The Half Decent Pharmaceutical Chemistry Blog

Summer is rapidly approaching and, apart from the warmer and sunnier days, if you’re a man, there’s a chance you have realised it by the sudden change in the diets of many women around you. Haven’t you noticed your girlfriend doesn’t you to take her out for dinner any more? Have you checked the ratio Diet Coke : Beer in her fridge? Haven’t most of your female colleagues at work started to sit around the table (where you have lunch all together) to eat, unlike you, carrots and yoghurt? Well, they are terrified, thinking about the day YOU will innocently propose to go the beach and they will have to squeeze themselves inside a microscopic bikini (perhaps, on that particular occasion they’d opt for a more forgiving swimming costume, pretending that it’s still too cold for them). Trust me, she hates you because you REALLY don’t care about your funny skin colour (after months in the lab, who could be bronzed?) and how fat you are.

Another common theme for a conversation among your female lab-mates (if you ever bothered listening) is the diet to try not to arrive…unprepared for the first Saturday at the beach. Magazines and tabloids are full of  last-minute, radical diets. Needless to say, most of these tips are for desperate, running out of time people and have no scientific rationale behind. So, they are useless and often dangerous.

Here at The Half Decent Pharmaceutical Chemistry Blog, we like to do things properly and, even when it comes to diet tips, we want to be taken seriously and put science behind what we say. So, for the diet I’m going to unveil for all the ladies reading this, I want you to believe me when I say I’ve tried it.

I’ve indeed what I call the Lowry diet on Wednesday and Thursday. In a few words, you skip the lunch break because you are in the middle of a protein extraction for a Western Blot: I know you’ll be thinking you should be in no hurry with the protease inhibitors and sodium orthovanadate and fluoride you have suspended your extract in. Don’t fool yourself, please: we all know you should be quick at boiling your samples and quantifying it (this, merely, because you’re curious to know how many microlitres you’ll have to load on your gel).
Whatever the reason, if you harvest at, say, midday (because it’s 24 hours after you performed a transfection), you’ll be right in the middle of your experiment, perhaps preparing your BSA standards for the calibration curve.
The Lowry assay, in fact, is a popular, maybe old-fashioned, certainly reliable and sensitive way of quantifying the concentration of proteins. It basically consists of two parts: in the former step you perform a Biuret reaction, which means you add an alkaline solution of Cu2+, that reacts with peptide bonds yielding Cu+ ions. Subsequently, Folin-Ciocalteau reagent is added: this mixture of phosphomolybdate and phosphotungstate undergoes a reduction to heteromolybdenum blue, which is coupled with the oxidation of aromatic amino acids in the presence of copper ions.

To sum up, the solution turns blue (picture to come soon, psi*psi) with an intensity that depends on the amount of tyrosine and trytophan and, therefore, the overall amount of proteins present in the cuvette. The precise concentration is determined by comparing the absorbance of the sample at 750 nm with those of standard solutions of BSA, used to draw a calibration curve.

This said, you generally need to wait 15 minutes after the addition of each of the two solutions, so you’ll be too busy to have lunch. It definitely works!

It’s July and I have begun to receive emails from worried, nerd undergraduates who ask me what they could do now that they are going to be kicked out of the lab.

I’m happy to announce, my nerdy friends, that I’ve got a perfect solution for you, which, believe it or not, will take you to sunny beaches so that you’ll all finally lose your ugly paleness, while giving you the opportunity of doing science.

It’s time to go crab hunting! In particular, this is the last month when you could hunt for Limulus Polyphemus!

This lovely animal is better known as horseshoe crab: these crabs evolved from sea scorpions in the Paleozoic era (fossils were found in Germany). They depose eggs in the sand of estuaries in April: there, trilobate larvae appear until, when the newborn is ready for living in the sea, high tide gently takes them out of the sand.

You shouldn’t bother about the offspring: concentrate on the parents. We need their blood! No, it’s not for a gory sacrifice or a delicious, Spanish plate: it’s all about saving lives, dear.

