Thursday, July 26, 2007

Behold cyclohexane!

Recently I was flipping through a magazine published by a learned society and my eyes were caught by this rather interesting picture. The aim of this photo is to encourage scientist to publish in certain society journals. Firstly, it seems rather lame that the teachers is pointing at cyclohexane that appears to be in its perfectly flat conformation? Is that the most acidic proton he's pointing at? Secondly, what is going on with the students. I have a feeling the guy to the left is a werewolf. Or maybe he's just split up with the girl to the far right which would explain why she looks like she's about to commit murder. And what's going on with the woman in the background? It's cyclohexane people and not Brevetoxin so stop looking at it like that. D!

Monday, July 23, 2007


We hardly finished talking about oxepans and Ferrier rearrangements when I spotted a paper on the synthesis of septanosides by a very similar route. This time the nucleophile is sodium methoxide and the rearrangement proceeds in an impressive yield to give a single anomer!

Interesting work by Ganesh and Jayaraman at the Indian Institute of Science in Bangalore. D!

Monday, July 16, 2007

Oxepane Nucleic Acids - Part II

Before I get started let me say that I think this is a good paper by Damha and that the compounds are genuinly interesting. However, I did find a number of things I believe could be improved.
In the preivious post I gave the Tm data for homo-adenine and homo-thymine Oxepane Nucelic Acids (ONA) and it was quite clear that the low melting temperatures renders ONA useless from a pharmaceutical point of view. However, as I said it has a high stability in serum and activates RNase H. To date only very few oligonucleotide analogues have activated RNase H. The authors seem to be of the opinion that only four RNase H activating oligonucleotide (ON) analogues have been reported to date. However, they seem to forget the very first and most famous analogue - Phosphorus monothioates (PS). PS have been well known since the early 80s and ISIS Pharmaceuticals tried deveoloping drugs based on this class of analogues for a loooong time (they may still be doing so for all that I know). Anyway, let's have a look at the chemistry. They choose a somewhat surprising starting material and do a very funky Vorbrüggen coupling. I've never seen anything quite like it (notice the counterintuitive stereochemical outcome). Very impressive although the yields are poor. They get a fair bit of the diene oxepan product and some of the alfa-anomer too. Nevertheless a nice piece of work. Apparently an adaption of some work by Hoberg (JOC, 1997, 62, p. 6615) that I haven't checked out. People who haven't worked with nucleosides probably don't realise how difficult even the simplest transformations can be a times. Thymine and Adenine are by far the two "easiest" nucleobases to deal with. Cytosin is bad news and Guanine can be an outright nightmare. This is probably the main reason why everyone tests T and A first. Anyway, after taking the protection group off they proceed to hydrogenate the olefin which goes well. However, from here on it's a bit nasty. Firstly, they decide to use monomethoxy trityl (MMT) rather than dimethoxy trityl (DMT) because they get better yields this way. Now different molecules behave differently but I have a hard time accepting that you can't get DMT on in a higher yield than they report for MMT protection. Very odd! I wish they informed what happens instead. Maybe di-protection is a problem for some reason? Furthermore, they proceed to synthesise the phosphoramidite using the classic phosphoramidochlorodite (PCl) reagent. I see this all the time. Virtually, everyone in the area is using this phosphitylation reagent instead of the superior phosphordiamidite (PN2) reagent (See below). Back in the Jurassic when I used to make phosphoramidites I always used PN2. As a consequence no chromatography was necessary and I got >95% yield. We published a paper on how to make LNA (pioneered by Wengel) phosphoramidites using this reagent some years ago (Synthesis, 2002, 6, p. 802). Anyone interested in improving their phosphoramidite synthesis can request a copy ( So later on they proceed to synthesise their oligonucleotides and only achieve coupling efficiencies of 98-99%. As always it is hard to know why the yield is reduced. I wonder if the use of MMT protection has anything to do with it. One final comment regarding their conclusion. They seem to spot a connection between RNase H activity of ON analogues and sugar conformer flexibility along the ON strand. However, they have just mentioned that alpha-L-LNA is a known ON analogue that activates RNase H. This analogue contains a highly constrained bicyclic sugar (See Figure) and hence doesn't support their conclusion. They could conceivably be right. Maybe some other mechanisms are at work with alpha-L-LNA but I think they should at least have mentioned this. Anyway, overall a good paper from this Canadian research group. Keep them coming. D!

