Diazomethane and the Arndt-Eistert Homologation

For the past year we have been starting peptidomimetic chemistry up as a new research area in our group. Many chemists believe that peptide chemistry is easy and that peptide chemists aren't "real chemists". However, let me tell you from personal experience that there is absolutely nothing trivial about peptide chemistry. Even short sequences with normal alpha amino acids can be a nightmare to make, troubleshooting is complicated, purification can be a major pain and yields that a small molecule chemist would consider a total fail is generally acceptable in this area of research. Some years ago I was working with a Post Doc that came from Dieter Seebach's lab at ETH. He introduced me to beta amino acids and ever since I have been fascinated by the use of these building blocks in peptidomimetic research. Inspired by the work of Samuel Gellman we are focusing on the use of beta-3 amino acids in combination with alpha amino acids. Consequently, we synthesise beta-3 amino acids to incorporate these in our peptides.

There is a number of ways to make beta-3 amino acids but from personal experience one method stands out as the best route to these molecules: the Arndt-Eistert homologation. In this classic approach an alpha amino acid is converted to a diazoketone followed by the Wolff rearrangement to provide beta-3 amino acids. The Arndt-Eistert homologation basically homologates a carboxylic acid with one methylene group as shown in the scheme below.

The last step, the Wolff rearrangement, is carried out by sonicating the diazoketone in the presence of a silver catalyst (in the dark). Because nitrogen is evolved during the course of the reaction we normally have an empty balloon fitted on the flask to avoid pressure build up. I rather like the feature that the balloon slowly gets inflated during the course of the reaction as shown in the picture below.

Silver catalysed Wolff rearragement in a sonicator. Left t = 0 hr; Right t = 2 hr.

However, as you may have noticed there is a down side to the Arndt-Eistert homologation: diazomethane! The reagent has a fearsome reputation and I have heard of a couple of guys who have managed to blow themselves up and gone deaf in the process. Allegedly, one chemist at our department even managed to set fire to himself! This was a long time a go when less attention was being paid to laboratory safety and the accidents were due to sloppiness and improper handling of diazomethane. If you are careful and use the correct glassware (with clear seal joints) there is (almost) nothing to worry about. We have purchased the setup shown on the picture below. This is a very nice diazomethane still consisting of only three pieces that will produce up to 40 mmol of diazomethane in approximately one hour. We only use hot water as the heating source and keep everything behind a blast shield just in case. Diazomethan is generated from Diazald  as shown in the scheme below and used immediately. The procedure it quite simple. In the separatory funnel you place a solution of Diazald in ether this is added dropwise to a heated mixture of aqueous potassium hydroxide, ether and a high boiling alcohol [commonly 2-(2-ethoxy-ethoxy)ethanol]. Diazald reacts with the base to produce diazomethane that is distilled with ether to the receiving flask.

Notice that diazomethane is always handled in solution. The neat stuff is known to explode unpredictably so don't even think about doing that. Because of the way that diazomethane is produced it is hard to add an exact number of equivalents to a reaction. For the synthesis of diazoketones we simply go for an excess of diazomethane (approximately 2-3 equivalents based on a 70% yield of diazomethane). We commonly distill the diazomethan directly into the reaction flask to minimise handling. For the synthesis of beta-3 amino acids the alpha amino acid is first transformed into a mixed anhydride which is exposed directly to an excess of diazomethane.

Diazoinsane clear seal distillation kit purchased from Sigma-Aldrich.

Unlike diazomethane, Diazald is reasonably stable and easy to handle yellow solid. Unfortunately, Diazald has obtained a rather bad reputation despite being relatively safe to deal with as long as you don't eat it, set fire to it, beat it with a hammer or something similarly stupid. Consequently, it can be rather hard to get hold of. When I worked in Australia it was particularly problematic as it can only be shipped by road and isn't produced in the country! Here in Denmark we get it from Germany but it does take a while because they don't send it with the regular shipments so you have to plan a bit ahead.

