Two kings of chirality

The infamy of thalidomide brought home the importance of understanding the optical activity of drug molecules. As two of the finest living analytical chemists – Laurence Barron and Werner Hug – call time on their illustrious careers we take a look back at how they cracked chirality.

THANKS to Laurence Barron and Werner Hug’s immense contribution to Raman vibrational spectroscopy, we have a valid technique for analysing the chirality of both simple and complex molecules and even viruses.

Professor Barron has already left his post at Glasgow University and Professor Hug is retiring from his post at Freibourg University, Switzerland, this summer. Both men have been credited with providing insights into Raman Optical Activity, and using it to create a spectroscopic technique which has helped reveal priceless information about the structure of both simple and complex molecules.

When the Nobel committee for chemistry casts around for potential candidates, members could do worse than give the work of Barron and Hug, some serious attention. Between them they have developed new instruments to help elucidate three-dimensional molecular structure.  They have inspired synthetic chemistry of the highest order and they have demonstrated that ab initio calculations by theoreticians and quantum chemists are within a whisper of ‘chemical truth’.
 
Southampton-born Laurence Barron spent most of his working life researching the phenomenon of ROA he discovered as a PhD student at Oxford. His legacy will continue as others carry his research forward and the instruments invented make inroads into the precise nature three-dimensional molecular structure or chirality.

He began his studies four decades ago and against all the odds his work has helped interpret the chirality of proteins, complex viruses and other biological molecules in real time.

Werner Hug, collaborated on the creation of the first ROA instrument and is a past winner of the Ruizika prize for chemistry.

Most people these days have some understanding of the concept of chirality – the fact that the image of a glove in a mirror is not the true image of the glove.  What’s in the mirror is the chiral opposite. This is not the case for say a toothbrush or fully symmetric object. This property of the glove is its chirality and it exists even in the simplest of molecules.

In nature all amino acids found on Earth are left-handed or homochiral a fact which has sparked speculation about the origins of life. John Cronin and Sandra Pizzarello have shown that some amino acids which may have fallen to Earth on meteorites are more left handed than right and therefore postulated that that we are made of L-amino acids which originated in space. Also the Bonner hypothesis, proposes that left handed radiation in space (from a rotating neutron star for example) could lead to left handed amino acids in space, which would explain the left handedness of amino acids in meteorites.

It is only in the last three of four decades that the implications of chirality have become apparent and that the properties of one enantiomer of a molecule might be different from the other.

For example with Limonene, a cyclic terpene (not to be confused with lemonene the disinfectant) the right (D)- naturally occurring enantiomer of the molecule has the aroma of orange while the (L)-enantiomer has a piney terpentine aroma.

The critical nature of chirality hit the headlines in the case of thalidomide. Used in the seventies to combat morning sickness in pregnant women one of the two enantiomers it was found that only one of the enantiomers was responsible for the severe deformities that developed in some foetuses.

Although it has been demonstrated that separating and using the harmless enantiomer only would not have prevented the tragedies because of the tendency of the single enantiomer to racemate to its chiral opposite version it was the ultimate lesson that on the molecular level drugs developers must fully understand chirality and conformation if the quest for designer drugs to fight other diseases was to be successful.

The discovery of Raman Optical Activity is now seen as a major step forward in the quest. Unlike X-ray crystallography, ROA can be applied to molecules in their natural state ¬– in aqueous solution.

It has a marked advantage over nuclear magnetic resonance (NMR) in that it is not limited by molecular weight. But perhaps its greatest contribution has been to the world of pharmaceutical research thanks to its ability to determine the absolute stereochemical conformation of small drug molecules.

ROA depends on a very delicate measurement of what has been described as a twist in the light. When beams of right and left circularly polarised light interact with an optically active or chiral molecule they produce vibrational Raman spectra with bands of slightly differing intensities. The difference between the bands is tiny because Raman scattering is itself a weak phenomenon which from the beginning made observing the optical activity effect experimentally a formidable challenge.

It required a measuring sensitivity of one part in a 1,000 to 100,000. As a result, the rate of development of ROA has been driven by advances in optical and solid state technology.

Barron had worked with the theoretician David Buckingham at Cambridge and decided to build an instrument to demonstrate the ROA experimentally. His first attempts failed and it looked hopeless but he was determined to make it work and eventually he succeeded. By then working in Glasgow he created a technique that could be used to distinguish not only between the enantiomers but also provide detailed stereochemical information.

Detecting ROA requires an Argon laser and some elegant optics and electronics. The laser beam is first converted via a polarised beam which oscillates between left and right. After passing through the sample, which may be in pure liquid form or in solution, the Raman scattered light is collected and measured.

