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HYDROGENATE ALIPHATIC AMIDES IN A FLOW REACTOR
Amide reduction is a common synthetic route to amines. Conventional amide-reduction protocols often involve highly reactive reducing agents and generate large amounts of waste that create safety and environmental problems.
Catalytic hydrogenation of amides, however, is highly atom-economic and generates little waste (water is the only byproduct). Inspired by developments in continuous-flow reaction systems, D. J. Cole-Hamilton and coauthors at the University of St. Andrews and Queen’s University (Belfast, both in the UK) designed and built a continuous-flow hydrogenation reactor for efficient, selective amide reduction with a heterogeneous catalytic system.
In the continuous-flow reactor, amide solutions, hydrogen, and supercritical CO2 (scCO2) are simultaneously fed through a preheater to a vertical-bed reactor packed with a 4% Pt−4% Re/TiO2 catalyst. With a hydrogen flow rate of 190 mL/min, 120ºC temperature, and 20 bar hydrogen pressure, N-methylpyrrolidin-2-one (1) in hexane (0.33 M, flow rate 0.06 mL/min) is reduced to N-methylpyrrolidine (2) with 100% conversion and selectivity. The concentration and flow rate of 1 in hexane can be increased to 0.67 M and 0.06 mL/min, respectively, without a significant decrease in conversion or selectivity. Similarly, N-methylpropanamide (3) can be converted to N-methylpropylamine (4) with up to 99% conversion and 86% selectivity.
This method has several advantages over the batch mode, including high mass recovery (with decane as the solvent) and minimal catalyst leaching. The scope of the catalytic system, however, is currently limited to aliphatic amides.
Xin Su, ChemCatChem, 5, 2843–2847 (2013)
American Chemical Society
UNIQUE CHANGE IN PROTEIN STRUCTURE GUIDES PRODUCTION OF RNA FROM DNA
Study sheds light on critical molecular process
One of biology’s most fundamental processes is something called transcription. It is just one step of many required to build proteins—and without it life would not exist. However, many aspects of transcription remain shrouded in mystery. But now, scientists at the Gladstone Institutes are shedding light on key aspects of transcription, and in so doing are coming even closer to understanding the importance of this process in the growth and development of cells—as well as what happens when this process goes awry.
In the latest issue of Molecular Cell, researchers in the laboratory of Gladstone Investigator Melanie Ott, MD, PhD, describe the intriguing behaviour of a protein called RNA polymerase II (RNAPII). The RNAPII protein is an enzyme, a catalyst that guides the transcription process by copying DNA into RNA, which forms a disposable blueprint for making proteins. Scientists have long known that RNAPII appears to stall or “pause” at specific genes early in transcription. But they were not sure as why.
“This so-called ‘polymerase pausing’ occurs when RNAPII literally stops soon after beginning transcription for a short period before starting up again,” explained Dr. Ott, who is also a professor of medicine at the University of California, San Francisco, with which Gladstone is affiliated. “All we knew was that this behaviour was important for the precise transcription of DNA into RNA, so we set out to understand how, when and—most importantly—why.”
The research team focused their efforts on a segment of RNAPII called the C-terminal domain, or CTD. This section is most intimately involved with transcription regulation. Previous research had found that CTD’s chemical structure is modified before and during transcription. However, the combinations of modifications as well as precisely how they influence or control transcription remained unclear. So in laboratory experiments on cells extracted from mammals, the researchers took a closer look.
The first breakthrough came when the research team identified a new type of modification, known as acetylation, which regulated transcription.
“Our next breakthrough occurred when we pinpointed the precise locations on the CTD where acetylation occurred—and realized it was unique to higher eukaryotes,” explained Sebastian Schröder, PhD, the paper’s first author. “We then wanted to see how this mammalian-specific acetylation fit into the realm of polymerase pausing.”
Now that the team knew where the CTD became acetylated, their next goal was to find out when. Clues to the timing of acetylation came in experiments where they mutated RNAPII so that CDT was unable to become acetylated. In these cases, the length of polymerase pausing dropped, and the necessary steps for the completion of transcription failed to occur. Additional experiments revealed the elusive timeline of acetylation and transcription.
