Bitopic Ligands and Epoxides

For most academics, research can be a somewhat slow process. From the conception of an idea to actually getting started can take a significant amount of time. The topic of this post started as an idea based on Dror et al.'s publication back in 2011 that provided some strong in silico evidence for the presence of so-called metastable binding sites (MBS). Explained in very basic terms the hypothesis is that ligands do not simply arrive in their binding pockets randomly but follow a path of low affininty binding sites that guide them to their destination. The report by Dror et al. provided some very compelling in silico evidence for the existence of MBS and planted the idea with us of making bitopic ligands that would simultanously target the orthosteric binding site (OBS) and a predicted MBS using the same pharmacophore. In principle this could lead to ligands with improved receptor subtype selectivity, higher affinity and slower off rates. We described the idea in a perspective paper in J. Med. Chem. in 2017 and you can also get a very basic idea of the principle in the figure below.

I was lucky enough to secure some funding from the Lundbeck Foundation Natural Sciences for developing these types of ligands back in 2015. A great funding scheme by the Lundbeck Foundation that they sadly stopped some years ago. Anyway, with the funding we managed to make this work take off and published our first paper on bitopic ligands this year in J. Med. Chem. From our study it is not clear if we have the predicted bitopic binding mode but we have some good indications that things are indeed working as hoped for. Even better we have another paper coming up in 2020 were we have very strong evidence for a bitopic binding mode with a MBS so I look forward to sharing that. The ligands that we synthesised in our paper were beta-blockers and they all have a classic beta-amino alcohol motif that is synthesised from glycidol as outlined below.

At first this may seem as a simple synthesis with a logical outcome. You activate the epoxide (optically active glycidol) with a sulfonyl leaving group, do a nucleophilc substitution with a phenolate, followed by ring-opening of the epoxide with isopropylamine. However, this only works with no stereochemical leakage thanks to Professor Barry Sharpless. In fact, it is rather tricky to make activated glycidol ring open strictly via a SN2 mechanism (= no stereochemical leakage) with no competitive SN2' reaction (= racemisation). Sharpless and co-workers solved this problem by screening various leaving groups and found that the meta-nosyl group did the trick. To my great pleasure Professor Erland Stevens from Davidson College noticed our publication and decided to use it for educational purposes posting a video on YouTube that explains the glycidol ring-opening reaction in detail. Great to see that our science can be used for educational purposes. D!

The Disconnection Approach - Automated!

In my time as a synthetic organic chemist the most important advance in the field was definitely the introduction of searchable databases such as SciFinder and Reaxys. Life before these involved spending days on end in the library flipping though dusty tomes of chemical abstracts and Beilstein. And at the end you weren't even sure if you missed something of critical importance. The introduction of open access LC-MS and high field NMR has also had a big impact for me by speeding things up considerably. However, besides these milestones I think that I have pretty much been doing chemistry the same way >25 years. Anyway, what I am getting at is "what will the next BIG thing be?" It's been a considerable time since we had a major technical breakthrough for the synthetic organic chemist. My colleagues and I have been discussing this for more than a decade and now I think I am spotting the next big thing - Chematica! Chematica was developed by Polish scientist Grzybowski and has been around for quite a while. Basically it is a computer programme that disconnects your molecule and suggests ways for you to synthesise it. What's changed since I first heard mention of Chematica ~5 years ago is that apparently now it works the way you would like it to. Recently Grzybowski and co-workers published an impressive paper where they synthesise 8 very different and rather challenging molecules. The abstract from the paper nicely summarises the achievement:

"Multistep synthetic routes to eight structurally diverse and medicinally relevant targets were planned autonomously by the Chematica computer program, which combines expert chemical knowledge with network-search and artificial intelligence algorithms. All of the proposed syntheses were successfully executed in the laboratory and offer substantial yield improvements and cost savings over previous approaches or provide the first documented route to a given target. These results provide the long-awaited validation of a computer program in practically relevant synthetic design."

You really should read this paper. These are not simple syntheses and would have taken quite some time to come up with if at all. Now it is still early days and Chematica is only for those with deep pockets. I am personally waiting for a quote right now and am very curious to see exactly how deep my pockets need to be. BUT it has started and I believe that now it's just a question of time (I'm guessing  less than 10 years) before you simply hit the disconnect button in ChemDraw, look through the suggestions that appear on your screen and pick the one that you like best.