If you think that playing around with beta-3 amino acids could be fun I can recommend the company Anand Chem based in Slovakia. They produce almost all beta-3 amino acids with the proteinogenic side chains of excellent quality at a highly competitive price. Depending on what they have in stock you may have to wait a couple of weeks for the stuff but it is worth the wait considering the quality and the price. D!

Anhydrous Solvents Part 3: Acetone and Molecular Sieves - Bad idea!

I have discussed anhydrous solvents a couple of times and have been advertising the use of molecular sieves (MS) quite strongly. During my MS crusade I have pointed out that MS are no good for drying THF but that pretty much all other standard solvents work well with sieves. As it turns out this is incorrect and I have received a terrible punishment from the MS God. It's all rather embarrassing as a PhD student in the lab was fully aware of the particular problem I'm getting to shortly. The deal with MS is that they are weakly basic. I take advantage of this by always adding some MS to my CDCl3 which keeps it dry and mops up any HCl formed by the slow decomposition of CDCl3. The other day I was running some of 1H NMR and to my great pleasure I had finally (after months of struggling) made a very important target molecule. I had split the fractions from a column up in three batches to be on the safe side. All three 1H NMR spectra were great so I decided to combine them in one flask. I was running NMR in acetone-d6 and decided to use some acetone for the transfer. I couldn't find the HPLC acetone we normally have standing around and was getting a bit frustrated when I remembered that about a year ago I had made a bottle of acetone over MS (This is were all the alarm bells go of with the experienced chemist). I managed to find the bottle and proceeded to transfer all my stuff into a new flask. However to my utter surprise I was unable to remove the solvent on the rotary evaporator. On the high vacuum pump with a fair bit of heating most of it came off but by TLC there was a new UV acitve (and quite polar) compound. I had to re-column my product but it still wasn't pure! Currently I am attempting to crystallise it from the impurities. A fair bit of yelling and acussing people of sabotage took place. Fortunately it was late and there was only one other person in the lab.

Before I proceeded to clean up my compound I decided to figure out what the source of the problem was and I quickly discovered that the acetone smelt funny. Initially, I thought it was contaminated with benzaldehyde but when more dilute it had a floral/perfume scent that reminded me of ketones/esters. At some point the PhD student in the lab realised that I had been adding MS to acetone and mentioned that as far as he knew that was a no go because it goes Aldol in the presence of the weakly basic MS. I cannot believe that this hadn't occurred to me. As it turns out it is well known that ketones go Aldol when exposed to MS and many different compounds are formed. A selection of possible products from acetone are shown in the Scheme above. The polar compound I removed by chromatography is the acetone trimer with one hydroxy group.
In my defence I'll say that acetone can be dried with MS provided you use the acetone within a few days. I successfully used the anhydrous acetone during a week of experiments back in July 2009. The take home message is to bin it after a week and not use it a year later as I did. In fact just don't add MS to acetone but instead dry it with MgSO4 as described here. D!

Butt-in-diol?

Old school 1,4-butynediol. A wondeful and cheap building block. D!

Gurrs Microscopical Stains and Reagents

Don't you just love the way they used to store chemicals back in the day when men where men and women were women? We use a fair bit of Rose Bengal as a triplet sensitiser for the photochemical synthesis of 1,2-dioxines from dienes and oxygen. At the moment we are using some top-quality Rose Bengal from the world renowned chemical supplier Gurr:

Chemicals just don't come in containers like this anymore. If Sherlock Holmes ever used Rose Bengal he would most certainly have been a Gurr-man. Unfortunately, I haven't been able to get a new catalogue from George T. Gurr. I did however find this add for his products in Journal of Physiology (March 1953, Vol. 119, No. 4):

I wonder what happened to goode olde George and his microscopical reagents. D!