Let’s begin with a bit of a history lesson. In the 1940, the Brits set a first official test to assay the content of pyrogens in parentally-administered drugs: they injected in three rabbits the product and, if the sum of their temperature rise was less than 1.15°C, there wasn’t any problem. Otherwise, whether the sum reached 2.65°c, that product had to be destroyed. Anything in between would require the assay to be repeated employing 6 rabbits (with different values, of course).

In 1960s, however, Dr. Frederik Bang, at the Marine Biological Laboratory in Woods Hole, Massachusetts, discovered the properties of horseshoe crabs.

You see, this lovely creatures present a granular amebocyte in their blood, where Cu replaces Fe. So you can really say this is a blue-blooded crab!
From May to July a third of hemolymph of Limulus Polyphemus is collected. The animal is therefore marked so that it won’t be used for the next two years or so and is released in the sea where, it has been proved, it goes on with its normal life as always. Perhaps it’ll look like an undergraduate preparing for exams among its peers…

However, this hemolymph is rather precious: in the presence of endotoxin G (typical of Gram- bacteria), it coagulates. That’s because endotoxins trigger the activation of factor C. This protein then acts on factor B, which in turn activates a proclotting enzyme, that converts coagulogen to coagulin. As a result a gel-clot is yielded.
Interestingly, this test works for yeasts and moulds too, which present β-1, 3 glucans on the surface, because this alternative substance activates factor G whose role is exactly the same as that of factor B.
It is interesting to highlight that the last 8 amino acids of the peptide C of coagulogen are the same of fibrinogen.

Briefly, the LAL test (Limulus Amebocyte Lysate) works as follows: physiologic coagulogen is substituted with a derivative which bears p-nitro aniline bound to an arginyl group, so that an aromatic amine is yielded. This is a great advantage as the concentration of this molecule can be easily detected through a colorimetric titration. To sum up, the concentration of endotoxins is determined using standard solutions whose pyrogen levels are known.

For what concerns the limits of endotoxins parentally administered, they are calculated through the K/M ratio, where K stands for the quantity of endotoxins that an average human being can receive without showing any pathological consequence (intravenous = 5.0 endotoxin units/kg/hour; intratechal = 0.2 EU/kg/h) and M is the maximum recommended human dose of that drug.

For more information, log on to this website (I’ve always wanted to say this phrase!). Hey, you! What are you waiting for?! You’re running out of time: go crab hunting! 

 

Movies, from ChemicalForums Blog, wrote, a few days ago, a brilliant article about quinine. In that post, he mentions that quinine is present in the tonic water we all use for preparing refreshing Gin & Tonics.

Apparently, he took inspiration from my recent post about LSD: he might have liked that little bit of history lesson I put right in the end of the article (as he says in his comment) or the link between drug and everyday life.

By the way, before writing on his blog, he told me I should have posted something dealing with quinine on mine (and in that occasion he mentioned tonic water as well).

As it turned out, he couldn't resist. Fortunately, however, since I'd have never been able to write such a masterpiece of organic chemistry, about cinchona alkaloids.

When I pointed this out he kindly suggested I could "complete" his synphony describing these drugs from a pharmacological point of view.
This will be done in future, but, today, I'm not going to talk about it.

Today, I'm going to do it: I'm going to check how much quinine is actually present is a normal can of tonic water!

To complete my task, I have to use fluorescence: the molecule needs no derivatization, as you can see from its structure, as it's already eligible this technique.
The assay is pretty quick, indeed.



Before we can determine the concentration of drug, we need to prepare a calibration curve: this means we need a standard solution of quinine that will be diluted to yield at least four different points.

To get a stock solution, 14.8 mg of quinine sulphate (MIND THE DIFFERENT MW!) standard are dissolved in a 50 mL volumetric flask with ethanol.
Then 1 mL of the solution is put in a 25 mL volumetric flask, plus 250 μL of acetic acid and water.

To find out the excitation wavelength (346 nm), I start with a UV spectrum.



Four dilutions are therefore prepared: 1:10, 1:20, 1:40 and 1:60. The second is immediately used to detect, at 346 nm, the wavelength at which the highest intensity of emission is observed: 456 nm.



Finally, we mesure the fluorescence emission intensities of the four solutions at 456 nm.



