Tuesday, September 18, 2012

Slow-gradient, sample-displacement chromatography

A universal, one-step method for the purification of large quantities of peptides

High performance liquid chromatography (HPLC) is one of the premier chromatographic techniques used world-wide for the purification of a wide variety of chemical compounds. It is essential for the isolation of new natural products, often used in the purification of synthetic molecules, ubiquitous in the peptide and protein synthesis lab, and indispensable for analytical chemistry. 

Figure 1. Schematic 
representation of molecule
elution profiles at different
loading levels: a) full peak
resolution, b) loss of 
resolution due to column
overload, c) displacement

For the most part, peak resolution is required to achieve purification by HPLC, regardless of which mode (e.g. normal phase, reversed phase) is being used.  This means the compound of interest has to be clearly separated from any impurities, as depicted schematically in Figure 1a.  This limits the quantities of compound that can be loaded onto the column, before resolution is lost due to sample overload and peak overlap (see Figure 1b).  Purification of large quantities of compound therefore requires either multiple, repetitive runs, or the use of large (and expensive) prep columns and large quantities of solvent.

Recently, researchers from the Brimble group at The University of Auckland, New Zealand, reported[1] the use of a slow-gradient, sample-displacement chromatography technique pioneered by Hodges et al.[2,3] for the successful purification of a wide variety of peptides.  The key advantage of this approach is that it allows the purification of large quantities (several hundred milligrams) of peptide in a single step.

Displacement chromatography is quite an old technique.  First proposed by Tiselius in 1943 and later developed further by Horváth,[4] it involves loading the sample onto a column, and then displacing it by a constant flow of a ‘displacer’ solution which contains a compound with higher affinity for the stationary phase than any of the components.  As the displacer travels down the column, it pushes the other components downstream giving consecutive areas of highly concentrated pure substances (see Figure 1c).  While this technique allows substantially higher sample loading, the requirement for identification of a suitable displacer and the need for its subsequent removal from the final product provide substantial drawbacks to this method.

Sample displacement chromatography removes these obstacles by using the sample components themselves as displacers. During loading of the sample mixture, which is done under overload conditions, the components compete for adsorption sites on the stationary phase.  The main separation occurs during the column loading phase: the components with higher affinity will compete for sites more successfully than those with lower affinity, which will be displaced further down the column.

Rather than requiring optimization of conditions for every different peptide or molecule, a generic slow gradient of 0.1% organic modifier per minute is used to then elute the components. This approach allows excellent separation and recoveries in a single chromatographic step, and even samples of very low purity, as shown in Figure 2a, can be 'rescued' by this technique.  As described by Harris et al., all 800 mgs of the crude peptide (Figure 2a) were loaded onto a semi-preparative, reversed-phased column and subjected to slow-gradient, sample-displacement purification (Figure 2b).  Remarkably, 70 mgs of pure (>99%) peptide (Figure 2c) were obtained in a single chromatographic step.  The detailed analysis of all the fractions, presented in Figure 2d, shows the impressive separation achieved by this method.

Figure 2. a) Analytical RP-HPLC of the crude peptide. The (*) refers to the desired product. b) Semi-preparative RP-HPLC of the crude peptide under slow-gradient, sample displacement conditions. c) Analytical RP-HPLC of the purified peptide. d) Comparison of all material eluted from the semi-preparative purification.  

A wide range of synthetic peptides, including those with non-natural modifications such as biotin and carboxyfluorescein, have been successfully purified in the Brimble lab using this one-step, universal method.  Harris et al. hope that "given the data presented here, this efficient mode of HPLC purification will be embraced by the wider peptide community."

[1]  Harris, P. W. R.; Lee, D. J.; Brimble, M. A., A slow gradient approach for the purification of synthetic polypeptides by reversed phase high performance liquid chromatographyJ. Pept. Sci. 2012, 18(9), 545-555.
[4]  Horváth, C.; Nahum, A.; Frenz, J. H., High-performance displacement chromatographyJ. Chromatogr., A 1981, 218, 365-393. 

Wednesday, September 12, 2012

100% Chemical Free

This is my winning entry for the 2011 Royal Society of New Zealand Manhire Prize for Creative Science Writing.

“How to create a safe and chemical-free home” advises the Queenstown Lakes district council.  Natural Pools NZ in turn, offers “chemical-free swimming”, and a “chemical-free cosmetic” is touted on our evening news. These are some of the many examples of what Chemical and Engineering News has dubbed as the age of “chemophobia”, or the irrational and unsubstantiated fear of chemistry and chemicals.

read the rest at The Listener

Tuesday, September 11, 2012

The Chemical History of Anaesthesia

Introduction: The Age of Agony

The accounts and recollections of surgery before the discovery of anaesthesia are gruesome and it is difficult to imagine what such surgery was truly like. One of the best descriptions of a pre-anaesthesia medical procedure was provided by Fanny Burney, an English author, in a letter to her sister describing her mastectomy: When the dreadful steel was plunged into the breast - cutting through veins - arteries - flesh - nerves - I needed no injunctions not to restrain my cries. I began a scream that lasted unintermittingly during the whole time of the incision - and I almost marvel that it rings not in my ears still! so excruciating was the agony.[1]

While there were some techniques used to provide a type of primitive anaesthesia that included the barbaric methods of nerve compression, deadly intoxication, exsanguination, refrigeration, carotid compression, and even concussion, ultimately a good surgeon was a fast surgeon.[2]
read the rest at Chemistry in New Zealand.