“RNAPII binds to DNA to prepare for transcription. Shortly after that we see polymerase pausing—at which point the CTD becomes highly acetylated,” continued Dr. Shröder. “Soon after the pause, CTD is then deacetylated—the original modification is reversed—and transcription continues without a hitch.”
Polymerase pausing is not unique to mammals—in fact it was characterized in HIV, the virus that causes AIDS, many years ago—but the fact that the CTD becomes acetylated just before or during the time when transcription is paused appears to be unique. Drs. Ott and Schröder argue that CTD acetylation is a stabilizer, preparing RNAPII for efficient completion of transcription and slowing down the process to make sure everything is functioning correctly—not unlike the final ‘systems check’ a pilot must perform before takeoff.
These findings offer important insight into the relationship between acetylation and transcription. And given the importance of transcription in the growth and maturation of cells in general, the team’s result stands to inform scientists about a variety of cellular processes. These include, for example, the mechanisms behind stem-cell development and what happens when normal cellular growth spirals out of control, such as in cancer.
“However, there is still much we don’t know about acetylation as it relates to transcription,” said Dr. Ott. “For example, if CTD acetylation is important for stabilizing transcriptional pausing, why do we also see CTD acetylation at non-paused genes, although at different locations? Further, we believe there may be other steps in the transcription cycle that depend upon acetylation. Our most immediate goal is to find them. By doing so, we hope to deepen our understanding of one of nature’s most elegant biological processes.”
Dr. Schröder performed this research at Gladstone while completing his PhD at the University of Heidelberg, Germany. Eva Herker, PhD, Sean Thomas, Phd, Katrin Kaehlcke, Sungyoo Cho, Katherine Pollard, PhD, John Capra, PhD and Benoit Bruneau, PhD, also participated in this research at Gladstone, which was supported by the National Institutes of Health, the National Institute of Environmental Health Sciences, the Boehringer Ingelheim Fonds, the Human Frontiers Science Program and an E.G.G. fellowship.
About the Gladstone Institutes
Gladstone is an independent and nonprofit biomedical-research organization dedicated to accelerating the pace of scientific discovery and innovation to prevent, treat and cure cardiovascular, viral and neurological diseases. Gladstone is affiliated with the University of California, San Francisco.
WHY STEM CELLS NEED TO STICK WITH THEIR FRIENDS
Scientists at University of Copenhagen and University of Edinburgh have identified a core set of functionally relevant factors which regulates embryonic stem cells’ ability for self-renewal. A key aspect is the protein Oct4 and how it makes stem cells stick together. The identification of these factors will be an important tool in devising better and safer ways of making specialised cells for future regenerative cell therapies for treatment of diseases like diabetes and Parkinson’s disease. The results have just been published in the scientific journal Current Biology.
Embryonic stem cells stick together (green, left image) while they express Oct4 protein in their nuclei (magenta, left). When Oct4 is removed their shape changes and their adhesion to each other is reduced (right panel, nuclei in blue). Scientists have known that the protein Oct4 plays a key role in maintaining the embryonic stem cells in pure form by turning on stem cell genes, however up until now it has not been know which of the 8.000 or more possible genes that Oct4 can choose from actually support self-renewal.
By comparing the evolution of stem cells in frogs, mice and humans, scientists at the Danish Stem Cell Center (DanStem) and The MRC Centre for Regenerative Medicine in Edinburgh have now been able to link the protein Oct4 with the ability of cells to stick together. They found that for embryonic stem cells to thrive they need to stick together and Oct4’s role is to make sure they stay that way.
“Embryonic stem cells can stay forever young unless they become grown-up cells with a specialised job in a process called differentiation. Our study shows that Oct4 prevents this process by pushing stem cells to stick to each other,” says Dr Alessandra Livigni, Research Fellow at the University of Edinburgh.
Identification of specific genesi
The research teams in Edinburgh and Copenhagen successfully identified 53 genes, out of more than 8.000 possible candidates that together with Oct4, functionally regulate cell adhesion. Almost like finding needles in a haystack the scientists have paved the way for a more efficient way of maintaining stem cells as stem cells.