Time to open our can of tonic water: 100 μL are diluted 1:100 with water (not gin, how sad...). So, you just register the spectrum and derive the concentration of quinine.



Our magic number is 55.77 μg/mL.

Time for a massive ingestion of gin&tonics, now! Seeya!

Sadly, last Sunday, the ninth series of Top Gear finished. But don't dispair, since here is what I've devised to cast away your sadness!

This is my idea: Bactrim and sulfonamides are annoyingly common drugs. However, we can live things up by assaying their most important feature: how fast they go around my track! I mean: How hard can it be?!

The track is a Luna Phenomenex C18 column for HPLC. However, because for the job in hand the molecules would be too hydrophilic for a traditional reverse phase HPLC, we are going to use a particular mobile phase: 4g of sodium heptanesulfonate are dissolved, together with 1 g of 1,10 diaminodecane, in water.
Concentrated phosphoric acid is, then, added and, finally, the pH is adjusted, with NaOH 1M, at 6.5.
This mixture is added to a smaller amount of acetonitrile (the "real" mobile phase), so that the final ratio is 83:17.

At this pH, all the basic centres of our molecules are protonated. So, once you've loaded the column, they will displace the diamine and react with the anionic end of heptanesulphonate, whose remaining lipophilic part is tightly bound to the stable phase.

While the charged groups of the analytes are engaged in ionic interactions, the rest of them binds (although less strongly) to the said lipophilic chains of heptanesulfonate.

As a result, the whole procedure looks like a normal reverse phase chromatography, with the most lipophilic element showing the highest retention time.

So, we test Bactrim (trimethoprim and sulfamethoxazole), sulfadiazine, sulfamethoxypyridazine and sulfadimethoxine.

And they're off!

 

 

 

Yes, Jeremy, trimethoprim has two positive charges, unlike the rest of the stuff here, but all that POWER, in this context, will actually slow trimethoprim: the double interaction with the stable phase leads to the highest retention time (8.493).

For the rest of the sulfonamides, their speed is the direct consequence of their interactions with the lipophilic side chains: consistently, sulfadimethoxine is the slowest (how pathetic is that 6.65?!) and most hydrophobic, whereas sulfadiazine sets our new record with a whopping 1.720 minutes retention time!

 

Look at these two molecules: obviously they are two xanthines and rather similar too. Still, one is Dyphylline (aka diprophylline) and the other is etofylline and, if you're asked to distinguish them, I'd better not be in a hurry and have good music with you.

The IR spectra, for example, look exactly the same. Annoyingly, even the melting point can't help you very much: both melt between 160°C and 165°C.

And don't fool yourself: yes, go on trying to see a different absorbance (273 v. 270) with the UV spectroscopy...rumour has it that someone is still in the lab hopelessly trying to detect any difference

These molecules are so similar to each other to make the job in hand look impossible.
Unless you fiercely fight the enemy here: water.

Your only option is acetic anhydride (99%). By the way, once again both are soluble in this aprotic solvent, but this is just the beginning.

The mixture boils for 15 minutes at 160°C. Meanwhile, the "magic" happens: both substances are capable of reacting with the anhydride, yielding acetylated derivatives, whose melting points, however, are fortunately different.



The derivative of dyphylline melts between 142 and 148 degrees, while acetyl etofylline needs approximately 120.

But this is just the end of the work. Before you could reach a verdict, the flask has to cool down to room temperature, so that a mixture (20:80) of ether and petroleum could be added.

Then, there is the trickiest part: you place the flask on ice for about 20 minutes. Meanwhile, you regularly stir the solution, increasing the chances of yielding a nice crystalline precipitate of the acetate by rubbing vigorously the inner of the flask every so often.

So you can't go out of the lab to check your inbox...

Finally, the precipitate is purified (through filtration), washed with the same mixture of ether and benzine, recrystallized from alcohol and dryed much as possible (which means this procedure takes 30 minutes, at least).

To sum up, the message here is rather direct: water is the enemy, ether is your best friend.

And you'd better take your MP3 player with you in such a circumstance, because the lab is cold and depressing when everybody has left.