

Hi all, this is my first post

I need to make isotopically enriched phosphoserine, but I am not so great with organic chemistry.  Although I can buy phosphoserine from any supplier, nobody sells it isotopically enriched so I must make it myself. 

I am having a hard time planning out what to do because there are 3 functional groups on serine, but I want to selectively phosphorylate the serine side chain hydroxyl.  In general, my current plan is the following:

1: N-boc protect L-serine
2: C-protect boc-L-serine with (I need help here, I want something that cleaves with TFA)
3: phosphorylate free hydroxyl using a phosphoramidite
4: oxidize phosphate with tBuOOH
5: cleave all protecting groups with TFA

Does anybody see any flaws with this general plan, or see any easier options? Am I missing anything that would allow me to selectively phosphorylate the serine hydroxyl?

thank you
-pigsknow

[discodermolide]

Hi all, this is my first post

I need to make isotopically enriched phosphoserine, but I am not so great with organic chemistry.  Although I can buy phosphoserine from any supplier, nobody sells it isotopically enriched so I must make it myself. 

I am having a hard time planning out what to do because there are 3 functional groups on serine, but I want to selectively phosphorylate the serine side chain hydroxyl.  In general, my current plan is the following:

1: N-boc protect L-serine
2: C-protect boc-L-serine with (I need help here, I want something that cleaves with TFA)
3: phosphorylate free hydroxyl using a phosphoramidite
4: oxidize phosphate with tBuOOH
5: cleave all protecting groups with TFA

Does anybody see any flaws with this general plan, or see any easier options? Am I missing anything that would allow me to selectively phosphorylate the serine hydroxyl?

thank you
-pigsknow

Use the t-Butyl ester this is cleavable with TFA. You make it using isobutylene and sulphuric acid in a glass hydrogenation apparatus, at RT.
I don't imagine you will be making large amounts, the oxidation with t-BuOOH is very exothermic. Can you even buy tBuOOH these days, I thought Aldrich stopped selling it.

[Babcock_Hall]

Have you looked up syntheses of this compound from the literature?  I have synthesized a few labeled amino acids myself, and I always found this was useful, even when I did not follow the synthesis that I found.  I seem to recall that John Perich worked in this general field at one point.

[orgopete]

Have you looked up syntheses of this compound from the literature?  I have synthesized a few labeled amino acids myself, and I always found this was useful, even when I did not follow the synthesis that I found.  I seem to recall that John Perich worked in this general field at one point.

That is how I would start also. It will give you reaction conditions for steps even if the reactants may be slightly different.

[pigsknow]

Have you looked up syntheses of this compound from the literature?  I have synthesized a few labeled amino acids myself, and I always found this was useful, even when I did not follow the synthesis that I found.  I seem to recall that John Perich worked in this general field at one point.

Thank you for your comments

yes I planned this synthesis using work from Perich, namely that I am currently trying to create a glycine-pSerine dipeptide on a solid support by coupling boc-serine to a Gly-Wang resin, phosphorylating using di-tertbutyl diethyl-phosphoramidite, and cleaving from the resin/deprotecting all boc groups at the same time (for my purpose, gly-pSer dipeptide serves the same purpose as pure pSer).  I have actually had some success so far

However, I feel like I am missing something:  is there an easy way to selectively phosphorylate a hydroxyl?  possibly with phosphorous pentachloride?  Or a more general question:  how would one best phosphorylate primary alcohols?

thank you!

[discodermolide]

Have you looked up syntheses of this compound from the literature?  I have synthesized a few labeled amino acids myself, and I always found this was useful, even when I did not follow the synthesis that I found.  I seem to recall that John Perich worked in this general field at one point.