The Ether Monument, the oldest statue in the
historic Boston Public Garden, possibly the
only statue to a chemical in the world.  It was
erected as an expression of gratitude for the
relief of human suffering occasioned by the
discovery of the anaesthetic properties of
sulphuric ether. It displays the description:
There shall be no more pain.

Monday, September 10, 2012

A new rainbow of colour for bioluminescence imaging

d-luciferin and several luciferin 
analogues synthesized that show 
varying colours of emitted light.
Luciferins are a class of molecules that are oxidized by the luciferase enzymes (for example firefly luciferase), producing oxyluciferin and energy which is released in the form of a photon of light (termed bioluminescence).[1]  Bioluminescence is a very sensitive imaging technique, making it one of the most popular methods for visualizing biological processes in vivo, especially in cancer biology research.[1]  Bioluminescencent imaging is preferred over the fluorescent counterpart because no external light source is required,[2] and the lack of endogenous bioluminescent reactions in mammalian tissue allows for near background-free imaging conditions.[3]  Unfortunately, luciferin-based bioluminescence imaging has been limited to monitoring one cell type or feature at a time, as nearly all the enzymes act on the same substrate (D-luciferin).  Additionally, light of wavelengths below 600 nm is absorbed and scattered by cells, which restricts the application of this technique to only superficial tissue depths.[1]

A range of luciferin analogues have been synthesized which show excellent bioluminescence properties and great potential in cell and tissue imaging. This series of luciferin analogues which absorb at different wavelengths has raised the possibility of multicomponent imaging using multiple colours.  Additionally, several of the analogues display red-shifted emission (>600nm) which give their signal better tissue penetration properties.

Researchers led by Stephen Miller from the University of Massachussets Medical School synthesized four alkylaminoluciferin substrates, which showed red-shifted and more intense light emissions than D-luciferin.[4]  They have also engineered several luciferase mutants that yield improved sustained light emission with aminoluciferins in both lysed and live mammalian cells.[5]

More recently, the Stanford University lab of William Moerner developed an analogue with a selenium atom in place of the native sulfur atom at position 1.  The resulting selenoluciferin emits 55% of its light above 600 nm.[6]

Soon after, Jennifer Prescher and co-workers at the University of California developed two further types of luciferin analogues, replacing the sulfur in either of the two heteroaromatic rings with nitrogen.[7]  One compound specifically shows the highest blue-shift of any luciferins.

Depending on the substitution pattern, the luciferin-emitted light can span a broad range, from deep in the red (>600 nm) up to bright blue (around 460 nm).  This is dependent on the identity and nature of the atoms that are substituted – for example, the more strongly electron-donating nature of the alkylamino group was hypothesized to red-shift the spectral properties.  The polar effect of the selenium atom was also predicted to red-shift the emission maximum; both assumptions turned out to be correct.  

While a palette of luciferin colours has now been developed, many of the analogues are still not ideal substrates.  The alkylaminoluciferins show a significant reduction in light output compared to D-luciferin, consistent with product inhibition and hence lower rate of enzymatic turnover.[4]  The selenocysteine analogue also has reduced light output, partly as a result of lower quantum yield.[6]  Some analogues synthesized displayed very limited or even no bioluminescence, making them of little use for imaging studies.[7]  Further tweaking of their structure will be required before luciferin-based multicomponent imaging is possible.

Luciferases and Fluorescent Proteins: Principles and Advances in Biotechnology and Bioimaging 2007, V. R. Viviani, Y. Ohmiya (eds). Transworld Research Network, 2007.

[1] Y.-Q. Sun, J. Liu, P. Wang, J. Zhang, W. Guo. d-Luciferin analogues: a multicolour toolbox for bioluminescence imaging Angew. Chem. Int. Ed. 2012, 51(34), 8428-8430
[2] M. Baker. A broader palette for luciferaseNat. Methods. 2012, 9(3), 225.
[3] D. M. Close, T. Xu, G. S. Sayler, S. Ripp.  In vivo bioluminescent imaging (BLI): noninvasive visualization and interrogation of biological processes in living animalsSensors 2011, 11(1), 180-206.
[4] G. R. Reddy, W. C. Thompson, S. C. Miller.  Robust light emission from cyclic alkylaminoluciferin substrates for firefly luciferaseJ. Am. Chem. Soc. 2010, 132(39), 13586-13587.
[5] K. R. Harwood, D. M. Mofford, G. R. Reddy, S. C. Miller.  Identification of mutant firefly luciferases that efficiently utilize aminoluciferinsChem. Biol. 2011, 18(12), 1649-1657.
[6] N. R. Conley, A. Dragulescu-Andrasi, J. Rao, W. E. Moerner.  A selenium analogue of firefly d-luciferin with red-shifted bioluminescence emissionAngew. Chem. Int. Ed. 2012, 51(14), 3350-3353.
[7] D. C. McCutcheon, M. A. Paley, R. C. Steinhardt, J. A. Prescher.  Expedient synthesis of electronically modified luciferins for bioluminescence imagingJ. Am. Chem. Soc. 2012, 134(18), 7604-7607.