Wednesday, July 11, 2007

Oxepane Nucleic Acids - Part I

The chemistry you start your career working with tends to stick to you. Stuff you work on later seems much easier to shake off. Anyway, I started as a nucleoside/oligonucleotide chemists and although what I do now is miles away from this area every time my eyes wander over a graphical abstract with a nucleoside I stop. I just can't help it. It happened again the other day. Oxepan Nucleic Acids (ONA). Can you believe that it hasn't been made before. Apparently, no one has gone beyond the six membered ring until now. Now ONA is not a great nucleoside analogue. The T15 and A15 ONA oligonucleotides (ON) have affinities less than 5 oC towards DNA, a very low affinity towards itself (ONA T15 + ONA A15 = 12 oC) and a similar Tm towards RNA. In other words ONA is not suitable for antisense purposes due to the very low Tm. However, ONA is very stable towards nucleases and importantly activates RNase H. Now before I continue I should explain what Tm, antisense and RNase H is to the uninitiated. Firstly, Tm is the temperature at which 50 % of a duplex has denatured, ie. high Tm = stable duplex. Secondly, antisense is a different approach to drug development targeting RNA rather than proteins. The idea is to knock the RNA out before it gets translated into protein (See figure). This is achieved by synthesising an antisense ON that is complementary to your RNA target. The mechanism of action for antisense is either:
(a) Inhibit the translation to protein by physically blocking the RNA strand making it impossible for ribosomes to translate it
(b) Activate the enzyme RNase H that specifically targets DNA-RNA duplexes and only degrades the RNA strand.
A lot of people in the field believe that antisense can only work effectively with RNase H activation and I tend to agree. The cell is amazingly efficient at making RNA and translating it to protein so if you have to get stoichiometric amounts of antisense ON to RNA into the cell you are likely to have a problem. The beauty with RNase H activation is that the system is catalytic. In other words the antisense ON gets released after RNA degradation and moves on to the next victim. The problem is that you cannot use regular DNA for antisense purposes as it has a very short half life in serum (~15 minutes). So you have to devise an analogue that is stable in serum, has high affinity towards RNA and activates RNase H. Now obviously this is no easy feat so why bother? The (theoretical) advantages when compared to traditional protein targeting drugs are:
(a) Complete selectivity only for the intended target
(b) You can target anything involving RNA
(c) The chemistry is the same every time. You just have to figure out what the sequence of your target is and synthesise the required ON
(d) Getting drugs to market is rapid because drug development is significantly faster
Obviously, things are much more complicated than this. Antisense was the big thing in the 80s. It was going to cure everything within the next decade but the reality is that only one product has made it to market. It's an ON called Vitravene (ISIS Pharmaceuticals) that prevents AIDS patients from going blind by targeting cytomegalovirus retinitis. That said a lot of advances have been made and there are numerous antisense ON in late stage clinical trials. Anyway, after this super condensed course in antisense ON I think we are ready for the actual paper. I'll let you off the hook for now. The next post should be up in a couple of days. D!

Friday, July 06, 2007

Still breathing

I happened to look at Curly Arrow the other's now been over a month since my last post! Not good, not good at all. Taitauwai even enquired about my well being. Well I'm still alive (sort of). My brain is slightly fried. I'm trying very hard to get some papers written whilst also attempting to set a new lab up, get my new projects going and phase new group members in and make their projects take off....yes I'm fairly busy. All my blogging time has effectively become paper writing time. Anyway, I have lots of things I would like to share at Curly Arrow and I'll try real hard to get some stuff posted. Whilst getting new gear for the lab I stumbled over an old box containing a virtually unused Vibro-Mischer - Das ideale rührwerk für Labor und Betrieb. Check out this nice poster I found in the box. That is one sexy model they picked to promote their products. If you click on the image you will get an enlarged version. D!