Thank you for your comments

yes I planned this synthesis using work from Perich, namely that I am currently trying to create a glycine-pSerine dipeptide on a solid support by coupling boc-serine to a Gly-Wang resin, phosphorylating using di-tertbutyl diethyl-phosphoramidite, and cleaving from the resin/deprotecting all boc groups at the same time (for my purpose, gly-pSer dipeptide serves the same purpose as pure pSer).  I have actually had some success so far

However, I feel like I am missing something:  is there an easy way to selectively phosphorylate a hydroxyl?  possibly with phosphorous pentachloride?  Or a more general question:  how would one best phosphorylate primary alcohols?

thank you!

The best way to phosphorylate primary alcohols is as you suggested, phosphoramidate then oxidation. Using any pentavalent phosphate with chlorine will form the primary chloride.
Your oxidation needs to be looked at, as I said I don't think you can buy tBuOOH anymore. Look at some of the more recent phospholipd literature and see how they did it.

[Babcock_Hall]

OPCl3 is a nice reagent for phosphorylating nucleosides.  It is selective for primary over secondary hydroxyl groups.  One might look up papers by Perry Frey and collaborators from some years ago.

[pigsknow]

OPCl3 is a nice reagent for phosphorylating nucleosides.  It is selective for primary over secondary hydroxyl groups.  One might look up papers by Perry Frey and collaborators from some years ago.

I found a reference from a chinese journal using POCl3, reacting twice with 2 eq water such that POCl3 forms PO(OH)2Cl and HCl gas, then use PO(OH)2Cl to react with 1 eq serine.  Maybe this is what I'm looking for?  Is this selective for primary alcohols over amines and acids as well as secondary hydroxyls?

I do have tBuOOH from Santa Cruz Biochemicals.  Would mcpba serve the same purpose?

[discodermolide]

OPCl3 is a nice reagent for phosphorylating nucleosides.  It is selective for primary over secondary hydroxyl groups.  One might look up papers by Perry Frey and collaborators from some years ago.

I found a reference from a chinese journal using POCl3, reacting twice with 2 eq water such that POCl3 forms PO(OH)2Cl and HCl gas, then use PO(OH)2Cl to react with 1 eq serine.  Maybe this is what I'm looking for?  Is this selective for primary alcohols over amines and acids as well as secondary hydroxyls?

I do have tBuOOH from Santa Cruz Biochemicals.  Would mcpba serve the same purpose?

I have always used the P(III) reagents followed by oxidation. You can try the Chinese chemistry. I would say it's selective for primary OH. In the phospholipid area I always had problems with such pentavalent phosphorus species such as the Chinese one.
The oxidation will go with any oxidizing agent, so m-CPBA will work just fine. You may have to use a two phase system in order to separate the m-chlorobenzoic acid.
Bleach is also good, but if you can get tBuOOH then use that. Just remember the reaction is very exothermic.

[Babcock_Hall]

I think this is the original reference for OPCl3 and nucleosides:  Yoshikawa, M.; Kato, T.; Takenishi, T. A novel method for phosphorylation   of   nucleosides   to   50 -nucleotides.   Tetrahedron Lett. 1967, 50, 5065–5068.  However, I am not trying to steer you away from P(III) reagents by any means.

[discodermolide]

I think this is the original reference for OPCl3 and nucleosides:  Yoshikawa, M.; Kato, T.; Takenishi, T. A novel method for phosphorylation   of   nucleosides   to   50 -nucleotides.   Tetrahedron Lett. 1967, 50, 5065–5068.  However, I am not trying to steer you away from P(III) reagents by any means.

Have you ever reacted P(O)Cl₃ with water? It's almost impossible to accurately especially on a small scale. You can't isolate the intermediate, you can't get rid of the HCl formed. You thus generate two strong acids which will probably remove all your protecting groups. It's really not to be recommended.

[pigsknow]

Thank you all, this is very helpful. 

If I use POCl3 and water to phosphorylate serine, I would not have any protecting groups -- I would attempt to directly phosphorylate serine.  I have recently had good success preparing a gly-pSer dipeptide on a Wang resin using a phosphoramidite followed by oxidation and cleavage, however I am not yet sure how my yields are.