Anhydrous solvents

Most organic chemists need dry solvents from time to time and almost every single day you bump into someone who's looking for dry DMF, ether, acetonitrile, THF etc. When I was working in Cambridge this was never a problem. They simply have a still for every single solvent you could imagine. Above there's a picture from the still room in Cambridge. Pretty nice innit? A massive fire hazard but quite handy as long as it doesn't blow up. Now I've moved on and where I work now we've just been inspected by OH&S (Occupational Health & Safety) and they don't like stills because of the fire hazard etc. and I have to say I completely agree with them. Nice as they may be they are dangerous and more or less completely redundant. The solvents you can buy nowadays are of super high quality and do not require distilling so it's basically only distilled in an effort to dry it. Now I will concede that there are stabilisers in many solvents, in particular ethers, but it is very rarely something that will affect your chemistry. So what should you do? The perfect setup that will provide guaranteed anhydrous solvents every day consists of good quality super activated molecular sieves (MS) and a Karl Fischer (KF) apparatus. MS are expensive but you can reduce the cost significantly by buying bulk quantities. We used to get ours from Grace Davison in big drums and they were very very good. The Karl Fischer apparatus (see picture below) can be purchased from Metrohm. All you have to do when you have this set up is add some 3 or 4 Å MS (depending on the solvent) to your solvent close the flask tightly and when you come back the next day take a small quantity out with a syringe and needle and squirt it into you KF apparatus. The display will now show you how many ppm's of water there was in the volume you just added. Everyone doing anhydrous chemistry should have this set up. It's safe, you are always confident about whether your solvent is dry or not and it requires close to no maintenance. Unfortunately, molecular sieves will not dry everybody's favourite solvent THF so you have to hold on to one still. Also you cannot add anything with acidic protons to a KF apparatus successfully. It will think it's all water. So for example acetone or methanol wouldn't work. Regarding MS I believe that you should never attempt to dry them yourself and never recycle them unless they are going into exactly the same solvent (just bin them when they are dead). Many chemists think they are saving money when they reactivate MS. However, I seriously doubt that is the case with the amount of energy not to mention time required to do so. So in other words buy good ones and bin them when they stop working. If you aren't sure whether the MS you've got are any good put a couple in the palm of your hand and add one drop of water. If they get really really hot they a very good and if they only warm up a little bit they are rubbish. If you are in a situation where you can't get good MS I guess you will have to dry them. Apparently one way of doing this is to throw them in a microwave and nuke them on max power until they start glowing. At this point you have to stop immediately unless you want the entire microwave to melt and transfer the MS to a desiccator that you stick on a high vacuum pump (I haven't tried this myself so no guarantees). If you don't feel like burning your department to the ground there is always the good old vacuum oven that most departments have. Just heat them under vacuum for a couple of days and then stick them in a desiccator attached to a high vacuum line. Drying solvents overnight using good 4 Å MS should get acetonitrile, DMF, DMSO, dichloromethane, toluene, ether, 1,2-dichloroethane, chloroform and hexane down to a water content of 10 ppm or less. If you check your freshly distilled THF on the KF it should be around 15 ppm. So for successful living convince your boss or department to get one of these babies and shut down all those damn stills for good. D!

