Org Prep Daily

September 24, 2013

O-selective acetylation of tyrosine

Filed under: procedures — milkshake @ 7:30 pm

1. HTyr(Ac)OH.MsOH:

Methanesulfonic acid 80mL (1.2 mol) solution in acetic acid 0.5L was added to L-tyrosine 181.2g (1.0 mol, Aldrich 97%+) in a 5L 29/42 joint flask and the mixture was stirred vigorously without cooling until complete tyrosine dissolution (about 2 hours). The flask was placed in +10C water bath and the mixture was stirred till the internal temperature was about 15C. Neat acetanhydride 105mL (1.1 mol) was added dropwise over a 30 min period with a vigorous stirring and cooling on cold water bath, then continued at 15-20C for additional 90 min, at which time a voluminous precipitate solidified the reaction mixture. Peroxide-free THF 1L was added to the crystalline mass, the mixture was mashed up with a large spatula to break the lumps and then stirred vigorously for 20 min. The precipitate was collected by filtration, washed thoroughly with THF, the product was dried by suction under N2 blanket and then in vacuo until only a faint smell of AcOH remained with the product (10 Torr, 1 day). Y=259.8g of white solid (84% th)
1H(d6-DMSO, 400MHz): 8.28(br s, 3H), 7.29(app d, 8.6Hz, 2H), 7.10(app d, 8.6Hz, 2H), 4.20(br t, 6.4Hz, 1H), 3.10(br d, 6.4Hz, 2H), 2.33(s, 3H), 2.26(s, 3H)

2. HTyr(Ac)OH:

The O-acetyl tyrosine mesylate salt from the first step was dissolved in D.I. water 0.5L in a 4L large beaker, a solution of triethylamine 118mL (0.84 mol; 1 eq.) in ethanol 0.5L was added rapidly with stirring, the crystallized mixture was combined with additional ethanol 1L and agitated with a large spatula, then stirred for 30 min and finally placed into a refrigerator (+ 4C) overnight. The precipitated product was collected by filtration, rinsed thoroughly with chilled 200-proof ethanol (0.5L, +4C) and then with few small portions of ambient ethanol (4x20mL), dried by suction under N2 blanket and then thoroughly dried in vacuo. Y=168.4g (79.5% overall) of a white fluffy solid.
1H(D2O 0.7mL/10mg, 400MHz): 7.36(app d, 8.4Hz, 2H), 7.14(app d, 8.4Hz, 2H), 4.79(s, 3H; HOD), 3.98(dd, 8.0Hz, 5.5Hz, 1H), 3.29(dd-ABX, 14.7Hz, 5.3Hz, 1H), 3.14(dd-ABX, 14.7Hz, 7.8Hz, 1H), 2.34(s, 3H)

Note:  Effective stirring and cooling during the Ac2O addition is important for achieving good results. Using a cheap grade of tyrosine (a yellowish muddy powder, with some impurities detectable on NMR in aromatic region) is tolerable – and MsOH from an old bottle that was already a bit dark worked fine in this procedure – but the used THF should be aldehyde+peroxide free.

22 Comments »

  1. How does this ‘suction under N2 blanket’ actually works? What’s the instrumental set up for it?

    Comment by lastpook — September 27, 2013 @ 2:05 pm

    • Easy: a big plastic funnel, connected to a nitrogen line, bottom up and put as a lid over a Buchner funnel. It is good for filtering hygroscopic materials from low-boiling solvents, i.e. amine hydrochlorides from ether. The flow of nitrogen needs to be adjusted so that it is higher than the suction flow.

      Comment by milkshake — September 27, 2013 @ 3:39 pm

  2. Milkshake, I would like your opinion on some chemistry we are trying in the lab. We are trying to convert a dimethylphenyl alcohol (made by methyl Grignard addition to benzoate) to the corresponding azide using the NaN3, TFA, CHCl3 method. The phenyl ring is strongly deactivated with a para-CF3 group and no reaction has occurred at room temperature. I am hesitant to suggest heating, even though I do find one reference detailing such a method (PCT Int. Appl., 2004024081). What do you think? Go for it, or throw it away and try something else? Thanks.

    Comment by PotStirrer — November 18, 2013 @ 5:52 am

    • Don’t worry…we dumped it. I don’t like the idea of maybe making triazidomethane. But what if we used a different solvent? Or tried BF3-OEt2 with TMSN3? Any thoughts?

      Comment by PotStirrer — November 18, 2013 @ 9:00 am

    • I would take the tertiary alcohol and treat it with neat oxalyl chloride, to obtain the corresponding tertiary chloride. I would pump on it, to get rid of the excess of oxalyl chloride and HCl. Then I would take the tertiary chloride and reflux it wit NaN3 in acetonitrile, similar to preparation of trityl azide. Trityl azide procedure is here: http://orgprepdaily.wordpress.com/2007/01/05/trityl-azide-is-lazy/

      Comment by milkshake — November 18, 2013 @ 12:08 pm

      • Have you ever bought morphine/codeine for research in gram scale from any vendors? Or do you know of any such place that sells? Looks like I can only buy 25mg from Fulka for the exuberant price? I wonder if its is really that costly!