Does anyone know much about the stability of phosphoramidites?  I am attempting to keep my two vials ($200 for 5gram vial...bleh) in the freezer under nitrogen, but I think one bottle has already gone bad.  I do not have access to a glove box, but I flush the vial with N2 whenever it is opened.

[discodermolide]

Thank you all, this is very helpful. 

If I use POCl3 and water to phosphorylate serine, I would not have any protecting groups -- I would attempt to directly phosphorylate serine.  I have recently had good success preparing a gly-pSer dipeptide on a Wang resin using a phosphoramidite followed by oxidation and cleavage, however I am not yet sure how my yields are.

Does anyone know much about the stability of phosphoramidites?  I am attempting to keep my two vials ($200 for 5gram vial...bleh) in the freezer under nitrogen, but I think one bottle has already gone bad.  I do not have access to a glove box, but I flush the vial with N2 whenever it is opened.

Well they are quite reactive but I don't think you need to keep them in the freezer, that can cause problems with water condensation.You are using a syringe?
You do not need a glove box. Get a large clear thin plastic bag and place all your stuff in that N₂ in N₂ out get someone to help you with your hands. Put them in and use rubber bands around your wrists, that should be ok. When the P(III) compound it in the flask and sealed you can get out of the bag.

[Babcock_Hall]

I am not thrilled with the idea of adding water to OPCl3, although I did add O-17 water to PCl5 one time.  I was surprised when I reread the 1967 Tetrahedron Letters paper that there was no base present, because I would have expected that acid would cleave the N-glycosidic bond.  However, I have seen papers from labs engaged in the synthesis of nucleotides cite this paper, so the work has probably been repeated many times.

I may have been thinking of a slightly different procedure.  From the abstract:  "Procedures are described for phosphorylating protected nucleotides, oligonucleotides and phosphoramidate oligonucleotide derivatives at the 3'-hydroxyl group. The conditions (phosphorylation with phosphorus oxychloride and pyridine in dioxane followed by hydrolysis with aqueous pyridine) are sufficiently mild that base labile (trifluoroacetylamino; beta-cyanoethyl phosphotriester) and acid labile (O-monomethoxytrityl; phosphoramidate) functions are retained intact..."  Nucleic Acids Res. 1974 Apr;1(4):615-27.  "Use of phosphorus oxychloride in synthesizing nucleotides and oligonucleotides."  Mungall WS, Greene GL, Miller PS, Letsinger RL.

[discodermolide]

I am not thrilled with the idea of adding water to OPCl3, although I did add O-17 water to PCl5 one time.  I was surprised when I reread the 1967 Tetrahedron Letters paper that there was no base present, because I would have expected that acid would cleave the N-glycosidic bond.  However, I have seen papers from labs engaged in the synthesis of nucleotides cite this paper, so the work has probably been repeated many times.

I may have been thinking of a slightly different procedure.  From the abstract:  "Procedures are described for phosphorylating protected nucleotides, oligonucleotides and phosphoramidate oligonucleotide derivatives at the 3'-hydroxyl group. The conditions (phosphorylation with phosphorus oxychloride and pyridine in dioxane followed by hydrolysis with aqueous pyridine) are sufficiently mild that base labile (trifluoroacetylamino; beta-cyanoethyl phosphotriester) and acid labile (O-monomethoxytrityl; phosphoramidate) functions are retained intact..."  Nucleic Acids Res. 1974 Apr;1(4):615-27.  "Use of phosphorus oxychloride in synthesizing nucleotides and oligonucleotides."  Mungall WS, Greene GL, Miller PS, Letsinger RL.

This may work, but I think the phosphoramidate route is better. I think people changed over to this route, (PIII) then oxidation, for the introduction of a phosphate into nucleotides oligos and phospho-lipids. Mainly because it is so much easier to control the reaction even although it is a two step procedure.