Tuesday, September 4, 2012

Tasty peptides

Taste is simple, right?  Table salt is salty.  Sugar is sweet.  Coffee is bitter.  And lemons are sour.

And of course we mustn't forget umami, the fifth and youngest (most recently agreed on) taste.  Described in 1908 by the Japanese scientist Kikunae Ikeda, it was only accepted quite recently, so that most languages do not even have their own name for it but have adopted the Japanese term.

Yet there is more to taste than simple salts (like NaCl), carbohydrates (like sucrose), alkaloids (like caffeine), and organic acids (like citric acid) or acid salts (monosodium glutamate).  Peptides, despite being subjected to intensive scrutiny of the many biological functions they perform, are rarely considered in light of their taste.  However, peptides essentially cover the whole range of the established tastes, and contribute significantly to the complex flavour of much of the food we eat every day.

In general, and not surprisingly, peptides with acidic residues such as aspartic or glutamic acid tend to have a sour taste.  Sourness is the taste that detects acidity, through the detection of protons (hydrogen ions) that are released when the carboxylic groups of the peptide dissociate.

Salty peptides are few and far between and are often accompanied by a bitter aftertaste. The 1980s saw a flurry of research around the newly discovered L-ornithine-taurine dipeptide, which was reported to have a salty taste without the presence of any sodium. Some controversy erupted over whether the taste was actually due to the peptide, or residual contaminant NaCl, and no salty peptide so far is being used as a salt substitute.

The hydrophobic amino acids phenylalanine, tryptophan, leucine, and tyrosine have bitter tastes, and similarly peptides rich in hydrophobic residues (especially if they are at the C-terminus) are bitter also.  One of the most bitter peptides described is the octapeptide Arg-Arg-Pro-Pro-Pro-Phe-Phe-Phe, with a bitterness comparable to that of strychnine (one of the most bitter molecules known).  While humans have an innate aversion to bitter tasting molecules (as protection from ingestion of poisonous substances, such as strychnine), the rejection of bitter foods is not absolute.  Foods such as beer, tea, and coffee can be highly bitter, yet are beloved world-wide.  In addition, bitter peptides are found in a variety of aged or fermented foodstuffs, including cheese and meaty products such as ham, and other foods containing fermented proteins.

The archetypal umami tastant is of course glutamate, but many peptides have been claimed to be "umami peptides".  The so-called "delicious peptide" - the octapeptide Lys-Gly-Asp-Glu-Glu-Ser-Leu-Ala - was suggested to have an umami potency higher than glutamate itself.  Unfortunately, upon re-examination of this peptide and its fragments, no real umami taste could be detected, casting the existence of umami peptides into doubt.  Yet they have not disappeared: recent research describes umami peptides from peanut hydrolysate and soybean paste.  If these results are confirmed, these umami peptides could be very desirable flavour-enhancing alternatives to the sometimes reviled monosodium glutamate.

But it is probably a sweet peptide that we are most familiar with - the dipeptide aspartame (L-aspartyl-L-phenylalanine methyl ester) is the most used non-caloric sweetener in the world.  Discovered accidentally when a researcher licked his (contaminated) finger to lift a piece of paper, aspartame was found to be 200 times sweeter than sucrose (table sugar).  Following on from this serendipitous discovery, scientists explored many alternatives more rigorously.  They found that Asp cannot be substituted by any other residue, whereas Phe can be replaced by some (but not all) hydrophobic amino acids.  Interestingly, they also found that all the other possible chiral isomers (D-L, L-D, and D-D) are not sweet at all, but quite bitter.  Other modifications, on the other hand, have led to the discovery of super-aspartame molecules, such as neotame.  It has a 3,3-dimethylbutyl group attached to the amino group of the aspartic acid, and is around 10,000 times as sweet as sucrose.  Most jurisdictions have now approved its use in food.

Proteins and peptides make up a large part of the foods we eat every day, and it is clear that they play a significant role in the complex chemical interplay that is taste.  Mostly, the peptides seem to contribute sweet, bitter, and sour tastes, but some evidence suggests salty and umami peptides exist also.  While the taste of the sour and salty peptides is probably simply due to the presence of the charged terminals and side chains, bitter and sweet receptors are clearly activated by specific electronic and conformational features of a specific peptide (as demonstrated by the various isomers of aspartame).  Peptides are therefore extremely useful tools for researching taste receptor function and leading to a better understanding of taste and taste perception.