        Comment by 1 — November 25, 2013 @ 6:30 pm

        • No, morphine or codeine is likely to be rather cheap but you will need to get the necessary permits to buy them in gram quantities. The same just like with amphetamines or cocaine. I am not expert on these regulatory subjects – try to talk to your research advisor / employer. Prepare yourself that it will take a long time to get the paperwork in place. Your best bet is to join a group that studies substance abuse in animal models (rats addicted to opioids and the like)

          Comment by milkshake — November 25, 2013 @ 6:50 pm

  3. Milkshake, I need your help! Do you know if organic (aliphatic) azides are bench-stable in dilute aqueous NaOH (~10 mM)? I have a stock solution of a compound that’s been on my bench for about 1 month. It doesn’t display the expected reactivity with a strained cyclooctyne, from what I can tell. I am thinking maybe the azide decomposed?

    Comment by Nuchemist — January 30, 2014 @ 11:14 pm

    • that’s strange. If you push on alkyl azide with a strong base like tBuOK in anh THF at reflux long enough (=overnight) , eventually you will eliminate azide to olefin but the elimination is rather slow and I don’t believe it would happen with diluted aqueous NaOH at pH=12. Perhaps you have some reducing agent like dithiothreitol in it?

      Comment by milkshake — January 31, 2014 @ 11:54 am

      • Yeah, I didn’t think the dilute NaOH would be a problem. There are definitely no reducing agents present. Essentially I polymerize the azide into nanoparticles, react with cyclooctyne-labeled biomolecule (1:1 mol ratio, 100uM each) and then filter 5x through by centrifuging the mixture through 50000 Da MWCO membrane filter. I am finding that the majority of my biomolecule passes through the filter unreacted, while the particles stay behind. I am sure my azide molecule has the correct identity (1H NMR, FTIR, and HRMS confirmed) as well as the biomolecule (MALDI-TOF confirmed). Maybe the sodium hydroxide is reacting with the cyclooctyne and I should carry out the reaction in a buffered solution (tris or PBS)? I’m really stumped here!

        Comment by Nuchemist — January 31, 2014 @ 1:54 pm

        • try to heat it up. Also, in my experience with making BCN-based reagents, cyclooctynes are quite sensitive to polymerization when in solution, in the presence of oxygen – even at room temp the decomposition to insoluble oligomeric stuff is fast enough to be noticeable but keeping it under Ar solves the decomposition problem

          Comment by milkshake — January 31, 2014 @ 3:21 pm

          • That’s interesting. We make dibenzocyclooctyne-labeled oligonucleotides on an automated synthesizer, purify by RP-HPLC, then lyophilize. They seem to be stable in water but I will look more closely into this degradation pathway you mentioned. Thanks!

            Comment by Nuchemist — January 31, 2014 @ 3:43 pm

          • Do you think aqueous ferric nitrate would catalyze hydration of the strained alkyne? I think this may be the problem as it’s present in the reaction mix… FTIR of the nanoparticles showed a strong azide signal (2095 cm^-1) and I still do not seem to be getting reaction of my cyclooctyne-biomolecule with the azide.

            Comment by Nuchemist — February 4, 2014 @ 5:25 pm

          • I think destruction of a highly-strained acetylene with Fe(III) nitrate is likely because it is fairly electrophilic (Fe(III)nitrate has been used for nitration of styrenes to beta-nitrostyrenes). With cyclooctyne around you have to avoid any oxidizer or electrophile, or acid, or Lewis acid, or Cu, Ag salts, etc – otherwise bad things will happen to it…

            Comment by milkshake — February 4, 2014 @ 6:46 pm

  4. Milkshake,
    Can you direct me to your post where you use a drop of some sort of silane or silanol to prevent water from bumping when being vacuum distilled?

    Thanks

    Comment by kjwx109 — February 1, 2014 @ 5:11 am

    • http://orgprepdaily.wordpress.com/2009/06/11/foaming/

      a drop of hexamethyldisilazane straight into the flask with the evaporated solution that was foaming and swirling with it around a bit for about a minute worked for me, but some people swear on pre-silanizing a clean flask (the typical mix is Me3SiCl + NEt3 in some anhydrous solvent, room temp, Me2SiCl2 supposedly also works)

      Comment by milkshake — February 1, 2014 @ 6:09 pm

  5. Hey Milkshake,

    A few questions if you don’t mind:

    1. How stable are t-butyl esters to aminolysis? Need to use aqueous ammonia for a substrate that contains a t-butyl benzoate with an o-methyl group (so it’s pretty hindered), but still not sure if heating it up to 60 C might start to kill it?
    2. On that note, would it be possible to do a lithium-halogen exchange at -78 C on the above substrate without BuLi eating away my t-butyl ester?
    3. Would it be possible to selectively reduce a methyl ester in the presence of a t-butyl ester? Was thinking of using lithium borohyride but that might be a tad too reactive? Perhaps lithium tri-tert-butoxyaluminum hydride might work better?