The spectrum shows the difference in intensity (lR-lL) between right and left circularly polarised light. This is compared with the spectrum showing the total Raman intensity (lR+lL). Mirror image enantiomers give spectra of opposite signs.

It wasn’t until the late eighties that Barron’s discoveries started to achieve their true potential.  Together with Lutz Hecht he built a new ROA spectrometer which took advantage of three basic technological advances made at the time.

First the instrument was configured to select back scattered light from the sample. ROA is maximised in the backward direction. The second was the introduction of a charge-coupled device CCD which provided a large increase in the sensitivity of detection. CCD can register simultaneously just a handful of photons over a wide range of wavelengths.

The use of new back-thinned CCDs increased the quantum efficiency to about 80%.The third timely breakthrough in hardware was the use of a holographic notch filter to block the Rayleigh line permitting a fast single grating spectrograph to be employed.

Laurence Nafie editor in chief of the Journal of Raman Spectroscopy who is based at the University of Syracuse has known Laurence Barron all his academic life.

He said: “In 1975, Laurence was working on ROA and my research focused on development of the sister technique called VCD, but I worked on ROA as well. We first met at the 1976 Conference on Raman Spectroscopy in Freiburg, Germany, and before returning to the US I visited Laurence at his home and laboratory in Glasgow.”

“In a nutshell, ROA was and is very difficult to measure with interfering artifacts, and it is only within the last decade, thanks to a design by Professor Hug that elimination of artifacts could be automated.

“Hug’s design is the basis of the commercial instrument from BioTools, the development of which in Syracuse, New York that I oversaw and continue guide in manufacture, improvement and production.”

The work has been an inspiration in other areas. It was a team of research students working under Hug who set a new standard in synthetic dexterity. Measuring Raman optical activity (ROA), they have confirmed the spatial arrangement of a molecule with almost impossibly subtle chirality: (R)-[2H1, 2H2, 2H3]-neopentane.

In the molecular world the two enantiomers of (R)-[2H1, 2H2, 2H3]-neopentane are so similar that it was thought to be impossible to confirm whether or not it has this important chemical property.

To establish its chirality, one of Werner Hug’s group, Jacques Haesler and colleagues from the University of Fribourg, Switzerland, first had to synthesise their tricky molecule. For its spatial arrangement to be detectable, they made a ‘chirally deuterated’ version, replacing six of its 12 hydrogens with deuterium.

Professor Hug pointed out at the time this process was such a challenge that, ‘despite the basic interest, it had never even been considered before.’ But this was only the beginning. The team then turned their compound into the ultimate test for their ultra-sensitive ROA measurement technique.

Laurence Barron said: “In some senses ROA is now the most powerful and incisive of any spectroscopic technique for obtaining structural information about chiral molecular structures. ROA can now be applied to the whole gamut of chiral molecules, ranging from this most subtle example through to the central molecules of life: proteins, carbohydrates and even intact viruses.”

Laurence Barron and Werner Hug can look back with deep satisfaction on their life’s work on ROA. Said Prof Barron: “Never in my wildest dreams did I think it would ever become so powerful and widely applicable.”

Despite this Werner Hug regrets the lack of enthusiasm at Freibourg for ROA work. “I could have stayed on for another two years but decided to rather spend my energy on helping to rebuild the Freibourg ROA instrument at a place where ROA is likely to have a big future, and that’s at Thomas Buergi’s laboratory in Heidelberg. The problem in Freibourg is that they have oriented themselves towards material sciences.  The place is just too small to also support spectroscopy.

“I will continue to work on computational aspects of VOA and vibrational spectroscopy in general. With some leftover industrial and with personal money I have put together, and gotten operational (with the help of my last Ph.D. student, a Linux freak – Linux is a free operating system) a small cluster during the past few months. It will allow me to do serious computations independent of any sort of institution, somewhat like science was done by noblemen in past centuries.”

Hug’s view on Thalidomide

Recently the British Government issued an apology to the victims of the thalidomide and agreed to pay victims a further £20m on top of the £28m paid out by the Distillers Company in the 1970s. Only Germany had more users than the UK and Grünenthal paid out much more to the victims there

Werner Hug said: “Greater interest at an earlier stage in studying the differing biochemical properties of enantiomers and their metabolites might have helped prevent what happened with thalidomide. More funding for spectroscopic work is needed now but I doubt that generous funding in the sixties would not have made a difference to the sad outcome.

“On the positive side some important subsequent funding in this area was doubtless stimulated by the thalidomide story.

“Work in this area might have profited considerably from better chir-optical techniques, but funding of chir-optical methods would not have led to a more careful attitude by the pharmaceutical industry.”

By Dermot Martin


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