And there is more. Another game changer is already here and everyone with a PC can do this: Machine Learning. My next post will be on this topic that we are already using to great effect at my work place. D!

How to Turn an Amine Into a Leaving Group

Leaving group activation of alcohols followed by nucleophilic substitution is routine stuff for the synthetic organic chemist. Just make the tosylate, nosylate, mesylate, triflate.... and things generally go according to plan. However, what if you are stuck with an amine and want to substitute it with a nucleophile. There are a number of ways to do this but it's not just a walk in the park. Until recently I had never had to do this but then one fine morning I wanted to do the reaction above. How does one go about doing this? Is there a simple method by which I could activate the amine and displace it with the anion of 2-nitropropane, followed by a simple reduction to get the amine I wanted? Well, as it turns out Katritzky and co-workers published a paper in 1979 introducing triphenylpyrylium salts that can convert amines to leaving groups. Granted, the atom economy in this process is (to say the least) poor. However, the required pyrylium salt is commertcially available at a resonable price. All you do is stir it up with the amine. Prior to adding the amine the suspension is pale yellow and then when you toss the amine in it becomes a deep red slurry. In the photo the amine has just been added. It's always exciting with a bit of colour if your an organic chemist. The product is isolated by filtration. In this case the pyridinium salt was isolated as a light brown solid in 63% yield, perfectly clean by NMR. Next the pyridinium salt was treated with deprotonated 2-nitropropane in hot DMSO to give the nitro compound that was reduced using old school conditions. Interestingly, we could not get any reduction AT ALL of the nitro compound by catalytic hydrogentaion (at atmospheric pressure). Very odd! I would have expected to see at least a few percent of the reduced stuff. Any ideas out there? Anyway, the amine was isolated in good yield over two steps after a short (2 cm tall) DCVC column. Yes a wastefull method but it is simple and fast. D!

The Skraup Reaction - How to Make a Quinoline

Recently, Derek Lowe was discussing reactions he hadn't done at his blog In the Pipeline. Among these were, in his own words "the widely disliked Skraup cyclization for quinolines". This was somewhat surprising to me. I have very limited experience with the Skraup reaction but it has worked for me and one of my former colleagues said it had always been a great reaction in his hands. Personally I was surprised how well it worked considering the reaction conditions. This is what I did: 

Easily the most extreme reaction conditions I have employed. Hardly surprising this is a lively reaction. Adding acrolein (boiling point of 53 oC) to a 70% sulfuric acid cocktail at 110 oC is rather exciting. Things are vaporising, spraying, hissing and instantly turns into jet black tarry goo. After 45 minutes the reaction is allowed to cool and then you attempt to work the black polymeric goo up with 25% aq. NaOH, brine and ethyl acetate. I suspect that the modest yield is due to loss of material during this annoying work-up. Scaling the reaction up is likely to improve the yield. The Skarup reaction is indeed performed on ridiculous industrial scale so it can't be all that bad. My system was rather elaborate containing two phenolic ethers and still I managed to pull out 47% and it was reproducible. Unfortunately, I cannot give full structural details as this was done in industry and I seem to recall some papers I signed explaining my life would end if I ever mentioned any of that stuff.

I should mention that it is rather important that you don't use too much acrolein as this will turn the whole thing into a rubbery solid (as I discovered) that is impossible to work with.

Anyway, the conditions I employed here a slightly different from the standard method so it may be worth giving a go if you are into quinolines. The full experimental details can be found here: C. O'Murcho, Synthesis, 1989, 880-882. By the way that's Zdenko Hans Skraup himself on the photo above. D!

How to make a primary amide - The Ley Way

Steven Ley's Group really produces some cracking results. This time they have developed a really simple method for preparing primary amides from carboxylic esters. This is very good news because this seemingly simple transformation is in fact not as simpel as one could have hoped for. In the past I have used ammonia in methanol to achieve this. The commercially available NH3 in MeOH is rather variable in quality so making it fresh by saturating methanol with ammonia using a gas cylinder is preferable. It's an annoying exercise making the solution and the actual reaction CO2R to CONH2 doesn't really work that well.So when Steven Ley comes up with a method that involves scooping solid magnesium nitride into a flask with your ester and some methanol, heating it to 80 oC for 24 hr, work up, filter, done! then that is really exciting good news to the synthetic organic chemist.