"Embryonic stem cells are characterized, among other things, by their ability to perpetuate themselves indefinitely and differentiate into all the cell types in the body – a trait called pluripotency. Though to be able to use them medically, we need to be able to maintain them as stem cells, until they're needed. When we want to turn a stem cell into a specific cell for example; an insulin producing beta cell, or a nerve cell like those in the brain, we'd like this process to occur accurately and efficiently. We cannot do this if we don't understand how to maintain stem cells as stem cells,” says Professor Joshua Brickman from DanStem, University of Copenhagen.
Future potential
As well as maintaining embryonic stem cells in their pure state more effectively, this new insight will also enable scientists to more efficiently manipulate adult cells to revert to a stem cell like stage known as induced pluripotent stem cells (iPS cells). These cells have many of the same traits and characteristics as embryonic stem cells but can be derived from the patients to both help study degenerative disease and eventually treat them.
“This research knowledge has the potential for us to change the way we grow stem cells, enabling us to use them in a less costly and more efficient way. It will help us devise better and safer ways to create specialised cells for future regenerative medicine therapies,” concludes Professor Joshua Brickman.
University of Copenhagen
Genevac’s new generation heat transfer plates for centrifugal evaporators enable almost any deep well plate to be dried up to 50 percent faster than previously possible. Evaporation from microplates, especially deep well plates, can be extremely slow. The design of deep well plates makes it particularly difficult for an evaporator system to transfer enough heat for rapid drying, especially with higher boiling point solvents such as water or DMSO. In addition, often the wells in deep-well plates are supported above the level of the skirt and need support to prevent the plate from deforming in a centrifugal evaporator. The new Genevac heat transfer plate design has a central flexible pad that deforms and moulds itself to the exact shape of your deep well plate. This intimate contact achieves the same level of heat transfer as from specially cut aluminium heat transfer plates but at a much lower cost. In addition the novel flexible heat transfer pad is proven to eliminate plate deformation. www.evaporatorinfo.com/info12_e.html NON-CONTACT OPTICAL THICKNESS SENSORS Precitec Optronik launches the newest member of its well established line of non-contact optical thickness sensors. Specifically designed to meet the measurement needs for manufacturers of plastic films, preforms, PET bottles, blisters (PET, PP, EVOH), balloons and many other plastic parts, the CHRocodile K is the key to quality in plastic manufacturing, as the company refers. The CHRocodile K truly is the key to quality control when it comes to plastics. The high measurement speed allows for 100 percent control, inline as well as offline. Variations during the production process are seen in real time, before they can have an impact on the production quality. Its compact optical probe requires access from only one side and can be easily integrated into a confined space. The robust measuring technology delivers excellent results in all environmental conditions. www.precitec.de ROCKET 4D RECEIVES POSITIVE CUSTOMER COMMENTARY Genevac has reported very positive feedback from early adopters of their Rocket 4D – a fully automated system for automatic drying or concentration of very large volumes up to 100 litres. Launched worldwide during the summer of 2013 – Genevac has received considerable interest, from laboratories drawn by the ease of use, automated productivity and performance of the Rocket 4D. A chemistry scale-up laboratory at a major Pharma company, running kilo scale preparative SFC, were drawn by the unattended automation offered by the Rocket 4D. Traditionally removing large volumes of methanol from their sample fractions using a number of rotary evaporators required a dedicated operator to top-up dry ice in cold traps, feed the systems with more product and watch the systems continually. The operator commented: "With the Rocket 4D you simply load your sample, select a method, press start, and walk away – the system will do the rest”. He added that he “got his life back” when the 4D arrived and now is able to spend his time much more productively. Compact in size the Rocket 4D allows you to dry or concentrate customers’ samples with complete confidence as it uses proprietary vacuum technology to suppress solvent bumping and foaming – problems associated with sample loss when using large scale rotary evaporators. The Rocket 4D comes as standard with a single, 5 litre 316 stainless steel vessel for drying or concentrating product. www.Genevac.com/Rocket4D SAFC® COMMERCIAL & CATSCI LTD Sigma-Aldrich Corporation has announced that SAFC® Commercial, its custom manufacturing services business unit, has entered into a services agreement with CatScI Ltd. SAFC customers will immediately benefit from the relationship through rapid access to new reactions, homogeneous and heterogeneous catalysis, biocatalysts, solvent selection and process development, in addition to metal removal, recovery and recycling services. www.sigma-aldrich.com/safc www.catsci.com