TEMPO - BAIB oxidation

Have you ever had to oxidise a primary alcohol to a carboxylic acid? Well as you know there is a ridiculous number of methods available. However, a common problem with many of the more traditional methods is that they are very harsh and could potentially rip your molecule apart. Just what you want after a 21-step linear synthesis innit? Moreover, many of the traditional metal-based oxidations can be a serious pain to work up so a mild and simple method would be kinda nice. Well one method that fulfills these criteria has been around for a while. I stumbled across it back in 1999 when I had to do one of these oxidations myself. A very nice piece of work on the oxidation of nucleosides: Now you have to admit that this oxidation uses the coolest reagents ever just judged by their abbreviations. BAIB should obviously be pronounced BABE. Anyway, at first I had no idea what TEMPO and BAIB were:Both TEMPO and BAIB are commercially available. Don't you just love stuff like TEMPO. A radical you just scoop out of the flask and throw into your RBF! So how does the reaction work? We'll TEMPO does the hard work of oxidising the alcohol. However, as it is only used in a catalytic amount a stoichiometric amount of BAIB is required to regenerate TEMPO. As far as I know no one has yet figured the exact mechanism out. However, for those interested there is a good review in Synthesis that takes a close look at the mechanism and shows the most plausible pathways: Nooy et al., Synthesis, 1996, pp. 1153 - 1174. The paper is worth looking up just to check out the photos of the guys who wrote it - absolutely priceless! Anyway, to get to the point the method is very user-friendly you basically just mix a big pile of BAIB with a small quantity of TEMPO add your alcohol and some acetonitrile and water and stir it for a couple of hours. The method is compatible with a whole range of functional groups (double and triple bonds, esters, ethers, acetals, epoxides, amides, halides, and azides) as well as protection groups (TBDMS, THP, MOM, Boc, Cbz, Benzyl and acetyl etc.). I have recommended the method to a number of people and they have all used it with great success even with very sensitive compounds so I suggest you give it a go if you are in an oxidising mood.

And finally a practical note. If you like me have managed to get stuck in the middle of nowhere and hence has to wait for 9-12 months to receive your BAIB by ship from the US you may consider just making it yourself. I haven't tried this myself but the guys in the lab do it frequently using a simple prep from Synthesis: Kazmierczak et al., Synthesis, 1998, pp. 1721 - 1723. The final stuff is supposed to be bright yellow but the guys assure me that the pseudo-yellowish stuff you for unknown reasons obtain sometimes works just as well. Here's a picture of the pseudo-yellowish BAIB one of the guys made a couple of weeks ago:

If you plan to hold on to it for a while it has to go in the freezer otherwise it goes off fast. Have fun, D!

Alcohols beware TBDMS-Cl is here!

Is there a better way to start our chem blog than to honour the late tenderbutton chem blog (That's Mr Stiles himself in the picture). Tenderbutton was the initiator for the present blog that aims to keep our brains busy by sharing chemistry related stuff.
Anyway, this post is about good old trusted TBDMS-Cl. You put it on and you take it off at your leisure just like underware. A very nice protection group that doesn't behave badly (most of the time). So just like all other synthetic organic chemists I keep coming back for a good time and every time my current supervisor has to bleed due to the high price of the stuff (2006 Aldrich catalogue price for 100 grams = 391 AU$). Somehow I managed to get through my PhD without using the stuff but now that I've grown up and moved away from home I'm using absolutely bucket loads of it. So what to do to save ones supervisor from financial ruin? Well Tenderbutton has the answer: Buy 1 kg from Oakwood Products in the US for next to nothing. Now I'm based in Adelaide, South Australia so it was obviously going to be a bit more complicated/expensive getting it down here. However, after negotiating with the Oakwood people for some time the final deal still makes it significantly cheaper to get from Oakwood despite the massive freight costs. A couple of days ago it arrived and mine is just as pretty as the one Tenderbutton got:
Nice innit? So how much does it cost to get a kilo of TBDMS-Cl sent to the middle of nowhere? Well the actual product only sets you back 353 AU$ which is pretty damn cheap. However, the freight expenses are a bit out of control and amount to 308 AU$ . So the total price is 661 AU$ meaning it's ~6 times cheaper than to buy it from Aldrich.

Interestingly, when I was looking at the invoices I couldn't help noticing how the courier had decided to describe the product:
Hmmm very interesting. I guess it makes it a lot easier to get it through customs if you very conveniently forget to mention that it's 1 kilo of a flammable and corrosive chemical substance. Moreover, I noticed that it hasn't been synthesised in the US as I naively assumed.

It seems that everything is made in China these days. Somewhat worryingly I think that my kids toys all smell a bit like TBDMS-Cl. I wonder if it's made in the same factory!
Anyway, anyone wanting to save a few bucks on their TBDMS-Cl get in touch with Oakwood and buy some. D!