Magnesium nitride is reasonably priced, e.g. Aldrich (#415111) 10 g for less than 40 Euros. D!

Asymmetric synthesis of vinylcyclopropanes

Apologies for the sluggish posting. Life is more complicated than usual as I've moved from one research group to another and as many of you will know this essentially means that you are working in two groups for a while. Finalising old stuff, cleaning up and writing papers on one topic whilst trying to start new projects in another lab....somewhat stressful and time consuming. Anyway, enough moaning. As you can see from the previous post I've sacked my fellow bloggers as they weren't blogging. So now it's all down to me (which it was anyway). I've been meaning to post this stuff since I read the paper in late December 2006. I have had a long lasting affair with cyclopropanes, in particular cyclopropane amino acids so I was very pleased to see this paper by Deng et al., DOI: 10.1021/ja056751o. These guys from Shanghai are doing some real cyclopropane magic using some easily obtainable camphor-derived sulfur ylides:The work is very throrough and makes up an 11 page JACS paper (not including any experimental). Many chemists would probably have split this work up in two papers. It's really nice to see these guys decided to stick the whole story in one paper. In brief these guys discover that they can make trisubstituted vinyl-cyclopropanes in high yield, diastereoselectivity and enantioselectivity. Moreover, they can make both enantiomers of cyclopropane selectively by switching from endo- to exo-sulfur ylides. This table from the paper illustrates how sweet this stuff is:Only "problem" here is that they are using stoichiometric sulfur ylide. However, they address this by developing a catalytic ylide cyclopropanation. The yields are not as impressive and the ee's are down to 50-80%. Still pretty cool and I bet these guys are working hard to improve the catalytic system. Finally, they decide to pull off a short and high yielding formal total synthesis of a known cyclopropane amino acid.
Obviously, they are making both enantiomers as well as both enantiomers of a diastereoisomer. And here I'm messing around trying to improve my lousy dr's on the racemic synthesis of the same target. Crap! D!

The Mannich Reaction revisited

The Mannich Reaction (Carl Ulrich Franz Mannich, 1877-1947) is yet another one of those reactions that look brilliant on paper. However, I have on many occasions heard chemists attempting the reaction moan a fair bit to say the least. The major problem seems to be that the reaction is sluggish requiring heating/reflux to get anywhere and that the reagents (and desired product) start polymerising. You can find Mannich's original paper here: Mannich, C.; Krosche, W. Arch. Pharm. 1912, 250, p. 647. There is a detailed entry in Wikipedia on the reaction for those not familiar with it. A good alternative to the classic Mannich conditions is to use Eschenmoser's salt which I've seen used successfully in a number of total syntheses. Anyway, recently a PhD student in my lab was bitching about his Mannich Reaction. He left the lab, did some reading and came back with this nice JOC Note by A. Erkkila and P. M. Pihko, DOI: 10.1021/jo052529q. When he started using this stuff all his problems were solved. Fortunately, he sorted all this out right before I had to do my first Mannich Reaction. It also worked as a charm for me so I warmly recommend this simple, and efficient Mannich protocol.

Now Erkkila and Pihko are quite concerned about reaction times because they are thinking of industry applications. However, for the average chemist that does a lot of work overnight (whilst at home in bed) it isn't essential that it's done in 1 hour. We found that if you do these reactions overnight no heating is required and the products are of very high purity. Very clean reactions indeed. Here's four examples from the paper:

As it turns out the chemistry works really well for most systems using catalyst 1. However, some aldehydes require catalyst 2 to give a good result, eg. entries 3 and 4. The only compounds tested in this paper that failed completely were aldehydes that exist predominantly in a hemiacetal form, eg. 5-hydroxy-valeraldehyde. So there you have it. Maybe something you should consider giving a go next time it's alpha-methylenation time. D!