    Thanks a lot!

    Comment by rh24 — February 6, 2014 @ 11:37 pm

    • I have done a selective hydrolysis of Et-ester in the presence of tBu ester (I think I used a stoechiometric quantity of LiOH.H2O in water+THF mix) and it worked reasonably well, there was only small quantity of diacid. With aminolysis, you should be fine in your case (hindered tBu ester of o-subst benzoic acid) but I would still occasionally monitor your reaction for conversion because the aminolysis goes on and on, since you have ammonia excess.

      tBu esters should be perfectly stable to Br/metal exchange using iPrMgCl+LiCl at -20C (Knochel protocol), I think it should be safer than BuLi. In case of iodide you don’t even need to add LiCl (and I/Mg exchange works at even lower temperatures and tolerates other esters too, but Br/Mg exchange works well only with tBu esters).

      With the respect to borohydride reduction: I think the first to try (and easiest to try) would be solid NaBH4 added slowly to the solution of your Me,tBu-diester in MeOH at 0C, it should be fairly selective to the Me ester- even with the excess of borohydride. But I would quench it with acetone before warming up, just to be sure.

      Comment by milkshake — February 7, 2014 @ 12:52 pm

  6. Hi Milkshake. Can I pick your brain again? Let’s say you’ve got an inhibitor that has a ketone carbonyl in it. You’d like to keep that carbon sp2 (mostly to avoid a chiral center), and want to improve plasma stability (presumably directly related to the carbonyl functionality) as well as solubility. Presumably you’ve got plenty of space to work with as this portion of your molecule is directed out of the target’s binding pocket toward solvent. Any ideas you’d be willing to share?

    Comment by PotStirrer — April 29, 2014 @ 7:45 am

    • would a simple tertiary amide like N.N-dimethylamide work for you? I would not add extra NH bonds unless needed because they can screw up cell penetration, but tertiary amides are generally OK and their plasma hydrolysis is typically slow.

      Also, since you already have the keto compound in hand, you could try to make some oximes – you might run into E/Z isomer formation problem, depending on the structure of your compound, but if it is easy enough for you to make and test these analogs I would do it.

      Another possibility would be to put there some 5-membered heterocycle – something that’s easy to make, i.e. pyrazole, thiazole, oxadiazole.

      Comment by milkshake — April 29, 2014 @ 10:31 am

  7. Hey Milkshake, I found an old procedure where a substiochiometric amount of copper-bronze is used in a Finkelstein reaction with sodium iodide. What the hell is copper bronze? In EROS I find an article where it has the same CAS as copper(0). I also found an article for copper(0). However, in none of them it says what copper bronze is supposed to be. So is it just sort of Rieke-copper or does it need to be spiked with any metal (basically dirty copper)?

    Comment by Young Padawan — May 2, 2014 @ 10:15 am

    • like you said, it is dirty copper powder with very fine particle size – a metallic bronze pigment used in paints, the composition varies but typically there is some tin in it so that it melts easier (it is made by “atomization” spraying of molten alloy followed by milling). It was probably cheaper than pure ultra-fine Cu powder, but not anymore (pure fine Cu is now widely used in powder metallurgy). Here is what procedures from OrgSyn say:

      “The copper bronze was supplied by BDH Chemicals Ltd. (Poole, England) as an extremely fine powder. The use of more granular electrolytic copper had no effect on the yield, but made magnetic stirring more difficult.”

      “The checkers used bronze powder obtained from George Benda, Inc., Boonton, New Jersey. Some varieties of copper powder tended to form a dense paste which did not disperse readily and resulted in lower yields”

      “Copper bronze, type 3310, obtained from U.S. Bronze Powder Works, Inc., Flemington, New Jersey, was used. The use of some grades of copper powder leads to a considerably lower yield.”

      “Ordinary copper bronze does not always give satisfactory results in the Ullmann reaction. More uniform results are obtained if the copper bronze is prepared as suggested by Kleiderer and Adams. The copper bronze 200g is treated with 2 l. of a 2% solution of iodine in acetone for 5–10 minutes. The product is then collected on a Büchner funnel, removed, washed by stirring into a slurry with 1 l. of a 1:1 solution of concentrated hydrochloric acid in acetone, and again filtered. The copper iodide dissolves, and the copper bronze remaining is separated by filtration and washed with acetone. It is then dried in a vacuum desiccator. It should be used immediately. “

      I don’t have any personal experience with using copper bronze but I would try maybe fine copper powder (as small particle size as you can get), and if it does not work I would then try the copper powder activation with I2 and HCl as described above

      Comment by milkshake — May 2, 2014 @ 11:05 am


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