Tethered aminohydroxylations Donohoe stylie

Back in 2002 I started on a project where we considered using the Sharpless asymmetric aminohydroxylation (AA) as a key step. However, the anticipation of major regioselectivity issues and the success we experienced using the Sharpless asymmetric dihydroxylation meant we abandoned this approach entirely. However, I clearly remember sitting at my desk drawing a tethered version of the AA reaction where the amine was attached to an allylic alcohol as a carbamate. I'm sure that hundreds of other guys where drawing similar stuff and scratching their heads, however, Timothy Donohoe, from Oxford University decided to put the pencil down and get some students to get on with it. I completely missed the first paper that came out in 2001 in Chem. Commun. (DOI: 10.1039/b107253f) and only picked up on what they were doing when they published a paper on their TA work in JACS in 2002 (DOI: 10.1021/ja0276117). Ever since I have been following the Donohoe groups progress closely. The reason that I'm posting this now is because they finally nailed the reaction down in a recent Org. Lett. paper (DOI:10.1021/ol070430v). Anyway, let's get down to business. Firstly, it's important to realise that the TA reaction isn't asymmetric. It is however, a stereospecific, stereo-, regio- and chemoselective process. In other words if you start with optically active substrates you are laughing. Here's the condensed version of the story so far:

(1) Donohoe et al., Chem Comm, 2001, pp 2078-2079 (DOI: 10.1039/b107253f)
TA of acyclic, allylic carbamates using tert-butyl hypochlorite as the reoxidant with 4 mol% osmium. Yields ranging from 41 to 61%. Here's a really nice example with a diene:

(2) Donohoe et al., JACS, 2002, pp 12934-12935 (DOI: 10.1021/ja0276117)
TA of cyclic, allylic carbamates using tert-butyl hypochlorite as the reoxidant with 4 mol% osmium. Yields ranging from 50 to 83%. Works for 6,7 and 8-membered rings but only 5-membered rings with exocyclic double bonds undergo aminohydroxylation. Here's another nice example making a protected amino-sugar:

(3) Donohoe et al., Org. Lett., 2004, pp 2583-2585 (DOI: 10.1021/ol049136i)
TA of chiral acyclic, allylic carbamates using tert-butyl hypochlorite as the reoxidant with 4 mol% osmium. Yields ranging from 57 to 74% with excellent syn-selectivity. Some very impressive examples of TA reactions in this paper, for example:

(4) Donohoe et al., JACS, 2006, pp 2514-2515 (DOI: 10.1021/ja057389g)

Finally, they manage to get rid of hypochlorite and NaOH by attaching a mesitylsulfonyl substituent to the carbamate nitrogen. As a consequence catalyst loading can go down to 1%, yields have improved (69-83%) and homo-allylic carbamates have become viable systems. Check this homo-allylic TA out:Nice stuff innit and it gets better.

(5) Donohoe et al., Org. Lett., 2007, pp. 1725-1728 (DOI: : 10.1021/ol070430v)

And finally the climax. This is the final, and very recent paper, from the Oxford lab. Previously some of the TAs just didn't work (with the mesitylsulfonyl N-substituent) for no apparent reason. So they screen a bunch of different N-leaving groups and discover that things take off big time when pentafluorobenzoyl is attached to the carbamate. Catalyst loading is now permanently down to 1 mol%, yields are up (71-98%) also for difficult homo-allylic substrates, and it works for both cyclic and acyclic systems. Here's a nice homo-allyic example:

So it took about 6 years to develop this methodology to the point where I believe it will start finding wide spread use in synthesis. I'm itching to try one of these for myself and I'm desperately looking for an excuse. If anyone has tried running some of these Donohoe TAs I would very much like to hear any comments - is it really as good as it looks on paper? D!

Tempo Oxidations Part II

I've previously mentioned the TEMPO/BAIB combo for oxidising alcohols to carboxylic acid. A very smooth and mild oxidation. Back when we discussed this particular reaction we had a brief discussion about stopping this reaction at the aldehyde stage. This is indeed what this particular reaction type was developed for originally and so when I recently had to oxidise a primary alcohol to an aldehyde I thought I'd give it a go. By chance Delfourne et al. (DOI: 10.1021/jm0308702) had previously made the same compound using a TEMPO oxidation. Too easy! According to the procedure the product was obtained in quantitative yield and purification wasn't required! This particular procedure involves a crazy cocktail of reagents. This is what I did:Fortunately, all the ingredients are reasonably affordable. Mixing it all up and adding the alcohol gives a biphasic reaction mixture that looks a bit like Schweppes Orange:
However, unlike Delfourne et al. my final product wasn't clean after a simple work up. Succinimide was simply precipitating everywhere and hence some silica was required. In the end a filtration through a silica plug proved sufficient to give clean product on a reasonably large scale (18 grams) in excellent yield (97%). So despite the fact that a simple work up wasn't sufficient to clean the product up this is an easy to do reaction that I would recommend to anyone who's tired of stinky old Swern. D!

Fun with singlet oxygen

So finally I'm back fresh and invigorated after numerous bottles of awesome South Australian Wine. I'd like to recommend Primo Estate/Joseph, Kay Brothers, Pertaringa and Leconfield/Richard Hamilton in McLaren Vale and Langmeil, Rockford and Richmond Grove in Barossa Valley. Anyway, first day back at work I did a photolysis as we often do were I work. In other words a diene is irradiated in the presence of oxygen and a triplet sensitiser (in this case Rose Bengal). I love this reaction because it's absolutely beautiful. Check it out man:
Now the above reaction is obviously totally irresponsible and was only on for less than a minute so that I could take the picture. We are dealing with a fancy piece of glassware that has oxygen bubbling through it whilst two flood lamps are hammering photons away generating singlet oxygen and in the process heating the fume hood up big time. Hence, aluminium foil on the bottom of the hood is required to literally avoid a melt down and I'll be hooking a pump up that sends ice water through the cooling jacket. Moreover, I'm synthesising an endoperoxide - AAaARRRrGGGHhHh PEROXIDE - yeah I know but they are really quite stable. In fact our friends at the defence force haven't been able to blow them up so we aren't too worried. However, to avoid any nasty surprises we try not to make more than 5-10 grams of endoperoxides at any time. So as I said totally irresponsible hence I proceed to wrap this beautiful reaction up in two blasts shields covered in aluminum foil, pull the sash down and hook a cooling box/pump up to the glass ware and the final set up looks like this:
Well at least I know it's beautiful behind all that plastic and aluminium foil. This particular day I was doing the following photolysis:

Reactions of this type generally work quite well giving yields in the 40-70% range and since dienes are easily accessible using classic Wittig chemistry we consider making endoperoxides quite trivial. So why was I making this particular endoperoxide? Well if I told you I would have to kill you. There should be a paper coming out later this year featuring amongst others this particular endoperoxide turning into a supa cool cyclopropane in one (yes one) synthetic step so keep your eyes open for that paper. Some of you are probably wondering what Rose Bengal is. Behold the halogenated beast:We tend to use the bis-triethyl ammonium salt (as shown) because it is nicely soluble in organic solvents such as dichloromethane. D!

Adelaide Synthetic Symposium 2006 Part II

As I mentioned previously Professor Mukund Sibi also presented at the symposium. His talk was entitled: "A New Dimension to Enantioselective Catalysis - Templates Come to the Rescue". Sibi is all about developing new methodologies for asymmetric synthesis. However, the approach is different from what other people in the area are doing. Basically, his concept is to attach a template to the molecule you would like to perform your asymmetric chemistry on. To achive asymmetric induction he now chucks some chiral Lewis acid into his flask followed by the reagent that is going to react with his substrate. The result is high yields and excellent ee's. Okay I think it's time for some structures to clarify matters. Sibi has done a whole bunch of asymmetric radical additions that goes along these lines:A very important detail is that the template is a simple, achiral unit. The sole purpose of the template is to coordinate the Lewis acid well, exert rotamer control and as a consequence give good facial selective for the incoming nucleophile. Now as I mentioned before this principal works very well for many reactions. The Lewis acid is used in sub-stoichiometric quantities (generally 10-20 mol%). The radical stuff that I outlined above is okay cool but I personally like his stuff on pericyclic reactions better. Back in 2001 he published a very interesting paper in JACS (DOI: 10.1021/ja016396b) on Diels-Alder reactions:

So this is taking things one step further by using a pyrazolidinone template with a substituted nitrogen. What they are achieving here is what can be described as relay induced enantioselectivity by nitrogen inversion. In other words, you use a achiral pyrazolidinone template and throw your chiral Lewis acid in that upon coordination will favour one asymmetric conformation of the template. Pretty funky stuff. You really need to check this paper out to get all the details. Anyway, it works very well. Here's some numbers:

Notice that template 11 with no relay unit is poor proving their point.

More recently Sibi has done some work on enantioselective [3+2] cycloaddition of nitrile imines (DOI: 10.1021/ja051650b). This time using their basic system with no relay. This stuff also works exceptionally well giving some heterocyclic compounds that might be appealing to people doing a bit of medicinal chemistry:

This time there's also regioselectivity issues. However, they solve this and all other associated problems elegantly producing the desired dihydropyrazoles in excellent yields and ee's.

I recommend reading these two JACS communications. Good thorough science and very well written papers. D!

Pd-catalysed Cross-Coupling Reactions of Heteroaromatic Carboxylic Acids

A while back Chris published a post on a Science paper entitled:

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Synthesis of Biaryls via Catalytic Decarboxylative Coupling (DOI: 10.1126/science.1128684).

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A very interesting piece of work. However, then one our readers known as aa posted the following comment:

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"Not sure if this is referenced in the Science paper (should be), but similar work was recently reported by a group at Boehringer Ingelheim in JACS. Check out JACS, 2006, 128, 11350-11351. They use palladium catalysis, and heteroaryl carboxylic acids, but the principle is identical."
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I finally did something about it and read the JACS paper (DOI:10.1021/ja063511f). Now first of all the German dudes who published their stuff in Science would have been hard pressed to cite the Boehringer Ingelheim group since their manuscript was submitted a month before the Boehringer people got round to it. The JACS paper is however very interesting. In fact, I find it even more exciting than the Science paper because these guys are making some pretty nice heteroaromatic systems that would make any pharma medchem person wet his pants. Moreover, they are doing it with excellent selectivity and in good yield. Most of the stuff in the paper is done under microwave conditions but they do one old school thermal example that works okay (See Scheme above).

So at first glance these two papers seem very similar. However the postulated mechanisms are quite different. Firstly, the stuff in the Science paper starts of with a decarboxylation / copper insertion followed by a transmetallation and so forth. So in other words all the action is happening where the new bond is being made, like this: However, the stuff in the JACS paper is different. Firstly, it only works if the carboxylic acid is adjacent to the heteroatom. Secondly, palladium adds adjacent to the carboxylic acid via an electrophilic palladation followed by palladium migration and concomitant decarboxylation. Finally the generated palladium species undergoes reductive elimination to form the desired product like this:

In other words quite a different mechanism that doesn't involve a transmetallation step.
Anyway, thanks to aa for the tip. This has been most educational. D!

How I learnt to love oxy-mercurations

Well maybe I'm pushing it a bit with the title. I'm not exactly loving oxy-mercurations but I have now added it to the list of synthetic transformations worth remembering. Now I have had a long standing disrespect for the goode olde oxy-mercuration. I have always erroneously assumed that you needed stoichiometric mercury for oxy-mercurations to work. However, oxy-mercurations work really well on paper and I teach the stuff to my undergrads when we do electrophilic addition to alkenes, I do however tell them that it has no place in the lab. Well not any more! Today I completed my first oxy-mercuration and it worked really well and guess what...it's catalytic in mercury! Now I hear that many Universities don't teach this stuff to their undergrads anymore so here's a quick little scheme if you don't have a clue what I'm talking about:

I've shown the reaction for an alkyne starting material but it also works for alkenes to give alcohols rather than ketones.

So why did I end up doing an oxy-mercuration? Well as it turns out we needed to get hold of a serious quantity of acetoxymethyl vinyl ketone and a quick search resulted in the following result:

Now these Scandos did a thorough job and wrote a fairly detailed experimental procedure:
A few small additions to the experimental procedure would be:
1) During the acetylation the reaction is indeed somewhat exothermic and unintentionally I proceeded to boil the crap out of it (boils at ~150 oC) for about 5 minutes.
2) If reactions go jet black and shite starts precipitating from them this should be included in the experimental procedure. This is exactly what happens when you start adding the alkyne to the mercury cocktail. At this stage I was convinced that I had fried my alkyne but apparently black crap means that the reactions is working smoothly! This is what it looks like after the addition of a small amount of the alkyne:3) Removing "part of the acetic acid" should be changed to: "remove most of the acetic acid". Otherwise, you'll end up doing nothing but adding sodium bicarbonate and filtering bucket loads of sodium acetate off for an entire day (just like I did!).

Anyway, to finish this oxy-mercuration business the chemistry works very well but man I was working my ass off for two days to get it all done. There's quite a few time consuming steps in this prep such as the adjusting of the pH to 8 followed by removal of endless quantities of sodium acetate not to mention a vacuum distillation at the very end. D!